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Isolation, purification and characterization of an extracellular proteolytic enzyme of Planococcus citreus

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Title:
Isolation, purification and characterization of an extracellular proteolytic enzyme of Planococcus citreus
Creator:
Alvarez, Ricardo Javier, 1954-
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English
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xiv, 131 leaves : ill. (some col.) ; 28 cm.

Subjects

Subjects / Keywords:
Broths ( jstor )
Enzyme activity ( jstor )
Enzyme substrates ( jstor )
Enzymes ( jstor )
Gelatins ( jstor )
Gels ( jstor )
Incubation ( jstor )
Molecular weight ( jstor )
Shrimp ( jstor )
Sodium ( jstor )
Dissertations, Academic -- Food Science and Human Nutrition -- UF
Food Science and Human Nutrition thesis Ph. D
Marine microbiology ( lcsh )
Planococcus citreus
Shrimps -- Microbiology ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1981.
Bibliography:
Bibliography: leaves 120-130.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Ricardo J. Alvarez.

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ISOLATION, PURIFICATION AND CHARACTERIZATION OF
AN EXTRACELLULAR PROTEOLYTIC ENZYME OF Planococcus citreus

BY

RICARDO J. ALVAREZ




























A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA

1981

































A mis queridos padres:

Gracias por la ayuda brindada, el amor,

el apoyo moral y la vision de avanzar en la vida.

Con todo mi amor
















ACKNOWLEDGMENTS

The author expresses his deepest gratitude to Dr. J. A. Koburger,

his major advisor, for his exceptional patience, guidance and encourage-

ment throughout the course of this work. The author also wishes to

acknowledge the advice, support and all the help given by Drs. R. H.

Schmidt, W. S. Otwell, J. L. Oblinger and G. Bitton as members of his

supervisory committee. Thanks go to Dean J. L. Fry for his under-

standing and support in delicate times.

Special appreciation is extended to Dr. J. R. Kirk for procuring a

much needed assistantship for the first two years of this research, to

Margie Summers for her beautiful graphic work, Beth Beville, Diane Dobsha,

Beth Johnsen and Mike Pyle for their patience in typing sections of this

dissertation. A very .special thank you goes to Melissa Michaels for the

typing of the final copy of this dissertation.

In addition, the author expresses thanks to Sam May for his help

and support around the laboratory and to Suzanne Davidson, Bridget

Walker and Janet Eastridge for their aid when needed. Thanks go to Dr.

L. D. Ingram for his constructive comments.

Recognition is also given to the faculty, staff and secretaries of

the Food Science and Human Nutrition Department for their cooperation

and to all fellow graduate students who shared with the author the years

at the University of Florida.




iii









Finally, he is deeply grateful to Mary Brannigan for her love,

patience and devotion, providing sentimental support and help through-

out all phases of his course work. He thanks her for her understanding

and provision of many reasons to pursue all achievements in life.



















































iv
















TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS .................................................. iii

LIST OF TABLES ................................................... vii

LIST OF FIGURES ................................................. ix

ABSTRACT ........................................................ xii

INTRODUCTION ............ .............. ..................... .. 1

LITERATURE REVIEW ............................................... 3

Shrimp Spoilage ..... ...................................... 3
Microbiological Characteristics of Shrimp .................. 4
Measurement of Shrimp Spoilage .............................. 7
Characteristics of Planococcus citreus ...................... 11
Proteolytic Enzymes ......................................... 17
Measuring Proteolytic Activity .............................. 19

MATERIALS AND METHODS ........................................... 25

Planococcus citreus Cultures ................................ 25
Determination of Proteolytic Activity ....................... 25
Efficacy of 5% Trichloroacetic Acid (TCA) ................... 29
Substrate Characteristics ................................... 33
Determination of Enzyme-Substrate Mixture Reaction Time ..... 33
Growth Medium and Enzyme Production ......................... 36
Optimization of Enzyme Activity to Growth and Cell Number ... 36
Effect of Incubation Temperature on Enzyme Production and
Activity .................................................. 38
Purification of the Extracellular Enzyme(s) ................. 39
Ammonium Sulfate Precipitation ......................... 39
Molecular Sieve Chromatography ......................... 39
Ion-Exchange Chromatography ............. ............... 40
Confirmation of Enzyme Purity ............................... 42
Characterization of the Proteolytic Enzyme(s) .............. 43
Molecular Weight Determination ......................... 43
Determination of the Purified Enzyme-Substrate Mixture
Reaction Time ........................ ....... ...... 43
Effect of Ionic Strength on Enzyme Activity ............ 44
Determination of Optimum pH .......................... 44
Determination of Optimum Temperature ................... 46
Thermal Stability ...................................... 46
Effect of Sodium Chloride Concentration ................ 47


v









TABLE OF CONTENTS (continued)

Page
Effect of Sodium Bisulfite Concentration ............... 47
Effect of Enzyme Concentration ......................... 47
Effect of Substrate Concentration .................. 47
Effect of Metal Ions on Enzyme Activity ................ 48
Effect of Various Reagents on Enzyme Activity .......... 48
Dipeptidase Activity ................................... 49
Enzyme Induction Studies .................................... 49

RESULTS AND DISCUSSION ........................................... 52

Proteolytic Activity of Cellular Fractions .................. 52
Growth Medium and Enzyme Production ......................... 55
Effect of Incubation Temperature on Enzyme Production and
Activity ................................................ 60
Purification of Extracellular Enzyme(s) ..................... 66
Purity of the Extracellular Proteolytic Enzyme .............. 71
Characterization of the Extracellular Proteolytic Enzyme .... 77
Molecular Weight Determination ......................... 77
Effect of Ionic Strength on Enzyme Activity ............ 80
Optimum pH Determination ............................... 82
Optimum Temperature Determination ...................... 84
Thermal Stability ...................................... 84
Effect of Sodium Chloride Concentration ............... 87
Effect of Sodium Bisulfite Concentration ............... 90
Effect of Enzyme Concentration ........................ 90
Effect of Substrate Concentration ........................ 93
Effect of Metal Ions on Enzyme Activity ................ 98
Effect of Various Reagents on Enzyme Activity .......... 101
Dipeptidase Activity ................................. 104
Enzyme Classification ................................... . 104
Enzyme Induction Studies .................................... 104

SUMMARY AND CONCLUSIONS ....................................... . 115

LITERATURE CITED ................ .............................. 120

BIOGRAPHICAL SKETCH ................................. ... ........ 131
















vi















LIST OF TABLES

TABLE Page

1 Chemical and physical tests available to measure shrimp
quality .................................................. 8

2 Hydrolysis of various protein sources by selected strains of
Planococcus citreus at 25 C ................................ 15

3 Proximate composition of the shrimp protein preparation .... 34

4 Composition of yeast carbon base medium .................... 50

5 Proteolytic activity at 35 C for 15 min (pH 8) of cellular
fractions obtained from Planococcus citreus grown in
Trypticase Soy Broth (TSB) using gelatin and shrimp protein
as substrates .............. ................... .... 53

6 Units of enzyme activity per cell per hour (m) of Planococcus
citreus grown in Trypticase Soy Broth (TSB), Plate Count
Broth (PCB) and Nutrient Broth (NB) at mid-log phase ....... 61

7 Enzyme activity measured at 5, 20 and 35 C (pH 8) of the
cell-free broths of Planococcus citreus grown in Trypticase
Soy Broth (TSB) at 5, 20 and 35 C for 108, 72 and 36 hrs,
respectively ...................... ..... ...... .............. 64

8 Purification of an extracellular proteolytic enzyme from
Planococcus citreus ....................................... 67

9 Proteolytic activity at 35 C for 15 min (pH 8) of various
ammonium sulfate fractions of the cell-free broth of
Planococcus citreus ..................................... 68

10 Effect of various metal ions on the activity (assayed at 35
C for 10 min (pH 8)) of the Planococcus citreus extracellu-
lar enzyme ................................................. 100

11 Effect of various reagents on the activity (assayed at 35 C
for 10 min (pH 8)) of the Planococcus citreus extracellular
enzyme ..................................................... 102

12 Dipeptidase activity of the Planococcus citreus extracellular
enzyme .............. ..................................... 105




vii








LIST OF TABLES (continued)

TABLE Page

13 Units of enzyme activity per cell per hour (m) of Planococcus
citreus grown in yeast carbon base supplemented with shrimp
protein and/or yeast extract at midlog phase ............... 111

14 Units of enzyme activity per cell per hour (m) of Planococcus
citreus grown in various media ............................. 114
















































vii















LIST OF FIGURES

FIGURE Page

1 Photomicrograph of Planococcus citreus cells showing
morphology and flagellation .............................. 14

2 Comparison of the Fluorescamine technique and the Lowry
procedure for determining protein concentration ........... 23

3 Effect of pH adjustment of gelatin-trichloroacetic acid
(TCA) filtrates on fluorescence intensity ............ ... 28

4 Excitation (curve A) and fluorescence (curve B) spectrum
for the reaction of a gelatin-trichloroacetic acid (TCA)
filtrate with fluorescamine at pH 8 ....................... 30

5 Excitation (curve A) and fluorescence (curve B) spectrum
for the reaction of a shrimp protein-trichloroacetic acid
(TCA) filtrate with fluorescamine at pH 8 ................. 31

6 Efficacy of trichloroacetic acid (TCA) in terminating the
enzyme-substrate reaction ................................. 32

7 Increase in fluorescence intensity using the shrimp protein
preparation as substrate after incubation with cell-free
broth for 1 hr at 35 C .................................... 35

8 Increase in fluorescence intensity using gelatin as
substrate and various amounts of cell-free broth after
incubation at 35 C for 1 hr .......... .................. 37

9 Outline of steps for the purification of the extracellular
protease(s) of Planococcus citreus ........................ 41

10 Increase in fluorescence intensity following incubation of
gelatin and shrimp protein substrate with purified enzyme
for up to 1 hr at 35 C .................. .................. 45

11 Spectrophotometric growth curves of Planococcus citreus in
Plate Count Broth, Nutrient broth and Trypticase Soy Broth
at 20 C ..............*................ .................... 56

12 Aerobic plate counts of Planococcus citreus incubated in
Plate Count Broth, Nutrient Broth and Trypticase Soy Broth
at 20 C for 96 hrs *...**.... ............*** ............... 58



ix









LIST OF FIGURES (continued)

FIGURE Page

13 Enzyme activity at 35 C for 15 min (pH 8) of the cell-free
broth of Planococcus citreus grown in Plate Count Broth,
Nutrient Broth and Trypticase Soy Broth for up to 96 hrs .. 59

14 Spectrophotometric growth curves of Planococcus citreus in
Trypticase Soy Broth incubated at 5, 20 and 35 C .......... 62

15 Separation of the proteins present in the 55-70% ammonium
sulfate fraction using Sephacryl S-200 ................... 70

16 Ion-exchange chromatography using DEAE-SephadexR A-50 of
the pooled active fractions obtained in the molecular sieve
chromatography purification step .......................... 72

17 Acrylamide gel electrophoresis of the purified enzyme of
Planococcus citreus ....................................... 74

18 Acrylamide gel electrophoresis of increasing concentration
of the purified extracellular enzyme of Planococcus citreus 75

19 Calibration curve used for the molecular weight estimation
of Planococcus citreus proteolytic enzyme using Sephacryl
S-200 column chromatography ............................ 78

20 Calibration curve used for the molecular weight estimation
of Planococcus citreus proteolytic enzyme using acrylamide
gel electrophoresis .....................9................ 79

21 Effect of ionic strength on the activity of the extracell-
ular proteolytic enzyme of Planococcus citreus ............ 81

22 Optimum pH of the extracellular proteolytic enzyme of
Planococcus citreus ..................................... 83

23 Temperature optimum of the extracellular proteolytic enzyme
of Planococcus citreus .................................... 85

24 Thermal stability of the enzymes in the cell-free broths of
Planococcus citreus grown at 5 and 35 C and of the
purified enzyme ............ . ............................. 86

25 Effect of sodium chloride concentration on enzyme activity 89

26 Effect of sodium bisulfite concentration on enzyme activity 91

27 Effect of enzyme concentration on enzyme activity ......... 92

28 Effect of gelatin concentration on the reaction rate of the
Planococcus citreus extracellular enzyme .................. 94



x









LIST OF FIGURES (continued)

FIGURE Page

29 Lineweaver-Burk plot of the Planococcus citreus extracell-
ular enzyme illustrating V and K values using gelatin
as substrate ................... .... ................... 95

30 Effect of shrimp protein concentration on the reaction rate
of the Planococcus citreus extracellular enzyme ........... 96

31 Lineweaver-Burk plot of the Planococcus citreus extracell-
ular enzyme illustrating V and K values using shrimp
max m
protein as substrate ..... ............ ......... 97

32 Growth of Planococcus' citreus in Yeast Carbon Base supple-
mented with shrimp protein and/or yeast extract at 20 C ... 107

33 Proteolytic enzyme activity of the cell-free broth of
Planococcus citreus grown in Yeast Carbon Base supplemented
with shrimp protein and/or yeast extract ................ 108

34 Growth of Planococcus citreus in Trypticase Soy Broth with
and without shrimp protein at 20 C ........................ 111

35 Proteolytic activity of the cell-free broth of Planococcus
citreus grown in Trypticase Soy Broth with and without
shrimp protein ................................ ..... . 112




























xi















Abstract of Dissertation Presented to the Graduate
Council of the University of Florida in Partial Fulfillment
of the Requirements for the Degree of Doctor of Philosophy

ISOLATION, PURIFICATION AND CHARACTERIZATION OF
AN EXTRACELLULAR PROTEOLYTIC ENZYME OF Planococcus citreus

By

Ricardo J. Alvarez

March 1981

Chairman: J. A. Koburger
Major Department: Food Science and Human Nutrition

Planococcus citreus is a gram-positive marine bacterium commonly

found in fresh and iced shrimp. Various studies have indicated that it

may contribute to spoilage of this valuable marine resource. In order

to understand the contribution of this organism to the degradation of

shrimp as well as other proteins, an investigation was undertaken to

study the extracellular proteolytic enzyme(s) of this organism. Results

indicated that the major portion (>95.0%) of the proteolytic activity

resided in the extracellular fraction.

Under the conditions tested, maximum extracellular enzyme produc-

tion occurred in Trypticase Soy Broth (TSB) as observed by the highest

m value (units of enzyme activity per cell per hour). In addition, the

cell-free broth obtained from P. citreus cells grown at 5 C for 108 hrs,

20 C for 72 hrs and 35 C for 36 hrs exhibited enzyme activity towards

shrimp protein at all three enzyme-substrate incubation temperatures (5,

20 and 35 C).




xii









P. citreus was grown in Trypticase Soy Broth at 20 C for 72 hrs.

Centrifugation, ammonium sulfate precipitation, SephacrylR S-200 Super-

fine molecular sieve chromatography, DEAE-SephadexR A-50 ion exchange

chromatography and acrylamide gel electrophoresis were used to purify

the extracellular enzyme(s). The enzyme was purified 26.50 fold (using

the fluorometric technique for activity measurement), and recovery of

the enzyme was above 49%. Gelatin and shrimp protein were used as sub-

strates throughout the study. The molecular weight of the purified

protease was approximately 29,000 as measured by SephacrylR S-200 column

chromatography and acrylamide gel electrophoresis.

Maximum activity of the enzyme was at pH 8 and 35 C. Ionic

strengths of above 0.83 (0.75 M NaCI) decreased the activity of the

extracellular enzyme. Heat treatment at 65 C for 15 min destroyed the

activity of the purified enzyme. However, 1.0% of the residual enzyme

activity was still present in the cell-free broth of P. citreus grown at

35 C for 36 hrs. In contrast, 15 min at 75 C were necessary to reduce

99.0% the activity of the enzymes in the cell-free broth of P. citreus

grown at 5 C for 108 hrs. When shrimp protein was used as substrate,

sodium chloride concentrations of 0.0-0.5% increased enzyme activity,

while concentrations of 0.5-1.5% decreased enzyme activity. However,

when gelatin substrate was used, NaCI concentrations of 0.0-1.5% had

no effect on enzyme activity. The activity of the purified enzyme

decreased as the concentration of sodium bisulfite increased. Michaelis-

Menten kinetics were followed when gelatin and shrimp protein preparation

were used as substrates. The apparent K values for gelatin and shrimp

protein were 0.98 mg/ml and 0.33 mg/ml, respectively. The apparent

V values were 666.67 and 431.03 units of activity for gelatin
max

xiii









and shrimp protein, respectively. Ferric chloride, mercuric chloride,

potassium chloride, ethylene diaminetetraacetic acid, citric acid,

cysteine, p-mercaptoethanol, potassium permanganate and formaldehyde

partially inactivated the enzyme. Calcium chloride increased the

activity of the extracellular proteolytic enzyme. Zinc chloride, p-

dioxane, manganese chloride and magnesium chloride had no effect on the

activity of the enzyme. The proteolytic enzyme exhibited peptidase

activity on various commercial synthetic dipeptides. The extracellular

proteolytic enzyme produced by P. citreus was apparently not induced by

the presence of shrimp protein in the medium of growth. Enzyme produc-

tion appeared to be related to the extent of growth of P. citreus in the

medium.



































xiv















INTRODUCTION

Quality deterioration and subsequent spoilage of shrimp during

storage are caused primarily by activities of indigenous tissue enzymes

and microbial enzymes (69). Various researchers (17,156) believe that

bacterial action plays a more important role than autolytic enzyme

release in causing spoilage of seafoods. During growth of the bacteria,

proteolysis of shrimp proteins and free amino acid formation by microbial

action has been observed. Enzymatic deamination and decarboxylation of

these amino acids from shrimp protein occur, resulting in the formation

of malodorous compounds (116).

Various types of bacteria have been reported to be present on

freshly caught shrimp. Numerous studies (3,42,43,45,116,142) have shown

the changes undergone by the bacterial flora of shrimp as the storage

period increases. Recent research (1,2,3,90) has noted the presence of

a gram-positive organism, Planococcus citreus, during shrimp storage.

The organism is described as a motile gram-positive coccus found in the

marine environment, capable of growing over a pH range of 7-10 between

5-35 C in broth containing 0.5-12% sodium chloride (NaC1), and the orga-

nism is capable of hydrolyzing gelatin, cottonseed, soy and shrimp

protein.

The potential of P. citreus as a "spoiler" of shrimp was shown by

the increase in pH and the rapid increase in the total volatile nitrogen/

amino acid-nitrogen ratio (TVN/AA-N) and trimethyl-amine nitrogen (TMN)

following growth of this organism on shrimp (4). The proteolytic


1




2



activity of this organism was further demonstrated by the decrease in

percent total extractable protein (percent TEP) in shrimp during storage

at 5 C (4,5) which had been inoculated with P. citreus.

The proteolytic activity exhibited by this organism deserves addi-

tional research in order to better understand the contribution of P.

citreus to the degradation of shrimp protein. A study was therefore

undertaken to study the enzyme(s) responsible for protein degradation.

The optimum medium and stage in the growth cycle of P. citreus were

determined for maximum extracellular enzyme(s) production. The effect

of incubation temperature (5, 20 and 35 C) on the growth of P. citreus

and proteolytic enzyme production was also investigated. Purification

of the extracellular enzyme(s) was achieved by precipitation and chro-

matographic techniques. Homogeneity of the enzyme was evaluated by gel

electrophoresis and chromatographic techniques. Optimum pH and tempera-

ture, ionic strength effect, thermal stability, molecular weight, sodium

chloride effect, sodium bisulfite effect, enzyme concentration, substrate

concentration, and the effect of metal ions and other reagents were

investigated. In addition, the potential of the P. citreus enzyme(s) to

degrade dipeptides and the possible effect of shrimp protein in the

growth medium inducing the extracellular enzyme(s) were studied.

Results obtained from this investigation indicate that P. citreus,

while growing on shrimp, may contribute to the overall decrease in shrimp

quality during iced or refrigerated storage. In addition, information

about the characteristics of the enzyme(s) produced by P. citreus will

be introduced.















LITERATURE REVIEW

In 1978, the shrimping industry was the most valuable fishery in

the United States (8). However, the quality of shrimp often falls short

of that expected by the consumer. Approximately 15-20% of the shrimp

landed is eventually lost due to quality deterioration. This deteriora-

tion of shrimp quality is usually attributed to rapid bacterial enzymatic

changes of the fresh shrimp resulting from mishandling and/or inadequate

processing. These changes, along with the chemical and physical methods

for measuring shrimp quality, are discussed as a basis for the

investigations presented in this dissertation.

Shrimp Spoilage

Quality changes in shrimp during storage on ice can lead to major

economic losses in the shrimp industry. Mechanical damage, bacterial

contamination and enzymatic activity may combine to cause undesirable

changes in the composition and quality of shrimp (20,34,38,39,43,45,53,

54,77,78,79,89,94,108,117,153,154).

The loss of acceptability of shrimp may be triggered by several

factors: 1) shrimp muscle enzymes, 2) direct microbial activity, 3)

microbial enzymes and/or 4) a combination of these factors. Defects

which may occur as a result of such reactions are formation of malodor-

ous substances, flavor deterioration, toughness, mushiness, juiciness,

dryness and discoloration (116).

Proteolytic enzymes play an important role in the spoilage of shrimp

by degrading muscle proteins and polypeptides, forming amino acids which


3




4



enrich the natural substrates and are thus available for the growth of

microorganisms. Enzymatic deamination and decarboxylation of amino acids

may also occur rapidly, resulting in the formation of spoilage products.

Pedraja (116) observed that from the moment a shrimp is taken out of the

water, its free amino acid pool is affected to some extent by osmoregula-

tion and also by the struggle during catching. Therefore, the onset of

enzymatic and bacterial actions will vary according to the factors

affecting the substrates available in shrimp muscle.

Another factor that can induce shrimp spoilage is mechanical damage.

Handling shrimp on the boats results in mechanical damage to the muscle,

which will accelerate microbial invasion. The expressible fluid with its

protein and amino acid content serves as an excellent medium for growth

and reproduction of invading microorganisms (116).

Microbiological Characteristics of Shrimp

The muscle tissue of freshly caught shrimp is generally regarded as

sterile (26); however, work by Lightner (97) showed bacteria in the gut,

gills and between muscle bundles of brown shrimp. Reports on the number

of bacteria found on freshly caught shrimp range from 2.5 x 102 to 2.0 x

106 organisms per gram (org/g) with Gulf coast shrimp averaging 1.0 x 104

org/g, whereas bay shrimp averaged 1.0 x 105 org/g (31,34,39,43,78,142).

Work completed in our laboratory has shown that fresh shrimp from the

Gulf of Mexico had bacterial counts ranging from 4.0 x 105 org/g to 2.0

x 106 org/g, while shrimp from the Atlantic coast had bacterial counts

ranging from 4.5 x 105 to 3.6 x 106 org/g (1).

Various kinds of bacteria have been reported on freshly caught

shrimp. Initially, the microbial flora is a mixture of organisms from

both the marine and terrestial environment. In the early 1950s, Campbell




5



and Williams (31) and Williams et al. (154) isolated species of Achromo-

bacter, Bacillus, Micrococcus, Flavobacterium and Pseudomonas from Gulf

coast shrimp. Vanderzant et al. (142) reported that the flora of shrimp

from the Gulf of Mexico consisted of coryneforms, Achromobacter, Flavo-

bacterium and Bacillus. In Pacific shrimp, Acinetobacter-Moraxella spe-

cies were predominant (80). Lee and Pfeifer (94) reported that the flora

of Pacific shrimp (Pandalus jordani) consisted of Moraxella, Pseudomonas,

Acinetobacter, Arthrobacter and Flavobacterium-Cytophaga species. Cann

(32) and Cann et al. (33) found that coryneform organisms were predomi-

nant in the bacterial flora of scampi, Nephrops norvegicus, with strains

of Achromobacter-Acinetobacter group and Pseudomonas, Cytophaga and

Micrococcus species also present. Koburger et al. (90) reported that

the Flavobacterium-Cytophaga group represented the majority of the

organisms of fresh rock shrimp (Sicyonia brevirostris), and Alvarez (1)

and Alvarez and Koburger (3) reported that Flavobacterium and Pseudomonas

were the predominant groups isolated from Penaeus shrimp from the East

and West coasts of Florida.

When shrimp are stored in ice, the number and kinds of bacteria

shift to a predominantly psychrotrophic flora (130). Psychrotrophs are

described as organisms having an optimal growth temperature of about 20

C. A comparatively longer storage life of iced shrimp from tropical

waters has been reported by Carrol et al. (34). Cann et al. (33) in

their review on tropical shrimp indicated that penaeid shrimp from the

Gulf of Thailand remained in acceptable condition for 12-16 days on ice,

whereas nontropical shrimp, such as Pandalus and Nephrops species, were

totally spoiled after 8-10 days. They attributed this difference to the

bacterial flora; the mesophilic flora on tropical shrimp are not active




6



at ice temperatures and little spoilage occurs until the psychrotrophic

flora develops. Cann et al. (33) stated that the amount of spoilage may

be related to the degree to which psychrotrophic strains are introduced

with the ice. Consequently, the rate of increase in bacterial growth

depends on the initial number of bacteria, handling on deck, and amount

and quality of ice used. Shewan (130) demonstrated that the action of

many psychrotrophic organisms resulted in rapid fish spoilage. The

principal organisms he mentioned were Pseudomonas, Aeromonas, Vibrio,

Moraxella, coryneforms and Flavobacterium. Castell and Mappleback (35)

concluded that Flavobacterium was among the most important of the fish-

spoilage bacteria. Flavobacterium is a frequently encountered bacterium

on fresh shrimp flesh.

The bacterial flora of shrimp undergoes marked changes as the stor-

age period increases. Campbell and Williams (31) showed Bacillus, Micro-

coccus and Flavobacterium made up over 50% of the flora initially,

whereas the Achromobacter-Pseudomonas group accounted for 98% of the

flora after 16 days of iced storage. In a study on the bacterial spoil-

age patterns of headless brown shrimp, Cook (45) noted that there was

only one consistent change in the bacterial types growing initially or

during the period of die-off. As the bacterial count began to rise,

Pseudomonas species became the predominant organism, accounting for 80-

100% of the bacterial types isolated. Vanderzant et al. (142) reported

that the predominant bacterial flora of fresh shrimp consisted of

coryneforms and that following storage Pseudomonas species predominated.

Cobb et al. (43) indicated that typical spoilage organisms of the genus

Pseudomonas are not usually found in freshly caught shrimp. It is not

until the shrimp are exposed to handling on board the vessel that this

organism becomes apparent.





7



Alvarez and Koburger (3) reported that the numbers of Moraxella,

Vibrio/Aeromonas and Planococcus species isolated from Penaeus shrimp

remained relatively constant throughout 10 days of ice storage. However,

Flavobacterium isolates increased until the fifth day, then decreased

rapidly. Pseudomonas species showed the opposite trend. They decreased

until the fifth day, then increased rapidly. Other workers have observed

the presence of Flavobacterium in raw shrimp (31,80,90,94,142,143) and

have noted this decrease in numbers during ice storage with a subsequent

increase in Pseudomonas species. Cook (45) was unable to produce typical

spoilage when shrimp were inoculated with Flavobacterium species, indi-

cating that they are probably an inert group of organisms found in

shrimp. In contrast, Pseudomonas species have been implicated as the

organisms primarily responsible for the spoilage of marine products

stored in ice (108,130).

Measurement of Shrimp Spoilage

Numerous methods for determining shrimp quality have been developed;

however, due to the complexity, time involved and inconsistent results of

many of these methods, only a few are routinely used by the industry and

then, only for internal quality control. In many of these chemical

tests, results can vary with the age of the shrimp, size, species, area

of catch and handling conditions. Many of the tests only indicate the

onset of spoilage (31,109). Table 1 lists the chemical and physical

tests that have been used to measure shrimp quality. Total volatile

nitrogen/amino acid-nitrogen (TVN/AA-N) ratio (40,41,42,43,64,75) is the

chemical test that shows the best correlation with organoleptic quality

measurements of shrimp. Moore and Eitenmiller (107) compared various

methods for measuring shrimp quality. They observed that a relatively






Table 1. Chemical and physical tests available to measure shrimp quality.



Test Parameter Measured Reference



acid-soluble orthophosphate trichloroacetic acid and soluble 14
orthophosphate

adenosine triphosphate and degradation of adenine dinucleotides 64
its degradation products to to hypoxanthine
hypoxanthine

alcoholic tumeric solution changes in percent transmission of 75
a yellow tumeric shrimp solution

amino-nitrogen changes in amine nitrogen content 14,19,62,93

ammonia ammonia content 75,143

B-vitamin content content of B-complex vitamins 15

cathecol ferric chloride change in percent transmission of 85
shrimp filtrate mixed with cathecol
ferric chloride

dimethylamine degradation of trimethyl amine oxide 31
by enzyme (TMO) to DMA

direct microscopic counts actively and non-actively 109
metabolizing bacteria

fluorescamine changes in free amine fractions 107

free fatty acids percent of free fatty acids 31

glycogen glycogen content 14,93






Table 1. (continued)


Test Parameter Measured Reference


hydrogen sulfide measure of H S presence 31

hydration capacity hydration of water insoluble protein 14,129,141

inosine monophosphate degradation of adenine dinucleotides 132
to IMP

indole utilization of tryptophane by 31,64,75,93
bacteria and its conversion
to indole

iodine titration presence of iodine 14,64,75

lactic acid lactic acid content 14

methylene blue reductase reduction of methylene blue by 111
bacteria

peroxide number determines peroxide oxygen which 31
has formed at the double bonds in
unsaturated fatty acids

pH hydrogen ion concentration 14,19,68,69,85,
93,141

phenol red test paper changes in pH 86

photoelectric reflection number changes in light transmission of 64
shrimp extract

picric acid turbidity of shrimp filtrates with 19
picric acid

skatole production of skatole 93






Table 1. (continued)


Test Parameter Measured Reference


total fat fat content 93

total nitrogen ammonia content 93

trimethylamine nitrogen degradation of trimethylamine oxide 14,19,31,44,64,67,
by enzyme (TMO) to TMA 75,85

total volatile nitrogen volatile nitrogen compounds (ammonia) 64,75

tyrosine free tyrosine levels 67

ultraviolet light-change ultraviolet absorption of 93
in fluorescence shrimp extracts

volatile acids volatile acidic compounds 19,31,46,62,68,69 o

volatile reducing substances measure of volatile nitrogen 62
compounds

volatile nitrogen volatile nitrogen containing 85
compounds (ammonia)









new method using fluorescamine primarily detected only the non-protein,

non-ammonia, small molecular weight amines in shrimp homogenates. They

proposed that fluorescamine analysis could be useful in determining

changes in the free amine fractions. The shrimp industry still depends

on visual observation, smell and bacteriological testing for evaluating

overall shrimp quality, whereas, the Food and Drug Administration (FDA)

uses decomposition, filth and odor for the evaluation of shrimp quality.

Characteristics of Planococcus citreus

Koburger et al. (90) noted the presence of a high percentage of

gram-positive organisms following iced storage of rock shrimp (Sicyonia

brevirostris). These organisms comprised up to 68% of the isolates. Of

these 40% were Planococcus citreus, an aerobic gram-positive motile

coccus of marine origin producing an orange or yellow pigment. Informa-

tion describing the isolation and characteristics of this organism is

limited. P. citreus was previously named Micrococcus citreus (27). The

8th Edition of the Bergey's Manual of Determinative Bacteriology (29)

does have a description of the organism; however, it is limited in scope.

Cook in 1970 (45) and previous researchers working with shrimp placed

all aerobic gram-positive to gram-variable coccoid shaped bacteria in

the genus Micrococcus. In addition, Cook (45) noted that many of these

organisms isolated from shrimp were pigmented orange or yellow and were

motile. According to Bergey's manual (29), the only genus in the family

Micrococcaceae that is pigmented, either yellow or orange, and motile,

is Planococcus. This change in the taxonomic status of this organism

and the difficulty of demonstrating motility are probably the reasons

why Planococcus has not been reported in previous studies.

The taxonomic status of Planococcus citreus has changed markedly

through the years. In 1894 and again in 1900, Migula (103,104) made a




12



recommendation that flagellated cocci be included either in the genus

Planococcus or Planosarcina. This suggestion was accepted by only a few

authors, e.g., Krasil'nikov in 1949 (92). The majority of the authors

have included the flagellated cocci in the genus Micrococcus (22,83),

mainly because these cocci could only be differentiated from the other

members of the genus by their motility. Most authors have considered

motility to be a minor characteristic for the recognition of a new genus.

The findings of Bohacek et al. (23,24) that the flagellated cocci differ

considerably in the guanosine-cytosine (GC) content of their deoxyribo-

nucleic acid (DNA) from other cocci shed new light on their taxonomic

position. It was proposed by Bohacek et al. (23) to include the flagel-

lated cocci with a GC content ranging from 40-50% in the genus Plano-

coccus. In 1970, Kocur et al. (91) revised and outlined the genus

Planococcus. However, according to Index Bergeyana (86), the Planococcus

genus includes nine species (P. agilis, P. casei, P. citreus, P. citro-

agilis, P. europeans, P. loffleii, P. luteus, P. ochrolencus and P.

roseus). Kocur et al. (91) evaluated the strains available in culture

and proposed that seven belong to one species, Planococcus citreus.

Although the remaining two species were closely related, he refrained

from giving a precise designation and labeled them only as Planococcus

species.

Schleifer and Kandler (126) found that the strains studied by

Bohacek et al. (23,24) and Kocur et al. (91) were uniform with respect

to the type of murein present in their cell walls and similar to that

of members of the genera Micrococcus and Staphylococcus. However,

serological investigation of P. citreus by Oeding in 1971 (112) revealed

no antigenic relationship to staphylococci or micrococci.





13



Thirkell and Summerfield (137,138) studied the effect of varying

the sea salt concentration on the chemical composition of a purified

membrane fraction of P. citreus. They concluded that the concentration

of salt in the medium affected the amount of membrane in the cell. Salt

concentrations above or below the normal 3% of sea water reduced the

amount of membrane material present. In addition, varying salt concen-

tration had no significant effect on the amount of total neutral lipid,

glycolipid or phospholipid in the P. citreus membrane preparations. But

a significant effect was observed on the amount of individual neutral

lipid or phospholipid classes present and on the number of individual

glycolipid components detected.

Our attention was directed toward this organism when, during a study

of the normal flora of rock shrimp (Sicyonia brevirostris), P. citreus

was consistently isolated and found to increase in numbers during iced

storage (90). In this study, 68% of the isolates recovered were gram-

positive cocci, with P. citreus increasing from 10% of the isolates on

the fresh rock shrimp to 40% on the ice stored rock shrimp. In recent

work (1,2,3), P. citreus has been found to be an important member of the

normal flora of Penaeus shrimp.

Alvarez and Koburger (5) described P. citreus as a motile gram-

positive coccus found in the marine environment, capable of growing over

a range of pH 7-10, 5-35 C, in broth containing 0.5-12% sodium chloride

(NaC1) and capable of hydrolyzing gelatin, cottonseed, soy and more

importantly to seafood microbiologists, shrimp protein. Figure 1 shows

a photomicrograph of P. citreus illustrating its morphology and flagella-

tion. Table 2 shows the capabilities of this organism to hydrolyze

various protein sources.








14

















jim^ .~, . ***H * y " - *- -* * .



" � *.-* pr i.^ * r ilq n . .I i.?


aaS& "'I Ir r**-i r" III " kL

i- iP P- -s .~g 4 ,~s . ..-

'- ^ r * * ' * ^ - .

Il < :. * * * , I - * . � -
-. ~ ~ �8s -6.'*. ~"~l a; �'. C a * -* . *
r""1 J1" � 6 .^l%(lb ^. ^




Fiue .Phtincogah fPlncccsciruscll hoig ophlg
and~ ~ ~~~~~~ . flglaio 6 manfctin90)





15






Table 2. Hydrolysis of various protein sources by selected strains of
Planococcus citreus at 25 C (4) (modification of Frazier (72)).


Protein Source2
CM

Isolate 4 : o 0
-4 o 0 CO i -4 0
o t oo o cc
0 9 0 Co (


A 17 +5 + + - + - - - + + -

E4 + + + - + - - - + - -

El + + + - + - - - + + -

E7 + + + - + - - - + + -

F9 + - + - + - - - + + -

F 15 + - + - + - - - + - -

F 18 + - + - + - - - + - -

KS-1 + - + - + - - - + - -

KS-2 + + + - + - - - + - -

KS-3 + - + - + - - - + - -

KS-4 + + + - + - - - + - -

CS-1 + + + - + - - - + - -


From Difco Laboratories, Detroit, MI.

Protein isolates obtained from Southern Utilization Research and
Development Division, New Orleans, LA.

Fresh samples were diluted 1:10 with 0.05 M phosphate buffer pH 7 and
ground in a Waring blender, dialyzed overnight with 10 volumes of the
same buffer (5 C) and lyophilized.

F. W. Knapp, Food Science and Human Nutrition Department, University
of Florida, Gainesville, FL.

5+ = hydrolysis; - = no hydrolysis.




16



The reports (4,5) by Alvarez and Koburger outline some observations

on the distribution of P. citreus in the marine environment. Of the 35

samples of marine origin examined for P. citreus, only 5 yielded this

organism. Four were shrimp samples and the fifth was a stuffed flounder

sample that had been prepared in a plant that processed predominantly

shrimp. One of the shrimp samples from which Planococcus was isolated

had been in frozen storage for over six years. Fresh seafood (trout,

sheephead, mackerel, crab and oysters) as well as Gulf Coast waters and

sediments from the vicinity of Suwannee, Florida, were also examined for

P. citreus without success. However, in more recent studies performed

by Mallory et al. (100), P. citreus was isolated from estuarine areas of

Chesapeake Bay in low numbers.

Since the isolation of gram-positive organisms from iced seafood is

uncommon, Alvarez and Koburger (5) studied the contribution of P. citreus

to the spoilage of Penaeus shrimp. They utilized gamma irradiation (600

Krads) to lower the number of bacteria in raw. shrimp and then inoculated

a portion of the shrimp with 5 x 103 P. citreus cells per gram of shrimp

in order to study the changes produced by this organism. P. citreus

counts increased in the inoculated shrimp from 5 x 103 bacteria/gram at
8
0 day to 1.9 x 10 bacteria/gram at the 16th day. The potential of P.

citreus as a "spoiler" of shrimp was shown by an increase in pH and the

rapid increase in total volatile nitrogen/amino acid-nitrogen ratio (TVN/

AA-N) and trimethyl-amine nitrogen (TMN) content. In 1973, Cobb et al.

(42) reported a high correlation between total volatile nitrogen/amino

acid-nitrogen ratio (TVN/AA-N) and quality of shrimp. Later work (41)

suggested that the TVN/AA-N ratio and the logarithm of bacterial counts

increased at approximately the same rate after the initial lag phase of





17



bacterial growth and that a TVN/AA-N ratio of 1.3 indicated a limited

shelf life of the shrimp. Alvarez and Koburger (4,5) showed that P.

citreus is capable of increasing the TVN/AA-N ratio at a similar rate as

the control sample (natural flora of shrimp). Thus, if the TVN/AA-N

ratio is an index of shrimp quality, P. citreus is capable of shortening

the shelf life of shrimp. The proteolytic activity of this organism was

demonstrated by a significant decrease in percent extractable protein

(% TEP) in the early days of storage. Maximal percent TEP decrease was

observed between the 4th and the 12th day of storage of shrimp at 5 C

(5).

Proteolytic Enzymes

Enzymes are proteins with highly specific catalytic activities. As

catalysts, enzymes have the following properties: 1) they are effective

in small concentrations; 2) they remain unchanged in the reaction; 3) if

present in small concentrations relative to the substrate, they speed

attainment of equilibrium as reflected by increases in the rate constants
K
K1 and K_I (A + B -- C + D, where A + B = reacting substances, C + D
K_1
= products of the A + B enzyme catalyzed reaction, K1 = rate constant of

the forward reaction, K_ = rate constant of the reverse reaction).

However, an enzyme does not change the ratio K1/K_1 = Keq (95).

Most living organisms possess the ability to degrade proteins to

more readily absorbed substances. Such attacks on the peptide bond are

made possible by the presence of proteolytic enzymes. Although proteo-

lytic enzymes from animal sources have been studied for more than a

century by both physiologists and biochemists, it was the work of Berg-

mann and Fruton (18) which led to a more complete understanding of the

mode of action of these enzymes. Their work established conclusively




18



that these enzymes exert a specificity toward the amino acids involved

in the peptide bonds which they attack. Bergmann and his students are

also responsible for the presently accepted classification of proteolytic

enzymes: They proposed that these enzymes be grouped into two classes--

endopeptidases and exopeptidases--depending upon whether they hydrolyzed

peptide bonds remote from, or near to, the end of the peptide chains of

their natural substrates. The former class includes such enzymes as

pepsin, trypsin and chymotrypsin, while the latter class contains the

dipeptidases and the amino and carboxy peptidases.

Proteinases in bacteria may be either intracellular or extracellular

depending upon whether they exert their activity within the cell or

whether they are excreted from the cell to attack proteins in the envi-

ronment (10,58). Also, enzymes may be classified according to their

location in, on or around the cell: a) cell-bound: 1) truly intracell-

ular, 2) surface-bound; and b) extracellular (58). Extracellular enzymes

are those enzymes which exist in the medium around the cell, having

originated from the cell without any alteration to cell structure greater

than that compatible with the cell's normal processes of growth and

reproduction. This distinction is not always clear and in some instances

it is entirely possible that autolysis of cells has permitted the escape

of intracellular enzymes into the culture filtrate. This is particularly

true when high proteolytic activity is dependent upon prolonged

incubation of the culture (74).

In 1964, the International Union of Biochemistry (54) recommended a

scheme for numbering enzymes, which is currently used for the classifi-

cation of enzymes. Enzymes are divided into groups on the basis of the

type of reaction catalyzed, and this, together with the name(s) of the




19



substrate(s), provides a basis for naming individual enzymes. Each

enzyme number contains four elements; the first element (1 through 6)

shows to which of the 6 main groups of enzymes the particular enzyme

belongs (the six main groups are made on the basis of the general chemi-

cal reaction catalyzed); the second and third elements show the subclass

and sub-subclass, respectively, thus defining the type of reaction; and

the fourth element is the serial number of the enzyme within its sub-sub-

class. Enzymes can be divided into six main groups: oxidoreductases,

transferases, hydrolases, lyases, isomerases and ligases.

Active extracellular proteinases are produced by numerous species of

Clostridium, Proteus, Bacillus, Pseudomonas, Micrococcus, Streptococcus,

Escherichia, Cytophaga and Staphylococcus (11,12,36,58,59,65,70,84,87,

105,110,113,114,118,136,139,155,157).

The continued study of these bacterial enzymes is important for at

least two reasons: (a) proteolysis by microorganisms plays an important

role in the biogeochemical cycles (74) and is responsible for numerous

environmental interrelationships; (b) the purification and the elucida-

tion of their bond specificities are certain to lead to the discovery of

new enzymes with new properties not previously known.

Measuring Proteolytic Activity

Many methods are available for measuring proteolytic activity. Some

are based on the measurement of increase in protein (or nitrogen) solu-

bility in the supernatant after centrifugation of the reaction mixture.

The most frequently cited method for measuring protein in solution is

that of Lowry et al. (98) in which the tyrosine-tryptophan groups of

proteins in solution, or precipitated with acid, are reacted with alka-

line Folin-phenol reagent after an alkaline copper treatment (71) to




20



produce a blue color that is measured in a spectrophotometer. Other

methods record proteolysis as the increase in ultraviolet absorption at

280 nm or the increase in absorbance (660 nm) of the tyrosine-tryptophan

filtrate after trichloroacetic acid (TCA) precipitation of the undigested

protein reacted with diluted (2:1) phenol reagent solution (9).

Schwabe (127) described a method which permited the assay of the

proteolytic enzyme activity on hemoglobin utilizing the fluorescamine

technique. The assay is about 100 times more sensitive than the Lowry

method, much faster and less complicated. He observed that the two main

obstacles for the successful use of fluorescamine in his assay system

were (1) the high blank produced by the reaction of e-amino groups of

the protein and (2) the fluorescent quenching effect of the hemoglobin.

The high blank of the hemoglobin he substantially suppressed by a chemi-

cal modification, i.e., succinylation. Hemoglobin is usually used as a

2% solution of which only 10 pl are pipetted into 2 ml of phosphate buf-

fer used for the reaction. He observed that the enzyme activity as mea-

sured by the fluorescamine method remained linear throughout thirty min-

utes while the Lowry method indicated a definite slowing of the reaction

beginning at about ten minutes. This was due to the fact that fluores-

camine detects an increase in free amino groups while the Lowry reagent

as well as the direct measurement of absorption at 280 nm depends on the

production of tyrosine or tryptophan containing peptides. A possible

explanation for this discrepancy is that the enzyme in its initial

attack on the hemoglobin molecule releases large peptides which are TCA

soluble and that subsequent enzyme action further degrades these large

peptides without significantly increasing the number of TCA-soluble

fragments containing tyrosine or tryptophan moieties. A reagent

depending upon primary amine groups is not subject to this error (125).




21



Fluorescamine is a new reagent for the detection of amino acids,

peptides, proteins and primary amines in the picomole range (18,133,140).

Its reaction with amines is almost instantaneous at room temperature in

aqueous media. The products are highly fluorescent, whereas the reagent

and its degradation products are nonfluorescent.

McCaman and Robins (101) introduced a fluorometric method now widely

used for assay of serum phenylalanine which is based on the interaction

of ninhydrin and peptides. Samejima et al. (124,125) found that it was

the phenylacetaldehyde formed on interaction with ninhydrin which com-

bined with additional ninhydrin and peptide or any other primary amine

to yield highly fluorescent products. The structure of these products

was subsequently elucidated by Weigele et al. (145), who then synthesized

a novel reagent (145). This reagent 4-phenylspiro (furan-2(3H),I'-

phthalan) 3,3'-dione (fluorescamine) reacts directly with primary amines

to form highly fluorescent products.

Several factors make fluorescamine suitable for assaying primary

amines, including amino acids, peptides and proteins. At pH 8-9, the

reaction with primary amines proceeds at room temperature (140) within a

fraction of a second. Excess reagent is concomitantly destroyed within

several seconds (140). Fluorescamine, as well as its hydrolysis prod-

ucts, is nonfluorescent. Studies with small peptides have shown that

the reaction goes to near completion (about 80% to 95% of theoretical

yield) even when fluorescamine is not present in excess. The following

is an example of the reaction of fluorescamine with an amine group

illustrating the product formed (fluorophor) and the rate of the reaction

(100-500 msec). In addition, the reaction of water with fluorescamine

with the formation of a nonfluorescent product is also shown.




22




R-





+ RNH2 OH
S100-500 msec 'C COOH


Fluorophor
t�
+ H26
Fluorescamine 2

(nonfluorescent) 5-10 sec Hydrolysis products

(nonfluorescent)




Primary amines are first buffered to an appropriate pH (7-8),and

then fluorescamine, dissolved in a water miscible, nonhydroxylic solvent

such as acetone or dioxane, is added. The reaction is complete,and in

less than a minute excess reagent is destroyed. The resulting fluores-

cence is proportional to the amine concentration, and the fluorophors

are stable over several hours. The above properties lend themselves

well to automation (123). It should be noted that fluorescamine does

not react with proline or hydroxyproline, which are not primary amines.

This disadvantage can be overcome by introducing an appropriate inter-

mediate step to convert these amino acids to primary amines (63,146).

An additional advantage of the fluoresamine assay is that comparatively

little fluorescence is developed with ammonia. Therefore, ammonia does

not interfere with an analysis to the extent that it does in the colori-

metric ninhydrin procedure. Figure 2 shows a comparison of the





23




S30- Fluorescamine
Z (Sample volume 5-10 ml)
LU


0 20

































Figure 2. Comparison between the Fluorescamine technique and the Lowry procedure
-J
LU
































(140) for determining protein concentration. a
sin monitored by the fluorescamine and the Lowry






Lowry
(Sample volume 50ml)
0.2
>-













02 40 60 80

FRACTION NUMBER
Figure 2. Comparison between the Fluorescamine technique and the Lowry procedure
(140) for determining protein concentration.
Chromatographyof a partially purified enzyme of guinea pig neuro-
physin monitored by the fluorescamine and the Lowry procedure.





24



fluorescamine technique with the standard Lowry procedure (97) for the

monitoring of protein in a column effluent. The volumes used for fluo-

rescamine assay were 10 to 20 percent of those used in the Lowry method,

and smaller amounts could have been used (140). Background interference

was negligible with the automated fluorescence method, and significant

peaks not discernible by the Lowry procedure were observed.

Due to the many advantages of the relatively new fluorometric tech-

nique, it was used to measure the proteolytic enzyme activity of P.

citreus. The ability of this organism to grow on shrimp as well as to

hydrolyze various protein preparations promoted investigations to iso-

late, purify and characterize the extracellular enzyme(s) produced by

P. citreus.















MATERIALS AND METHODS

Unless otherwise specified, Difco (55,56) or Baltimore Biological

Laboratories (BBL) (16) products were used for all microbiological

analyses. Serial dilutions used Butterfield's Phosphate buffer and fol-

lowed the procedures outlined in the Compendium of Methods for the

Microbiological Examination of Foods (6). All chemicals used were

reagent grade meeting American Chemical Society specifications. All

media and glassware were autoclaved for 15 min at 121 C unless label

directions specified otherwise.

Planococcus citreus Cultures

The culture of P. citreus used in this study, A-17, was isolated

from rock shrimp (Sicyonia brevirostris) (90). The culture chosen was

able to grow well in shrimp during iced storage and showed strong pro-

teolytic activity toward various protein preparations. The isolate used

for the study was grown on Plate Count Agar slants (Difco) with 0.5%

sodium chloride (NaC1) added and incubated at 20 C for 72 hrs (4).

Appropriate dilutions in buffer were made to obtain a concentration of

approximately 5 x 103 organisms per ml. The A-17 isolate used was

capable of hydrolyzing gelatin, whey, cottonseed, soy, hog blood and

shrimp protein preparations (4). It also grew well in 0.5% to 16% NaCI

and pH 7.0 to 10.9.

Determination of Proteolytic Activity

A modified fluorescamine fluorescent (fluorometric) technique (129)

was used to measure enzyme activity. FluorescamineR is capable of


25




26



detection of amino acids, peptides, proteins and primary amines in the

picomole range (140).

P. citreus cells were grown in various media throughout the study.

After incubation, the cultures were centrifuged in a RC-5 Superspeed

Refrigerated Centrifuge (Sorval, Dupont Co. Instruments, Newtown, CT) at

a force of 20,000 x g for 30 min. The supernatant (cell-free broth) was

used for further investigations involving extracellular enzymes. The

cell pellet was washed twice with 0.05 M phosphate buffer (pH 8). The

whole cells were then resuspended with 10-20 ml of the same buffer,

transferred to a dry ice chilled Eaton pressure cell (60) and allowed to

freeze under dry ice for 3 hrs. The frozen microbial cells were disinte-

grated using the Eaton pressure cell at a constant pressure of 7.03 x 106

kg/m2 on a Carver hydraulic laboratory press (F. S. Carver, Inc., Summit,

NJ). The ruptured cell extract was fractionated into intracellular solu-

ble and particulate fractions by centrifugation at a force of 12,000 x g

for 15 min. The particulate fraction was resuspended in 10 ml of 0.05 M

phosphate buffer prior to enzyme activity determinations of all frac-

tions. Five milliliters of the substrate (gelatin or shrimp protein)

were reacted with 1 ml of each of the above fractions for 15 min at 35 C.

The reactions were terminated by adding 10 ml of 5% TCA. Zero time

blanks were prepared by adding the trichloroacetic acid (TCA, Fisher

Scientific Co., Fairlanes, NJ) before the incubation period (see latter

part of this section).

One milliliter of the cell-free broth or 100 pl of the purified

enzyme was reacted with 5 ml of substrate (gelatin or shrimp protein) for

the appropriate reaction time (to be determined) at 35 C, pH 8. The

enzyme-substrate reaction was stopped by precipitating the mixture with





27



10 ml of 5% TCA. After 5 to 10 min, to allow the proteins to settle,

the solution was filtered through Whatman #1 filter paper. Two hundred

microliters of the TCA filtrate were transferred to a 13 x 100 mm test

tube (Dispo culture tubes, Scientific Products, McGraw Park, IL) and the

volume brought to 1.5 ml with 0.5 M sodium phosphate buffer, pH 8.

While the test tube was vigorously mixed in a Vortex Mixture (Scientific

Products, Evanston, NY), 0.5 ml of fluorescamine in dioxane (30 mg/100

ml, Eastman Kodak Corp., Rochester, NY) was rapidly added to the buffered

protein solution. A model 204-A Fluorescence Spectrophotometer (Perkin

Elmer Corp., Norwalk, CT) was used to measure fluorescence intensity.

Zero time blanks were prepared by adding 10 ml of 5% TCA after adding the

enzyme and prior incubation of the mixture. This blank represented the

background activity present in the mixture at zero time. Zero time

fluorescence reading was subtracted from the reading of the substrate-

enzyme mixture after the appropriate incubation time.

Total enzyme activity was expressed as the change in 0.1 fluores-

cence units of the TCA filtrate per milliliter of enzyme per minute.

Specific activity was expressed as the units of total enzyme activity/mg

of protein present (units of activity/mg of protein).

Previous research involving the use of the fluorescamine technique

(47,127,140) indicated that pH affected fluorescence intensity. Buffers

of pH from 2 to 10 (see buffers described on pg. 44) were used to deter-

mine the effect of varying the pH of the buffer on fluorescence intens-

ity. TCA filtrates (0.2 ml) were reacted with 1.3 ml of the various

buffers (pH 2-pH 10) before addition of the fluorescamine reagent.

Figure 3 indicates that addition of pH 8 buffer resulted in the highest

fluorescence itensity. Consequently, pH 8 buffer was used for the

remainder of the research.




28




150-












100








LUJ

Ct
50-
50













2 4 6 8 10
pH

Figure 3. Effect of pH adjustment of gelatin-trichloroacetic acid (TCA) .filtrates
on'r:flaorescence intensity.




29



In order to determine the reaction spectrum of our working enzyme

solution when gelatin and shrimp protein were used as substrates, the

excitation and emission (fluorescent) wavelengths were scanned (48).

Figure 4 shows the excitation (curve A) and fluorescence (curve B)

spectra for TCA filtrates of the gelatin substrate. The excitation

spectrum has a maximum of 360 nm and a secondary peak at 390 nm. The

secondary peak at 390 nm was chosen because it results in minimal zero

time blank fluorescence values. The fluorescence emission maximum with

the excitation wavelength at 390 nm was at 475 nm. Figure 5 shows the

excitation (curve A) and fluorescence (curve B) emission spectra for TCA

filtrates of the shrimp protein substrate. The excitation spectrum has

a maximum peak at 375 nm and a secondary peak at 390 nm. Again, the

secondary peak was chosen. The fluorescence emission maximum with the

excitation wavelength at 390 nm was at 490 nm.

Efficacy of 5% Trichloroacetic Acid (TCA)

In order to determine the efficacy of 10 ml of 5% trichloroacetic

acid (TCA) in terminating the enzyme-substrate reaction, 5 ml aliquots

of substrate (gelatin) were incubated with 1 ml of cell-free broth and

10 ml of 5% TCA for 0, 10, 15, 30, 45 and 60 min at 35 C. A positive

control was done by incubating the enzyme-substrate mixture at 35 C for

0, 10, 15, 30, 45 and 60 min before adding the TCA. After the incubation

period, the positive control was terminated by adding 10 ml of 5% TCA.

Data in Figure 6 shows that 10 ml of 5% TCA were adequate for inhibiting

the enzyme substrate reaction effectively since there was no increase in

fluorescence intensity. The fluorescence intensity, observed when TCA

is immediately reacted with the enzyme and substrate, represents the

background fluorescence of the assay mixture.









100- A B




S75-

W

z
I-J



W25





360 390 475
WAVELENGTH (nm)
Figure 4. Exitation (curve A) and fluorescence (curve B) spectrum for the reaction of a gelatin-trichloro-
acetic acid (TCA) filtrate with fluorescamine at pH 8.









100-
SA B



75-
I--

z
U
C-
i 50-
W

-J
U 25-
Cr_



---,----- --,----~
375 390 490
WAVELENGTH (nm)
Figure 5. Exitation (curve A) and fluorescence (curve B) spectrum for the reaction of a shrimp protein-
trichloroacetic acid (TCA) filtrate with fluorescamine pH 8.







100
--- TCA Control
- Positive Control
90

80

U)70-

l-_
2 60

S50-


20-

_)
20-


I0
I0


0 10 15 30 45 60
INCUBATION TIME AT 350 C (min)
Figure 6. Efficacy of trichloroacetic acid (TCA) in terminating the enzyme-substrate reaction.




33



Substrate Characteristics

Two substrates were used throughout the study: gelatin (Difco)

(1.2 mg/ml) and a shrimp protein preparation (0.6 mg/ml). Higher concen-

trations of the shrimp protein preparation were not used because of

solubility problems in the buffer systems used. The shrimp protein prep-

aration was prepared as follows: fresh raw shrimp meat ground in a War-

ing blender with 0.05 M phosphate buffer (pH 7, 1:10 dilution), dialyzed

overnight with four changes of the same buffer at 5 C and lyophylized for

preservation (Virtis Freeze Dryer, Gardiner, NY). Protein, fat, moisture

and ash were determined for the shrimp protein preparation. Protein was

determined by the AOAC standard micro-Kjeldahl method (13). Crude fat

was determined by a modification of the AOAC method (13) using the Gold-

fisch solvent chamber. Approximately 2 grams of sample were extracted

overnight with petroleum ether. Moisture was determined in a vacuum

oven at 70 C for 12 hrs. Ashing was done in a muffle furnace at 600 C

for 8 hrs. Table 3 shows that the shrimp protein preparation consisted

of 77.44% protein, 5.40% fat, 8.95% moisture, 6.50% ash and 1.71%

carbohydrate (calculated by difference).

Determination of Enzyme-Substrate Mixture Reaction Time

Five milliliters of substrate and an aliquot of cell-free broth were

incubated at 35 C for 0, 5, 10, 15, 20, 30 and 60 min in order to deter-

mine the time course of enzyme activity and apparent optimum reaction

time. In experiments involving gelatin, 0.5, 1.0 and 2.0 ml of cell-free

broth were used while 1.0 ml of cell-free broth was used with the shrimp

protein substrate. An incubation time of 15 min was an appropriate

enzyme-substrate contact reaction time when shrimp were used as substrate

(Figure 7). In addition, when gelatin was used as a substrate and




34



Table 3. Proximate composition of the shrimp protein preparation.a



Percent (%)



Protein 77.44

Fat 5.40

Moisture 8.95

Ash 6.50

Carbohydrateb 1.71



aAverage of two determinations

Calculated by difference




35











70-
-- Shrimp


60-



50-
LJ


040-
U)
UJ
�r
0
D30-


20-



10-




0 5 10 15 20 30 60
INCUBATION TIME AT 35 C (min)

Figure 7. Increase in fluorescence intensity using the shrimp protein
preparation as substrate after incubation with cell-free
broth for up to 1 hr at 35 C.




36



various amounts of cell-free broth were reacted with gelatin, a 15 min

reaction time was also a sufficient substrate reaction time (Figure 8).

This reaction time was used for the remainder of the study.

Growth Medium and Enzyme Production

Various media were used to determine growth rates and production of

extracellular enzyme(s) by P. citreus. Three hundred milliliters of

Plate Count Broth (PCB) + 0.5% NaC1, Nutrient Broth (NB) + 0.5% NaCl and

Trypticase Soy Broth (TSB) were used to grow the organism. Incubation

was at 20 C for up to 96 hrs. Samples were drawn at 0, 12, 24, 48, 72

and 96 hrs. All samples were assayed for growth by measuring optical

density at 600 nm in a Spectronic-20 Spectrophotometer (Bausch and Lomb,

Rochester, NY) and by plating in Plate Count Agar (PCA) with incubation

at 20 C for 5 days. Cultures were centrifuged in a RC-5 Superspeed

Refrigerated Centrifuge at a force of 20,000 x g for 30 min. The sedi-

ment was discarded. One milliliter of the cell-free broth was assayed

for enzyme activity with gelatin substrate. P. citreus growth and

enzyme analyses were done three times and each time in duplicate.

Optimization of Enzyme Activity to Growth and Cell Number

The optimum time for cell harvesting along the logarithmic section

of the growth curve (approximately midlog) of the organism was selected.

The logarithm of the cell count was plotted against the incubation time.

The specific growth rate (k) of each medium was calculated using the

equation In B = In B + k (where B = bacterial count at time x, B =
x o x o
bacterial count at time 0 (both in midlog growth phase), k = specific

growth rate, t = time, hrs). The units of enzyme activity per cell per

hour (m) were then calculated using the formula (58,124): Mt - Mo =I

(Bx - B ) (where Mt = enzyme activity at cell number Bx, M = enzyme







90 -0- 0.5 ml Cell Free Brotha
--- 1.0 ml Cell Free Brothb
8 -A- 2.0 ml Cell Free Broth

70-
U
060-
z
O50
l


LL
30-


20-





0 5 10 15 20 30 60
INCUBATION TIME AT 350 C (min)
a Average of 3 observations
b Average of 6 observations
Figure 8. Increase in fluorescence intensity using gelatin as substrate and various amounts of the cell-
free broth after incubation at 35 C for 1 hr.




38



activity at cell number B , k = specific growth rate). This formula was
dm kt
obtained through the integration of the following equation: d- = m B e
dt o

(where dm/dt = change in enzyme activity over time). The calculated m's

were compared for the three media used. A test to observe any difference

between the values for the units of enzyme activity per cell per hour (m)

in each medium was designed using the Statistical Analysis System (SAS)

program package (15) for analysis of variance. A completely randomized

design (102,132) was used in that the major source of error to be

considered was due to the nutritional differences between media.

Effect of Incubation Temperature on Enzyme Production and Activity

The effect of incubation temperature (5, 20 and 35 C) on the growth

of P. citreus and its ability to produce an active extracellular enzyme

was investigated. Five-hundred-milliliter Erlenmeyer flasks containing

100 ml of medium were inoculated with approximately 5 x 10 P. citreus

and incubated at the three temperatures. All samples were assayed for

growth by measuring optical density at 600 nm in a Spectronic-20 spectro-

photometer and for proteolytic activity using the fluorometric technique.

For the 35 C grown cells, samples were drawn at 0, 6, 12, 24, 48, 72 and

96 hrs. For the 20 C grown cells, samples were drawn at 0, 12, 24, 48,

72, 96 and 120 hrs. Finally, for the 5 C grown cells, samples were drawn

at 0, 24, 48, 72, 96, 120 and 144 hrs. After determining the midlog

phase of growth for P. citreus at each temperature, P. citreus cells were

then harvested at this stage. Enzyme activity determinations were done

using the cell-free broth obtained from growing the organism at the three

temperatures until midlog phase. Five milliliters of the shrimp protein

preparation were incubated with 1 ml of each cell-free broth at 5 C for

60 min, 20 C for 30 min and 35 C for 15 min. Analyses were done three

times and each time in duplicate.




39



In addition, direct microscopic observations of the cells grown at

5, 20 and 35 C were conducted. Any morphological change due to growth

temperature was observed and recorded.

Purification of the Extracellular Enzyme(s)

Planococcus citreus was grown in the selected medium at 20 C until

midlog phase. The cells were then centrifuged at a force of 20,000 x g

for 30 min. This cell-free broth was used in the purification procedure.

Ammonium Sulfate Precipitation

Fractional precipitation of the enzyme(s) in the cell-free broth was

accomplished with 0-55%, 55-70% and 70-100% ammonium sulfate saturation

(Mallinckrodt, Inc., Paris, KY). The required amount of ammonium sulfate

was added with stirring until dissolved (88). The mixture was allowed to

equilibrate for 1 hr at 4 C and centrifuged at a force of 20,000 x g for

20 min. The precipitate was resuspended with 10 ml of Butterfield's

phosphate buffer at pH 8 and dialyzed for 16 hrs (dialysis tubing #24,

Scientific Products, McGraw Park, IL) against 500 ml of 0.05 M phosphate

buffer pH 8 (108,153). Each ammonium sulfate fraction was then assayed

for proteolytic activity using gelatin as substrate.

Molecular Sieve Chromatography

SephacrylR S-200 Superfine (Pharmacia Fine Chemicals, Uppsala,

Sweden), a high resolution chromatographic medium for gel filtration was

used to separate the enzyme solution according to molecular weight after

the ammonium sulfate precipitation step. A 30 x 2.5 cm column was packed

with SephacrylR S-200 Superfine gel and a PharmaciaR peristaltic pump

(p-3) was used to pack the column at a speed of 40 ml/cm/hr (120). The

enzyme solution was eluted using reverse flow at a speed of 30 ml/cm/hr.

Five milliliter fractions were collected in each tube with 0.02 M phos-




40



phate buffer pH 7 as the eluting agent using a Gilson Fraction collector

(Model FC-220K Fractionator, Gilson Medical Electronics, Inc., Middle-

town, WI). Enzyme activity of each fraction collected was then measured

using gelatin as substrate. The protein present in the fractions was

determined by following absorbance at 280 nm using a Beckman Model 25

spectrophotometer (Beckman Instruments, Inc., Fullerton, CA).

Ion-Exchange Chromatography

The active fractions recovered from the gel filtration step were

pooled and further separated by ion-exchange chromatography using DEAE-

SephadexR A-50 (Pharmacia Fine Chemicals, Uppsala, Sweden). The column

was prepared following the procedures given by Pharmacia Fine Chemicals

(119). A 40 x 2.5 cm column was prepared and the protein eluted with

0.02 M phosphate buffer with a linear gradient of tris 0.01 M NaCl-tris

0.15 M NaCl (47) at a rate of 25 ml/hr (1.5 reading in the peristaltic

pump) and collected in 10 ml fractions. Two hundred fifty milliliters

of 0.01 M and 0.15 M NaCl solutions were placed in each vessel for the

linear gradient. The protein present in the fractions was followed by

reading the absorbance at 280 nm using a Beckman Model 25 spectrophotom-

eter. Enzyme activity of each fraction was measured with gelatin as the

substrate. All column studies were duplicated.

The protein content of each fraction eluted using the SephacrylR

S-200 Superfine and the DEAE-SephadexR A-50 was also analyzed by the

Lowry (98) method for protein with Bovine Serum Albumin (Sigma Chemical

Co., St. Louis, MO) as the standard. Protein content is expressed as

mg/ml. Figure 9 summarizes the steps followed in the purification of

the extracellular proteolytic enzyme(s) of P. citreus.




41



STERILE
TRYPTICASE SOY BROTH
inoculated with
5 x 103 P. citreus/ 100 ml


harvest cells at
mid-log phase DEAE- SEPHADEX A-50

CENTRIFUGE ION EXCHANGE ROMATOGRAPHY

( 20,000 x g/ 30 min)


cell-free broth
PURITY DETERMINATION
(NH ) SO FRACTIONAL STDY CHARACTERIZATION
4 2 4 STUDIES
PRECIPITATION




SEPHACRYL S-200 SUPERFINE
CHROMATOGRAPHY







Figure 9: Outline of steps for the purification of the extracellular
protease(s) of Planococcus citreus.




42



Confirmation of Enzyme Purity

A modification of the Weber and Osborn (144) method for sodium

dodecyl sulfate-poly acrylamide gel (SDS-PAG) gel electrophoresis was

used. A Buchler 3-1500 electrophoresis apparatus (Buchler Instruments

Corp., Fort Lee, NJ) was used to evaluate the purity of the isolated

extracellular enzyme.

A 10% acrylamide:BIS, 30:0.8 gel was prepared and allowed to poly-

merize for 2 hrs. A sample of the purified enzyme was diluted 1:1 with

the sample buffer. The sample buffer consisted of 0.01 M sodium phos-

phate (pH 7), 10% sodium dodecyl sulfate, 0.1% dithiothrietol, 10% glyc-

erol and 0.001% bromocresol blue. The protein solutions were placed onto

the gels (50 ug protein/gel, 100 pg/gel, 150 ug/gel and 200 pg/gel) and

were layered carefully with electrode buffer (pH 8.3) to the top of each

tube. The lower electrode chamber was then 2/3 filled with electrode

buffer. The tubes in the apparatus were then lowered into the electrode

chamber. The upper chamber was filled with water to approximately 1 inch

over the tube top. The water jacket was connected and the electrode

wires from the power source were also connected. A constant current of

1-1.5 mAmps/gel was applied until the marker dye band just exited from

the gels (approximately 3 hrs). The gels were immediately removed from

their tubes. The gels were fixed overnight in a fresh 50% TCA solution.

The fixed gels were then stained 1-2 hrs with 0.1% Coomassie brilliant

blue solution made up fresh in 50% TCA at 37 C in a water bath. The gels

were further diffusion-destained by repeated washings in 7% acetic acid

(17-72 hrs). Gels were then stored in 7% acetic acid (82).




43



Characterization of the Proteolytic Enzyme(s)

Molecular Weight Determination

Two methods (7,21,150) were used to estimate the molecular weight

of the enzyme(s).

A 2.5 x 30 cm column packed with SephacrylR S-200 (Pharmacia Fine

Chemicals, Uppsala, Sweden) was used and the following standards applied:

Ribonuclease A (13,000 MW), Trypsin (23,500 MW), Pepsin (45,000 MW),

Bovine Serum Albumin (70,000 MW) and Aldolase (158,000 MW) (Pharmacia

Fine Chemicals, Piscataway, NJ) following the procedures suggested by

Pharmacia Fine Chemicals (116). The K value of each protein sample was
av
calculated and plotted against the corresponding molecular weight
V - V
e o
(Kav = -- V where V = column void volume, V = elution volumes and
t 0o
Vt = total column volume). Protein was monitored at 280 nm using a

Beckman Model 25 spectrophotometer (Beckman Instruments, Inc., Fullerton,

CA).

Bio-RadR Low Molecular Weight Protein Standards (10,000-100,000) for

SDS Gel Electrophoresis were also used for molecular weight determination

using SDS-PAG gel electrophoresis. The instructions outlined by Bio-RadR

(Bio-Rad Laboratories, Richmond, CA) were followed (21). The proteins

included were Phosphorylase B, Bovine Serum Albumin, Ovalbumin, Carbonic

Anhydrase, Soybean Trypsin Inhibitor, Lysozyme and the purified enzyme.

The motility of the enzyme was then compared to the relative motility

(Rm) of the standards.

Determination of the Purified Enzyme-Substrate Mixture Reaction Time

Five milliliters of substrate (gelatin or shrimp protein) and 100 pi

of the purified enzyme were incubated at 35 C for 0, 5, 10, 15, 30 and 60

min in order to determine the apparent optimum reaction time.




44



Enzyme activity was measured using the Fluorescamine technique. Figure

10 shows that 10 min was the optimum reaction time for the purified

enzyme-substrate (gelatin or shrimp protein) reaction mixture. This

optimum reaction time was used for the remainder of the characterization

of the extracellular proteolytic enzyme.

Effect of Ionic Strength on Enzyme Activity

The effect of ionic strength on enzyme activity was investigated.

Gelatin (1.2 mg/ml) was dissolved in the following solutions of sodium

chloride (NaC1): 0.05 M (v = 0.13), 0.08 M (P = 0.16), 0.18 M (u =

0.26), 0.25 M (P = 0.35), 0.34 M (P = 0.42), 0.51 M (P = 0.59), 0.75 M

(0 = 0.83), 1.00 M (0 = 1.08) and 1.5 M (P = 1.58). The NaC1 was dis-

solved in 0.05 M phosphate buffer (pH 8). Five milliliters of this mix-

ture were reacted with 100 Pl of the purified enzyme and incubated at

35 C for the selected reaction time (10 min).

Determination of Optimum pH

Buffers of varying pH from pH 2 to pH 10 were used to determine the

optimum pH for the proteolytic activity of the enzyme(s). The following

buffers were used:

Ionic Strength

pH 2 0.1 M citric acid 0.25

3 47.0 ml of 0.1 M citric acid + 3.5 ml of 0.35
0.1 M sodium citrate

4 33.0 ml of 0.1 M citric acid + 17.0 ml of 0.45
0.1 M sodium citrate

5 20.5 ml of 0.1 M citric acid + 29.5 ml of 0.45
0.1 M sodium citrate

6 88 ml of 0.2 M monobasic sodium phosphate + 0.45
12.5 ml dibasic sodium phosphate

7 39.0 ml of 0.2 M monobasic sodium phosphate + 0.35
61.0 ml of 0.2 M dibasic sodium phosphate




45















250-
-250 Gelatin
---- Shrimp

200-
ULJ


05 0 O






050-
-j







0 5 10 15 30 60
INCUBATION TIME AT 355�C (min)

Figure 10. Increase in fluorescence intensity following incubation of
gelatin and shrimp protein substrate with purified enzyme
for up to 1 hr at 35 C.




46



Ionic Strength

8 5.3 ml of 0.2 M monobasic sodium phosphate 0.25
+ 95.0 ml of 0.2 M dibasic sodium phosphate

9 50 ml of 0.2 M glycine + 8.8 ml of 0.2 M NaOH 0.22

10 50 ml of 0.2 M glycine + 32.0 M NaOH 0.20

Gelatin (1.2 mg/ml) or shrimp protein (0.6 mg/ml) were dissolved in

the various buffers. Any pH adjustments due to the addition of the sub-

strates were done using 10 mM HC1 or 10 mM NaOH. Five milliliters of

this mixture were reacted with 100 ip of the purified enzyme and incu-

bated at 35 C for the selected reaction time (10 min).

Determination of Optimum Temperature

Five milliliters of gelatin or shrimp protein substrate and 100 pi

of the purified enzyme were incubated at 5, 10, 20, 35, 45, 55 and 65 C

for 10 min at the optimum pH determined in the previous section.

Thermal Stability

P. citreus was incubated at 5 and 35 C in 300 ml of Trypticase Soy

Broth (TSB). Cell-free broths obtained at midlog phase, 108 and 36 hrs

for the 5 and 35 C grown cells, respectively, were used in this study.

Five milliliters of the cell-free broths were incubated at 35, 45, 55,

65, 75 and 85 C for 15 min. The heat treated cell-free broths solutions

were rapidly cooled (87), and their activity was assayed at 35 C for 15

min using gelatin as substrate. The residual activities at each solution

were compared to the activity observed when the cell-free broths were

incubated with the substrate at 35 C for 15 min.

In addition, 1 ml of the purified enzyme was also incubated at 35,

45, 55, 65, 75 and 85 C for 10 min. The heat treated purified enzyme

solution was cooled, and its activity assayed at 35 C for 10 min using




47



gelatin as substrate. The residual activities of each solution were

compared to the activity observed when the purified enzyme was incubated

with gelatin at 35 C for 10 min.

Effect of Sodium Chloride Concentration

Various concentrations of NaCl were tested for their effect on

enzyme activity. Concentrations of 0.00, 0.25, 0.50, 0.75, 1.00, 1.25

and 1.50% were used. NaCl was dissolved in 0.05 M phosphate buffer pH 8.

The shrimp protein (0.6 mg/ml) and gelatin (1.2 mg/ml) were dissolved in

the NaC1 solutions. Five milliliters of the NaC1 solutions were incu-

bated with 100 pl of the purified enzyme at 35 C for 10 min.

Effect of Sodium Bisulfite Concentration

Various concentrations of sodium bisulfite (NaHSO3) were tested for

their effect on enzyme activity. Concentrations of 0.0, 0.5, 1.0, 2.0

and 3.0% were tested. NaHSO3 (J. T. Baker Chemical Co., Phillipsburg,

NJ) was dissolved in 0.05 M phosphate buffer pH 8. The shrimp substrate

was dissolved in these NaHSO3 solutions (0.6 mg/ml). Five milliliters

of the NaHSO3 solutions were incubated with 100 Ul of the purified

enzyme at 35 C for 10 min.

Effect of Enzyme Concentration

Various quantities of enzyme (from 0 to 200 ~1) were tested to

observe the effect of enzyme concentration on enzyme activity. Five mil-

liliters of substrate (gelatin or shrimp protein) were incubated with 0,

50, 75, 100 and 200 P1 of enzyme at 35 C for 10 min.

Effect of Substrate Concentration

The enzyme was incubated with various concentrations of gelatin and

shrimp protein in order to determine substrate saturation conditions.

For the gelatin substrate, 0.00, 0.15, 0.30, 0.45, 0.60 and 1.20 mg/ml




48



were tested. However, for the shrimp substrate, 0.000, 0.075, 0.100,

0.125, 0.150, 0.300 and 0.600 mg/ml were tested. Five milliliters of

each substrate solution were reacted with 100 pl of the purified enzyme

at 35 C for 10 min. From these data, Lineweaver-Burk plots were derived,

and K and V values for each substrate were extrapolated from these
m max
plots (95,152).

Effect of Metal Ions on Enzyme Activity

Calcium chloride (10, 20 mM), ferric chloride (1, 20 mM), magnesium

chloride (10, 20 mM), mercurous chloride (1, 20 mM), zinc chloride (10,

20 mM), manganese chloride (10, 20 mM) and potassium chloride (5, 20 mM)

were tested for their effect on enzyme activity (all metals were dis-

solved in 0.05 tris-HCl buffer). For the control, a buffer with no

metal ions added was used (76,87). Five milliliters of substrate (gela-

tin) 100 pl of enzyme and 1 ml of the metal ion buffer solution were

reacted for 10 min at 35 C. The fluorometer reading of the control

sample was compared to the reading of the metal ion samples.

Effect of Various Reagents on Enzyme Activity

Ethylene diaminetetraacetic acid (EDTA) (10, 20 mM), citric acid

(10, 20 mM), formaldehyde (1, 20 mM), potassium cyanide (KCN) (1, 20 mM),

potassium permanganate (KMnO4) (1, 20 mM), cysteine (1, 20 mM), 2-

mercaptoethanol (1, 20 mM), p-dioxane (10, 20 mM) and trichloroacetic

acid (TCA) (5, 10%) were tested for their effect on the proteolytic

activity of the P. citreus enzyme (76,87). All reagents were dissolved

and/or mixed with 0.05 M tris-HCl buffer. Five milliliters of substrate

(gelatin), 100 il of enzyme and 1 ml of the appropriate reagent buffer

solution were reacted at 35 C for 10 min. A control with no reagent

added was used and the fluorometer reading from the various reagents was

compared to the control.




49



Dipeptidase Activity

The potential of the P. citreus enzyme to degrade peptides was

investigated. DL-leucylglycine, DL-leucyl-DL-alanine, glycyl-DL-leucine,

DL-alanylglycine and L-leucyl-l-tryptrophan (Sigma Chemical Co., St.

Louis, MO) were used in this study. Fifty milligrams of each dipeptide

were dissolved in 50 ml of phosphate buffer, pH 8. Five milliliters of

the dipeptide solutions were incubated with 100 .l of the purified

enzyme at 35 C for 10 min. The reaction was terminated by adding 10 ml

of 5% TCA. Zero time blanks were done by adding the TCA to the enzyme-

peptide mixture before the incubation period.

Enzyme Induction Studies

P. citreus was grown in various media in order to determine if the

extracellular proteolytic enzyme produced by this organism is induced by

shrimp protein. Three-hundred milliliters of the following were used:

(1) Yeast Carbon Base (YCB) (control)

(2) YCB + 1.0% Shrimp Protein

(3) YCB + 0.1% Yeast Extract

(4) YCB + 0.1% Yeast Extract + 1.0% Shrimp Protein

Table 4 shows the composition of the Yeast Carbon Base medium (YCB).

P. citreus growth and enzyme activity were analyzed at 0, 24, 48, 72

and 96 hrs following incubation at 20 C. Cell numbers were determined

by pour plating into Trypticase Soy Agar (TSA) with incubation at 20 C

for 5 days. Five milliliters of the shrimp substrate were incubated

with 1 ml of the cell-free broth from each culture for 15 min at 35 C.

The reaction was terminated by adding 10 ml of 5% TCA. Zero time blanks

were done by adding the TCA to the cell-free broth-substrate mixture

before the incubation period. This study was done twice in duplicate.




50





Table 4. Composition of yeast carbon base medium (56).



Formula in Grams per Liter of Distilled Water


Boric Acid 0.500 mg.
Copper Sulfate 0.040
Potassium Iodide 0.100
Ferric Chloride 0.200
Manganese Sulfate 0.400
Sodium Molybdate 0.200
Zinc Sulfate 0.400
Biotin 0.002 mg.
Calcium Pantothenate 0.400
Folic Acid 0.002
Inositol 2.000
Niacin 0.400
p-Aminobenzoic Acid 0.200
Pyridoxine 0.400
Riboflavin 0.200
Thiamine HC1 0.400
L-Histidine HC1 0.001 g.
DL-Methionine 0.002
DL-Tryptophan 0.002
Potassium Phosphate 1.000 g.
Magnesium Sulfate 0.500
Sodium Chloride 0.100
Calcium Chloride 0.100
Dextrose 10.000
Final pH of the base adjusted to 7.5




51


The data was analyzed in a similar manner as the Growth Medium and

Enzyme Activity data. Again, a comparison of the m's for each medium

used (m = units of enzyme activity/cell/hr) was attempted using the SAS

program package for analysis of variance (15).

The Duncan's New Multiple-Range Test (pg. 187-190 (132)) was used to

compare any difference in the calculated means of the data obtained after

analysis of variance in the "Optimization of enzyme activity to growth

and cell number" section (pgs. 36 and 38), "Optimum pH determination"

section (pg. 82), "Optimum temperature determination" section (pg. 84)

and "Enzyme induction study" section (pg. 105-115). The Duncan's New

Multiple-Range Test was done using the SAS program package (15).















RESULTS AND DISCUSSION

The ability of Planococcus citreus to grow in shrimp during ice

storage raised the question as to whether this organism could contribute

to the spoilage of shrimp. Various studies (3,4,5) have indicated that

this organism may contribute to the spoilage of this valuable marine

resource. In order to more clearly understand the contribution this

organism makes to the degradation of shrimp, an investigation was under-

taken to study the extracellular proteolytic enzyme(s) produced by this

organism.

Proteolytic Activity of Cellular Fractions

The proteolytic activity of cellular fractions of P. citreus cells

grown in Trypticase Soy Broth (TSB) was investigated in order to deter-

mine the distribution of the enzyme activity in the isolated fractions.

In addition to the cell-free broth (extracellular fraction), whole cells,

washings of the whole cells, soluble intracellular and the cellular

particulate fraction were examined. Table 5 shows the total activity

(units of activity), protein content (mg/ml), specific activity (units

of activity/mg of total protein) and distribution of activity (%) for

all the fractions tested using both gelatin and shrimp protein sub-

strates. The extracellular fraction showed the highest specific activ-

ity, 29.450 units of activity/mg of protein and 27.540 units of activ-

ity/mg of protein towards gelatin and shrimp protein, respectively.

This represented 95.9 and 95.8% of the total activity present in all of

the fractions towards gelatin and shrimp protein, respectively.

52







Table 5. Proteolytic activity at 35 C for 15 min (pH 8) of cellular fractions obtained from
Planococcus citreus grown in Trypticase Soy Broth (TSB) using gelatin and shrimp as
substrates.



Total Enzyme Specific Activityb Distribution of
Activity (units) Proteina (units/mg total protein) Activity (%)
Fractions Gelatin Shrimp (mg/ml) Gelatin Shrimp Gelatin Shrimp


Whole cells 386.7 395.6 23.40 0.136 0.139 0.4 0.5

Extracellular 131,100.0 122,590.0 44.50 29.450 27.540 95.0 95.8

1st washing 251.1 253.3 6.26 0.401 0.406 1.3 1.4

2nd washing 163.3 133.3 3.26 0.500 0.408 1.6 1.4

Intracellular 101.1 81.1 10.33 0.098 0.079 0.4 0.3

Particulates 171.1 192.2 11.26 0.152 0.171 0.6 0.6


bAverage of duplicate samples
Total activity/Total protein = specific activity (units of activity/mg protein)




54



The whole cell fraction (cell bound fraction), both whole cell washings

(loosely bound to cell wall fraction) and the particulate fraction

exhibited low specific activity towards both high molecular weight sub-

strates. The intracellular soluble fraction (the soluble fraction after

the differential centrifugation of ruptured cells) exhibited the lowest

specific activity when gelatin and shrimp protein were used as substrates

(Table 5). These results show that the major portion (>95.0%) of the

active enzyme towards these two high molecular weight substrates resides

in the extracellular fraction.

Most microorganisms can synthesize various enzymes within their

cell structure. Each enzyme system may have its own unique characteris-

tics, and these characteristics will vary depending on the enzyme, the

substrate and conditions during the enzyme-substrate reaction. In addi-

tion, the location of the proteinase(s) within the bacterial cell may

vary markedly between microorganisms. Various researchers (53,115,135,

139) have studied the location of particular bacterial proteinases

within the cell and how this location relates to the function of the

enzyme. Thomas et al. (139), using gentle procedures for cell fraction-

ation, suggested two criteria for the location of a proteinase produced

by Streptococcus lactis. The two criteria they suggested were: 1)

intact cells (whole cells) possessed substantial proteinase activity

when incubated with a high molecular weight substrate; 2) most of the

cell-bound proteinase activity was released during spheroplast formation.

The solubilized cell wall, plasma membrane and cytoplasm fractions con-

tained 84%, 0% and 16% activity, respectively, of the total proteinase

activity with casein as substrate (139). In the results presented in

this dissertation, the whole cell and cellular particulate fractions of




55



the P. citreus cells showed little enzyme activity towards gelatin and

shrimp protein (both high molecular weight substrates).

Thomas et al. (139) also concluded that the cell wall proteinase

may serve a similar nutritional role in nature as the surface-bound pro-

teinases discussed by Payne and Gilvarg (115) and Sussman and Gilvarg

(135). Gilvarg and his co-workers stated that surface-bound protein-

ase(s) appear to serve a nutritional role by hydrolyzing proteins to

amino acids or peptides that are small enough to enter the cell. In

turn, Payne, Sussman and Gilvarg (115,135) also suggested that the

intracellular peptidases could further hydrolyze the peptides formed and

release their constituent amino acids, thus, permitting the utilization

of the protein substrate for growth. In Table 5, we can observe that

certain P. citreus fractions (whole cells, intracellular and cellular

particulate) had substantial amounts of protein present. Perhaps some

of the protein present in these fractions include other enzymes (i.e.,

peptidases) that can utilize the peptides produced by the action of the

extracellular protease(s) that later may enter the P. citreus cell. In

this manner, P. citreus cells could fully utilize the protein available

(i.e., shrimp protein as well as other proteins) for their growth.

Growth Medium and Enzyme Production

Trypticase Soy Broth (TSB), Plate Count Broth (PCB) + 0.5% NaCI and

Nutrient Broth (NB) + 0.5% NaC1 were used to determine growth rates and

production of extracellular proteolytic enzyme(s) by P. citreus. Figure

11 shows the growth of P. citreus, as measured by the increase in optical

density (600 nm), in the three media used. In all three media, P.

citreus exhibited a 12 hr lag phase in which an increase in optical den-

sity was not evident. After this lag period, TSB supported the most




56






-*- Plate Count Broth
-- Nutrient Broth
.30 - - Trypticase Soy Broth




.25-

C
0
0
O
.20-

U-

Ld
c .15




a_
O .10-




.05




II I I I
0 12 24 48 72 96
INCUBATION TIME AT 200C (hr)

Figure 11. Spectrophotometric growth curves of Planococcus citreus in
Plate Count Broth, Nutrient Broth and Trypticase Soy Broth
at 20 C.




57



rapid growth of P. citreus. The optical density after 96 hours of incu-

bation at 20 C was .314, .200 and .072 for TSB, PCB and NB, respectively.

Figure 12 shows a similar trend; however, P. citreus growth was measured

by the Aerobic Plate Count technique (6). Again, we can observe that

the P. citreus log count per ml increases slightly during the first

12 hours of incubation at 20 C. After 96 hrs of incubation, the P.

citreus log count for TSB, PCB and NB was 6.02, 5.10 and 4.17, respec-

tively. Consequently, TSB allowed for the "optimum" growth of P.

citreus when grown at 20 C.

Nutritional components present in the growth medium are of utmost

importance for gram-positive microorganisms which are generally more

fastidious in its nutrient requirements than gram-negative bacteria (29).

Realizing these growth requirements of gram-positive microorganisms, the

results from this section are not surprising. TSB contains tryptone,

soytone, dextrose, sodium chloride and dipotassium phosphate (16). This

combination of nutrients provide an adequate nitrogen, carbohydrate,

vitamin and overall nutrient supply for the growth of P. citreus. In

contrast, PCB and NB are not as nutritionally complex.

Figure 13 shows the enzyme activity of the cell-free broth of P.

citreus cells grown in TSB, NB and PCB for 96 hrs at 20 C. An active

extracellular enzyme fraction was produced by P. citreus in all three

media. However, after 96 hrs of growth, the amount of enzyme produced

by this organism in TSB is greater than that produced when grown in PCB

of NB. The enzyme activity after 96 hrs of incubation of the cell-free

broth of P. citreus grown in TSB, PCB and NB was 323, 270 and 200 units

of activity, respectively. However, if the cells are harvested at

approximately midlog phase, the difference in the amount of enzyme




58







-- Plate Count Broth
-A- Nutrient Broth
6- -A- Trypticase Soy Broth




5-

I)




Z)
()
OD











I-






0 12 24 48 72 96

INCUBATION TIME AT 200 C (hr)

Figure 12. Aerobic plate counts of Planococcus citreus incubated in
Plate Count Broth, Nutrient Broth and Trypticase Soy Broth
at 20 C for 96 hrs.




59



35-
-*- Plate Count Broth
--- Nutrient Broth
-A- Trypticase Soy Broth

30




25-

20

>-20-
F--




10-
N






5-





0 12 24 48 72 96
INCUBATION TIME AT 200C (hr)
Figure 13. Enzyme activity at 35 C for 15 min (pH 8) of the cell-free broth
of Planococcus citreus cells grown in Plate Count Broth, Nutrient
Broth and Trypticase Soy Broth for up to 96 hrs.




60



produced (as measured by enzyme activity) is more clearly observed.

After 48 hrs of incubation, the enzyme activity for the P. citreus grown

in TSB, PCB and NB was 200, 100 and 85.5 units of activity, respectively.

Under the conditions tested, P. citreus exhibited maximum enzyme produc-

tion when grown in TSB.

The units of activity per cell per hr (m) was calculated for each

medium used and the results are shown in Table 6. The average m value

for TSB, PCB and NB was 168.50, 105.67 and 59.32, respectively. These

data show that the amount of enzyme produced by actively growing P.

citreus cells (midlog phase) in TSB is higher than that produced when

P. citreus is grown in PCB or NB. The analysis of the data supports

this observation. A significant difference (a = 0.05 level) was

observed between the m values of TSB, PCB and NB (Table 6).

Thus, after evaluating the results from this section, TSB was cho-

sen as the best medium for P. citreus growth and enzyme production and

was used for the remainder of the study. The combination of nutrients

in TSB allowed for the rapid growth of P. citreus and by doing so, per-

mitted the production of more extracellular proteolytic enzyme. In

addition to the combination of nutrients in TSB, the presence of 0.25%

dextrose may play a role in extracellular enzyme production. Dextrose

has been suggested as a possible inducer of a variety of enzymes (57),

although this effect was not specifically tested in these experiments.

Effect of Incubation Temperature on Enzyme Production and Activity

The ability of P. citreus to produce an active extracellular enzyme

at 5, 20 and 35 C was investigated in order to determine the ability of

the extracellular enzyme(s) to affect shrimp protein at refrigeration

(5 C) or iced temperatures. Figure 14 shows the increase in optical




61





Table 6. Units of enzyme activity per cell per hour (m) of Planococcus
citreus grown in Trypticase Soy Broth (TSB), Plate C unt
Broth (PCB) and Nutrient Broth (NB) at midlog phase.


Medium mean m value2


TSB 148.50a

PCB 105.67b

NB 59.32c


ICells were grown at 20 C and enzyme activity was measured at 35 C
2for 15 min (pH 8).
average of 6 observations
Means followed by the same letter do not differ significantly at the
a = 0.05 (r from Anova table 0.984)




62







1.7-
---- 350 C
1.6 - - 200 C

1.5 -A- 5 C

1.4-

'.3
0I .2-

01.-
O
0
I.0 -

>0.9-
U)
. 0.8-

C3 0.7-
uJ

F J-
0.6-

n0.5 -
0
0.4-

0.3-

0.2-

0.1


0 12 24 48 72 96 120 144
INCUBATION TIME (hr)

Figure 14. Spectrophotometric growth curves of Planococcus citreus in
Trypticase Soy Broth incubated at 5, 20 and 35 C.




63



density of P. citreus cultures grown at 5 C for 144 hrs, 20 C for 120

hrs and 35 C for 96 hrs. After different time intervals for growth

adaptation, P. citreus grew at all three temperatures. Direct micro-

scopic observation of P. citreus cells growing at the three temperatures

revealed a difference in cell arrangements. When cells were grown at 35

C, the predominant morphology present was clusters of gram-positive cocci.

At this temperature, the cells are rapidly growing and dividing, thus,

possibly accounting for the observed predominance of clusters. At 20 C

a mixture of tetrads, pairs and single cells were observed which is the

predominant morphology exhibited by this organism, as described in

Bergey's Manual of Determinative Bacteriology (29). At 5 C the predomi-

nant cell arrangement observed was single cells. This particular morpho-

logical structure might predominate because of the slow metabolic rate

at this temperature, although these rates were not investigated. Indi-

vidual cells have more surface area for the uptake of nutrients. At 5 C,

the movement of nutrients within the cell is slow. Consequently, the

increase in surface area is particularly desirable from a nutritional

standpoint.

Table 7 illustrates the enzyme activity of the cell-free broth of

P. citreus grown at 5 C for 108 hrs, 20 C for 72 hrs and 35 C for 36

hrs (midlog at each temperature) and incubated with shrimp protein at 5,

20 and 35 C for 60, 30 and 15 min, respectively. P. citreus produced an

active extracellular enzyme(s) when grown at all temperatures. In addi-

tion, the cell-free broth obtained from the three temperatures of growth

exhibited activity at all three enzyme-substrate incubation temperatures

(5, 20 and 35 C). As the temperature of growth increased from 5 to 35

C, the enzyme activity increased at a similar rate at the three enzyme-







Table 7. Enzyme activity measured at 5, 20 and 35 C (pH 8) of the cell-free broths of Planococcus
citreus grown in Trypticase Soy Broth (TSB) at 5, 20 and 35 C for 108, 72 and 36 hrs,
respectively.


Enzyme-Substrate Temperature of Growth (C)
Incubation Temperature (C) 5 20 35


5 24.18 � 1.11a 58.18 � 2.08 116.17 � 8.40

20 25.65 � 1.20 72.30 � 2.04 139.67 � 7.88

35 28.56 � 1.35 98.55 � 3.10 178.93 t 7.68


aAverage of 6 observations � standard deviation




65



substrate incubation temperatures. A higher P. citreus count was

observed at 35 C and the production of extracellular enzyme(s) was also

higher at all three enzyme-substrate incubation temperatures. This

indicates that the amount of enzyme produced by P. citreus is related to

the amount of growth of the organism in the medium. In addition, as the

enzyme-substrate incubation temperature increased from 5 to 35 C, the

enzyme activity of the cell-free broths increased. Although enzyme

activity is present at the lower temperatures, the data presented indi-

cate that the optimum temperature of the extracellular protease system

may be close to 35 C. Consequently, the results indicate that P. citreus

can indeed produce an active extracellular enzyme(s) capable of utilizing

the protein in shrimp when shrimp is stored at refrigerated or iced

temperatures.

The effect of refrigeration temperatures on enzyme activity has

been studied (50,51). In most of the research, the majority of the

enzymes studied lost activity when incubated at low temperatures. Stud-

ies have shown that lactic streptococci characteristically produced less

acid after storage at refrigerated temperatures. Such stored cells also

show a diminished residual proteinase activity (49,50,51,52,149). The

researchers stated that after storage at 3 C, the enzyme showed gross

structural alterations with a concomitant loss of activity. Gel filtra-

tion and sedimentation velocity data indicate that inactivation of the

enzyme was a result of aggregation to higher molecular weight forms (50).

However, several investigators (49,52,131) previously suggested that

storage inactivation of enzymes may be caused by induced conformational

or structural changes. Scutton and Utter (128) and Havir et al. (81)

observed that inactivation of various enzymes by low temperature storage




66



was due to dissociation of the molecules into subunits. The inactivated

enzymes could be reactivated by warming to room temperature.

Purification of the Extracellular Enzyme(s)

Planococcus citreus was grown in Trypticase Soy Broth (TSB) at 20

C for 72 hrs. The cell-free broth was used in the isolation of the

extracellular enzyme(s) of this organism. The cell-free broth had a

total activity of 1.31 x 105 units of activity (total enzyme activity =

change in 0.1 fluorescence units of the TCA filtrate per milliliter of

enzyme per minute), 44.52 mg/ml of protein and a specific activity of

29.45 units of activity/mg protein (Table 8). The cell-free broth was

then fractionated with 0-55%, 55-70% and 70-100% ammonium sulfate

((NH4)2S04).

After overnight dialysis (16 hrs) in phosphate buffer pH 8, the

activity of the 0-55%, 55-70% and 70-100% ammonium sulfate precipitates

was measured. Table 9 shows the proteolytic activity of the various

fractions examined. The specific activity of each fraction was 4.59,

52.39 and 3.99 units of activity/mg of protein for the 0-55%, 55-70% and

70-100% fractions, respectively. Eighty-six percent of the activity was

present in the 55-70% fraction. This is compared to 7.5 and 6.5% for

the 0-55% and 70-100% fractions, respectively (Table 9). Ammonium sul-

fate precipitation is a common method used to precipitate proteins for

their purification. As the ammonium sulfate concentration is raised

from zero, the solubility of a given protein at first usually increases

but then the "salting-in" effect comes to an end and as the salt concen-

tration is raised to higher values a "salting-out" effect is observed

and the protein becomes progressively less soluble (65). The major por-

tion of the extracellular proteolytic enzyme(s) of P. citreus was salted







Table 8. Purification of an extracellular proteolytic enzyme from Planococcus citreus.



Total Activitya Proteinb Specific Activityc Recovery
Fraction Volume (units x 10 ) (mg/ml) (units/mg total protein) Purification (%)


Cell-free broth 900 113.3 44.52 29.45 100.0

70% (NH ) SO
precipitation 350 102.7 19.60 52.39 1.78x 78

SephacrylR
S-200 Superfine 115 65.3 1.42 461.65 15.67x 50

DEAE-SephadexR
A-50 90 64.0 0.32 780.37 26.50x 49
aa

aDetermined using gelatin as substrate
Average of duplicate samples
cTotal activity/Total protein = specific activity (units of activity/mg protein)







Table 9. Proteolytic activity at 35 C for 15 min (pH 8) of various ammonium sulfate
fractions of the cell-free broth of Planococcus citreus.



Total Activitya Proteinb Specific Activityc Distribution of
Fraction (units x 10 ) (mg/ml) (units/mg total protein) Activity (%)


0-55% 8.31 18.07 4.59 7.5

55-70% 102.70 19.60 52.39 86.0

70-100% 5.00 12.50 3.99 6.5


aDetermined using gelatin as substrate
Average of duplicate samples
cTotal activit/Total rotein ecific activity (units of activit/m protein)
Total activity/Total protein = specific activity (units of activity/mg protein) 00




69



out between 55-70% ammonium sulfate saturation. Table 9 shows that 86%

of the activity towards gelatin is observed in this fraction. Table 8

shows that the activity of the 55-70% ammonium sulfate fraction was 1.78

times greater in specific activity than the cell-free broth. A 78%

recovery of the extracellular enzyme(s) was achieved in this step of

the enzyme purification.

SephacrylR S-200 Superfine, a high resolution chromatographic

medium for gel filtration of proteins, nucleic acids, polysaccharides

and biopolymers (120), was used to separate the enzyme(s) present in the

55-70% ammonium sulfate fraction according to molecular weight. Figure

15 shows that four protein peaks were recovered after the elution of the

enzyme fraction through the SephacrylR S-200 column. However, when the

proteolytic activity was measured, the majority of the activity was pre-

sent in protein peak C (third peak in Figure 15). Peak C had a specific

activity of 651.0 units. The enzyme(s) was purified 15.67 times and 50%

of the enzyme was recovered in this step (Table 8). The fractions com-

prising peak C were pooled for further purification. The percent recov-

ery of the extracellular proteolytic enzyme of P. citreus after molecular

sieve chromatography using SephacrylR S-200 Superfine was within the

range of most of the enzymes recovered when the more traditional

SephadexR gels have been used (70,113,114,136).

The pooled fractions of peak C were further rechromatographed using

DEAE-SephadexR A-50 (functional group -C2H4N+(C2H5)2H. A-50 gels are

usually used for low and medium molecular weight proteins (up to

200,000). Ion exchange chromatography may be defined as the reversible

exchange of ions in solution with ions electrostatically bound to an

insoluble support medium. The ion exchanger is the inert support medium












v.25 -50_
O
E
0.20- -40>-

15 F-
00 -30


CrI
0.IO 20>-
c) N
m Z

.05- -I 0

.02-


10 20 30 40 50 60 70
FRACTION NUMBER (5ml fraction)
Figure 15. Separation of proteins present in the 55-70 % ammonium sulfate fraction using SephacrylR
S-200. a
a30 x 2.5 cm column, eluted with 0.02 M phosphate buffer pH 7.




71



to which is covalently bound positive (in the case of the anionic

exchanger) or negative (in the case of a cation exchanger) functional

groups (48). A sodium chloride (NaC1) gradient (range of ionic strength,

P = 0.11 - 0.23) was used with the ion exchange column to elute the pro-

tein components. A gradient is a physical method of constantly changing

the salt concentration of a solution that is being passed through the

column creating a constant and linear increase in concentration (48).

Figure 16 shows one major peak after ion exchange of the pooled active

fractions from peak C. The isolated peak exhibited a specific activity

of 780.37 units (Table 8). The proteolytic enzyme was purified 26.50

times and 49% recovery was achieved (Table 8). Fractions 17 to 21

(Figure 16) were pooled for future characterization.

Purity of the Extracellular Proteolytic Enzyme

Many methods can be used to establish the purity of an enzyme prep-

aration. However, the best indication of purity of an enzyme prepara-

tion is by the consistent failure to detect heterogeneity when several

analytical techniques are used (i.e., a single peak in chromatographic

systems, a single band on electrophoresis, a single band after isoelec-

tric focusing and/or one component in solubility or precipitation tests).

However, the final criterion for purity is the demonstration of a unique

amino acid sequence (61,65) but this is rarely done in order to

demonstrate purity.

The recovery of the isolated peak (Figure 16) as a single entity

with homogeneous activity after DEAE-SephadexR A-50 ion-exchange chroma-

tography was the first indication that the major extracellular

proteolytic activity of P. citreus was isolated in a purified form.









.5 -100

-90*
.4- -800 -.20z
E O
11 ' 70 v


2- _70



C > -<30 2
00.3





0 N )
0 5 10 15 20 25 30 35 40
> Z
Lij - 50 -d





FRACTION NUMBER (10 ml fraction)





Figure 16. Ion-exchange chromatography using DEAE-Sephadex A-50 of the pooled active fractions obtained
in the molecular sieve chromatography step.
0 30 >-









40 x 2.5 column, eluted with 0.02 M phosphate buffer pH 7 with a linear
gradient of 0.01 M NaC- tris 0.15 NaC at a rate of 25 ml/hr.25
0 5 10 15 20 25 30 35 40
FRACTION NUMBER (10 ml fraction)
Figure 16. Ion-exchange chromatography using DEAE-SephadexR A-50 of the pooled active fractions obtained
in the molecular sieve chromatography step.a
a40 x 2.5 column, eluted with 0.02 M phosphate buffer pH 7 with a linear
gradient of 0.01 M NaCI- tris 0.15 M NaCl at a rate of 25 ml/hr.




73



According to Cooper (48) electrophoretic techniques have become

principal tools for characterizing macromolecules and for assaying their

purity. Figure 17 shows a single band after SDS-PAG electrophoresis

using 50 il of the purified enzyme. A single homogeneous band is indic-

ative of the presence of only one enzyme, i.e., the purity of the extra-

cellular enzyme of P. citreus. In addition, as an additional test for

purity, increasing amounts of the purified extracellular enzyme were

added to the gels. Enzyme concentrations of 50 .g protein/gel, 100 ug/

gel, 150 pg/gel and 200 pg/gel were used. Figure 18 shows that a single

band is recovered after SDS-PAG electrophoresis of each protein fraction.

Thus, these results add to the evidence indicating the purity of the

extracellular proteolytic enzyme of P. citreus. Consequently, an extra-

cellular proteolytic enzyme produced by P. citreus was purified 26.50

times using the procedures outlined previously with 49.0% of the enzyme

being recovered (Table 8). The specific activity of the enzyme was

780.37 units of activity/mg protein.

Schwabe (127) reported the use of the fluorescamine reagent to mea-

sure proteolytic enzyme activity of cathepsin enzymes using hemoglobin

a substrate. He stated that while the fluorescamine reagent has been

used successfully for quantitative amino acid analysis, protein and pep-

tide determination, it has also beneficial applications in enzymology.

In addition, Schwabe (127) compared the fluorometric technique with the

Lowry method (98). He concluded that the fluorometric method was 100

times more sensitive than the Lowry method, much faster and less com-

plicated. The fluorometric technique proved to be an efficient method

for the measurement of proteolytic enzyme activity.





74
















































Figure 17. Acrylamide gel electrophoresis of the purified enzyme of
Planococcus citreus.




75



































50 pg/gel' 100 pg/gel 150 vg/gel 200 pg/gel


Figure 18. Acrylamide gel electrophoresis of increasing concentrations
of the purified extracellular enzyme of Planococcus citreus.




76



With the presence of the relatively new Fluorometric technique,

that appears to be more sensitive and reproducible than the traditional

methods available for measuring proteolytic enzyme activity, the results

of various previous research with extracellular enzymes (36,78,84,88,

136) using the Anson method (9) could have possibly resulted in higher

recoveries and higher measurable total enzyme activity. The following

investigators are some of those who used the Anson method to study the

various enzymes. Tarrant et al. (136) working with Pseudomonas fragi in

pig muscle isolated an extracellular proteolytic enzyme with only 18%

recovery after partial purification. Husein and McDonald (84) character-

ized an extracellular proteinase from Micrococcus freudenreichii using

casein as substrate with 23% recovery after partial purification.

Christison and Martin (36) isolated and preliminarily characterized an

extracellular protease of Cytophaga spp. using casein, hemoglobin and
R
azocoll as substrates. After chromatography with DEAE-Cellulose only

26% of the enzyme was recovered. Khan et al. (88), looking at the

extracellular proteases of Mucor pusillus, isolated and characterized

two fractions. However, after DEAE-SephadexR A-50 only 29.3% of the

milk-clotting fraction was recovered and 47.0% of the fraction with pro-

tease activity toward hemoglobin was recovered. Gnosspelius (76) puri-

fied an extracellular protease from Myxococcus virescens using phosphate

precipitation, gel exclusion and ion exchange chromatography. Only

20.1% was recovered after the chromatographic step. In the work reported

in this dissertation, following DEAE-SephadexR A-50, 49.0% of the extra-

cellular enzyme of P. citreus was recovered when the Fluorometric method

was used to measure proteolytic activity.





77



Characterization of the Extracellular Proteolytic Enzyme

The fractions collected (17-21) from peak B (Figure 16) were pooled

and used for the characterization of the extracellular enzyme of P.

citreus.

Molecular Weight Determination

Two methods were used to determine the molecular weight of the

enzyme, column chromatography (SephacrylR S-200 Superfine) and acrylamide

gel electrophoresis. Standards ranging from a molecular weight of 10,000

to 200,000 were used to determine the molecular weight of the P. citreus

enzyme. Using both techniques, the molecular weight of the extracellular

enzyme of this organism was approximately 29,000. Figures 19 and 20 show

the molecular weight determination using SephacrylR S-200 and acrylamide

gel electrophoresis, respectively. Different standards were used in each

case to assure that the molecular weight was estimated correctly.

A search of the literature was done in order to compare the molecu-

lar weight of the extracellular proteolytic enzyme of P. citreus with

extracellular proteases from other microorganisms. Pacaud and Uriel

(113) estimated the molecular weight of a protease from Escherichia coli

using electrophoresis on polyacrylamide gels and sucrose-density gradient

centrifugation to be about 43,000. Four years later, Pacaud and Richaud

(114) estimated the molecular weight of a second protease of E. coli

using gel filtration and SDA-acrylamide gels to be 58,000. Drapeau et

al. (59) estimated the molecular weight of an extracellular protease of

Staphylococcus aureus to be approximately 12,000 using sedimentation

equilibrium and gel electrophoresis studies. Arvidson et al. (12)

reported the molecular weight of an extracellular (alkaline protease)

enzyme from S. aureus to be approximately 12,500. Later, he reported




78












1.6-


1.4-
Ribonuclease A
1.2Trypsin
* P citreus (29,000)
Pepsin *
Bovine Serum Albumin

.8- -

.6-
* Aldolase
.4-

.2


I " I I
0 2 4 6 8 10 12 14 16 18 20 22
MOLECULAR WEIGHT (x O4)
Figure 19. Calibration curve for the molecular weight estimation o
Planococcus citreus proteolytic enzyme using Sephacryl
S-200 column chromatography.
40 x 2.5 column, eluted with 0.02 M phosphate
buffer at a rate of 15 ml/hr.






100,000
1Phosphorylase B


(_ Bovine Serum Albumin


50,000-
c< 0 Ovalbumin
40,000

30,000 Carbonic Anhydrase P, citre (29,000)

20,000-- Soybean Trypsin Inhibitor 0
Lysozyme
10,000

I I I I I
0 1 2 3 4 5 6
Relative Mobility (cm)
Figure 20. Calibration curve for the molecular weight estimation of Planococcus citreus proteolytic
enzyme using acrylamide gel electrophoresis.
a10 % acrylamide: Bis 30:0.8 gel, 1-1.5 mAMP/ gel for 3 hrs.





80



(11) the molecular weight of a EDTA-sensitive S. aureus protease as

28,000. Recently, Hoshida et al. (137) estimated the molecular weight

of a proteolytic enzyme from Bacillus sphaericus to be about 26,000.

Gnosspelius (76) working with an extracellular enzyme of Myxococcus

virescens reported its molecular weight as 26,000. Thus, the apparent

molecular weight of the extracellular enzyme of P. citreus (MW 29,000)

is within the range of other extracellular proteolytic enzymes reported

in the literature.

Effect of Ionic Strength on Enzyme Activity

The effect of salts on the solubility of proteins is well known.

The solubility is usually a function of the ionic strength. In condi-

tions of high ionic strength, the ions attract around themselves the

polarizable water molecules, making less water available for the pro-

teins since, at high salt concentrations, the number of charged groups

contributed by the salts is enormous compared with those of the proteins.

Consequently, the solubility of the proteins decreases (152). In addi-

tion, any change in the charges of an enzyme may cause various transfor-

mations in structure or active site configuration that could affect its

activity towards the substrate. Figure 21 shows that ionic strengths (P)

of 0.15-0.83 did not alter the attraction of the P. citreus extracellular

enzyme towards gelatin substrate. However, as the ionic strength was

increased the activity of the enzyme decreased. An ionic strength of

1.60 (1.5 M NaC1) caused a decrease in enzyme activity of approximately

60%. Thus, if ionic strengths above 0.83 (0.75 M NaC1) are used they

may cause a change in solubility of the enzyme, charged groups, confor-

mation of the enzyme, active site stability and/or active site availa-

bility to the substrate. Gnosspelius (76) stated that variations in the







50-




140-
O



30-



20- --- Gelatin
1UJ 20-
>-
N
Z
LLJ
10-





0.10 0.38 0.75 1.13 1.6
IONIC STRENGTH (t.)
Figure 21. Effect of ionic strenght on the activity of the extracellular proteolytic enzyme of
Planococcus citreus.
aActivity assayed at 35 C for 10 min, p1 8.




82



ionic strength did not signficantly influence the activity of Myxococcus

virescens when casein was used as the substrate. However, the actual

data for this observation were not presented in the literature.

In that the activity of the proteolytic enzyme was not affected by

ionic strengths of p=0.83 or lower, the buffers shown in page 44,

(Determination of Optimum pH) were considered acceptable and were used

for the determination of the optimum pH of the P. citreus enzyme.

Optimum pH Determination

The pH optimum of an enzyme is dependent upon a number of experi-

mental parameters. Changes in pH may cause changes in the ionization

of prototropic groups (groups capable of ionization) in the active site

of an enzyme. These prototropic groups in the active site may be

involved in maintaining the proper configuration of the site, in binding

a substrate to enzyme and/or in transformation of substrate to products

(133). However, there is usually a zone of maximum ion stability in

which enzyme activity is maximal. Enzyme inactivation also increases on

the acid and alkaline sides of this maximum activity zone. Observing

Figure 22, enzyme activity was maximum at pH 8 when both gelatin and

shrimp protein were used as substrates. The activity dropped as the pH

became more acidic or alkaline. Although not statistically significant,

a slightly higher activity was evident at the alkaline pH's (9 and 10)

when shrimp protein was used as substrate.

The majority of the bacterial enzymes studied have shown maximum

proteolytic activity at neutral pH's (57,65,151). The enzyme isolated

in this study resembles the bacterial proteolytic enzyme from Proteus

vulgaris (105), Bacillus sphaericus (155), Staphylococcus aureus (11,12),

Serratia marcescens (106) and Pseudomonas spp. (87) in that they all




83






60-
-*- Gelatin
- - Shrimp


50




-40-

>-


P30-
O /



N20-
LU


10-





2 3 4 5 6 7 8 9 10
pH
Figure 22. Optimum pH of the extracellular proteolytic enzyme of
Planococcus citreus.a
a
Gelatin and shrimp protein substrate incubated
at 35 C for 10 min, pH 8.




84



require a slightly alkaline pH for optimum activity. Considerable

activity is present at neutral pH's; the pH of freshly caught shrimp is

around neutrality. During shrimp storage, the pH of shrimp will

increase (5).

Optimum Temperature Determination

Changes in temperature may affect enzymatic reactions in a number

of ways. Some of these effects may include: a) stability of the enzyme;

b) affinity of enzymes for activators and inhibitors; c) ionization of

prototropic groups; d) enzyme-substrate affinity; and e) velocity of

breakdown of enzyme-substrate complex (131). The optimum temperature of

the P. citreus extracellular enzyme when both shrimp protein and gelatin

were used as substrates was 35 C (Figure 23). Although not statistically

signficant, a slightly higher enzyme activity was observed at the lower

temperatures (5 and 10 C) using shrimp protein as substrate as compared

to gelatin. However, at the higher temperatures (45 and 55 C) the

reverse was evident. Thus, as previously observed with the cell-free

broth (enzyme crude extract), the purified enzyme of P. citreus can

exhibit activity at the temperatures of refrigerated shrimp.

Thermal Stability

The cell-free broths obtained from P. citreus cells grown in Trypti-

case Soy Broth (TSB) at 5 and 35 C for 108 and 36 hrs, respectively, and

the P. citreus purified enzyme were incubated at 35, 45, 55, 65, 75 and

85 C for 15 min in order to study the various temperatures effects on

stability. The enzyme activity of the cell-free broths and purified

enzyme remaining after the various heat treatments was assayed using

gelatin as the substrate and compared to the activity observed with the

unheated cell-free broths and purified enzyme. Figure 24 shows the







50-
--- Gelatin
-*- Shrimp


40-




>- 30
F-


UJ20-



L-
lo
N








5 10 20 35 45 55 65
TEMPERATURE (oC)
Figure 23. Temperature optimum of the extracellular proteolytic enzyme of Planococcus citreus.a
Activity assayed at 35 C for 10 min, pH 8.
















CD

N
fD


� REMAINING ENZYME ACTIVITY (%)
N C~l - ch 1 n w 0 5





. ia a iiNa a a a. a .f a a
.1t
oo
)D 0 0 0 0 0 0 0 0 0 0










fnO
(D
0Cl I-C (D




o9 I- S. ..









o0 1
N i- _____







* r "! ( '-*



( rt




D.. f.. B
0

:3 m 0




0 0
P-c . 0 0 o







0








<"<
0 0












98N




Full Text
130
143. Ward, D. R., G. Finne and R. Nickelson. 1979. Use of a specific
ion electrode (ammonia) in determining the quality of shrimp. J.
Food Sci. 44:1052-1057.
144. Weber, K. and M. Osborn. 1969. The reliability of molecular
weight determination by dodecylsulfate polyacrylamide gel
electrophoresis. J. Biol. Chem. 244:4406-4410.
145. Weigele, M., S. L. DeBernardo and W. Leimgruber. 1973. Fluoro-
metric assay of secondary amino acids. Biochem. Biophys. Res.
Comm. 50:352-356.
146. Weigele, M., S. L. DeBernardo, J. P. Teugi and W. Leimgruber.
1972. A novel reagent for the fluorometric assay of primary
amines. J. Amer. Chem. Soc. 94:5927-5928.
147. Weil, L., W. Kocholaty and L. D. Smith. 1939. CV III. Studies
on proteinases of some anaerobic and aerobic microorganisms.
Biochem. J. 33:893-897.
148. Weingartner, K. E., J. A. Koburger, J. L. Oblinger and F. W. Knapp.
1977. Residual bisulfite in iced Penaeus shrimp. J. Food Prot.
40:234-235.
149. Westhoff, D. C., R. A. Cowman and M. L. Speck. 1970. Effect of
storage at 3 C on the proteinase enzyme systems of slow and fast
strains of lactic streptococci. J. Dairy Sci. 53:1023-1027.
150. Whitaker, J. R. 1963. Determination of molecular weights of
proteins by gel filtration on Sephadex. Anal. Chem. 35:1950-1960.
151. Whitaker, J. R. 1972. Principles of Enzymology for the Food
Sciences. 1st Ed. Marcel Dekker, Inc., New York, NY.
152. White, A., P. Handler and E. L. Smith. 1973. Principles of
Biochemistry. 5th Ed. McGraw-Hill, Inc., New York, NY.
153. Williams, 0. B. 1949. Microbiological examination of shrimp.
J. Milk Food Technol. 12:109-110.
154. Williams, 0. B., L. L. Campbell, Jr. and H. B. Rees, Jr. 1952.
The bacteriology of Gulf Coast shrimp. II. Qualitative observa
tions on the external flora. Texas J. Sci. 4:43-54.
155. Willms, C. R. 1960. Studies on an extracellular proteolytic
enzyme from a marine bacterial species. Ph.D. Dissertation.
Agricultural and Mechanical College of Texas.
156. Wood, A. J., G. J. Sigundsson and W. J. Dyer. 1942. The surface
concept in measurements of fish spoilage. J. Fish. Res. Brd.
Can. 6:53-62.
157. Yoshida, K., H. Hidaka, S. Miyado, U. Shibata, K. Saito and Y.
Yamada. 1977. Purification and some properties of Bacillus
sphaericus protease. Agrie. Biol. Chem. 41:745-754.


106
presence of a specific substrate (28,57,73). A substantial number of
bacterial exo-enzymes appear to be induced by their substrate or closely
related compounds (122) while others are continuously being synthesized
by the microorganisms during their growth. According to Pollock (121),
enzyme induction does not introduce a new pattern of protein structure
into the cell. Whether constituent or induced, and whatever inducer is
used, the enzyme formed appears to be identical (121).
Yeast Carbon Base (YCB), a minimal substrate level medium, was used
in this study (see Table 4) and was fortified with shrimp protein and/or
yeast extract. In addition, a study with Trypticase Soy Broth (TSB)
fortified with shrimp protein was also done.
Figure 32 shows the growth of _P. citreus in the various media fol
lowing 96 hrs of incubation at 20 C. Overall, _P. citreus grew poorly in
all four media tested. About a 1.15 log increase in _P. citreus cell
number was observed in YCB, a 1.20 log increase in YCB + 1.0% shrimp
protein (YCBS), a 1.40. log increase in YCB + 0.1% yeast extract (YCBY)
and a 1.60 log increase in YCB + 1.0% shrimp protein + 0.1% yeast extract
(YCBSY). Therefore, as the nutrients in the growth medium increased,
improved growth of _P. citreus was observed.
The proteolytic activity of the cell-free broth of _P. citreus grown
in Yeast Carbon Base supplemented with shrimp protein and/or yeast
extract is shown in Figure 33. After 96 hrs at 20 C, the proteolytic
enzyme activity of the cell-free broths from each medium was 141, 114,
90 and 77.5 units for YCBSY, YCBY, YCBS and YCB, respectively. Thus, as
observed with the _P. citreus growth data (Figure 32), as the nutritional
composition of the growth medium increased, the amount of enzyme produced
also increased (as measured by an increase in total enzyme activity).


14
Figure 1. Photomicrograph of Planococcus citreus cells showing morphology
and flagellation (6) (magnification 950X).


54
The whole cell fraction (cell bound fraction), both whole cell washings
(loosely bound to cell wall fraction) and the particulate fraction
exhibited low specific activity towards both high molecular weight sub
strates. The intracellular soluble fraction (the soluble fraction after
the differential centrifugation of ruptured cells) exhibited the lowest
specific activity when gelatin and shrimp protein were used as substrates
(Table 5). These results show that the major portion (>95.0%) of the
active enzyme towards these two high molecular weight substrates resides
in the extracellular fraction.
Most microorganisms can synthesize various enzymes within their
cell structure. Each enzyme system may have its own unique characteris
tics, and these characteristics will vary depending on the enzyme, the
substrate and conditions during the enzyme-substrate reaction. In addi
tion, the location of the proteinase(s) within the bacterial cell may
vary markedly between microorganisms. Various researchers (53,115,135,
139) have studied the location of particular bacterial proteinases
within the cell and how this location relates to the function of the
enzyme. Thomas et al. (139), using gentle procedures for cell fraction
ation, suggested two criteria for the location of a proteinase produced
by Streptococcus lactis. The two criteria they suggested were: 1)
intact cells (whole cells) possessed substantial proteinase activity
when incubated with a high molecular weight substrate; 2) most of the
cell-bound proteinase activity was released during spheroplast formation.
The solubilized cell wall, plasma membrane and cytoplasm fractions con
tained 84%, 0% and 16% activity, respectively, of the total proteinase
activity with casein as substrate (139). In the results presented in
this dissertation, the whole cell and cellular particulate fractions of


A FLUORESCENCE
35
Figure 7. Increase in fluorescence intensity using the shrimp protein
preparation as substrate after incubation with cell-free
broth for up to 1 hr at 35 C.


LIST OF FIGURES (continued)
FIGURE Page
29 Lineweaver-Burk plot of the Planococcus citreus extracell
ular enzyme illustrating V and K values using gelatin
, .' max m 66 qr
as substrate ?->
30 Effect of shrimp protein concentration on the reaction rate
of the Planococcus citreus extracellular enzyme 96
31 Lineweaver-Burk plot of the Planococcus citreus extracell
ular enzyme illustrating V and K values using shrimp
. max m 07
protein as substrate 7/
32 Growth of Planococcus^ citreus in Yeast Carbon Base supple
mented with shrimp protein and/or yeast extract at 20 C ... 1-07
33 Proteolytic enzyme activity of the cell-free broth of
Planococcus citreus grown in Yeast Carbon Base supplemented
with shrimp protein and/or yeast extract 108
34 Growth of Planococcus citreus in Trypticase Soy Broth with
and without shrimp protein at 20 C HI
35 Proteolytic activity of the cell-free broth of Planococcus
citreus grown in Trypticase Soy Broth with and without
shrimp protein 112


40
phate buffer pH 7 as the eluting agent using a Gilson Fraction collector
(Model FC-220K Fractionator, Gilson Medical Electronics, Inc., Middle-
town, WI). Enzyme activity of each fraction collected was then measured
using gelatin as substrate. The protein present in the fractions was
determined by following absorbance at 280 nm using a Beckman Model 25
spectrophotometer (Beckman Instruments, Inc., Fullerton, CA).
Ion-Exchange Chromatography
The active fractions recovered from the gel filtration step were
pooled and further separated by ion-exchange chromatography using DEAE-
Sephadex A-50 (Pharmacia Fine Chemicals, Uppsala, Sweden). The column
was prepared following the procedures given by Pharmacia Fine Chemicals
(119). A 40 x 2.5 cm column was prepared and the protein eluted with
0.02 M phosphate buffer with a linear gradient of tris 0.01 M NaCl-tris
0.15 M NaCl (47) at a rate of 25 ml/hr (1.5 reading in the peristaltic
pump) and collected in 10 ml fractions. Two hundred fifty milliliters
of 0.01 M and 0.15 M NaCl solutions were placed in each vessel for the
linear gradient. The protein present in the fractions was followed by
reading the absorbance at 280 nm using a Beckman Model 25 spectrophotom
eter. Enzyme activity of each fraction was measured with gelatin as the
substrate. All column studies were duplicated.
The protein content of each fraction eluted using the Sephacryl
S-200 Superfine and the DEAE-Sephadex^ A-50 was also analyzed by the
Lowry (98) method for protein with Bovine Serum Albumin (Sigma Chemical
Co., St. Louis, MO) as the standard. Protein content is expressed as
mg/ml. Figure 9 summarizes the steps followed in the purification of
the extracellular proteolytic enzyme(s) of P. citreus.



PAGE 1

ISOLATION, PURIFICATION AND CHARACTERIZATION OF AN EXTRACELLULAR PROTEOLYTIC ENZYME OF Planococcus citreus BY RICARDO J. ALVAREZ A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1981

PAGE 2

A mis queridos padres: Gracias por la ayuda brindada, el amor, el apoyo moral y la vision de avanzar en la vida. Con todo mi amor

PAGE 3

ACKNOWLEDGMENTS The author expresses his deepest gratitude to Dr. J. A. Koburger, his major advisor, for his exceptional patience, guidance and encouragement throughout the course of this work. The author also wishes to acknowledge the advice, support and all the help given by Drs, R. H. Schmidt, W. S. Otwell, J. L. Oblinger and G. Bitton as members of his supervisory committee. Thanks go to Dean J. L. Fry for his understanding and support in delicate times. Special appreciation is extended to Dr. J. R. Kirk for procuring a much needed assistantship for the first two years of this research, to Margie Summers for her beautiful graphic work, Beth Beville, Diane Dobsha, Beth Johnsen and Mike Pyle for their patience in typing sections of this dissertation. A very ^special thank you goes to Melissa Michaels for the typing of the final copy of this dissertation. In addition, the author expresses thanks to Sam May for his help and support around the laboratory and to Suzanne Davidson, Bridget Walker and Janet Eastridge for their aid when needed. Thanks go to Dr. L. D. Ingram for his constructive comments. Recognition is also given to the faculty, staff and secretaries of the Food Science and Human Nutrition Department for their cooperation and to all fellow graduate students who shared with the author the years at the University of Florida.

PAGE 4

Finally, he is deeply grateful to Mary Brannigan for her love, patience and devotion, providing sentimental support and help throughout all phases of his course work. He thanks her for her understanding and provision of many reasons to pursue all achievements in life. iv

PAGE 5

TABLE OF CONTENTS Page ACKNOWLEDGMENTS iii LIST OF TABLES vii LIST OF FIGURES , ix ABSTRACT xii INTRODUCTION 1 LITERATURE REVIEW 3 Shrimp Spoilage 3 Microbiological Characteristics of Shrimp 4 Measurement of Shrimp Spoilage 7 Characteristics of Planococcus citreus 11 Proteolytic Enzymes 17 Measuring Proteolytic Activity 19 MATERIALS AND METHODS 25 Planococcus citreus Cultures 25 Determination of Proteolytic Activity 25 Efficacy of 5% Trichloroacetic Acid (TCA) 29 Substrate Characteristics 33 Determination of Enzjnne-Substrate Mixture Reaction Time 33 Growth Medium and Enzjnne Production , 36 Optimization of Enzyme Activity to Growth and Cell Number ... 36 Effect of Incubation Temperature on Enzyme Production and Activity 38 Purification of the Extracellular Enzyme (s) 39 Ammonium Sulfate Precipitation 39 Molecular Sieve Chromatography 39 Ion-Exchange Chromatography 40 Confirmation of Enzyme Purity 42 Characterization of the Proteolytic Enzyme (s) 43 Molecular Weight Determination 43 Determination of the Purified Enzyme-Substrate Mixture Reaction Time 43 Effect of Ionic Strength on Enzjnne Activity 44 Determination of Optimum pH 44 Determination of Optimum Temperature 46 Thermal Stability 46 Effect of Sodium Chloride Concentration , 47 v

PAGE 6

— .yv -» , > ^ -TJ. _TABLE OF CONTENTS (continued) Page Effect of Sodium Bisulfite Concentration 47 Effect of Enzyme Concentration 47 Effect of Substrate Concentration 47 Effect of Metal Ions on Enz3nne Activity 48 Effect of Various Reagents on Enzyme Activity 48 Dipeptidase Activity 49 Enzjme Induction Studies 49 RESULTS AND DISCUSSION 52 Proteolytic Activity of Cellular Fractions 52 Growth Medium and Enzyme Production 55 Effect of Incubation Temperature on Enzyme Production and Activity 60 Purification of Extracellular Enzyme(s) 66 Purity of the Extracellular Proteolytic Enzyme 71 Characterization of the Extracellular Proteolytic Enzyme .... 77 Molecular Weight Determination 77 Effect of Ionic Strength on Enzyme Activity 80 Optimum pH Determination 82 Optimum Temperature Determination 84 Thermal Stability 84 Effect of Sodium Chloride Concentration 87 Effect of Sodium Bisulfite Concentration 90 Effect of Enzjmie Concentration 90 Effect of Substrate Concentration 93 Effect of Metal Ions on Enzyme Activity 98 Effect of Various Reagents on Enzjnne Activity 101 Dipeptidase Activity 104 Enzyme Classification 104 Enzyme Induction Studies 104 SUMMARY AND CONCLUSIONS 115 LITERATURE CITED 120 BIOGRAPHICAL SKETCH 131 vi

PAGE 7

LIST OF TABLES TABLE Page 1 Chemical and physical tests available to measure shrimp quality S 2 Hydrolysis of various protein sources by selected strains of Planococcus citreus at 25 C 15 3 Proximate composition of the shrimp protein preparation .... 34 4 Composition of yeast carbon base medium 50 5 Proteolytic activity at 35 C for 15 min (pH 8) of cellular fractions obtained from Planococcus citreus grovm in Trypticase Soy Broth (TSB) using gelatin and shrimp protein as substrates 53 6 Units of enzyme activity per cell per hour (m) of Planococcus citreus grown in Trypticase Soy Broth (TSB), Plate Count Broth (PCB) and Nutrient Broth (NB) at mid-log phase 61 7 Enzyme activity measured at 5, 20 and 35 C (pH 8) of the cell-free broths of Planococcus citreus grown in Trypticase Soy Broth (TSB) at 5, 20 and 35 C for 108, 72 and 36 hrs, respectively 64 8 Purification of an extracellular proteolytic enzyme from Planococcus citreus 67 9 Proteolytic activity at 35 C for 15 min (pH 8) of various ammonium sulfate fractions of the cell-free broth of Planococcus citreus 68 10 Effect of various metal ions on the activity (assayed at 35 C for 10 min (pH 8)) of the Planococcus citreus extracellular enzyme 100 11 Effect of various reagents on the activity (assayed at 35 C for 10 min (pH 8)) of the Planococcus citreus extracellular enzyme 102 12 Dipeptidase activity of the Planococcus citreus extracellular enzyme 105 vii

PAGE 8

LIST OF TABLES (continued) TABLE Page 13 Units of enzyme activity per cell per hour (m) of Planococcus citreus grown in yeast carbon base supplemented with shrimp protein and/or yeast extract at midlog phase HI 14 Units of enzjnne activity per cell per hour (m) of Planococcus citreus grown in various media 114 vii

PAGE 9

LIST OF FIGURES FIGURE Page 1 Photomicrograph of Planococcus citreus cells showing morphology and flagellation 14 2 Comparison of the Fluorescamine technique and the Lowry procedure for determining protein concentration 23 3 Effect of pH adjustment of gelatin-trichloroacetic acid (TCA) filtrates on fluorescence intensity 28 4 Excitation (curve A) and fluorescence (curve B) spectrum for the reaction of a gelatin-trichloroacetic acid (TCA) filtrate with fluorescamine at pH 8 30 5 Excitation (curve A) and fluorescence (curve B) spectrum for the reaction of a shrimp protein-trichloroacetic acid (TCA) filtrate with fluorescamine at pH 8 31 6 Efficacy of trichloroacetic acid (TCA) in terminating the enzyme-substrate reaction 32 7 Increase in fluorescence intensity using the shrimp protein preparation as substrate after incubation with cell-free broth for 1 hr at 35 C 35 8 Increase in fluorescence intensity using gelatin as substrate and various amounts of cell-free broth after incubation at 35 C for 1 hr 37 9 Outline of steps for the purification of the extracellular protease(s) of Planococcus citreus 41 10 Increase in fluorescence intensity following incubation of gelatin and shrimp protein substrate with purified enzyme for up to 1 hr at 35 C 45 11 Spectrophotometric growth curves of Planococcus citreus in Plate Count Broth, Nutrient broth and Trypticase Soy Broth at 20 C 55 12 Aerobic plate counts of Planococcus citreus incubated in Plate Count Broth, Nutrient Broth and Trypticase Soy Broth at 20 C for 96 hrs 58 ix

PAGE 10

LIST OF FIGURES (continued) FIGURE Page 13 Enz3mie activity at 35 C for 15 min (pH 8) of the cell-free broth of Planococcus citreus grown in Plate Count Broth, Nutrient Broth and Trypticase Soy Broth for up to 96 hrs ..59 14 Spectrophotometric growth curves of Planococcus citreus in Trypticase Soy Broth incubated at 5, 20 and 35 C 62 15 Separation of the proteins present in the 55-70% ammonium sulfate fraction using Sephacryl S-200 70 16 Ion-exchange chromatography using DEAE-Sephadex A-50 of ^ the pooled active fractions obtained in the molecular sieve chromatography purification step 72 17 Acrylamide gel electrophoresis of the purified enz}nne of Planococcus citreus 74 18 Acrylamide gel electrophoresis of increasing concentration of the purified extracellular enzyme of Planococcus citreus 75 19 Calibration curve used for the molecular weight estimatiog of Planococcus citreus proteolytic enzjnne using Sephacryl S-200 column chromatography , 78 20 Calibration curve used for the molecular weight estimation of Planococcus citreus proteolytic enzyme using acrylamide gel electrophorjesis 79 21 Effect of ionic strength on the activity of the extracellular proteolytic enzyme of Planococcus citreus 81 22 Optimum pH of the extracellular proteolytic enzyme of Planococcus citreus 83 23 Temperature optimum of the extracellular proteolytic enzyme of Planococcus citreus 85 24 Thermal stability of the enzymes in the cell-free broths of Planococcus citreus grown at 5 and 35 C and of the purified enzyme 86 25 Effect of sodium chloride concentration on enzjnne activity 89 26 Effect of sodium bisulfite concentration on enzyme activity 91 27 Effect of enzyme concentration on enzyme activity 92 28 Effect of gelatin concentration on the reaction rate of the Planococcus citreus extracellular enzyme 94 x

PAGE 11

LIST OF FIGURES (continued) FIGURE Page 29 Lineweaver-Burk plot of the Planococcus cltreus extracellular enz)nne illustrating V and K values using gelatin as substrate ? 95 30 Effect of shrimp protein concentration on the reaction rate of the Planococcus citreus extracellular enzyme 96 31 Lineweaver-Burk plot of the Planococcus citreus extracellular enzyme illustrating V and K values using shrimp protein as substrate ? 97 32 Growth of Planococcus' citreus in Yeast Carbon Base supplemented with shrimp protein and/or yeast extract at 20 C ... 107 33 Proteolytic enzyme activity of the cell-free broth of Planococcus citreus grown in Yeast Carbon Base supplemented with shrimp protein and/or yeast extract 108 34 Growth of Planococcus citreus in Trypticase Soy Broth with and without shrimp protein at 20 C Ill 35 Proteolytic activity of the cell-free broth of Planococcus citreus grown in Trypticase Soy Broth with and without shrimp protein 112 xi

PAGE 12

Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ISOLATION, PURIFICATION AND CHARACTERIZATION OF AN EXTRACELLULAR PROTEOLYTIC ENZYME OF Planococcus citreus By Ricardo J. Alvarez March 1981 Chairman: J, A. Koburger Major Department: Food Science and Human Nutrition Planococcus citreus is a gram-positive marine bacterium commonly found in fresh and iced shrimp. Various studies have indicated that it may contribute to spoilage of this valuable marine resource. In order to understand the contribution of this organism to the degradation of shrimp as well as other proteins, an investigation was undertaken to study the extracellular proteolytic enzyme(s) of this organism. Results indicated that the major portion (>95,0%) of the proteolytic activity resided in the extracellular fraction. Under the conditions tested, maximum extracellular enzjnne production occurred in Trypticase Soy Broth (TSB) as observed by the highest m value (units of enzyme activity per cell per hour). In addition, the cell-free broth obtained from _P. citreus cells grown at 5 C for 108 hrs, 20 C for 72 hrs and 35 C for 36 hrs exhibited enzyme activity towards shrimp protein at all three enzyme-substrate incubation temperatures (5, 20 and 35 C) , xii

PAGE 13

p. citreus was grown in Trypticase Soy Broth at 20 C for 72 hrs. Centrifugation, ammonium sulfate precipitation, Sephacryl S-200 Superfine molecular sieve chromatography, DEAE-Sephadex A-50 ion exchange chromatography and acrylamide gel electrophoresis were used to purify the extracellular enzyme(s). The enzyme was purified 26.50 fold (using the fluorometric technique for activity measurement), and recovery of the enzjnne was above 49%. Gelatin and shrimp protein were used as substrates throughout the study. The molecular weight of the purified protease was approximately 29,000 as measured by Sephacryl S-200 column chromatography and acrylamide gel electrophoresis. Maximum activity of the enz}nne was at pH 8 and 35 C. Ionic strengths of above 0.83 (0.75 M NaCl) decreased the activity of the extracellular enzyme. Heat treatment at 65 C for 15 min destroyed the activity of the purified enzyme. However, 1.0% of the residual enzyme activity was still present in the cell-free broth of P^. citreus grown at 35 C for 36 hrs. In contrast, 15 min at 75 C were necessary to reduce 99.0% the activity of the enzymes in the cell-free broth of P^. citreus grown at 5 C for 108 hrs. When shrimp protein was used as substrate, sodium chloride concentrations of 0.0-0.5% increased enzyme activity, while concentrations of 0.5-1.5% decreased enzyme activity. However, when gelatin substrate was used, NaCl concentrations of 0.0-1.5% had no effect on enzyme activity. The activity of the purified enzyme decreased as the concentration of sodium bisulfite increased. MichaelisMenten kinetics were followed when gelatin and shrimp protein preparation were used as substrates. The apparent values for gelatin and shrimp protein were 0.98 mg/ml and 0.33 mg/ml, respectively. The apparent V ^ values were 666.67 and 431.03 units of activity for gelatin max ' ° xiii

PAGE 14

and shrimp protein, respectively. Ferric chloride, mercuric chloride, potassium chloride, ethylene diaminetetraacetic acid, citric acid, cysteine, p-mercaptoethanol, potassium permanganate and formaldehyde partially inactivated the enzyme. Calcium chloride increased the activity of the extracellular proteolytic enzjmie. Zinc chloride, pdioxane, manganese chloride and magnesium chloride had no effect on the activity of the enzyme. The proteolytic enzyme exhibited peptidase activity on various commercial synthetic dipeptides. The extracellular proteolytic enzyme produced by _P. citreus was apparently not induced by the presence of shrimp protein in the medium of growth. Enzyme production appeared to be related to the extent of growth of _P. citreus in the medium. XiV

PAGE 15

INTRODUCTION Quality deterioration and subsequent spoilage of shrimp during storage are caused primarily by activities of indigenous tissue enzymes and microbial enzymes (69). Various researchers (17,156) believe that bacterial action plays a more important role than autolytic enzyme release in causing spoilage of seafoods. During growth of the bacteria, proteolysis of shrimp proteins and free amino acid formation by microbial action has been observed. Enzymatic deamination and decarboxylation of these amino acids from shrimp protein occur, resulting in the formation of malodorous compounds (116). Various types of bacteria have been reported to be present on freshly caught shrimp. Numerous studies (3,42,43,45,116,142) have shown the changes undergone by the bacterial flora of shrimp as the storage period increases. Recent research (1,2,3,90) has noted the presence of a gram-positive organism, Planococcus citreus , during shrimp storage. The organism is described as a motile gram-positive coccus found in the marine environment, capable of growing over a pH range of 7-10 between 5-35 C in broth containing 0.5-12% sodium chloride (NaCl) , and the organism is capable of hydrolyzing gelatin, cottonseed, soy and shrimp protein. The potential of _P. citreus as a "spoiler" of shrimp was shown by the increase in pH and the rapid increase in the total volatile nitrogen/ amino acid-nitrogen ratio (TVN/AA-N) and trimethyl-amine nitrogen (TMN) following growth of this organism on shrimp (4). The proteolytic

PAGE 16

activity of this organism was further demonstrated by the decrease in percent total extractable protein (percent TEP) in shrimp during storage at 5 C (4,5) which had been inoculated with P. citreus. The proteolytic activity exhibited by this organism deserves additional research in order to better understand the contribution of P. citreus to the degradation of shrimp protein. A study was therefore undertaken to study the enzyme (s) responsible for protein degradation. The optimum medium and stage in the growth cycle of P. citreus were determined for maximum extracellular enz3rme(s) production. The effect of incubation temperature (5, 20 and 35 C) on the growth of citreus and proteolytic enzyme production was also investigated. Purification of the extracellular enz3mie(s) was achieved by precipitation and chromatographic techniques. Homogeneity of the enzyme was evaluated by gel electrophoresis and chromatographic techniques. Optimum pH and temperature, ionic strength effect, thermal stability, molecular weight, sodium chloride effect, sodium bisulfite effect, enzyme concentration, substrate concentration, and the effect of metal ions and other reagents were investigated. In addition, the potential of the citreus enz3rme(s) to degrade dipeptides and the possible effect of shrimp protein in the growth medium inducing the extracellular enzyme (s) were studied. Results obtained from this investigation indicate that P. citreus, while growing on shrimp, may contribute to the overall decrease in shrimp quality during iced or refrigerated storage. In addition, information about the characteristics of the enzyme(s) produced by P. citreus will be introduced.

PAGE 17

LITERATURE REVIEW In 1978, the shrimping industry was the most valuable fishery in the United States (8). However, the quality of shrimp often falls short of that expected by the consumer. Approximately 15-20% of the shrimp landed is eventually lost due to quality deterioration. This deterioration of shrimp quality is usually attributed to rapid bacterial enzymatic changes of the fresh shrimp resulting from mishandling and/or inadequate processing. These changes, along with the chemical and physical methods for measuring shrimp quality, are discussed as a basis for the investigations presented in this dissertation. Shrimp Spoilage Quality changes in shrimp during storage on ice can lead to major economic losses in the shrimp industry. Mechanical damage, bacterial contamination and enzymatic activity may combine to cause undesirable changes in the composition and quality of shrimp (20,34,38,39,43,45,53, 54,77,78,79,89,94,108,117,153,154). The loss of acceptability of shrimp may be triggered by several factors: 1) shrimp muscle enzymes, 2) direct microbial activity, 3) microbial enzymes and/or 4) a combination of these factors. Defects which may occur as a result of such reactions are formation of malodorous substances, flavor deterioration, toughness, mushiness, juiciness, dryness and discoloration (116). Proteolytic enzjanes play an important role in the spoilage of shrimp by degrading muscle proteins and polypeptides, forming amino acids which 3

PAGE 18

4 enrich the natural substrates and are thus available for the growth of microorganisms. Enzymatic deamination and decarboxylation of amino acids may also occur rapidly, resulting in the formation of spoilage products. Pedraja (116) observed that from the moment a shrimp is taken out of the water, its free amino acid pool is affected to some extent by osmoregulation and also by the struggle during catching. Therefore, the onset of enzymatic and bacterial actions will vary according to the factors affecting the substrates available in shrimp muscle. Another factor that can induce shrimp spoilage is mechanical damage. Handling shrimp on the boats results in mechanical damage to the muscle, which will accelerate microbial invasion. The expressible fluid with its protein and amino acid content serves as an excellent medium for growth and reproduction of invading microorganisms (116). Microbiological Characteristics of Shrimp The muscle tissue of freshly caught shrimp is generally regarded as sterile (26); however, work by Lightner (97) showed bacteria in the gut, gills and between muscle bundles of brown shrimp. Reports on the number of bacteria found on freshly caught shrimp range from 2.5 x 10 to 2.0 x 10^ organisms per gram (org/g) with Gulf coast shrimp averaging 1.0 x 10^ org/g, whereas bay shrimp averaged 1.0 x 10^ org/g (31,34,39,43,78,142). Work completed in our laboratory has shown that fresh shrimp from the Gulf of Mexico had bacterial counts ranging from 4.0 x 10^ org/g to 2.0 X 10^ org/g, while shrimp from the Atlantic coast had bacterial counts ranging from 4.5 x 10^ to 3.6 x 10^ org/g (1). Various kinds of bacteria have been reported on freshly caught shrimp. Initially, the microbial flora is a mixture of organisms from both the marine and terrestial environment. In the early 1950s, Campbell

PAGE 19

5 and Williams (31) and Williams et al. (154) isolated species of Achromobacter . Bacillus , Micrococcus , Flavobacterium and Pseudomonas from Gulf coast shrimp. Vanderzant et al. (142) reported that the flora of shrimp from the Gulf of Mexico consisted of coryneforms, Achromobacter , Flavobacterium and Bacillus . In Pacific shrimp, Acinetobacter-Moraxella species were predominant (80). Lee and Pfeifer (94) reported that the flora of Pacific shrimp ( Panda lus jordani ) consisted of Mo r axe 11a , Pseudomonas . Acinetobacter , Arthrobacter and Flavobacterium-Cytophaga species. Cann (32) and Cann et al. (33) found that coryneform organisms were predominant in the bacterial flora of scampi, Nephrops norvegicus , with strains of Achromobacter-Acinetobacter group and Pseudomonas , Cytophaga and Micrococcus species also present. Koburger et al. (90) reported that the Flavobac t er ium-Cy t ophaga group represented the majority of the organisms of fresh rock shrimp ( Sicyonia brevirostris ) , and Alvarez (1) and Alvarez and Koburger (3) reported that Flavobacterium and Pseudomonas were the predominant groups isolated from Penaeus shrimp from the East and West coasts of Florida. When shrimp are stored in ice, the number and kinds of bacteria shift to a predominantly psychrotrophic flora (130). Psychrotrophs are described as organisms having an optimal growth temperature of about 20 C. A comparatively longer storage life of iced shrimp from tropical waters has been reported by Carrol et al. (34). Cann et al. (33) in their review on tropical shrimp indicated that penaeid shrimp from the Gulf of Thailand remained in acceptable condition for 12-16 days on ice, whereas nontropical shrimp, such as Pandalus and Nephrops species, were totally spoiled after 8-10 days. They attributed this difference to the bacterial flora; the mesophilic flora on tropical shrimp are not active

PAGE 20

6 at ice temperatures and little spoilage occurs until the psychrotrophic flora develops. Cann et al. (33) stated that the amount of spoilage may be related to the degree to which psychrotrophic strains are introduced with the ice. Consequently, the rate of increase in bacterial growth depends on the initial number of bacteria, handling on deck, and amount and quality of ice used, Shewan (130) demonstrated that the action of many psychrotrophic organisms resulted in rapid fish spoilage. The principal organisms he mentioned were Pseudomonas , Aeromonas , Vibrio , Moraxella , coryneforms and Flavobacterium . Castell and Mappleback (35) concluded that Flavobacterium was among the most important of the fishspoilage bacteria. Flavobacterium is a frequently encountered bacterium on fresh shrimp flesh. The bacterial flora of shrimp undergoes marked changes as the storage period increases. Campbell and Williams (31) showed Bacillus , Micrococcus and Flavobacterium made up over 50% of the flora initially, whereas the Achromobacter-Pseudomonas group accounted for 98% of the flora after 16 days of iced storage. In a study on the bacterial spoilage patterns of headless brown shrimp. Cook (45) noted that there was only one consistent change in the bacterial types growing initially or during the period of die-off. As the bacterial count began to rise, Pseudomonas species became the predominant organism, accounting for 80100% of the bacterial types isolated. Vanderzant et al. (142) reported that the predominant bacterial flora of fresh shrimp consisted of coryneforms and that following storage Pseudomonas species predominated, Cobb et al. (43) indicated that typical spoilage organisms of the genus Pseudomonas are not usually found in freshly caught shrimp. It is not until the shrimp are exposed to handling on board the vessel that this organism becomes apparent.

PAGE 21

7 Alvarez and Koburger (3) reported that the numbers of Moraxella , Vibrio/ Aeromonas and Planococcus species isolated from Penaeus shrimp remained relatively constant throughout 10 days of ice storage. However, Flavobacterium isolates increased until the fifth day, then decreased rapidly. Pseudomonas species showed the opposite trend. They decreased until the fifth day, then increased rapidly. Other workers have observed the presence of Flavobacterium in raw shrimp (31,80,90,94,142,143) and have noted this decrease in numbers during ice storage with a subsequent increase in Pseudomonas species. Cook (45) was unable to produce typical spoilage when shrimp were inoculated with Flavobacterium species, indicating that they are probably an inert group of organisms found in shrimp. In contrast, Pseudomonas species have been implicated as the organisms primarily responsible for the spoilage of marine products stored in ice (108,130). Measurement of Shrimp Spoilage Numerous methods for determining shrimp quality have been developed; however, due to the complexity, time involved and inconsistent results of many of these methods, only a few are routinely used by the industry and then, only for internal quality control. In many of these chemical tests, results can vary with the age of the shrimp, size, species, area of catch and handling conditions. Many of the tests only indicate the onset of spoilage (31,109). Table 1 lists the chemical and physical tests that have been used to measure shrimp quality. Total volatile nitrogen/amino acid-nitrogen (TVN/AA-N) ratio (40,41,42,43,64,75) is the chemical test that shows the best correlation with organoleptic quality measurements of shrimp. Moore and Eitenmiller (107) compared various methods for measuring shrimp quality. They observed that a relatively

PAGE 22

8 o c 0) l-l (U vo 0^ •> i-H in 00 o o -> ^ CO T3 C > c u •H U CO T3 4-> ^ •H to Qi E>% a •H 4-1 U 4-1 M •H C 4-> •H c J= •H dJ 4-» X s O U tA 4-) o (U 4-t CO c iH •H •H O to 1 Q) e CO CO •V e a 4-1 E )-< 4-J *J to «W CO 0) CJ cu E c 4-1 c O o o •H c 4-1 o 0) 4-l O c 0) tu (U *> 4-1 o •H (U B cu 1 CO •H °g l-l u 4-1 c o 4= c 4-1 3 e a 4-1 O c ^ c c o CO & o c c 4-1 c o 1-1 1—1 o CO H c U-l o 0 CO •H CO •H •H o o c •H •H V N •H O u o 4-1 X 3 •H 14-1 c o ,c CO o 03 o CO CO 4-1 « ^ 1-1 CO 4-> (U 1-1 a T3 o. (1) iH ^ ao .-1 60 C 60 (U O a U c c > )-i u 0) o J= e o x; tu Q) >^ o cu (U 1-1 4-1 o T3 4-1 o CO CO a o CO to B o a 60 CO (U H (U O 4J 73 4-1 n CO C o J= CO CO •H a 4-1 4-1 CO 0) u 3 o +J 3 iH j= CO o a X o CO o CO O. o 4-1 o •H u o a. o (U c •H "H E (U 0) )-l 4-1 (U 3 60 .-1 4-' CO C 4-1 o V-i 3 OJ CO J= u 4-1 i-H a u *j •H •H O •H 60 C 1-H B CO CO 01 CO o 1 O T3 X x: o Tt c o o c •H lU CO CI. o •H O T3 4-1 >^ iH B to CO -H CO CO c o i CO B CU 4-1 B o a B CO +J •H > I P3 0) O o 0) O a CU 4-> CO o cu B •H E CO 1—1 >^ 0 9 O O Ck. CO o •o o •H CO CU O o B CO •H o E >^ •H to 4J E o 4-1 E CO CO (U 4-1 cu <4-l 60 CJ u O a> o CU O u 3 •H u 1—1 Xl u-l u-l 60

PAGE 23

9 o c 0) (U <4-l 0) 0) 3 C C o o CO H J3 <3\ CM r-i on 0^ «300 VO 00 VO VO 00 VO CO CO -l CO o 4-) •H c B •H O c o 4J CO c (1) •A cu CiO +J c (U (U O o to •H c •H u CO o •H -P X. O TD •a a +-I >^ c C O to •H •H •H 1—1 t-l (U ,J0 E 3 C 3J -a •H o u M a CO a CO o c cu X. D. u « I B o u (U )-l )-l 4-1 a 0) o CJ CO CJ CO <3v CO •H (U a Kit by •H CD c OS 0) o 43 3 C o « •a 3 c c O 3 1-1 U M-l 01 cu •H •H 4-> c (U O o 4-1 rH CO C a o 4-1 rH 0) CO c u a (U 0) o (U c T) (U 4-1 44 lU I-I (U •o V4 c (U 4= •H s o 4-> a 4-> CO CO •H 4-1 4J > 73 c O 4-" 4-1 o 4= 43 CO CO s 14-1 (4-1 •H O o V4 CO o 00 4J CO CO CM o o •H o 0) 4-1 VM 33 iH U M-l M CM T3 14-1 Ow to c -H CO <4-l 0 O c c C M-l T3 O o I-I 0 -o CLi VM o o CO O •H to T3 « iH c C 4-1 1-1 c O c •H •H (U o c (U (U 4-1 iH 1-1 X 0 0 o 4-1 4-) CO iH -H CO 4J 3 4J 0) to c C 1-1 X U •T3 CO •a 00 •H O 0) y T3 O 4J CO CO T3 CO CO )-4 u 0 0 a; 0) o 4-1 CO O CO 0) to 0) to c >^ 4= 4: X 3 -H M E 4J 3-43 4-" rH M 43 T3 45 3 43 y U CO 4-< 0. a, rH O 4-1 2 CO

PAGE 24

10 0) u c 14-4 ON SO NO • 00 vO * sim VO 0k CO VO 1— t vO Oi CO — 1 ON m CO l-H 00 »i #> o\ 1-4 CM VO 00 M CO to -i •H o C 4-1 CO J= < o. c 3 •H 4-1 4-> S a o O c c 1^ o CO •H a o o f-i 4-1 a CJ /-V •H O 0) O. o I-l CO h 4-1 c > u o •H C -H 4-1 01 o 'W (u c 4-1 60 I-H CO CO u CO 60 O c (4-1 O o Xi 4-1 •H iH O E (U t-l (U CO u T3 O e 4-1 4-> c CO •H > 4-) CO +-1 c c ^ •H •H 'M u V •H w c o o c CO (U CO VM c to <0 ^ (U t-l o 0) (U 0) -o c iH >, •H 1— 1 0) c '-^ C o •a N t-l 4-> > a •H 3 •H 3 o c to G 4-1 CO a 4J 3 O 4-1 O o u > o iH J= O (U o 9 ° 4-4 T) -O > 3 CO > a y > CJ 3 C C O o CO 0) H to (U e c 60 cu <1) C 60 60 CO O O •c U CJ 4-1 4-) 1 •H •H 4-1 C C J= 60 n (U Oi tH o CO ^ O O n •H o rH 4-1 •H > 3 •H iH rH CO to I-H 4-1 CO CO a CO O t4H to 4-1 4-1 •H 4-1 U 4-1 rH o o M O >s rH c o 4-1 4J 4-1 4-1 4-1 3 •H > OS 0) u fi CO 4J OD .fi 9 CO 60 c •H CJ 3 TS (U u o > c 60 O U o >

PAGE 25

11 new method using f luorescamine primarily detected only the non-protein, non-ammonia, small molecular weight amines in shrimp homogenates. They proposed that f luorescamine analysis could be useful in determining changes in the free amine fractions. The shrimp industry still depends on visual observation, smell and bacteriological testing for evaluating overall shrimp quality, whereas, the Food and Drug Administration (FDA) uses decomposition, filth and odor for the evaluation of shrimp quality. Characteristics of Planococcus citreus Koburger et al. (90) noted the presence of a high percentage of gram-positive organisms following iced storage of rock shrimp (Sicyonia brevirostris ) . These organisms comprised up to 68% of the isolates. Of these 40% were Planococcus citreus , an aerobic gram-positive motile coccus of marine origin producing an orange or yellow pigment. Information describing the isolation and characteristics of this organism is limited. _P. citreus was previously named Micrococcus citreus (27). The 8th Edition of the Bergey's Manual of Determinative Bacteriology (29) does have a description of the organism; however, it is limited in scope. Cook in 1970 (45) and previous researchers working with shrimp placed all aerobic gram-positive to gram-variable coccoid shaped bacteria in the genus Micrococcus . In addition. Cook (45) noted that many of these organisms isolated from shrimp were pigmented orange or yellow and were motile. According to Bergey's manual (29), the only genus in the family Micrococcaceae that is pigmented, either yellow or orange, and motile, is Planococcus . This change in the taxonomic status of this organism and the difficulty of demonstrating motility are probably the reasons why Planococcus has not been reported in previous studies. The taxonomic status of Planococcus citreus has changed markedly through the years. In 1894 and again in 1900, Migula (103,104) made a

PAGE 26

12 recommendation that flagellated cocci be included either in the genus Planococcus or Planosarcina . This suggestion was accepted by only a few authors, e.g., Krasil'nikov in 1949 (92). The majority of the authors have included the flagellated cocci in the genus Micrococcus (22,83), mainly because these cocci could only be differentiated from the other members of the genus by their motility. Most authors have considered motility to be a minor characteristic for the recognition of a new genus. The findings of Bohacek et al. (23,24) that the flagellated cocci differ considerably in the guanos ine-cytosine (GC) content of their deoxyribonucleic acid (DNA) from other cocci shed new light on their taxonomic position. It was proposed by Bohacek et al. (23) to include the flagellated cocci with a GC content ranging from 40-50% in the genus Pianococcus . In 1970, Kocur et al. (91) revised and outlined the genus Planococcus . However, according to Index Bergeyana (86), the Planococcus genus includes nine species (P. agilis , casei , P^. citreus , _P. citroagilis , _P. europeans , P. loffleii, luteus , 2« ochrolencus and ?, roseus). Kocur et al. (91) evaluated the strains available in culture and proposed that seven belong to one species, Planococcus citreus . Although the remaining two species were closely related, he refrained from giving a precise designation and labeled them only as Planococcus species. Schleifer and Kandler (126) found that the strains studied by Bohacek et al. (23,24) and Kocur et al. (91) were uniform with respect to the type of murein present in their cell walls and similar to that of members of the genera Micrococcus and Staphylococcus . However, serological investigation of _P. citreus by Ceding in 1971 (112) revealed no antigenic relationship to staphylococci or micrococci.

PAGE 27

13 Thirkell and Summerfield (137,138) studied the effect of varying the sea salt concentration on the chemical composition of a purified membrane fraction of _P. citreus . They concluded that the concentration of salt in the medium affected the amount of membrane in the cell. Salt concentrations above or below the normal 3% of sea water reduced the amount of membrane material present. In addition, varying salt concentration had no significant effect on the amount of total neutral lipid, glycolipid or phospholipid in the citreus membrane preparations. But a significant effect was observed on the amount of individual neutral lipid or phospholipid classes present and on the number of individual glycolipid components detected. Our attention was directed toward this organism when, during a study of the normal flora of rock shrimp ( Sicyonia brevlrostris ) , P. citreus was consistently isolated and found to increase in numbers during iced storage (90) . In this study, 68% of the isolates recovered were grampositive cocci, with F. citreus increasing from 10% of the isolates on the fresh rock shrimp to 40% on the ice stored rock shrimp. In recent work (1,2,3), _P. citreus has been found to be an important member, of the nonnal flora of Penaeus shrimp. Alvarez and Koburger (5) described citreus as a motile grampositive coccus found in the marine environment, capable of growing over a range of pH 7-10, 5-35 C, in broth containing 0.5-12% sodium chloride (NaCl) and capable of hydrolyzing gelatin, cottonseed, soy and more importantly to seafood microbiologists, shrimp protein. Figure 1 shows a photomicrograph of _P. citreus illustrating its morphology and flagellation. Table 2 shows the capabilities of this organism to hydrolyze various protein sources.

PAGE 28

14 Figure 1. Phototnicrograph of Planococcus citreus cells showing morphology and flagellation (6) (magnification 950X) .

PAGE 29

15 Table 2. Hydrolysis of various protein sources by selected strains of Planococcus citreus at 25 C (4) (modification of Frazier (72)). Protein Source Isolate c •H to iH 0) fsl (U 2 Cottonseed 1— H c *H 0) cn Cfl 3 Shrimp tn •H 2 Peanut 2 Corn germ >^ O Hog blood isolate^ 2 Barley A 17 + + + + + E 4 + + + + + E 1 + + + + + + E 7 + + + + + + F 9 + + + + + F 15 + — + + + F 18 + + + + KS-1 + + + + KS-2 + + + + + KS-3 + + + + KS-4 + + + + + CS-1 + + + + + From Difco Laboratories, Detroit, MI. 'Protein isolates obtained from Southern Utilization Research and Development Division, New Orleans, LA. 'Presh samples were diluted 1:10 with 0.05 M phosphate buffer pH 7 and ground in a Waring blender, dialyzed overnight with 10 volumes of the same buffer (5 C) and lyophilized. F. W. Knapp, Food Science and Human Nutrition Department, University of Florida, Gainesville, FL. + = hydrolysis; = no hydrolysis.

PAGE 30

16 The reports (4,5) by Alvarez and Koburger outline some observations on the distribution of 2« citreus in the marine environment. Of the 35 samples of marine origin examined for _P. citreus , only 5 yielded this organism. Four were shrimp samples and the fifth was a stuffed flounder sample that had been prepared in a plant that processed predominantly shrimp. One of the shrimp samples from which Planococcus was isolated had been in frozen storage for over six years. Fresh seafood (trout, sheephead, mackerel, crab and oysters) as well as Gulf Coast waters and sediments from the vicinity of Suwannee, Florida, were also examined for _P. citreus without success. However, in more recent studies performed by Mallory et al. (100), ?. citreus was isolated from estuarine areas of Chesapeake Bay in low numbers. Since the isolation of gram-positive organisms from iced seafood is uncommon, Alvarez and Koburger (5) studied the contribution of citreus to the spoilage of Penaeus shrimp. They utilized gamma irradiation (600 Krads) to lower the number of bacteria in raw. shrimp and then inoculated a portion of the shrimp with 5 x 10^ P. citreus cells per gram of shrimp in order to study the changes produced by this organism. citreus counts increased in the inoculated shrimp from 5 x 10^ bacteria/gram at 0 day to 1.9 X 10 bacteria/gram at the 16th day. The potential of _P. ^^^^^^s as a "spoiler" of shrimp was shown by an increase in pH and the rapid increase in total volatile nitrogen/amino acid-nitrogen ratio (TVN/ AA-N) and trimethyl-amine nitrogen (TMN) content. In 1973, Cobb et al. (42) reported a high correlation between total volatile nitrogen/amino acid-nitrogen ratio (TVN/AA-N) and quality of shrimp. Later work (41) suggested that the TVN/AA-N ratio and the logarithm of bacterial counts increased at approximately the same rate after the initial lag phase of

PAGE 31

bacterial growth and that a TVN/AA-N ratio of 1.3 indicated a limited shelf life of the shrimp. Alvarez and Koburger (4,5) showed that P. citreus is capable of increasing the TVN/AA-N ratio at a similar rate as the control sample (natural flora of shrimp). Thus, if the TVN/AA-N ratio is an index of shrimp quality, _P. citreus is capable of shortening the shelf life of shrimp. The proteolytic activity of this organism was demonstrated by a significant decrease in percent extractable protein (% TEP) in the early days of storage. Maximal percent TEP decrease was observed between the 4th and the 12th day of storage of shrimp at 5 C (5) . Proteolytic Enzymes Enzymes are proteins with highly specific catalytic activities. As catalysts, enzymes have the following properties: 1) they are effective in small concentrations; 2) they remain unchanged in the reaction; 3) if present in small concentrations relative to the substrate, they speed attainment of equilibrium as reflected by increases in the rate constants h and (A + B ^ > C + D, where A + B = reacting substances, C + D ^-1 = products of the A + B enzyme catalyzed reaction, = rate constant of the forward reaction, = rate constant of the reverse reaction). However, an enzyme does not change the ratio K,/K = K (95). 1 —1 eq Most living organisms possess the ability to degrade proteins to more readily absorbed substances. Such attacks on the peptide bond are made possible by the presence of proteolytic enzymes. Although proteolytic enzymes from animal sources have been studied for more than a century by both physiologists and biochemists, it was the work of Bergmann and Fruton (18) which led to a more complete understanding of the mode of action of these enzymes. Their work established conclusively

PAGE 32

18 that these enzymes exert a specificity toward the amino acids involved in the peptide bonds which they attack. Bergmann and his students are also responsible for the presently accepted classification of proteolytic enzymes: They proposed that these enzjnnes be grouped into two classes — endopeptidases and exopeptidases — depending upon whether they hydrolyzed peptide bonds remote from, or near to, the end of the peptide chains of their natural substrates. The former class includes such enzymes as pepsin, trypsin and chjmio trypsin, while the latter class contains the dipeptidases and the amino and carboxy peptidases. Proteinases in bacteria may be either intracellular or extracellular depending upon whether they exert their activity within the cell or whether they are excreted from the cell to attack proteins in the environment (10,58), Also, enzymes may be classified according to their location in, on or around the cell: a) cell-bound: 1) truly intracellular, 2) surface-bound; and b) extracellular (58). Extracellular enzymes are those enzymes which exist in the medium around the cell, having originated from the cell without any alteration to cell structure greater than that compatible with the cell's normal processes of growth and reproduction. This distinction is not always clear and in some instances it is entirely possible that autolysis of cells has permitted the escape of intracellular enzymes into the culture filtrate. This is particularly true when high proteolytic activity is dependent upon prolonged incubation of the culture (74). In 1964, the International Union of Biochemistry (54) recommended a scheme for numbering enzymes, which is currently used for the classification of enzymes. Enzymes are divided into groups on the basis of the type of reaction catalyzed, and this, together with the name(s) of the

PAGE 33

19 substrate(s) , provides a basis for naming individual enzymes. Each enzyme number contains four elements; the first element (1 through 6) shows to which of the 6 main groups of enz3nnes the particular enzjrme belongs (the six main groups are made on the basis of the general chemical reaction catalyzed); the second and third elements show the subclass and sub-subclass, respectively, thus defining the type of reaction; and the fourth element is the serial number of the enzyme within its sub-subclass. Enz3nnes can be divided into six main groups: oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. Active extracellular proteinases are produced by numerous species of Clostridium . Proteus , Bacillus , Pseudomonas , Micrococcus . Streptococcus . Escherichia . Cytophaga and Staphylococcus (11,12,36,58,59,65,70,84,87, 105,110,113,114,118,136,139,155,157). The continued study of these bacterial enz5nnes is important for at least two reasons: (a) proteolysis by microorganisms plays an important role in the biogeochemical cycles (74) and is responsible for numerous environmental interrelationships; (b) the purification and the elucidation of their bond specificities are certain to lead to the discovery of new enzymes with new properties not previously known. Measuring Proteolytic Activity Many methods are available for measuring proteolytic activity. Some are based on the measurement of increase in protein (or nitrogen) solubility in the supernatant after centrifugation of the reaction mixture. The most frequently cited method for measuring protein in solution is that of Lowry et al. (98) in which the tyrosine-tryptophan groups of proteins in solution, or precipitated with acid, are reacted with alkaline Folin-phenol reagent after an alkaline copper treatment (71) to

PAGE 34

20 produce a blue color that is measured in a spectrophotometer. Other methods record proteolysis as the increase in ultraviolet absorption at 280 nm or the increase in absorbance (660 nm) of the tyrosine-tryptophan filtrate after trichloroacetic acid (TCA) precipitation of the undigested protein reacted with diluted (2:1) phenol reagent solution (9). Schwabe (127) described a method which permited the assay of the proteolytic enzjmie activity on hemoglobin utilizing the f luorescamine technique. The assay is about 100 times more sensitive than the Lowry method, much faster and less complicated. He observed that the two main obstacles for the successful use of f luorescamine in his assay system were (1) the high blank produced by the reaction of e-amino groups of the protein and (2) the fluorescent quenching effect of the hemoglobin. The high blank of the hemoglobin he substantially suppressed by a chemical modification, i.e., succinylation. Hemoglobin is usually used as a 2% solution of which only 10 yl are pipetted into 2 ml of phosphate buffer used for the reaction. He observed that the enzyme activity as measured by the f luorescamine method remained linear throughout thirty minutes while the Lowry method indicated a definite slowing of the reaction beginning at about ten minutes. This was due to the fact that f luorescamine detects an increase in free amino groups while the Lowry reagent as well as the direct measurement of absorption at 280 nm depends on the production of tyrosine or tryptophan containing peptides. A possible explanation for this discrepancy is that the enzjone in its initial attack on the hemoglobin molecule releases large peptides which are TCA soluble and that subsequent enzyme action further degrades these large peptides without significantly increasing the number of TCA-soluble fragments containing tyrosine or tryptophan moieties. A reagent depending upon primary amine groups is not subject to this error (125).

PAGE 35

21 Fluorescamine is a new reagent for the detection of amino acids, peptides, proteins and primary amines in the picomole range (18,133,140). Its reaction with amines is almost instantaneous at room temperature in aqueous media. The products are highly fluorescent, whereas the reagent and its degradation products are nonf luorescent. McCaman and Robins (101) introduced a fluorometric method now widely used for assay of serum phenylalanine which is based on the interaction of ninhydrin and peptides. Samejima et al. (124,125) found that it was the phenylacetaldehyde formed on interaction with ninhydrin which combined with additional ninhydrin and peptide or any other primary amine to yield highly fluorescent products. The structure of these products was subsequently elucidated by Weigele et al. (145), who then synthesized a novel reagent (145). This reagent 4-phenylspiro (furan-2(3H) ,1'phthalan) 3,3'-dione (fluorescamine) reacts directly with primary amines to form highly fluorescent products. Several factors make fluorescamine suitable for assaying primary amines, including amino acids, peptides and proteins. At pH 8-9, the reaction with primary amines proceeds at room temperature (140) within a fraction of a second. Excess reagent is concomitantly destroyed within ' several seconds (140). Fluorescamine, as well as its hydrolysis products, is nonf luorescent. Studies with small peptides have shown that the reaction goes to near completion (about 80% to 95% of theoretical yield) even when fluorescamine is not present in excess. The following is an example of the reaction of fluorescamine with an amine group illustrating the product formed (fluorophor) and the rate of the reaction (100-500 msec). In addition, the reaction of water with fluorescamine with the formation of a nonf luorescent product is also shown.

PAGE 36

22 (nonf luorescent) Hydrolysis products (nonf luorescent) Primary amines are first buffered to an appropriate pH (7-8), and then fluorescamine, dissolved in a water miscible, nonhydroxylic solvent such as acetone or diojcane, is added. The reaction is complete, and in less than a minute excess reagent is destroyed. The resulting fluorescence is proportional to the amine concentration, and the fluorophors are stable over several hours. The above properties lend themselves well to automation (123). It should be noted that fluorescamine does not react with proline or hydroxyproline, which are not primary amines. This disadvantage can be overcome by introducing an appropriate intermediate step to convert these amino acids to primary amines (63,146). An additional advantage of the f luoresamine assay is that comparatively little fluorescence is developed with ammonia. Therefore, ammonia does not interfere with an analysis to the extent that it does in the colorimetric ninhydrin procedure. Figure 2 shows a comparison of the

PAGE 37

23 Figure 2, 20 4 0 5 0 FRACTION NUMBER 80 Comparison between the Fluorescamine technique and the Lowry procedure ^ a^°^ determining protein concentration. ^ Chromatography of a partially purified enzyme of guinea pig neurophysin monitored by the fluorescamine and the Lowry procedure.

PAGE 38

24 f luorescamine technique with the standard Lowry procedure (97) for the monitoring of protein in a column effluent. The volumes used for fluorescamine assay were 10 to 20 percent of those used in the Lowry method, and smaller amounts could have been used (140). Background interference was negligible with the automated fluorescence method, and significant peaks not discernible by the Lowry procedure were observed. Due to the many advantages of the relatively new fluorometric technique, it was used to measure the proteolytic enzjnne activity of P. citreus . The ability of this organism to grow on shrimp as well as to hydrolyze various protein preparations promoted investigations to isolate, purify and characterize the extracellular enzyme (s) produced by P. citreus.

PAGE 39

MATERIALS AND METHODS Unless otherwise specified, Difco (55,56) or Baltimore Biological Laboratories (BBL) (16) products were used for all microbiological analyses. Serial dilutions used Butterf ield's Phosphate buffer and followed the procedures outlined in the Compendium of Methods for the Microbiological Examination of Foods (6), All chemicals used were reagent grade meeting American Chemical Society specifications. All media and glassware were autoclaved for 15 min at 121 C unless label directions specified otherwise, Planococcus citreus Cultures The culture of _P« citreus used in this study, A-17, was isolated from rock shrimp (Sicyonia brevirostris ) (90) . The culture chosen was able to grow well in shrimp during iced storage and showed strong proteolytic activity toward various protein preparations. The isolate used for the study was grown on Plate Count Agar slants (Difco) with 0.5% sodium chloride (NaCl) added and incubated at 20 C for 72 hrs (4). Appropriate dilutions in buffer were made to obtain a concentration of 3 approximately 5 x 10 organisms per ml. The A-17 isolate used was capable of hydrolyzing gelatin, whey, cottonseed, soy, hog blood and shrimp protein preparations (4). It also grew well in 0.5% to 16% NaCl and pH 7.0 to 10.9. Determination of Proteolytic Activity A modified f luorescamine fluorescent (f luorometric) technique (129) was used to measure enzyme activity. Fluorescamine'^ is capable of 25

PAGE 40

26 detection of amino acids, peptides, proteins and primary amines in the picomole range (140). _P. citreus cells were grown in various media throughout the study. After incubation, the cultures were centrifuged in a RC-5 Superspeed Refrigerated Centrifuge (Sorval, Dupont Co. Instruments, Newtown, CT) at a force of 20,000 x g for 30 min. The supernatant (cell-free broth) was used for further investigations involving extracellular enzjmies. The cell pellet was washed twice with 0.05 M phosphate buffer (pH 8). The whole cells were then resuspended with 10-20 ml of the same buffer, transferred to a dry ice chilled Eaton pressure cell (60) and allowed to freeze under dry ice for 3 hrs. The frozen microbial cells were disintegrated using the Eaton pressure cell at a constant pressure of 7.03 x 10^ kg/m=^ on a Carver hydraulic laboratory press (F. S. Carver, Inc., Summit, NJ). The ruptured cell extract was fractionated into intracellular soluble and particulate fractions by centrifugation at a force of 12,000 x g for 15 min. The particulate fraction was resuspended in 10 ml of 0.05 M phosphate buffer prior to enzyme activity determinations of all fractions. Five milliliters of the substrate (gelatin or shrimp protein) were reacted with 1 ml of each of the above fractions for 15 min at 35 C. The reactions were terminated by adding 10 ml of 5% TCA. Zero time blanks were prepared by adding the trichloroacetic acid (TCA, Fisher Scientific Co., Fairlanes, NJ) before the incubation period (see latter part of this section). One milliliter of the cellfree broth or 100 yl of the purified enzyme was reacted with 5 ml of substrate (gelatin or shrimp protein) for the appropriate reaction time (to be determined) at 35 C, pH 8, The enzyme-substrate reaction was stopped by precipitating the mixture with

PAGE 41

27 10 ml of 5% TCA. After 5 to 10 min, to allow the proteins to settle, the solution was filtered through Whatman #1 filter paper. Two hundred microliters of the TCA filtrate were transferred to a 13 x 100 mm test tube (Dispo culture tubes. Scientific Products, McGraw Park, IL) and the volume brought to 1.5 ml with 0.5 M sodium phosphate buffer, pH 8. While the test tube was vigorously mixed in a Vortex Mixture (Scientific Products, Evanston, NY), 0.5 ml of f luorescamine in dioxane (30 mg/100 ml, Eastman Kodak Corp., Rochester, NY) was rapidly added to the buffered protein solution. A model 204-A Fluorescence Spectrophotometer (Perkin Elmer Corp., Norwalk, CT) was used to measure fluorescence intensity. Zero time blanks were prepared by adding 10 ml of 5% TCA after adding the enzjme and prior incubation of the mixture. This blank represented the background activity present in the mixture at zero time. Zero time fluorescence reading was subtracted from the reading of the substrateenzyme mixture after the appropriate incubation time. Total enzyme activity was expressed as the change in 0.1 fluorescence units of the TCA filtrate per milliliter of enzyme per minute. Specific activity was expressed as the units of total enzyme activity/mg of protein present (units of activity/mg of protein). Previous research involving the use of the f luorescamine technique (47,127,140) indicated that pH affected fluorescence intensity. Buffers of pH from 2 to 10 (see buffers described on pg. 44) were used to determine the effect of varying the pH of the buffer on fluorescence intensity. TCA filtrates (0.2 ml) were reacted with 1.3 ml of the various buffers (pH 2-pH 10) before addition of the f luorescamine reagent. Figure 3 indicates that addition of pH 8 buffer resulted in the highest fluorescence itensity. Consequently, pH 8 buffer was used for the remainder of the research.

PAGE 42

28 I50n gure 3. Effect of oH adjustment of gelatin-trichloroacetic acid (TCA) filtrat ohvflQotescence intensity.

PAGE 43

29 In order to determine the reaction spectrum of our working enzyme solution when gelatin and shrimp protein were used as substrates, the excitation and emission (fluorescent) wavelengths were scanned (48). Figure 4 shows the excitation (curve A) and fluorescence (curve B) spectra for TCA filtrates of the gelatin substrate. The excitation spectrum has a maximum of 360 nm and a secondary peak at 390 nm. The secondary peak at 390 nm was chosen because it results in minimal zero time blank fluorescence values. The fluorescence emission maximum with the excitation wavelength at 390 nm was at 475 nm. Figure 5 shows the excitation (curve A) and fluorescence (cut-ve B) emission spectra for TCA filtrates of the shrimp protein substrate. The excitation spectrum has a maximum peak at 375 nm and a secondary peak at 390 nm. Again, the secondary peak was chosen. The fluorescence emission maximum with the excitation wavelength at 390 nm was at 490 nm. Efficacy of 5% Trichloroacetic Acid (TCA) In order to determine the efficacy of 10 ml of 5Z trichloroacetic acid (TCA) in terminating the enzyme-substrate reaction, 5 ml aliquots of substrate (gelatin) were incubated with 1 ml of cell-free broth and 10 ml of 5% TCA for 0, 10, 15, 30, 45 and 60 min at 35 C. A positive control was done by incubating the enzyme-substrate mixture at 35 C for 0, 10, 15, 30, 45 and 60 min before adding the TCA. After the incubation period, the positive control was terminated by adding 10 ml of 5% TCA. Data in Figure 6 shows that 10 ml of 5% TCA were adequate for inhibiting the enzyme substrate reaction effectively since there was no increase in fluorescence intensity. The fluorescence intensity, observed when TCA is immediately reacted with the enzyme and substrate, represents the background fluorescence of the assay mixture.

PAGE 44

30 I o u o iH XI o H U 4J I c 1-1 0) 00 G O •H O CO « u u o 14-1 e 3 • S-i 00 u e CO a cj to u 3 o CO CU 4J O S 3 rH 01 CO C U CO tH > U 3 w a c o A1ISN31NI 3AllVn3d 4J O CO -H W 4J •H 0) X O U CO 0) 3 to

PAGE 45

31 A11SN31NI 3AllVn3d R i 4) U O a 1 h Ji a CO of o o 03 u tH 3 4-1 O OJ > — y a cd c o o 1-1 •H o iH cd O •H •H X u 4-1 • in 0) u 0 60 •H

PAGE 46

32

PAGE 47

33 Substrate Characteristics Two substrates were used throughout the study: gelatin (Difco) (1.2 mg/ml) and a shrimp protein preparation (0.6 mg/ml) . Higher concentrations of the shrimp protein preparation were not used because of solubility problems in the buffer systems used. The shrimp protein preparation was prepared as follows: fresh raw shrimp meat ground in a Waring blender with 0.05 M phosphate buffer (pH 7, 1:10 dilution), dialyzed overnight with four changes of the same buffer at 5 C and lyophylized for preservation (Virtis Freeze Dryer, Gardiner, NY). Protein, fat, moisture and ash were determined for the shrimp protein preparation. Protein was determined by the AOAC standard micro-Kjeldahl method (13). Crude fat was determined by a modification of the AOAC method (13) using the Goldfisch solvent chamber. Approximately 2 grams of sample were extracted overnight with petroleum ether. Moisture was determined in a vacuum oven at 70 C for 12 hrs. Ashing was done in a muffle furnace at 600 C for 8 hrs. Table 3 shows that the shrimp protein preparation consisted of 77.44% protein, 5.40% fat, 8.95% moisture, 6.50% ash and 1.71% carbohydrate (calculated by difference) . Determination of Enzyme-Substrate Mixture Reaction Time Five milliliters of substrate and an aliquot of cell-free broth were incubated at 35 C for 0, 5, 10, 15, 20, 30 and 60 min in order to determine the time course of enzyme activity and apparent optimum reaction time. In experiments involving gelatin, 0.5, 1.0 and 2,0 ml of cell-free broth were used while 1.0 ml of cell-free broth was used with the shrimp protein substrate. An incubation time of 15 min was an appropriate enzyme-substrate contact reaction time when shrimp were used as substrate (Figure 7). In addition, when gelatin was used as a substrate and

PAGE 48

34 Table 3. Proximate composition of the shrimp protein preparation.^ Percent (%) Protein 77.44 Fat 5.40 Moisture 8.95 Ash 6.50 Carbohydrate 1.71 Average of two determinations ^Calculated by difference

PAGE 49

35 70-1 INCUBATION TIME AT 35° C (min) re 7. Increase in fluorescence intensity using the shrimp protein preparation as substrate after incubation with cell-free broth for up to 1 hr at 35 C.

PAGE 50

36 various amounts of cell-free broth were reacted with gelatin, a 15 min reaction time was also a sufficient substrate reaction time (Figure 8). This reaction time was used for the remainder of the study. Growth Medium and En23nne Production Various media were used to determine growth rates and production of extracellular enzyme (s) by JP. citreus . Three hundred milliliters of Plate Count Broth (PCB) + 0.5% NaCl, Nutrient Broth (NB) + 0.5% NaCl and Trypticase Soy Broth (TSB) were used to grow the organism. Incubation was at 20 C for up to 96 hrs. Samples were drawn at 0, 12, 24, 48, 72 and 96 hrs. All samples were assayed for growth by measuring optical density at 600 nm in a Spectronic-20 Spectrophotometer (Bausch and Lomb, Rochester, NY) and by plating in Plate Count Agar (PCA) with incubation at 20 C for 5 days. Cultures were centrifuged in a RC-5 Superspeed Refrigerated Centrifuge at a force of 20,000 x g for 30 min. The sediment was discarded. One milliliter of the cell-free broth was assayed for enzyme activity with gelatin substrate. _P« citreus growth and enzyme analyses were done three times and each time in duplicate. Optimizat ion of Enzyme Activity to Growth and Cell Number The optimum time for cell harvesting along the logarithmic section of the growth curve (approximately midlog) of the organism was selected. The logarithm of the cell count was plotted against the incubation time. The specific growth rate (k) of each medium was calculated using the equation In B^ = In B^ + k' (where B = bacterial count at time x, B = o bacterial count at time 0 (both in midlog growth phase), k = specific growth rate, t = time, hrs). The units of enzyme activity per cell per hour (m) were then calculated using the formula (58,124): M M = t ok (B^ (where = enzyme activity at cell number B^, = enzyme

PAGE 51

37 a -o o Brol Bro1 Brol 0) 0) (U k_ Li_ Ll. Cell Cell Cell E £ e If) d q o O O c mmtm J o O C/) t/5 in c c .2 UJ c/) CO O ^ -Q I— ro (D goo — OJ 0) hCP ^ < D O ^ ^ ^ O < < O CX) O \ o o o o o CO «n ''J" oj 30N30S3dOnnd V iH fH 0) U 0) 43 4J «M O a 3 2 i a 9 O >H > c OJ 4J n) i-i CO .a 3 to CO CO c •H 4J CO rH to M C 'r-l •H o 03 14-1 3 u in •H CO c 0) CO 4J C C •H O •H (U 4J o CO c
PAGE 52

38 activity at cell number B^, k = specific growth rate). This formula was obtained through the integration of the following equation: = m e^ (where dm/dt = change in enzyme activity over time). The calculated m's were compared for the three media used. A test to observe any difference between the values for the units of enzyme activity per cell per hour (m) in each medium was designed using the Statistical Analysis System (SAS) program package (15) for analysis of variance. A completely randomized design (102,132) was used in that the major source of error to be considered was due to the nutritional differences between media. Effect of Incubation Temperature on Enzyme Production and Activity The effect of incubation temperature (5, 20 and 35 C) on the growth of _P. citreus and its ability to produce an active extracellular enzyme was investigated. Five-hundred-milliliter Erlenmeyer flasks containing 100 ml of medium were inoculated with approximately 5 x 10 P. citreus and incubated at the three temperatures. All samples were assayed for growth by measuring op^tical density at 600 nm in a Spectronic-20 spectrophotometer and for proteolytic activity using the fluorometric technique. For the 35 C grown cells, samples were drawn at 0, 6, 12, 24, 48, 72 and 96 hrs. For the 20 C grown cells, samples were drawn at 0, 12, 24, 48, 72, 96 and 120 hrs. Finally, for the 5 C grown cells, samples were drawn at 0, 24, 48, 72, 96, 120 and 144 hrs. After determining the midlog phase of growth for JP. citreus at each temperature, P_, citreus cells were then harvested at this stage. Enzyme activity determinations were done using the cell-free broth obtained from growing the organism at the three temperatures until midlog phase. Five milliliters of the shrimp protein preparation were incubated with 1 ml of each cell-free broth at 5 C for 60 min, 20 C for 30 min and 35 C for 15 min. Analyses were done three times and each time in duplicate.

PAGE 53

39 In addition, direct microscopic observations of the cells grown at 5, 20 and 35 C were conducted. Any morphological change due to growth temperature was observed and recorded. Purification of the Extracellular Enzyme (s) Planococcus citreus was grown in the selected medium at 20 C until midlog phase. The cells were then centrifuged at a force of 20,000 x g for 30 min. This cell-free broth was used in the purification procedure. Ammonium Sulfate Precipitation Fractional precipitation of the enzyme (s) in the cell-free broth was accomplished with 0-55%, 55-70% and 70-100% ammonium sulfate saturation (Mallinckrodt , Inc., Paris, KY) . The required amount of ammonium sulfate was added with stirring until dissolved (88). The mixture was allowed to equilibrate for 1 hr at 4 C and centrifuged at a force of 20,000 x g for 20 min. The precipitate was resuspended with 10 ml of Butterf ield's phosphate buffer at pH 8 and dialyzed for 16 hrs (dialysis tubing #24, Scientific Products, McGraw Park, IL) against 500 ml of 0.05 M phosphate buffer pH 8 (108,153). Each ammonium sulfate fraction was then assayed for proteolytic activity using gelatin as substrate. Molecular Sieve Chromatography Sephacryl S-200 Superfine (Pharmacia Fine Chemicals, Uppsala, Sweden) , a high resolution chromatographic medium for gel filtration was used to separate the enzyme solution according to molecular weight after the ammonium sulfate precipitation step. A 30 x 2.5 cm column was packed with Sephacryl^ S-200 Superfine gel and a Pharmacia^ peristaltic pump (p-3) was used to pack the column at a speed of 40 ml/cm/hr (120). The enzyme solution was eluted using reverse flow at a speed of 30 ml/cm/hr. Five milliliter fractions were collected in each tube with 0.02 M phos-

PAGE 54

40 phate buffer pH 7 as the eluting agent using a Gilson Fraction collector (Model FC-220K Fractionator , Gilson Medical Electronics, Inc., Middletown, WI) . Enzyme activity of each fraction collected was then measured using gelatin as substrate. The protein present in the fractions was determined by following absorbance at 280 nm using a Beckman Model 25 spectrophotometer (Beckman Instruments, Inc., Fullerton, CA) . Ion-Exchange Chromatography The active fractions recovered from the gel filtration step were pooled and further separated by ion-exchange chromatography using DEAESephadex A-50 (Pharmacia Fine Chemicals, Uppsala, Sweden). The column was prepared following the procedures given by Pharmacia Fine Chemicals (119). A 40 X 2.5 cm column was prepared and the protein eluted with 0.02 M phosphate buffer with a linear gradient of tris 0.01 M NaCl-tris 0.15 M NaCl (47) at a rate of 25 ml/hr (1.5 reading in the peristaltic pump) and collected in 10 ml fractions. Two hundred fifty milliliters of 0.01 M and 0.15 M NaCl solutions were placed in each vessel for the linear gradient. The protein present in the fractions was followed by reading the absorbance at 280 nm using a Beckman Model 25 spectrophotometer. Enzyme activity of each fraction was measured with gelatin as the substrate. All column studies were duplicated. The protein content of each fraction eluted using the Sephacryl^ S-200 Superfine and the DEAE-Sephadex^ A-50 was also analyzed by the Lowry (98) method for protein with Bovine Serum Albumin (Sigma Chemical Co., St. Louis, MO) as the standard. Protein content is expressed as mg/ml. Figure 9 summarizes the steps followed in the purification of the extracellular proteolytic enzyme(s) of P. citreus.

PAGE 55

41 STERILE TRYPTICASE SOY BROTH inoculated with 3 5 X 10 P^. citreus/ lOO ml harvest cells at mid-log phase V CENTRIFUGE ( 20,000 X g/ 30 min) cell-free broth (NH^)2S0^ FRACTIONAL PRECIPITATION T / SEPHACRYL S-200 SUPERFINE Figure 9: Outline of steps for the purification of the extracellular protease(s) of Planococcus citreus. DEAESEPHADEX A-50 ION EXCHANGE CHROMATOGRAPHY CHARACTERIZATION STUDIES

PAGE 56

42 Confirmation of Enzyme Purity A modification of the Weber and Osborn (144) method for sodium dodecyl sulfate-poly acrylamide gel (SDS-PAG) gel electrophoresis was used. A Buchler 3-1500 electrophoresis apparatus (Buchler Instruments Corp., Fort Lee, NJ) was used to evaluate the purity of the isolated extracellular enzjmie. A 10% acrylamide: BIS, 30:0.8 gel was prepared and allowed to polymerize for 2 hrs. A sample of the purified enzyme was diluted 1:1 with the sample buffer. The sample buffer consisted of 0.01 M sodium phosphate (pH 7), 10% sodium dodecyl sulfate, 0.1% dithiothrietol , 10% glycerol and 0.001% bromocresol blue. The protein solutions were placed onto the gels (50 ug protein/gel, 100 yg/gel, 150 yg/gel and 200 ug/gel) and were layered carefully with electrode buffer (pH 8.3) to the top of each tube. The lower electrode chamber was then 2/3 filled with electrode buffer. The tubes in the apparatus were then lowered into the electrode chamber. The upper chamber was filled with water to approximately 1 inch over the tube top. The water jacket was connected and the electrode wires from the power source were also connected. A constant current of 1-1.5 mAmps/gel was applied until the marker dye band just exited from the gels (approximately 3 hrs). The gels were immediately removed from their tubes. The gels were fixed overnight in a fresh 50% TCA solution. The fixed gels were then stained 1-2 hrs with 0.1% Coomassie brilliant blue solution made up fresh in 50% TCA at 37 C in a water bath. The gels were further dif f usion-destained by repeated washings in 7% acetic acid (17-72 hrs). Gels were then stored in 7% acetic acid (82).

PAGE 57

43 Characterization of the Proteolytic Enzyme(s) Molecular Weight Determination Two methods (7,21,150) were used to estimate the molecular weight of the enzyme (s), A 2.5 X 30 cm column packed with Sephacryl S-200 (Pharmacia Fine Chemicals, Uppsala, Sweden) was used and the following standards applied: Ribonuclease A (13,000 MW) , Trypsin (23,500 MW) , Pepsin (45,000 MW) , Bovine Serum Albumin (70,000 MW) and Aldolase (158,000 MW) (Pharmacia Fine Chemicals, Piscataway, NJ) following the procedures suggested by Pharmacia Fine Chemicals (116). The K^^ value of each protein sample was calculated and plotted against the corresponding molecular weight V V e o ~ u v~ where V = column void volume, V = elution volumes and a V t ~ o ^ = total column volume) . Protein was monitored at 280 nm using a Beckman Model 25 spectrophotometer (Beckman Instruments, Inc., Fullerton, CA). Bio-Rad Low Molecular Weight Protein Standards (10,000-100,000) for SDS Gel Electrophoresis were also used for molecular weight determination using SDS-PAG gel electrophoresis. The instructions outlined by Bio-Rad^ (Bio-Rad Laboratories, Richmond, CA) were followed (21). The proteins included were Phosphorylase B, Bovine Serum Albumin, Ovalbumin, Carbonic Anhydrase, Soybean Trypsin Inhibitor, Lysozyme and the purified enzyme. The motility of the enzyme was then compared to the relative motility (Rm) of the standards. Determination of the Purified Enzyme-Substrate Mixture Reaction Time Five milliliters of substrate (gelatin or shrimp protein) and 100 yl of the purified enzyme were incubated at 35 C for 0, 5, 10, 15, 30 and 60 min in order to determine the apparent optimum reaction time.

PAGE 58

44 Enzyme activity was measured using the Fluorescamine technique. Figure 10 shows that 10 min was the optimum reaction time for the purified enzyme-substrate (gelatin or shrimp protein) reaction mixture. This optimum reaction time was used for the remainder of the characterization of the extracellular proteolytic enzyme. Effect of Ionic Strength on Enz3rme Activity The effect of ionic strength on enz}rme activity was investigated. Gelatin (1.2 mg/ml) was dissolved in the following solutions of sodium chloride (NaCl) : 0.05 M (y = 0.13), 0.08 M (p = 0.16), 0.18 M (m = 0.26), 0.25 M (P = 0.35), 0.34 M (y = 0.42), 0.51 M (y = 0.59), 0.75 M (y = 0.83), 1.00 M (y = 1.08) and 1.5 M (y = 1.58). The NaCl was dissolved in 0.05 M phosphate buffer (pH 8). Five milliliters of this mixture were reacted with 100 yl of the purified enzyme and incubated at 35 C for the selected reaction time (10 min). Determination of Optimum pH Buffers of varying pH from pH 2 to pH 10 were used to determine the optimum pH for the proteolytic activity of the enzyme(s). The following buffers were used: Ionic Strength pH 2 0.1 M citric acid 0.25 3 47,0 ml of 0.1 M citric acid + 3.5 ml of 0.35 0.1 M sodium citrate 4 33.0 ml of 0.1 M citric acid + 17.0 ml of O.45 0.1 M sodium citrate 5 20.5 ml of 0.1 M citric acid + 29.5 ml of 0.45 0.1 M sodium citrate 6 88 ml of 0.2 M monobasic sodium phosphate + 0.45 12.5 ml dibasic sodium phosphate 7 39.0 ml of 0.2 M monobasic sodium phosphate + 0.35 61.0 ml of 0.2 M dibasic sodium phosphate

PAGE 59

45 250n UJ o LlI O (f) Ld cr o Ll. 200500050Gelatin — Shrimp 0 10 15 30 60 INCUBATION TIME AT 35° C (min) gure 10. Increase in fluorescence intensity following incubation of gelatin and shrimp protein substrate with purified enzyme for up to 1 hr at 35 C.

PAGE 60

46 Ionic Strength 8 5.3 ml of 0.2 M monobasic sodium phosphate 0.25 + 95.0 ml of 0.2 M dibasic sodium phosphate 9 50 ml of 0.2 M glycine + 8.8 ml of 0.2 M NaOH 0.22 10 50 ml of 0.2 M glycine + 32.0 M NaOH • 0.20 Gelatin (1.2 mg/ml) or shrimp protein (0.6 mg/ml) were dissolved in the various buffers. Any pH adjustments due to the addition of the substrates were done using 10 mM HCl or 10 mM NaOH. Five milliliters of this mixture were reacted with 100 yl of the purified enzyme and incubated at 35 C for the selected reaction time (10 min). Determination of Optimum Temperature Five milliliters of gelatin or shrimp protein substrate and 100 pi of the purified enzyme were incubated at 5, 10, 20, 35, 45, 55 and 65 C for 10 min at the optimum pH determined in the previous section. Thermal Stability P_, citreus was incubated at 5 and 35 C in 300 ml of Trypticase Soy Broth (TSB). Cell-free broths obtained at midlog phase, 108 and 36 hrs for the 5 and 35 C grown cells, respectively, were used in this study. Five milliliters of the cell-free broths were incubated at 35, 45, 55, 65, 75 and 85 C for 15 min. The heat treated cell-free broths solutions were rapidly cooled (87), and their activity was assayed at 35 C for 15 min using gelatin as substrate. The residual activities at each solution were compared to the activity observed when the cell-free broths were incubated with the substrate at 35 C for 15 min. In addition, 1 ml of the purified enzyme was also incubated at 35, 45, 55, 65, 75 and 85 C for 10 min. The heat treated purified enzyme solution was cooled, and its activity assayed at 35 C for 10 min using

PAGE 61

47 gelatin as substrate. The residual activities of each solution were compared to the activity observed when the purified enzyme was incubated with gelatin at 35 C for 10 min. Effect of Sodium Chloride Concentration Various concentrations of NaCl were tested for their effect on enzyme activity. Concentrations of 0.00, 0.25, 0.50, 0.75, 1,00, 1.25 and 1.50% were used. NaCl was dissolved in 0.05 M phosphate buffer pH 8. The shrimp protein (0.6 mg/ml) and gelatin (1.2 mg/ml) were dissolved in the NaCl solutions. Five milliliters of the NaCl solutions were incubated with 100 ul of the purified enzjnne at 35 C for 10 min. Effect of Sodium Bisulfite Concentration Various concentrations of sodium bisulfite (NaHSO^) were tested for their effect on enzyme activity. Concentrations of 0,0, 0.5, 1.0, 2.0 and 3,0% were tested. NaHSO^ (J. T. Baker Chemical Co., Phillipsburg, NJ) was dissolved in 0.05 M phosphate buffer pH 8. The shrimp substrate was dissolved in these NaHSO^ solutions (0.6 mg/ml). Five milliliters of the NaHSO^ solutions were incubated with 100 ul of the purified enzyme at 35 C for 10 min. Effect of Enzyme Concentration Various quantities of enzyme (from 0 to 200 Ml) were tested to observe the effect of enzyme concentration on enzyme activity. Five milliliters of substrate (gelatin or shrimp protein) were incubated with 0, 50, 75, 100 and 200 ul of enzyme at 35 C for 10 min. Effect of Substrate Concentration The enzyme was incubated with various concentrations of gelatin and shrimp protein in order to determine substrate saturation conditions. For the gelatin substrate, 0.00, 0.15, 0.30, 0.45, 0.60 and 1.20 mg/ml

PAGE 62

48 were tested. However, for the shrimp substrate, 0.000, 0.075, 0.100, 0.125, 0.150, 0.300 and 0.600 mg/ml were tested. Five milliliters of each substrate solution were reacted with 100 yl of the purified enzyme at 35 C for 10 min. From these data, Lineweaver-Burk plots were derived, and K and V values for each substrate were extrapolated from these plots (95,152). Effect of Metal Ions on Enzjone Activity > Calcium chloride (10, 20 mM) , ferric chloride (1, 20 mM) , magnesium chloride (10, 20 mM) , mercurous chloride (1, 20 mM) , zinc chloride (10, 20 mM) , manganese chloride (10, 20 mM) and potassium chloride (5, 20 mM) were tested for their effect on enzjnne activity (all metals were dissolved in 0.05 tris-HCl buffer). For the control, a buffer with no metal ions added was used (76,87). Five milliliters of substrate (gelatin) 100 yl of enzyme and 1 ml of the metal ion buffer solution were reacted for 10 min at 35 C. The fluorometer reading of the control sample was compared to, the reading of the metal ion samples. Effect of Various Reagents on Enzyme Activity Ethylene diaminetetraacetic acid (EDTA) (10, 20 mM) , citric acid (10, 20 mM), formaldehyde (1, 20 mM) , potassium cyanide (KCN) (1, 20 mM) , potassium permanganate (KMnO^) (1, 20 mM) , cysteine (1, 20 mM) , 2mercaptoethanol (1, 20 mM) , p-dioxane (10, 20 mM) and trichloroacetic acid (TCA) (5, 10%) were tested for their effect on the proteolytic activity of the _P. citreus enzyme (76,87). All reagents were dissolved and/or mixed with 0.05 M tris-HCl buffer. Five milliliters of substrate (gelatin), 100 yl of enzyme and 1 ml of the appropriate reagent buffer solution were reacted at 35 C for 10 min. A control with no reagent added was used and the fluorometer reading from the various reagents was compared to the control.

PAGE 63

49 Dipeptidase Activity The potential of the _P. citreus enzyme to degrade peptides was investigated. DL-leucylglycine, DL-leucyl-DL-alanine , glycyl-DL-leucine, DL-alanylglycine and L-leucyl-l-tryptrophan (Sigma Chemical Co., St. Louis, MO) were used in this study. Fifty milligrams of each dipeptide were dissolved in 50 ml of phosphate buffer, pH 8. Five milliliters of the dipeptide solutions were incubated with 100 Ml of the purified enz3Tne at 35 C for 10 min. The reaction was terminated by adding 10 ml of 5% TCA. Zero time blanks were done by adding the TCA to the enzymepeptide mixture before the incubation period. Enzyme Induction Studies _P. citreus was grown in various media in order to determine if the extracellular proteolytic enzyme produced by this organism is induced by shrimp protein. Three-hundred milliliters of the following were used: (1) Yeast Carbon Base (YCB) (control) (2) YCB + 1.0% Shrimp Protein (3) YCB + 0.1% Yeast Extract (4) YCB + 0.1% Yeast Extract + 1.0% Shrimp Protein Table 4 shows the composition of the Yeast Carbon Base medium (YCB). _P. citreus growth and enzyme activity were analyzed at 0, 24, 48, 72 and 96 hrs following incubation at 20 C. Cell numbers were determined by pour plating into Trypticase Soy Agar (TSA) with incubation at 20 C for 5 days. Five milliliters of the shrimp substrate were incubated with 1 ml of the cell-free broth from each culture for 15 min at 35 C. The reaction was terminated by adding 10 ml of 5% TCA. Zero time blanks were done by adding the TCA to the cell-free broth-substrate mixture before the incubation period. This study was done twice in duplicate.

PAGE 64

50 Table 4. Composition of yeast carbon base medium (56). Formula in Grams per Liter of Distilled Water Boric Acid 0.500 mg. Copper Sulfate 0.040 Potassium Iodide 0.100 Ferric Chloride 0.200 Manganese Sulfate 0.400 Sodium Molybdate 0.200 Zinc bulrate 0.400 Biotin 0,002 mg. Calcium Pantothenate 0.400 rollc Acid 0.002 Inos itol 2. 000 Niacin 0.400 p-Aminobenzoic Acid 0.200 Pyridoxine 0.400 Riboflavin 0.200 Thiamine HCl 0.400 L-Histidine HCl 0.001 g. DL-Methionine 0.002 DL-Tryptophan 0.002 Potassium Phosphate 1.000 g. Magnesium Sulfate 0.500 Sodium Chloride 0.100 Calcium Chloride 0.100 Dextrose 10.000 Final pH of the base adjusted to 7.5

PAGE 65

51 The data was analyzed in a similar manner as the Growth Medium and Enzjrme Activity data. Again, a comparison of the m's for each medium used (m = units of enzyme activity/cell/hr) was attempted using the SAS program package for analysis of variance (15). The Duncan's New Multiple-Range Test (pg. 187-190 (132)) was used to compare any difference in the calculated means of the data obtained after analysis of variance in the "Optimization of enz3nne activity to growth and cell number" section (pgs. 36 and 38), "Optimum pH determination" section (pg. 82), "Optimum temperature determination" section (pg. 84) and "Enzyme induction study" section (pg. 105-115). The Duncan's New MultipleRange Test was done using the SAS program package (15).

PAGE 66

RESULTS AND DISCUSSION The ability of Planococcus citreus to grow in shrimp during ice storage raised the question as to whether this organism could contribut to the spoilage of shrimp. Various studies (3,4,5) have indicated that this organism may contribute to the spoilage of this valuable marine resource. In order to more clearly understand the contribution this organism makes to the degradation of shrimp, an investigation was under taken to study the extracellular proteolytic enzyme (s) produced by this organism. Proteolytic Activity of Cellular Fractions The proteolytic activity of cellular fractions of citreus cells grown in Trypticase Soy Broth (TSB) was investigated in order to determine the distribution of the enzyme activity in the isolated fractions. In addition to the cell-free broth (extracellular fraction), whole celli washings of the whole cells, soluble intracellular and the cellular particulate fraction were examined. Table 5 shows the total activity (units of activity), protein content (mg/ml), specific activity (units of activity/mg of total protein) and distribution of activity (%) for all the fractions tested using both gelatin and shrimp protein substrates. The extracellular fraction showed the highest specific activity, 29.450 units of activity/mg of protein and 27.540 units of activity/mg of protein towards gelatin and shrimp protein, respectively. This represented 95.9 and 95.8% of the total activity present in all of the fractions towards gelatin and shrimp protein, respectively. 52

PAGE 67

53 CO E to o M 0 CB u 00 CO « 3 r-l 3 n CO H U J= O O ^ — \ 00 PQ BC a. 0 w to c a> to E to o m •H *— < 4-1 a u o H H d, o CO o > •H •H c •H •4-1 a •t-i CO <: to •H O Ge 4-« 4J o •H M > a j: a to o O 4-1 •H (4-1 C>0 •H E o oj CO (X 4-1 CO -H c 3 •H ^ 01 a 4-1 O 60 04 ^ OJ 4-1 N 3 c ^ ^ 4-1 CO -H •M > O -H H +J O < a. e •H CO CO C o o to u in CO • • • • • • o in a\ •—1 o o o m CO • • • • r-4 o o ON o 00 ON 1—1 ro o o i-H St o I-H • • • • • • o O o o o CM o I-H o CO CM m o o tj\ m sr m o • • • • • • o ON CN o o o o o O cn vO sr m CN tN CN • • • • • • ro CO o CN 1— 1 o ro ro CN • • • • • • m o ro ro 1—1 CN CJN CJN in m 00 t3N ro m CN I-H «> CM CN •—1 h~ o i-H ro f-H v-H vo O ( ro 1— ( I-H 00 o in vo o fO 1— 1 CN 1— 1 I-H fO u u CO CO CO CO l-( 60 rH (U 3 c c 3 +J -H •H •H I-H to N 3 4-) •O •H > lU •H o 4-1 U 0) CO 60 CO •-I CO (1) 4-> o H

PAGE 68

54 The whole cell fraction (cell bound fraction), both whole cell washings (loosely bound to cell wall fraction) and the particulate fraction exhibited low specific activity towards both high molecular weight substrates. The intracellular soluble fraction (the soluble fraction after the differential centrifugation of ruptured cells) exhibited the lowest specific activity when gelatin and shrimp protein were used as substrates (Table 5). These results show that the major portion (>95.0%) of the active enzyme towards these two high molecular weight substrates resides in the extracellular fraction. Most microorganisms can synthesize various enzymes within their cell structure. Each enzyme system may have its own unique characteristics, and these characteristics will vary depending on the enzyme, the substrate and conditions during the enzyme-substrate reaction. In addition, the location of the proteinase (s) within the bacterial cell may vary markedly between microorganisms. Various researchers (53,115,135, 139) have studied the location of particular bacterial proteinases within the cell and how this location relates to the function of the enzyme. Thomas et al. (139), using gentle procedures for cell fractionation, suggested two criteria for the location of a proteinase produced by Streptococcus lactis . The two criteria they suggested were: 1) intact cells (whole cells) possessed substantial proteinase activity when incubated with a high molecular weight substrate; 2) most of the cell-bound proteinase activity was released during spheroplast formation. The solubilized cell wall, plasma membrane and cytoplasm fractions contained 84%, 0% and 16% activity, respectively, of the total proteinase activity with casein as substrate (139). In the results presented in this dissertation, the whole cell and cellular particulate fractions of

PAGE 69

55 the _P. citreus cells showed little enzjrme activity towards gelatin and shrimp protein (both high molecular weight substrates) . Thomas et al. (139) also concluded that the cell wall proteinase may serve a similar nutritional role in nature as the surface-bound proteinases discussed by Payne and Gilvarg (115) and Sussraan and Gilvarg (135). Gilvarg and his co-workers stated that surface-bound proteinase (s) appear to serve a nutritional role by hydrolyzing proteins to amino acids or peptides that are small enough to enter the cell. In turn, Payne, Sussman and Gilvarg (115,135) also suggested that the intracellular peptidases could further hydrolyze the peptides formed and release their constituent amino acids, thus, permitting the utilization of the protein substrate for growth. In Table 5, we can observe that certain citreus fractions (whole cells, intracellular and cellular particulate) had substantial amounts of protein present. Perhaps some of the protein present in these fractions include other enzymes (i.e., peptidases) that can utilize the peptides produced by the action of the extracellular protease (s) that later may enter the ?. citreus cell. In this manner, citreus cells could fully utilize the protein available (i.e., shrimp protein as well as other proteins) for their growth. Growth Medium and Enzyme Production Trypticase Soy Broth (TSB) , Plate Count Broth (PCB) + 0.5% NaCl and Nutrient Broth (NB) +0.5% NaCl were used to determine growth rates and production of extracellular proteolytic enzyme(s) hy ?. citreus . Figure 11 shows the growth of _P. citreus , as measured by the increase in optical density (600 nm) , in the three media used. In all three media, _P. citreus exhibited a 12 hr lag phase in which an increase in optical density was not evident. After this lag period, TSB supported the most

PAGE 70

56 Plate Count Broth Nutrient Broth Trypticase Soy Broth Figure 11, 24 48 72 96 INCUBATION TIME AT 20°C(hr) Spectrophotometric growth curves of Planococcus citreus in Plate Count Broth, Nutrient Broth and Trypticase Soy Broth 3.ti 20 C •

PAGE 71

57 rapid growth of _P. citreus . The optical density after 96 hours of incubation at 20 C was .31A, .200 and .072 for TSB, PCB and NB, respectively. Figure 12 shows a similar trend; however, _P. citreus growth was measured by the Aerobic Plate Count technique (6). Again, we can observe that the 2,' citreus log count per ml increases slightly during the first 12 hours of incubation at 20 C. After 96 hrs of incubation, the P. citreus log count for TSB, PCB and NB was 6.02, 5.10 and 4.17, respectively. Consequently, TSB allowed for the "optimum" growth of P. citreus when grown at 20 C, Nutritional components present in the growth medium are of utmost importance for gram-positive microorganisms which are generally more fastidious in its nutrient requirements than gram-negative bacteria (29). Realizing these growth requirements of gram-positive microorganisms, the results from this section are not surprising. TSB contains tryptone, soytone, dextrose, sodium chloride and dipotassium phosphate (16). This combination of nutrients provide an adequate nitrogen, carbohydrate, vitamin and overall nutrient supply for the growth of citreus . In contrast, PCB and NB are not as nutritionally complex. Figure 13 shows the enz3nne activity of the cell-free broth of P. citreus cells grown in TSB, NB and PCB for 96 hrs at 20 C. An active extracellular enzyme fraction was produced by P_. citreus in all three media. However, after 96 hrs of growth, the amount of enzyme produced by this organism in TSB is greater than that produced when grown in PCB of NB. The enzyme activity after 96 hrs of incubation of the cell-free broth of _P. citreus grown in TSB, PCB and NB was 323, 270 and 200 units of activity, respectively. However, if the cells are harvested at approximately midlog phase, the difference in the amount of enzyme

PAGE 72

58 CO Z) LU cr o in ZD O o o o o < _l _ o o 6Plate Count Broth Nutrient Broth Trypticase Soy Broth 12 24 48 72 96 INCUBATION TIME AT 20^ C (hr) Figure 12. Aerobic plate counts of Planococcus citreus incubated in Plate Count Broth, Nutrient Broth and Trypticase Soy Broth at 20 C for 96 hrs .

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59 35-1 INCUBATION TIME AT 20°C (hr) igure 13. Enzyme activity at 35 C for 15 min (pH 8) of the cell-free broth of Planococcus citreus cells grown in Plate Count Broth, Nutrient Broth and Trypticase Soy Broth for up to 96 hrs.

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60 I produced (as measured by enzjmie activity) is more clearly observed. After 48 hrs of incubation, the enzyme activity for the _P. citreus grown in TSB, PCB and NB was 200, 100 and 85.5 units of activity, respectively. Under the conditions tested, citreus exhibited maximum enzyme production when grown in TSB, The units of activity per cell per hr (m) was calculated for each medium used and the results are shown in Table 6. The average m value for TSB, PCB and NB was 168.50, 105.67 and 59.32, respectively. These data show that the amount of enz3niie produced by actively growing P. citreus cells (midlog phase) in TSB is higher than that produced when _P. citreus is grown in PCB or NB. The analysis of the data supports this observation. A significant difference (a = 0.05 level) was observed between the m values of TSB, PCB and NB (Table 6). Thus, after evaluating the results from this section, TSB was chosen as the best medium for _P. citreus growth and enz3nne production and was used for the remainder of the study. The combination of nutrients in TSB allowed for the rapid growth of _P. citreus and by doing so, permitted the production of more extracellular proteolytic enzyme. In addition to the combination of nutrients in TSB, the presence of 0.25% dextrose may play a role in extracellular enzyme production. Dextrose has been suggested as a possible inducer of a variety of enzymes (57), although this effect was not specifically tested in these experiments. Effect of Incubation Temperature on Enz3nne Production and Activity The ability of _P. citreus to produce an active extracellular enzyme at 5, 20 and 35 C was investigated in order to determine the ability of the extracellular enzyme (s) to affect shrimp protein at refrigeration (5 C) or iced temperatures. Figure 14 shows the increase in optical

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61 Table 6. Units of enzyme activity per cell per hour (m) of Planococcus citreus grown in Trypticase Soy Broth (TSB) , Plate Count Broth (PCB) and Nutrient Broth (NB) at midlog phase. 2 Medium mean m value TSB 148.50^ PCB 105.67^ NB 59.32'^ Cells were grown at 20 C and enzyme activity was measured at 35 C ,for 15 min (pH 8). average of 6 observations Means followed by the same letter do not differ significantly at the a = 0.05 (r from Anova table 0.984)

PAGE 76

62 2 24 x 48 72 96 120 INCUBATION TIME(hr) 144 Figure 14. Spectrophotometric growth curves of Planococcus citreus in Trypticase Soy Broth incubated at 5, 20 and 35 C.

PAGE 77

63 density of citreus cultures grown at 5 C for 144 hrs, 20 C for 120 hrs and 35 C for 96 hrs. After different time intervals for growth adaptation, _P. citreus grew at all three temperatures. Direct microscopic observation of citreus cells growing at the three temperatures revealed a difference in cell arrangements. When cells were grown at 35 C, the predominant morphology present was clusters of gram-positive cocci. At this temperature, the cells are rapidly growing and dividing, thus, possibly accounting for the observed predominance of clusters. At 20 C a mixture of tetrads, pairs and single cells were observed which is the predominant morphology exhibited by this organism, as described in Sergey's M anual of Determinative Bacteriology (29). At 5 C the predominant cell arrangement observed was single cells. This particular morphological structure might predominate because of the slow metabolic rate at this temperature, although these rates were not investigated. Individual cells have more surface area for the uptake of nutrients. At 5 C, the movement of nutrients within the cell is slow. Consequently, the increase in surface area is particularly desirable from a nutritional standpoint. Table 7 illustrates the enzyme activity of the cell-free broth of P. citreus grown at 5 C for 108 hrs, 20 C for 72 hrs and 35 C for 36 hrs (midlog at each temperature) and incubated with shrimp protein at 5, 20 and 35 C for 60, 30 and 15 min, respectively. P. citreus produced an active extracellular enzymeCs) when grown at all temperatures. In addition, the cell-free broth obtained from the three temperatures of growth exhibited activity at all three enzyme-substrate incubation temperatures (5, 20 and 35 C) . As the temperature of growth increased from 5 to 35 C, the enzyme activity increased at a similar rate at the three enzyme-

PAGE 78

• > •H O iH U 60 > to •H CO 4J 3 O I 0) •O CO T3 C CO +1 CO c o CO <

PAGE 79

65 substrate incubation temperatures. A higher _P. citreus count was observed at 35 C and the production of extracellular enzjnne(s) was also higher at all three enzjmie-substrate incubation temperatures. This indicates that the amount of enzjnne produced by P. citreus is related to the amount of growth of the organism in the medium. In addition, as the enzyme-substrate incubation temperature increased from 5 to 35 C, the enzyme activity of the cell-free broths increased. Although enzyme activity is present at the lower temperatures, the data presented indicate that the optimum temperature of the extracellular protease system may be close to 35 C. Consequently, the results indicate that _P« citreus can indeed produce an active extracellular enzyme (s) capable of utilizing the protein in shrimp when shrimp is stored at refrigerated or iced temperatures. The effect of refrigeration temperatures on enzyme activity has been studied (50,51). In most of the research, the majority of the enzymes studied lost activity when incubated at low temperatures. Studies have shown that lactic streptococci characteristically produced less acid after storage at refrigerated temperatures. Such stored cells also show a diminished residual proteinase activity (49,50,51,52,149). The researchers stated that after storage at 3 C, the enzyme showed gross structural alterations with a concomitant loss of activity. Gel filtration and sedimentation velocity data indicate that inactivation of the enzyme was a result of aggregation to higher molecular weight forms (50). However, several investigators (49,52,131) previously suggested that storage inactivation of enzymes may be caused by induced conformational or structural changes. Scutton and Utter (128) and Havir et al. (81) observed that inactivation of various enzymes by low temperature storage

PAGE 80

66 was due to dissociation of the molecules into subunits. The inactivated enzymes could be reactivated by warming to room temperature. Purification of the Extracellular Enzyme (s) Planococcus citreus was grown in Trypticase Soy Broth (TSB) at 20 C for 72 hrs. The cell-free broth was used in the isolation of the extracellular enzyme (s) of this organism. The cell-free broth had a total activity of 1.31 x 10^ units of activity (total enzyme activity = change in 0.1 fluorescence units of the TCA filtrate per milliliter of enzyme per minute), 44.52 mg/ml of protein and a specific activity of 29.45 units of activity/mg protein (Table 8). The cell-free broth was then fractionated with 0-55%, 55-70% and 70-100% ammonium sulfate ((NH^)2S0^). After overnight dialysis (16 hrs) in phosphate buffer pH 8, the activity of the 0-55%, 55-70% and 70-100% ammonium sulfate precipitates was measured. Table 9 shows the proteolytic activity of the various fractions examined. The specific activity of each fraction was 4.59, 52.39 and 3.99 units of activity/mg of protein for the 0-55%, 55-70% and 70-100% fractions, respectively. Eighty-six percent of the activity was present in the 55-70% fraction. This is compared to 7.5 and 6.5% for the 0-55% and 70-100% fractions, respectively (Table 9). Ammonium sulfate precipitation is a common method used to precipitate proteins for their purification. As the ammonium sulfate concentration is raised from zero, the solubility of a given protein at first usually increases but then the "salting-in" effect comes to an end and as the salt concentration is raised to higher values a "salting-out" effect is observed and the protein becomes progressively less soluble (65). The major portion of the extracellular proteolytic enzyme (s) of P. citreus was salted

PAGE 81

67 u (U > ^ o c o 3 — s c •H o (U M O •H l-i > a •H 4-1 iH O to < 4-1 o o 4-1 •H U-l 60 •H e O OJ CO O. 4-1 to •H c 3 ^ — •H CJ <; CO c 3 I 3 O > c o o CO U In O O <3^ -ao o 00 CM m o CM O O m to o ON VO o CM VO sr cn • o 00 CM sr CM CO VO VO o CJv c OS O •H X u o o CM J-4-« >^ 3 a. IB -H l-l C/l (U VM z a CJ CO ^ T-i to o iH CJ J= o u o •H Q, CM <«: (U O Vi rH (U 4-1 CO 00 to 4-> u O (JO -H H c fH •H a >^ CO 3 4-> 3 •o vi "O •H 0) o 4-> c O •H cu CO s 00 to -H (U u to 4-1 4-1
PAGE 82

68 CO 4-1 •H 14-1 O o CO 3 00 O o o a y s ' o c c to •H .H B m U-l 1—1 o M O 4^ y-i o u o m (U m 0) V4 4-1 14-1 cu 1 iH >, ^ 4-1 0) •H a > > c tH 0 o •H l-i -H 4-> 4-1 CO U •H <: ^\ c •H u 0) 4>l O •H > a •H 4-1 O to < 4-1 o O 4-> •H U-( 00 •H e a — 0 to 4-m •H O > •H 4-1 X a <; CO 4J to c 4-< 3 o ^ H c o o to u o • • • CO ON ON ON • • • m o o O u-l • • • (X) ON CM i-H 1-H o o o • • • 00 CN O u-l O &^ O O u-l iT, 1 1 1 o o u-l >*-( 4-1 •H to O u 0) 4-1 CU CO CO J3 3 II CO CO n CO N CO 3 4-> 3 T3 •H T3
PAGE 83

69 out between 55-70% annnonium sulfate saturation. Table 9 shows that 86% of the activity towards gelatin is observed in this fraction. Table 8 shows that the activity of the 55-70% ammonium sulfate fraction was 1.78 times greater in specific activity than the cell-free broth. A 78% recovery of the extracellular enzyme (s) was achieved in this step of the enzyme purification. Sephacryl S-200 Superfine, a high resolution chromatographic medium for gel filtration of proteins, nucleic acids, polysaccharides and biopol)nners (120), was used to separate the enzyTne(s) present in the 55-70% ammonium sulfate fraction according to molecular weight. Figure 15 shows that four protein peaks were recovered after the elution of the enzyme fraction through the Sephacryl S-200 column. However, when the proteolytic activity was measured, the majority of the activity was present in protein peak C (third peak in Figure 15). Peak C had a specific activity of 651.0 units. The enzyme(s) was purified 15.67 times and 50% of the enzyme was recovered in this step (Table 8). The fractions comprising peak C were pooled for further purification. The percent recovery of the extracellular proteolytic enzyme of _P. citreus after molecular sieve chromatography using Sephacryl S-200 Superfine was within the range of most of the enzymes recovered when the more traditional Sephadex gels have been used (70,113,114,136). The pooled fractions of peak C were further rechromatographed using DEAE-Sephadex A-50 (functional group -C2H^N+(C2H^) A-50 gels are usually used for low and medium molecular weight proteins (up to 200,000). Ion exchange chromatography may be defined as the reversible exchange of ions in solution with ions electrostatically bound to an insoluble support medium. The ion exchanger is the inert support medium

PAGE 84

70 {-m,om A1IA113V 3IA1AZN3 o o o ro I O CM _J_ OS O 1^ >. u a to XI c 0) C/3 OO c •H , « cn C = • — o 08^) 30Nvadosav cd . u o CO O a. CM 0) I
PAGE 85

71 to which is covalently bound positive (in the case of the anionic exchanger) or negative (in the case of a cation exchanger) functional groups (48). A sodium chloride (NaCl) gradient (range of ionic strength, y = 0.11 0.23) was used with the ion exchange column to elute the protein components. A gradient is a physical method of constantly changing the salt concentration of a solution that is being passed through the column creating a constant and linear increase in concentration (48). Figure 16 shows one major peak after ion exchange of the pooled active fractions from peak C. The isolated peak exhibited a specific activity of 780.37 units (Table 8). The proteolytic enzyme was purified 26.50 times and 49% recovery was achieved (Table 8). Fractions 17 to 21 (Figure 16) were pooled for future characterization. Purity of the Extracellular Proteolytic Enzyme Many methods can be used to establish the purity of an enzyme preparation. However, the best indication of purity of an enzyme preparation is by the consistent failure to detect heterogeneity when several analytical techniques are used (i.e., a single peak in chromatographic systems, a single band on electrophoresis, a single band after isoelectric focusing and/or one component in solubility or precipitation tests). However, the final criterion for purity is the demonstration of a unique amino acid sequence (61,65) but this is rarely done in order to demonstrate purity. The recovery of the isolated peak (Figure 16) as a single entity with homogeneous activity after DEAE-Sephadex^ A-50 ion-exchange chromatography was the first indication that the major extracellular proteolytic activity of _P. citreus was isolated in a purified form.

PAGE 86

72 N0llVdlN30N00 IQDN O lO o CVJ — — • • -J I in CM f»-,0ix) AllAllOV 3mZh\3 082) 30Nvadosav \J Q) i-t ' CD vu Q) ft rH rn UJ w u -p I X. s: 1 1 4-1 •H e in CS! 14-1 O 0) OJ frt 14-4 4J (4-1 CO 3 >^ 111 XI rH CO Q 0) w 4-1 CO CO d) CO CJ 4-» o CO z 1 1 1 in CM o • • o o cn XI •H (11 4J 5-1 •H 4J 1 rH d) d) CD 4J CO * rn 3 Z w rH <<< cu i-H c o e CM 3 d ~ U 1-H CO +J o rrl o o cS in u c CM (U CI] O •H X rtl CO U »-> o U 4-J flJ
PAGE 87

73 According to Cooper (48) electrophoretic techniques have become principal tools for characterizing macromolecules and for assaying their purity. Figure 17 shows a single band after SDS-PAG electrophoresis using 50 yl of the purified enzyme. A single homogeneous band is indicative of the presence of only one enzyme, i.e., the purity of the extracellular enzyme of _P. citreus . In addition, as an additional test for purity, increasing amounts of the purified extracellular enzyme were added to the gels. Enzjrme concentrations of 50 pg protein/gel, 100 pg/ gel, 150 pg/gel and 200 yg/gel were used. Figure 18 shows that a single band is recovered after SDS-PAG electrophoresis of each protein fraction. Thus, these results add to the evidence indicating the purity of the extracellular proteolytic enzyme of £. citreus . Consequently, an extracellular proteolytic enzyme produced by P^. citreus was purified 26.50 times using the procedures outlined previously with 49.0% of the enzyme being recovered (Table 8). The specific activity of the enzyme was 780.37 units of activity/mg protein, Schwabe (127) reported the use of the f luorescamine reagent to measure proteolytic enzyme activity of cathepsin enzymes using hemoglobin a substrate. He stated that while the f luorescamine reagent has been used successfully for quantitative amino acid analysis, protein and peptide determination, it has also beneficial applications in enzymology. In addition, Schwabe (127) compared the fluorometric technique with the Lowry method (98). He concluded that the fluorometric method was 100 times more sensitive than the Lowry method, much faster and less complicated. The fluorometric technique proved to be an efficient method for the measurement of proteolytic enzjmie activity.

PAGE 88

74 Figure 17. Acrylamide gel electrophoresis of the purified enzyme of Planococcus citreus.

PAGE 89

75 50 yg/gel' 100 yg/gel 150 yg/gel 200 ug/gel Figure 18. Acrylamide gel electrophoresis of increasing concentrations of the purified extracellular enzjrme of Planococcus citreus

PAGE 90

76 With the presence of the relatively new Fluorometric technique, that appears to be more sensitive and reproducible than the traditional methods available for measuring proteolytic enzjone activity, the results of various previous research with extracellular enz3nnes (36,78,84,88, 136) using the Anson method (9) could have possibly resulted in higher recoveries and higher measurable total enzyme activity. The following investigators are some of those who used the Anson method to study the various enzymes. Tarrant et al. (136) working with Pseudomonas fragi in pig muscle isolated an extracellular proteolytic enzyme with only 18% recovery after partial purification. Husein and McDonald (84) characterized an extracellular proteinase from Micrococcus f reudenreichii using casein as substrate with 23% recovery after partial purification. Christison and Martin (36) isolated and preliminarily characterized an extracellular protease of Cytophaga spp. using casein, hemoglobin and azocoll as substrates. After chromatography with DEAE-Cellulose^ only 26% of the enzyme was recovered. Khan et al. (88) , looking at the extracellular proteases of Mucor pusillus , isolated and characterized two fractions. However, after DEAE-Sephadex^ A-50 only 29.3% of the milk-clotting fraction was recovered and 47.0% of the fraction with protease activity toward hemoglobin was recovered. Gnosspelius (76) purified an extracellular protease from Myxococcus virescens using phosphate precipitation, gel exclusion and ion exchange chromatography. Only 20.1% was recovered after the chromatographic step. In the work reported in this dissertation, following DEAE-Sephadex^ A-50, 49.0% of the extracellular enzyme of P. citreus was recovered when the Fluorometric method was used to measure proteolytic activity.

PAGE 91

77 Characterization of the Extracellular Proteolytic Enzyme The fractions collected (17-21) from peak B (Figure 16) were pooled and used for the characterization of the extracellular enzyme of P. citreus . Molecular Weight Determination Two methods were used to determine the molecular weight of the enzyme, column chromatography (Sephacryl^ S-200 Superfine) and acrylamide gel electrophoresis. Standards ranging from a molecular weight of 10,000 to 200,000 were used to determine the molecular weight of the P. citreus enzyme. Using both techniques, the molecular weight of the extracellular enzyme of this organism was approximately 29,000. Figures 19 and 20 show the molecular weight determination using Sephacryl^ S-200 and acrylamide gel electrophoresis, respectively. Different standards were used in each case to assure that the molecular weight was estimated correctly. A search of the literature was done in order to compare the molecular weight of the extracellular proteolytic enzyme of citreus with extracellular proteases from other microorganisms. Pacaud and Uriel (113) estimated the molecular weight of a protease from Escherichia coli using electrophoresis on polyacrylamide gels and sucrose-density gradient centrifugation to be about 43,000. Four years later, Pacaud and Richaud (114) estimated the molecular weight of a second protease of E, coli using gel filtration and SDA-acrylamide gels to be 58,000. Drapeau et al. (59) estimated the molecular weight of an extracellular protease of Staphylococcus aureus to be approximately 12,000 using sedimentation equilibrium and gel electrophoresis studies. Arvidson et al. (12) reported the molecular weight of an extracellular (alkaline protease) enzyme from S. aureus to be approximately 12,500. Later, he reported

PAGE 92

78 v^^Ribonuclease A Trypsin P citreus (29,000) Pepsin Bovine Serum Albumin Aldolase + + 4+ + H \ h18 20 22 4 6 8 10 12 14 16 MOLECULAR WEIGHT (x 10"^) ure 19. Calibration curve for the molecular weight estimation of Planococcus citreus proteolytic enzyme using Sephacryl S-200 column chromatography.^ 40 X 2.5 column, eluted with 0.02 M phosphate buffer at a rate of 15 ml/hr.

PAGE 93

79 -in •H u rH O 0) 4-1 o a to 3 CO 3 O a o o o c + O o Q o o o Q. o o o o o fO o o o o CJ CO J3 u o 60 o o o o c tz o m c o 1—1 1 •H 1—1 n) 1 e 1-1 • — • •H cu U 60 COCO OJ • CX) o to 4J JS 60 esi CD O •H u in >. cu 60 U CJ de CO u •H 5^5 fo lam O t— 1 to > a 3 CO a 60 c c o •H •H CO U 3 to M (U J3 B •H >, tH t\i to C u tu 1H9GM dVnn03"I01AI o CM tu u 3 60

PAGE 94

80 (11) the molecular weight of a EDTA-sensitive S. aureus protease as 28,000. Recently, Hoshida et al. (137) estimated the molecular weight of a proteolytic enzyme from Bacillus sphaericus to be about 26,000. Gnosspelius (76) working with an extracellular enz3mie of M3^ococcus virescens reported its molecular weight as 26,000. Thus, the apparent molecular weight of the extracellular enzyme of _P. citreus (MW 29,000) is within the range of other extracellular proteolytic enzjmes reported in the literature. Effect of Ionic Strength on Enzyme Activity The effect of salts on the solubility of proteins is well known. The solubility is usually a function of the ionic strength. In conditions of high ionic strength, the ions attract around themselves the polarizable water molecules, making less water available for the proteins since, at high salt concentrations, the number of charged groups contributed by the salts is enormous compared with those of the proteins. Consequently, the solubility of the proteins decreases (152). In addition, any change in the charges of an enzyme may cause various transformations in structure or active site configuration that could affect its activity towards the substrate. Figure 21 shows that ionic strengths (P) of 0.15-0.83 did not alter the attraction of the JP. citreus extracellular enzyme towards gelatin substrate. However, as the ionic strength was increased the activity of the enzyme decreased. An ionic strength of 1.60 (1.5 M NaCl) caused a decrease in enzyme activity of approximately 60%. Thus, if ionic strengths above 0.83 (0.75 M NaCl) are used they may cause a change in solubility of the enzyme, charged groups, conformation of the enzyme, active site stability and/or active site availability to the substrate. Gnosspelius (76) stated that variations in the

PAGE 95

81

PAGE 96

82 ionic strength did not signficantly influence the activity of Myxococcus virescens when casein was used as the substrate. However, the actual data for this observation were not presented in the literature. In that the activity of the proteolytic enz}Tne was not affected by ionic strengths of p=0.83 or lower, the buffers shown in page 44, (Determination of Optimum pH) were considered acceptable and were used for the determination of the optimum pH of the _P. citreus enzyme. Optimum pH Determination The pH optimum of an enzyme is dependent upon a number of experimental parameters. Changes in pH may cause changes in the ionization of prototropic groups (groups capable of ionization) in the active site of an enzyme. These prototropic groups in the active site may be involved in maintaining the proper configuration of the site, in binding a substrate to enzyme and/or in transformation of substrate to products (133). However, there is usually a zone of maximum ion stability in which enzyme activity is maximal. Enzyme inactivation also increases on the acid and alkaline sides of this maximum activity zone. Observing Figure 22, enzyme activity was maximum at pH 8 when both gelatin and shrimp protein were used as substrates. The activity dropped as the pH became more acidic or alkaline. Although not statistically significant, a slightly higher activity was evident at the alkaline pH's (9 and 10) when shrimp protein was used as substrate. The majority of the bacterial enzymes studied have shown maximum proteolytic activity at neutral pH's (57,65,151). The enzyme isolated in this study resembles the bacterial proteolytic enzyme from Proteus vulgaris (105), Bacillus sphaericus (155), Staphylococcus aureus (11,12), Serratia marcescens (106) and Pseudomonas spp. (87) in that they all

PAGE 97

83 PH gure 22. Optimum pH of the extracellular proteolytic enzyme of Planococcus citreus a Gelatin and shrimp protein substrate incubated at 35 C for 10 min, pK 8.

PAGE 98

84 require a slightly alkaline pH for optimum activity. Considerable activity is present at neutral pH's; the pH of freshly caught shrimp is around neutrality. During shrimp storage, the pH of shrimp will increase (5) . Optimum Temperature Determination Changes in temperature may affect enzjnmatic reactions in a number of ways. Some of these effects may include: a) stability of the enzyme; b) affinity of enzymes for activators and inhibitors; c) ionization of prototropic groups; d) enzyme-substrate affinity; and e) velocity of breakdown of enzyme-substrate complex (131). The optimum temperature of the _P, citreus extracellular enz)rme when both shrimp protein and gelatin were used as substrates was 35 C (Figure 23). Although not statistically signficant, a slightly higher enz3nne activity was observed at the lower temperatures (5 and 10 C) using shrimp protein as substrate as compared to gelatin. However, at the higher temperatures (45 and 55 C) the reverse was evident. Thus, as previously observed with the cell-free broth (enzyme crude extract), the purified enzyme of _P. citreus can exhibit activity at the temperatures of refrigerated shrimp. Thermal Stability The cell-free broths obtained from 2« citreus cells grown in Trypticase Soy Broth (TSB) at 5 and 35 C for 108 and 36 hrs, respectively, and the _P. citreus purified enzyme were incubated at 35, 45, 55, 65, 75 and 85 C for 15 min in order to study the various temperatures effects on stability. The enzyme activity of the cell-free broths and purified enzyme remaining after the various heat treatments was assayed using gelatin as the substrate and compared to the activity observed with the unheated cell-free broths and purified enzyme. Figure 24 shows the

PAGE 99

85

PAGE 100

86 SI 0> o O CD oi a> t 2. UN U c _ — Ld 0) O o lO 3 lO ro Q_ o o o o SI o o 00 CVJ CVJ _ T i T tn r» n 1 com '•• T i <> . 1 .1 ....... o o I I I I 1 1 1 1 r (7o)AllA110V 3mZN3 9NlNIVl^J3d in 00 in 1^ in O Ld in in < cr LU Q_ LU H in in ro 4J o u 05 s: o u , •H N c cn 0) ^"S N -H (U -H ^1 x: a (U U O •H H T) •H C XI to CO •U U CO CO e -o ^1 c i > to CO U cn O CO CO cO C 4J r 1 cQ <^ r-H 1-t rH tu H /-* \J Tl w QJ rri I ^ c3 CJ AJ *H QJ rl QJ r; XJ ni Ta p3 0) •H 4-1 •H U ^ — s 3 CO Ci. -^3 1 1, 1 C CO c (11 •H CO e c D. ^ CJ c c o •H CO e x: u •u o LO o u CJ 14-1 CO }-l T3 4-1 , .H CO iH cn QJ tn to CO

PAGE 101

I 87 thermal stablity of the cell-free broth of P_. cltreus cells grown at 5 and 35 C and of the purified enzjnne. In general, as the temperature increased, the activity of all three fractions decreased. After 15 min of incubation at 65 C, all the activity was lost in the purified enzyme fraction and only 1% was left in the 35 C cell-free broth fraction. However, 28% of the activity still remained in the 5 C cell-free broth fraction. After 15 min at 75 C, 1% of the activity of the 5 C cell-free broth fraction still remained. Perhaps the enzymes in the cell-free broth of P^. citreus grown and stored at 5 C have undergone a structural change (50,51) or have a slightly different structure than the enzyme produced at 35 C. This change could result in an enzyme conformation with an active site that is more protected from increased temperatures. Usually, an enzyme is more stable to temperature changes in an intact tissue or in an homogenate where its structure is protected by the presence of other colloidal material (i.e. proteins, carbohydrates, etc.) than it is in a purified form (147,151). However, in general, those enzymes which have molecular weights ranging from 12,000 to 50,000 are composed of single polypeptide chains and having disulfide bonds are usually more resistant to heat treatment. The larger the enzyme and the more complex its structure the more susceptible it is to increases in temperature (151). Figure 24 shows that the cell-free broths are more stable to heat than the purified enzyme. Effect of Sodium Chloride Concentration ^ The effect of increasing sodium chloride (NaCl) concentration on enzyme activity was examined. Concentrations of 0-1.50% NaCl were investigated. When shrimp protein was used as the substrate, the activity of the extracellular enzyme increased until 0.50 g/100 ml NaCl (0.5%)

PAGE 102

88 was reached, then the activity started decreasing. However, when gelatin was used as the substrate, salt concentration (0-1.50%) had no apparent effect on the activity of the enzjnne (Figure 25) . The concentrations of the NaCl solutions used in this study, 0-0.26 M NaCl, have an ionic strength of ij=0.15-0.34 and are not within the ionic strength range that resulted in decreased enzyme activity (Figure 21), The effect observed when shrimp protein is used as substrate is probably due to an initial increase in solubility of the substrate due to the increase in salt concentration. Structurally, gelatin is a small protein when compared to shrimp protein. Perhaps the increase in solubility allowed an easier enzyme-substrate interaction, thus, accounting for the initial increase in enzyme activity. The effect of higher concentrations of NaCl on enzyme activity was not investigated. However, Figure 21 illustrates that an ionic strength of 0.83 (0.75 M NaCl) or above resulted in decreased enzyme activity. Reversible inactivation of the enzyme and substrate effects due to higher NaCl concentrations were not investigated. Arvidson and coworkers (11,12) showed that the activity of both extracellular proteases I and II from Staphylococcus aureus (neutral and alkaline protease, respectively) was reduced by concentrations of 0.5 M NaCl or above. Gnosspelius (76) stated that an NaCl concentration of 0.2 M in the assay mixture had no effect on the activity of the Myxococcus virescens extracellular enzyme. However, higher NaCl concentrations decreased the proteolytic activity. During the storage of Penaeus shrimp on ice, the salt concentration will decrease due to the leaching of the salt as the ice melts or percolates through the shrimp. As the NaCl concentration decreases in shrimp, the activity of the _P. citreus enzyme will be enhanced.

PAGE 103

89 i / / r / / r C Q. O CO I f \ \ \ \ \ -r o o T" O (,0I X) AllAllOV 31AIAZN3 -O o i-lO in CO > •H U U to e N d a> C o o § < cr LU O O in • CJ o in CVJ u u C (U o c o o •a •H U o o e o a (U ^ 9 •H CO o U-l u in CO 03 cn CO •a -H o > to -H

PAGE 104

90 Effects of Sodium Bisulfite Concentration To control black spotting in shrimp (148), sodium bisulfite (NaHSO^) is used to inhibit enz)miatic oxidation of both tyrosine and dihydroxyphenylalanine thereby preventing darkening of the shell (66,96). Since 1956, agencies such as the former Florida State Department of Conservation (30), now the Department of Natural Resources, have recommended dipping shrimp in a 1.25% sodium bisulfite solution for 1 min to control black spot development. Therefore, the effect of sodium bisulfite concentration (0 to 3%) on the activity of the extracellular proteolytic enzyme of citreus was investigated. Figure 26 shows that as the concentration of sodium bisulfite increases, the activity of the enzyme decreases. When sodium bisulfite dissociates in water, it may affect enzyme activity by reducing disulfite (-S=S-) linkages (57). The activity of the enzyme in the presence of 1.25% sodium bisulfite was approximately 240 units of activity. Thus, with the addition of 1.25% sodium bisulfite, approximately 47% of the activity of the proteolytic enzyme was lost. However, as the concentration of sodium bisulfite decreases (leaches out in the melt water) (148) the activity of the enzyme should be less affected. Effect of Enzyme Concentration Rates of enzyme-catalyzed reactions are directly dependent on enzyme concentration (151). The effect of enzyme concentration (0 to 200 yl) on the activity of the P. citreus enzyme when gelatin and shrimp protein were used as substrates was investigated. By observing Figure 27, the enzyme preparations used for characterization followed a linear relationship with increasing levels of enzyme. According to Dixon and Webb (57) when the plot passes through the origin, inhibitors are usually

PAGE 105

91

PAGE 106

92 ENZYME CONCENTRATION (^1) ect of enzyme concentration on enzyme activity.^ Increasing enzyme concentrations were incubated with the substrates at 35 C for 10 min (pH 8).

PAGE 107

93 absent from the preparation. Thus, looking at Figure 27, we can observe that inhibitors were not present in the preparation. Effect of Substrate Concentration Substrate concentration is one of the most important factors which determine the velocity of enz3nne reactions. Figures 28 and 30 illustrate the effect of substrate concentration on the velocity of the reaction when gelatin (0 to 1.2 mg/ml) and shrimp protein (0 to 0.6 mg/ml) substrates, respectively, were used. Both enzyme-substrate reactions followed Michaelis-Menten kinetics. That is, the enzjrme E first combines with the substrate S to form the enzyme substrate complex ES; the latter then breaks down in a second step to form the free enzyme E and the product P: E + S ^ E + P. Figures 28 and 30 follow the traditional Michaelis-Menten shape curve (95). Enzyme kinetic calculations (95,152) were done in order to add to the information about the P. citreus extracellular enzyme and to determine substrate saturation conditions. The values (Michael is-Ment en constant) calculated in this study are apparent K^'s. Whole protein substrates (either gelatin or shrimp protein) rather than specific synthetic amide substrates were used. True values are calculated using specific substrates and having a definite knowledge of the enzyme's active site (57). Additional research is required to demonstrate the active site of the _P. citreus extracellular enzyme. Apparent and V^^^ values were calculated by transforming the data in Figures 28 and 30. Double-reciprocal plots (Lineweaver-Burk plots) were done and they are shown in Figures 29 and 31 for gelatin and shrimp protein substrates, respectively. The apparent K values m for the gelatin and shrimp protein substrates were 0.98 mg/ml and 0.33

PAGE 108

94 50n O 40> 30> 1O < 20UJ > ZN 1 0LU 15 .3 .45 .6 12 GELATIN CONCENTRATION (mg/ml) Figure 28. Effect of gelatin concentration on the reaction rate of the Planococcus citreus extracellular enzyme.^ Increasing gelatin concentrations were incubated with the enzyme at 35 C for 10 min (pH 3) .

PAGE 109

95 1 6 > fcO (3 •H 4-1 nj V-l U CO 3 r— ( iH •H (U 6 >i N c -i 4-1 4-) 03 03 3 XI > ID 0) c •H c cd CM 0) 3 SO •H fn

PAGE 110

96

PAGE 111

97 \ • • ^ \ \\ \ %\ • 8.06.0o o_\ ^' ^"-1 \ £-01 XA/I I' \ ro \ ro \ II E _ OJ -00 t. c CO -CD C o CL Q. E o e CVJ _ CVJ I B > c •H 4J (0 u u CO 3 r— 1 •H E M c OJ re 3 0) iH re 0) > c •H 0) 3 C^ •H o CO OJ l-l 3 GO •H 0) 4J c cu CO
PAGE 112

98 mg/ml, respectively. The apparent V^^^ values for gelatin and shrimp protein were 666.67 and 431.03 units of activity, respectively. Since is defined as the rate of the disappearance of the enzyme-substrate (ES) complex to the appearance of the ES complex, it appears that gelatin has a higher affinity for the enzyme than shrimp protein. Consequently, V is lower with shrimp protein as substrate. This is max shown in Figures 29 and 31. _P. citreus extracellular proteolytic enzyme can utilize and degrade gelatin at a more rapid rate than shrimp protein. Various factors could be responsible for observing a higher V when max gelatin is used as substrate. Gelatin, with a molecular weight of 90,000 (152), is a simple protein when compared to shrimp protein. Four amino acids comprise 70% of the total amino acid composition. Glycine and alanine add to approximately 50% of the amino acids present in gelatin (152). Possibly, the relative simplicity of gelatin makes this protein more available to the action of the _P. citreus extracellular enzyme. In addition, gelatin showed higher solubility than shrimp protein in the buffer system used. Perhaps, this increased solubility allowed for an easier enzyme-substrate interaction. However, shrimp protein has a more complex primary structure, and as it was prepared in this study, it is probably a mixture of proteins. Eighteen amino acids comprise approximately 60% of the total amino acid composition (53). The composition of the shrimp protein preparation probably makes it a more complex substrate for the ?. citreus extracellular enzyme. Consequently, shrimp protein is not as easily available for the reaction with the enzyme. Effect of Metal Ions on Enzyme Activity The effect of various metal ions on the activity of the extracellular proteolytic enzyme of citreus was investigated. The effect of

PAGE 113

99 two concentrations of each metal ion is shown in Table 10. Calcium chloride (CaCl2) increased the activity of the proteolytic enzyme, while ferric chloride (FeCl^) , mercuric chloride (HgCl^) and potassium chloride . (KCl) all suppressed the activity of the enzyme to some extent. Zinc chloride (ZnCl2), chloride (MgCl2) manganese chloride (MnCl^) had little effect on enzyme activity. Divalent cations, except CaCl2, had no appreciable effect on the activity of the extracellular enzyme. The ionic radius of divalent cations is such that one of their primary functions is to coordinate the substrate to the active site (37). Mg and Ca, the alkaline earth cations, participate in the formation of the catalytically active conformation. Monovalent ions are usually involved in transport mechanisms in the cell (37), thus, usually not as an integral part of the active center. Possibly, K ions could be displacing a required ion resulting in the suppression of enzyme activity. In contrast. Figure 25 showed that NaCl concentrations had no effect on enzyme activity when gelatin was used as the substrate. This apparent effect of k"*" but not of Na"^, possibly related to the larger ionic radius of ions, should be the subject of additional investigations. Salts of heavy metals, such as silver, copper, mercury and lead, react rapidly and at low concentrations with sulfhydryl groups; however, they also react with other groups including the imidazole, carboxyl and peptide groups (151). At high concentrations, heavy metals can inhibit by neutralizing charges on the protein and/or by forming cross linkages between protein molecules (57). Iron salts have been found to activate some enzymes but suppress others (57). Some metal ions can activate enzymes by: 1) becoming an integral part of the active site; 2) linking

PAGE 114

100 CO *w d) >^ Ct3 CO CO • CO E >^ N C •H OJ > )^ to o f-i (0 3 rH c to <4-l o Bu p 0) o to 3 T) •H CO (U cc! •H > •H 4-1 U < 9) I N c c o to 4-1 c O c o u c o o U1 in O CO o 00 r— 1 CM 00 O '001 O f-H o CM ro CO m CM •OOT CM O .— ( CM C7> ON 00 00 ON C7\ CM vD sim 00 o rm f-H sr m o\ CJN o o • m 00 00 < r-~ 00 CM 00 CO CM 1—1 CM CM CM CM CM CM o CM CM VO +1 +1 n +1 +1 •t-l +1 +1 HI +1 +1 +1 i-i +1 +1 a\ 1— I 1— ( CO so i-H I-H so U-I o CM CO • CO v£> vO CO CJN o so 00 CM CO CO m O O m CO O 00 a\ m ^» rv m CJN tn o vD 00 CO o CO 00 - o < u to XI

PAGE 115

101 the enzyme with the substrate; 3) changing the equilibrium constant of enzyme reaction; 4) changing surface charge of enzyme; 5) removing inhibitors; and 6) inducing a more active enzjnne conformation (151). Kato et al. (87) reported that calcium chloride (CaCl2) and magnesium chloride (MgCl2) ^'^^^'^^^^'^ enzyme of a marine-psychrotrophic bacterium ( Pseudomonas spp.) and mercuric chloride (HgCl^) and ferric sulfate (FeSO^) suppressed the enzyme. Arvidson (11) reported that magnesium chloride (MgCl2) , ^^"'^ chloride (ZnCl2) calcium chloride (CaCl2) activated a protease from Staphylococcus aureus . However, Pacaud and Uriel (112) stated that calcium chloride (CaCl^) , manganese chloride (MnCl2) ferric chloride (FeCl2) ^^^i^^^^^ enzjnne from Escherichia coli but magnesium chloride (MgCl2) , zinc chloride (ZnCl2) and mercuric chloride (HgCl2) "° ^^^^^^ °" activity. The results of this study indicate the diversity of effects ions can have on the activity of the enzyme produced by _P. citreus . Effect of Various Reagents on Enzyme Activity Various reagents were tested to observe their effect on the activity of the extracellular proteolytic enz3mie of citreus . None of the reagents tested activated the enzyme (Table 11). Dioxane, one of the reagents used in the enzyme activity assay, had no apparent effect on the activity of the enzyme at either concentration examined (10 and 20 mM). The percent residual activity observed was 98.9 and 98.6%, respectively. The results observed with trichloroacetic acid (TCA, 5 and 10%) indicate that both concentrations can terminate the activity of the enzyme (2.0 and 0.0% residual activity, respectively). These results are comparable to those observed in Figure 6.

PAGE 116

102 c •H B O u o m CO CO •o ^ ^ N c >, (U 4-1 •H M > (0 •H iH J 3 O >H «0 iH (U 0) U ^2 N 4J C X (U (U (U CO u C •M O -H y 03 CO 3 OJ u 60 O to O Q) O U O a CO CO 3 -H O •H M (U to j: > to 3 -O •H CO (U Di 6^ •H > N a Cd o to u 4-1 e u c o c (U 60 to o o o IT) O o o u-i O o o fsl o o en o t— 1 O o o o 00 O O o CM o o o vO 'GOT 1— ( CO CM m en m vO (» O 00 «N o O vO O < m 00 C^N 00 0^ cn m O OS m O VO O m *— ( o CO 00 O o U1 • vO m 00 m iri 00 00 m O ov CM m CM CM o m CM CM ro CM CM CM m t-H O .—1 o \o \o CO CO +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 m CM O CM CM CO CJN 00 o iri 1^ O u-i CJ^ • u-i ro u-i 00 <3CM o 00 O 00 a\ I-H o I-H 00 o 0^ vO 0^ C7^ ro 0^ o 00 m 0^ 1— 1 sr lO CO , a: 0) a to e o o < H CO >^ CJ O s « o a u 0) g o H Q I a B O > 9) •T3 CO Ta c to 4-1 (U CO O. +1 6 to CO CO C 0 iH •H O 4J U CO 4-1 > C U O 01 CJ CO O ^ 4-1 VO O •T3 (U (U 60 U CO CO IH Cu (U S > o < u to ^

PAGE 117

103 The enzyme was inactivated by citric acid and EDTA, which act as metal ion chelators. Considerable activity was lost by the enzyme when cysteine and p-mercaptoethanol were added. These reagents reduce and interchange with sulfhydryl groups (SH groups) in proteins resulting in a possible reorganization of the enz3nne structure (98). In addition, cysteine can also bind and remove metals (57,155). Possibly, these two compounds might be binding a required trace metal resulting in the observed suppression of enzyme activity. The enzyme lost practically all the activity when 1 mM potassium permanganate (KMnO^) was added (2.10% residual activity) and all the activity when 20 mM KMnO, was 4 added. When 1 mM or 20 mM of formaldehyde was added, 63.6 and 53.2% residual activity, respectively, was observed. These results are comparable to those observed when p-mercaptoethanol was added. Formaldehyde can react with SH groups forming methylene bridges between amino acids and amide groups. However, it can also link a-amino acids (73), possibly making them unavailable for the reaction with the Fluoram^. reagent. Potassium cyanide (KCN) affected the activity of the enzyme to a small degree. Cyanide groups are known to combine with cofactors (metal ions) in substrates of enzymes when a C=0 group is involved (37). Thus, CN is usually a carbonyl group inhibitor. Numerous researchers (12,36,87,113,119,157) have reported on the effect of various reagents on extracellular proteases. Kato et al. (87) reported that KMnO^, EDTA and citrate inactivated a Pseudomonas spp. protease while KCN and p-Dioxane did not affect the activity of the enzyme. Yoshida et al. (157), Christison and Martin (36) and Arvidson (12) stated that EDTA and citrate inhibited the proteolytic enzymes from Bacillus sphaericus. Cytophaga spp. and Staphylococcus aureus . However,

PAGE 118

104 a protease from Escherichia coli (113) was not inhibited by EDTA, cysteine nor p-mercaptoethanol. The results of this study indicate that the P, citreus enzyme may contain a metal cof actor and possibly sulfhydryl groups. Additional work should be conducted to confirm these findings, Dipeptidase Activity Table 12 shows the activity of the purified _P, citreus extracellular enzyme towards five synthetic dipeptides. Although lower when compared to the activity on the whole protein substrates, enzyme activity was observed in all five peptides. The highest activity was observed with DL-alanylglycine (47.67 units of activity). In order to make predictions on the specificity of this enzyme, additional peptides should be investigated. Consequently, the extracellular enzyme of _P. citreus can utilize and degrade dipeptides to their constituent amino acids. Enzyme Classification According to the International Union of Biochemistry scheme (54) for numbering enzymes, the _P. citreus proteolytic enz3mie would be classified as: 3.4.1 (acting on peptide bonds, an a-amino-acyl-peptide hydrolase). The data presented in this study point to the possibility of having an aminopeptidase enzjnne; however, further studies with synthetic peptides are necessary for the complete classification of the citreus enzyme. In addition, studies need to be conducted to determine if the 2« citreus enzjrme exhibits endo or exopeptidase activity. Enz3rme Induction Studies Various media were used in order to determine if the extracellular proteolytic enzyme produced by _P. citreus was induced by shrimp protein. Induction is the complete de novo synthesis of enzyme molecules in the

PAGE 119

105 Table 12. Dipeptldase activity of the Planococcus citreus extracellular enzyme. Dipeptide Enzyme Activity DL-leucylglycine 11.67 + 0.89 DL-leucyl-DL-alanine 22.94 + 0.44 L-leucyl-L-Tryptophane 32.61 + 1.98 glycyl-DL-leucine 11.56 + 0.18 DL-alanylglycine 47.67 + 2.48 Average of 6 observations ± standard deviation

PAGE 120

106 presence of a specific substrate (28,57,73). A substantial number of bacterial exo-enzymes appear to be induced by their substrate or closely related compounds (122) while others are continuously being synthesized by the microorganisms during their growth. According to Pollock (121), enzyme induction does not introduce a new pattern of protein structure into the cell. Whether constituent or induced, and whatever inducer is used, the enzyme formed appears to be identical (121). Yeast Carbon Base (YCB) , a minimal substrate level medium, was used in this study (see Table 4) and was fortified with shrimp protein and/or yeast extract. In addition, a study with Trypticase Soy Broth (TSB) fortified with shrimp protein was also done. Figure 32 shows the growth of _P. citreus in the various media following 96 hrs of incubation at 20 C. Overall, _P. citreus grew poorly in all four media tested. About a 1.15 log increase in _P. citreus cell number was observed in YCB, a 1.20 log increase in YCB +1.0% shrimp protein (YCBS) , a l.AQ log increase in YCB + 0.1% yeast extract (YCBY) and a 1.60 log increase in YCB + 1.0% shrimp protein + 0.1% yeast extract (YCBSY). Therefore, as the nutrients in the growth medium increased, improved growth of P_. citreus was observed. The proteolytic activity of the cell-free broth of _P. citreus grown in Yeast Carbon Base supplemented with shrimp protein and/or yeast extract is shown in Figure 33. After 96 hrs at 20 C, the proteolytic enzyme activity of the cell-free broths from each medium was 141, 114, 90 and 77.5 units for YCBSY, YCBY, YCBS and YCB, respectively. Thus, as observed with the P. citreus growth data (Figure 32), as the nutritional composition of the growth medium increased, the amount of enzyme produced also increased (as measured by an increase in total enzyme activity).

PAGE 121

AT 107 6n CO O O O O o CD Q \ 5H CO UJ cr. H O 432Yeast Carbon Base Yeast Carbon Base + 1% Shrinnp Protein Yeast Carbon Base + 0. 1 % Yeast Extract Yeast Carbon 5ase+ 0.1% Yeast Extract + l7o Shrimp Protein r 0 24 48 72 96 INCUBATION TIME AT 20°C (hr) Figure 32, Growth of Planococcus citreus in Yeast Carbon Base supplemented with shrimp protein and/or yeast extract at 20 C. I

PAGE 122

108 -•Yeast Carbon Base 200 24 48 72 96 INCUBATION TIME AT 20°C (hr) Figure 33. Proteolytic enzyme activity of the cell-free broth of Planococ <^^^^e^s grown in Yeast Carbon Base supplemented with shrimp protein and/or yeast extract.

PAGE 123

109 The units of enzyme activity per cell per hr (m) was calculated for each medium used. Table 13 shows that the average m value for YCB, YCBS, YCBY and YCBSY was 4.77, 7.62, 27.82 and 37.12, respectively. These data agree with the results in Figures 32 and 33. As the nutrients in the growth medium increased, more enzyme was produced per P. citreus cell. Thus, it seems that the nutritional composition of the medium of growth, not the mere presence of shrimp protein, influences enzyme production. No significant difference (a=0.05 level) was found between YCB (control) and YCB with shrimp protein added (Table 13). The results presented indicate that the enzyme produced by £. citreus is constantly being produced as long as there is cell growth. Consequently, the enzyme is constituent. The results shown in these induction studies reflect the production of an enzyme by _P. citreus in a minimal medium in which the maximum growth attained was 1.6 logs after 96 hrs of incubation at 20 C. Figure 34 shows the growth of , _P . citreus cells in Trypticase Soy Broth (TSB) with and without shrimp protein added. TSB was previously chosen as the best medium for _P. citreus growth and enzyme production (see Growth Medium and Enzyme Production section). P. citreus log count increased approximately 3.75 and 3.80 logs in TSB and TSB +1.0% shrimp protein, respectively. Thus, P. citreus grew equally as well as in media with or without shrimp protein added. Figure 35 shows the proteolytic activity of the cell-free broth of P. citreus cells grown in TSB with and without shrimp protein. After 96 hrs at 20 C, the proteolytic enzyme activity of the cell-free broths was 297 and 295 units of activity for TSB and TSB +1.0% shrimp protein, respectively. Thus, these data support the previous results in showing

PAGE 124

110 Table 13. Units of enzyme activity per cell per hr (m) of Planococcus citreus grown in Yeast Carbon Base supplemented with shrimp protein and/or yeast extract at midlog phase. Medium Mean m^ Hm / / Yeast Carbon Base + 1.0% shrimp protein (YCBS) 7.62^ Yeast Carbon Base + 0.1% yeast extract (YCBY) 27.82^ Yeast Carbon Base + 1.0% shrimp protein + 0.1% yeast extract (YCBSY) 37.12^ ^average of 6 observations Means followed by the same letter do not differ 0=0.05 level (r from Anova table 0.978) significantly at the

PAGE 125

Ill — Trypticase Soy Broth Trypticase Soy Broth +1.0% Shrimp Protein 7£ INCUBATION TIME AT 20° C (hr) gure 34. Growth of Planococcus citreus in Trypticase Soy Broth with and without shrimp protein at 20 C.

PAGE 126

112 — •— Trypticase Soy Broth • Trypticase Soy Broth + l.07o Shrimp Protein ^ 1— 1 1 r 0 12 24 48 72 96 INCUBATION TIME AT 20° C (hr) 35. Proteolytic enzyme activity of the cell-free broth of Planococcus citreus grown in Trypticase Soy Broth with and without shrimp protein.

PAGE 127

113 that _P. cltreus can produce an extracellular enzyme (s) in the presence or absence of shrimp protein in the growth medium. The units of enzyme activity per cell per hr (m) of the enzyme produced in TSB and TSB + 1.0% shrimp protein were 147.04 and 146.58, respectively. These values are not significantly different (a=0.05 level). However, when compared to the m values in Table 13, both of these m values are significantly different (a=0.05 level). Consequently, these data appear to indicate that the extracellular proteolytic enzyme produced by _P. citreus is not induced by the presence of shrimp protein in the growth medium. The enzyme is produced in minimal media and its activity per cell per hr increases as the nutrient composition in the medium increases. _P« citreus produces the extracellular enzyme even in the presence of surplus nutrients. Table 14 illustrates these findings more clearly. There appears to be no effect, e.g., gene repression, by any of the factors present in the various media used. Consequently, the production of the extracellular proteolytic enzyme by 2. citreus appears to be related more to growth of the organism than to the presence of any specific nutrient.

PAGE 128

114 Table 14. Units of enzyme activity per cell per hr (m) of Planococcus citreus grown in various media. Medium i' J.CClt.A 111 V CI ^ Trypticase Soy Broth (TSB) 147 68^ Trypticase Soy Broth + 1,0% shrimp protein 146.58^ Plate Count Broth (PCB) lOS 67^ Nutrient Broth (NB) 59.32*^ Yeast Carbon Base + 1.0% shrimp protein + 0.1% yeast extract (YCBSY) 37.11** Yeast Carbon Base + 0.1% yeast extract (YCBY) 27.82^ Yeast Carbon Base + 1.0% shrimp protein (YCBS) 7.62^ Yeast Carbon Base (YCB) 4.77^ Average of 10 observations for TSB, 4 for TSB + 1.0% shrimp protein and 6 for the other media used Means followed ^y the same letter do not differ significantly at the a=0.05 level (r from Anova table 0.994)

PAGE 129

SUMMARY AND CONCLUSIONS An extracellular proteolytic enzyme from a marine organism. Pianococcus citreus , was isolated, purified and characterized. Ammonium sulfate precipitation, Sephacryl S-200 Superfine chromatography and DEAESephadex A-50 ion exchange chromatography were used to purify the enzyme. A single band present after acrylamide gel electrophoresis, as well as chromatography, showed the purity of the extracellular proteolytic enzyme. In addition, the fluorometric technique proved to be an efficient, fast and economical (less enz5nne is required) method for the measurement of enzyme activity. Each fraction of the ?_. citreus cells studied (extracellular, whole cells, washings of whole cells, soluble intracellular and cellular particulate) exhibited proteolytic activity. However, the major portion, greater than 95.0%, of the active enzjmie towards the high molecular weight substrates (gelatin and shrimp protein) was recovered in the extracellular fraction. Trypticase Soy Broth (TSB) , a highly nutritious medium, proved to be the best medium for culturing citreus cells and for the production of the extracellular proteolytic enzyme. A highly significant difference (a=0.05 level) was observed between the units of enzyme activity produced per cell per hr (m) by _P. citreus grown in Trypticase Soy Broth, Plate Count Broth and Nutrient Broth. The following properties were characteristic of the P. citreus extracellular proteolytic enz}Tne: 115

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116 1) The cell-free broth obtained from P_. citreus cells grown at 5 C for 108 hrs, 20 C for 72 hrs and 35 C for 36 hrs exhibited enzyme activity towards shrimp protein at all three enzjrme-substrate incubation temperatures (5, 20 and 35 C) . Thus, citreus when grown at 5 C produces an extracellular enzyme capable of utilizing the protein in shrimp stored either at refrigeration or higher temperatures. 2) The major portion of the extracellular proteolytic enzjrme of citreus was recovered at an ammonium sulfate concentration between 55-70% saturation. Eighty-six percent of the total activity was recovered in this fraction. 3) Using the fluorometric method for activity measurements, the protease was purified 26.50 fold with a recovery of approximately 49%. The specific activity of the purified enzyme was 780.37 (units of activity/mg of protein) . 4) Purity of the enzjnne was demonstrated by the presence of a single band after acrylamide gel electrophoresis using various protein concentrations as well as by the presence of a single peak with homogeneous activity after ion-exchange chromatography. 5) The molecular weight of the _P. citreus enzjone was approximately 29,000 according to column chromatography using Sephacryl S-200 and acrylamide gel electrophoresis. 6) Ionic strengths of 0,15-0.83 had no effect on the activity of the extracellular enzyme. 7) pH optimum of the proteolytic enzjrme was 8. Activity of the enzjnne decreased as the pH deviated from this optimum, 8) The optimum temperature for the citreus enzyme was 35 C; however, activity was observed at 5 C,

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117 9) After 15 min of incubation of the purified enzyme at 65 C, no activity was observed and only 1.0% activity remained in the cell-free broth of _P. citreus grown at 35 C for 36 hrs. However, after 15 min at 75 C, 1,0% activity still remained in the cell-free broth of P. citreus cells grown at 5 C for 108 hrs. Results ind icated that the enzyme system in the crude preparations (cell-free broths) was less affected by temperature changes than the purified enz3rme. In addition, the enzyme system produced by _P» citreus grown at 5 C was more stable to changes in temperature than the 35 C crude enz3Tne preparation. Perhaps the enz}Tnes present in the 5 C crude extract have an enzyme configurat ion that better protects the active site from temperature changes. However, when shrimp is processed (boiled, canned or broiled), the enzjnne should be easily inactivated. 10) When shrimp protein was used as substrate, the activity of the enz3mie increased as the sodium chloride (NaCl) concentration increased up to 0.5% NaCl. Enzyme activity decreased with higher concentrations of NaCl (0.5-1.5%). When gelatin was used as the substrate, NaCl concentrations (1-1.5%) had no effect on enz3mie activity. The increase in NaCl concentration up to 0.5% might have caused an increase in shrimp protein solubility, thus, making shrimp protein more available for the _P. citreus enzyme. 11) As the concentration of sodium bisulfite (NaHSO^) was increased from 0 to 3%, the activity of the protease decreased (first-order reaction) . Approximately 47% of the activity was lost when 1.25% sodium bisulfite was present in the medium. 12) As the concentration of enzyme increased (0-200 yl) , the rate of the reaction increased when gelatin and shrimp protein were used as substrates.

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118 13) Michaelis-Menten kinetics were followed when gelatin and shrimp protein were used a substrates. 14) The apparent K values for gelatin and shrimp protein were m 0,98 mg/ml and 0.33 mg/ml, respectively. The apparent V^^^ values were 666,67 and 431.03 (units of activity), respectively. This indicates that the _P. citreus extracellular enzyme can degrade gelatin faster than shrimp protein. 15) FeCl^, HgCl2 ^^'^ inhibited the enzyme to some extent, while CaCl2 activated the extracellular enz3rme. ZnCl2, ^^^'•2 MnCl^ had no appreciable effect on the activity of the proteolytic +3 +2 enzyme. The repressing effect of Fe and Hg on the activity of this extracellular enzyme may indicate that the enzjnne contains sulfhydryl groups. p-Dioxane had no effect on the activity of the proteolytic enzyme. EDTA, citric acid, cysteine, p-mercaptoethanol, potassium permangate and formaldehyde inactivated the enzyme to different degrees indicating that the extracellular enzyme was affected by metal chelators. These results indicate that the enzjnne may contain a metal ion as cof actor and possibly sulfhydryl groups. 16) The proteolytic enzjrme of P_. citreus exhibited activity against the following dipeptides: DL-leucylglycine, DL-leucyl-DL-alanine, Lleucyl-L-tryptophane, glycyl-DL-leucine and DL-alanylglycine. The highest activity was observed with DL-alanylglycine. 17) Preliminary classification of the enzyme shows that it is an a-amino-acyl-peptide hydrolase (3.4.1), Additional studies with synthetic peptides are necessary for complete classification. 18) The extracellular proteolytic enzyme produced by P, citreus was not induced by the presence of shrimp protein in the growth medium.

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119 The enzyme appears to be produced continuously during growth of the organism. Results obtained under the conditions of these investigations indicate that Planococcus citreus produces an extracellular enzyme which is active at refrigerated temperatures and capable of degrading shrimp protein. In addition, this enzyme is capable of cleaving dipeptides to their constituent amino acids. Consequently, the production of this enzyme by _P. citreus while growing on shrimp may contribute to the overall decrease in shrimp quality during iced or refrigerated storage. The function of the extracellular enzyme appears to be one of supplying nitrogenous compounds to the cell. 2* citreus does not actively utilize carbohydrates for growth, rather its metabolism is directed towards the utilization of proteins. In a nutrient limited marine environment, it appears advantageous from an evolutionary standpoint to produce a single extracellular enzyme of broad specificity. This dependence upon nitrogen compounds by _P. citreus could be one reason for the unique ecological association that exits between P. citreus and shrimp in nature.

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128 115. Payne, J. W. and C. Gilvarg. 1971. Peptide transport. Adv. in Enzymol. 35:187-244. 116. Pedraja, R. R. 1970. Change in composition of shrimp and other marine animals during processing. Food Technol. 24:1355-1360. 117. Peterson, A. C. and M. F. Gunderson. 1960. Some characteristics of proteolytic enz3mies from Pseudomonas fluorescens . Appl. Microbiol. 8:98-103. 118. Peplow, A. J., J. A. Koburger and H. Appledorf. Effect of ice storage on the total weight, proximate composition and mineral content of shrimp. Proc. of the Third Annual Tropical and Subtropical Fisheries Technological Conference, New Orleans, LA 3:92-102. 119. Pharmacia Fine Chemicals . 1975. Sephadex Ion Exchangers: A Guide to Ion Exchange Chromatography. Uppsala, Sweden. 120. Pharmacia Fine Chemicals . 1976. Sephacryl S-200 Superfine: For High Performance Gel Filtration. Uppsala, Sweden. 121. Pollock, M. R. 1960. Induced formations of enzymes. In: The Enzymes . Vol. I, 2nd Ed. Eds. P. D. Boger, H. Hardy and K. Myrback. Academic Press, Inc., New York, NY. 122. Pollock, M. R. 1962. Exoenzymes. In: The Bacteria : Volume IV: The Physiology of Growth. Eds. I. C. Gunsalus and R. Y. Stainer. Academic Press, Inc., New York, NY. 123. Preston, K. R. 1976. An automated fluorometric assay for proteolytic activity in wheat. Cereal Chem. 52:451-458. 124. Samejima, K., W. Dairman and S. Udenfriend. 1971. Condensation of ninhydrin with aldehydes and primary amines to yield highly fluorescent ternary compounds. 1. Studies of the mechanisms of the reaction and some characteristics of the condensation products. Anal. Biochem. 42:222-236. 125. Samejima, K., W. Dairman, J. Stone and S. Udenfriend. 1971. Condensation of ninhydrin with aldehydes and primary amines to yield highly fluorescent ternary compounds. 2. Application to the detection and assay of peptides, amino acids, amines and amino sugars. Anal. Biochem. 42:237-247. 126. Schleifer, K. H. and D. Kandler. 1970. Amino acid sequence of the murein of Planococcus and other Micrococcaceae. J. Bacteriol 103:387-392. 127. Schwabe, C. 1973. A fluorescent assay for proteolytic enzymes. Anal. Biochem. 53:484-490. 128. Scrutton, M. C. and M. F. Utter. 1965. Pyruvate carboxylase. III. Some physical and chemical properties of the highly purified enzyme. J. Biol. Chem. 240:1-9.

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129 129. Shelef, L. A. and J. M. Jay. 1971. Hydration capacity as an index of shrimp microbial quality. J. Food Sci. 36:994-997. 130. Shewan, J. M. 1971. The microbiology of fish and fishery products a progress report. J. Appl. Bacteriol. 34:299-315. 131. Shuster, C. W. and M. Doudoroff . 1962. A cold-sensitive D(-)beta-hydroxybutyric acid dehydrogenase from Rhodospirillum rubrum. J. Biol. Chem. 237:603-607. 132. Steele, R. G. D. and J. H. Torrie. 1960. Principles and Procedures for Statisticians . McGraw-Hill Co., Inc., New York, NY. 133. Stein, S., P. Bohlen, J. Stone, W. Dairman and S. Udenfriend. 1973. Amino acid analysis with f luorescamine at the picomole level. Arch. Biochem.Biophys. 155:202-212. 134. Stone, F. E. 1971. Inosine monophosphate (IMP) and hypoxanthin formation in three species of shrimp held on ice. J. Milk Food Technol. 34:354-356. 135. Sussman, A. J. and C. Gilvarg. 1971. Peptide transport and metabolism in bacteria. Ann. Rev. Biochem. 40:397-408. 136. Tarrant, P. J. V., N. Jenkins, A. M. Pearson and T. R. Outson. 1973. Proteolytic enzyme preparation from Pseudomonas fragi : Its action on pig muscle. Appl. Microbiol. 25:996-1005. 137. Thirkell, D. and M. Summerfield. 1977. The effect of varying sea salt concentration in the growth medium on the chemical composition of a purified membrane fraction from Planococcus citreus Migula. Anton, van Leeuwenhoek 43:37-42. ' 138. Thirkell, D. and M. Summerfield. 1977. The membrane lipids of Planococcus citreus Migula from cells grown in the presence of three different concentrations of sea salt added to a basic medium. Anton, van Leeuwenhoek 43:43-54. 139. Thomas, T. D., E. D. W. Jarvis and N. A. Skipper. 1974. Localization of proteinase (s) near the cell surface of Streptococcus lactis. J. Bacteriol. 118:329-333. 140. Udenfriend, S., S. Stein, P. Bohlen and W. Dairman. 1972. Fluorescamine: A reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science 178:871-872. 141. Vanderzant, C. and R. Nickelson. 1971. Comparison of extractrelease volume, pH and agar Plate Count of shrimp. J. Milk Food Technol. 34:115-118. 142. Vanderzant, C, E. Mroz and R. Nickelson. 1970. Microbial flora ot Gulf of Mexico and pond shrimp. J. Milk Food Technol. 33:346J 3 u •

PAGE 144

130 143. Ward, D. R., G. Finne and R. Nickelson. 1979. Use of a specific ion electrode (ammonia) in determining the quality of shrimp. J. Food Sci. 44:1052-1057. 144. Weber, K. and M. Osborn. 1969. The reliability of molecular weight determination by dodecylsulf ate polyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4410. 145. Weigele, M. , S. L. DeBernardo and W. Leimgruber. 1973. Fluorometric assay of secondary amino acids. Biochem. Biophys. Res, Comm. 50:352-356. 146. Weigele, M. , S. L. DeBernardo, J. P. Teugi and W. Leimgruber. 1972. A novel reagent for the fluorometric assay of primary amines. J. Amer. Chem. Soc. 94:5927-5928. 147. Weil, L., W. Kocholaty and L. D. Smith. 1939. CV III. Studies on proteinases of some anaerobic and aerobic microorganisms. Biochem. J. 33:893-897. 148. Weingartner, K. E., J. A. Koburger, J. L. Oblinger and F. W. Knapp. 1977. Residual bisulfite in iced Penaeus shrimp. J. Food Prot, 40:234-235. 149. Westhoff, D. C, R. A. Cowman and M. L. Speck. 1970. Effect of storage at 3 C on the proteinase enzjmie systems of slow and fast strains of lactic streptococci. J. Dairy Sci. 53:1023-1027. 150. Whitaker, J. R. 1963. Determination of molecular weights of proteins by gel filtration on Sephadex. Anal. Chem. 35:1950-1960. 151. Whitaker, J. R. 1972. Principles of Enzymology for the Food Sciences . 1st Ed. Marcel Dekker, Inc., New York, NY. 152. White, A., P. Handler and E. L. Smith. 1973. Principles of Biochemistry . 5th Ed. McGraw-Hill, Inc., New York, NY. 153. Williams, 0. B. 1949. Microbiological examination of shrimp. J. Milk Food Technol. 12:109-110. 154. Williams, 0. B., L. L. Campbell, Jr. and H. B. Rees, Jr. 1952. The bacteriology of Gulf Coast shrimp. II. Qualitative observations on the external flora. Texas J. Sci. 4:43-54. 155. Willms, C. R. 1960. Studies on an extracellular proteolytic enz3mie from a marine bacterial species. Ph.D. Dissertation. Agricultural and Mechanical College of Texas. 156. Wood, A. J., G. J. Sigundsson and W. J. Dyer. 1942. The surface concept in measurements of fish spoilage. J. Fish. Res. Brd. Can. 6:53-62. 157. Yoshida, K., H. Hidaka, S. Miyado, U. Shibata, K. Saito and Y. Yamada. 1977. Purification and some properties of Bacillus sphaericus protease. Agric. Biol. Chem. 41:745-754.

PAGE 145

BIOGRAPHICAL SKETCH Ricardo Javier Alvarez was born on November 4, 1954, in Santiago, Chile. In June, 1972, he graduated from Colegio San Ignacio de Loyola in San Juan, Puerto Rico. He attended the University of South Florida, and in June, 1976, he received his Bachelor of Science in microbiology. He enrolled as a graduate student in the Food Science and Human Nutrition Department, University of Florida, in January, 1977. He received his Master of Science degree in June, 1978. He anticipates receiving his Ph.D. degree in food science and human nutrition with a minor in environmental engineering in March, 1981. 131

PAGE 146

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Human Nutrition I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Dr. J. L. Oblinger Associate Professor of Food Science and Human Nutrition I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Dr/^, Bitton Associate Professor of Environmental Engineering Sciences

PAGE 147

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Dr. R. H. Schmidt Associate Professor of Food Science and Human Nutrition I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Dr. W. S. Otwell ' Assistant Professor of Food Science and Human Nutrition This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. Dean for Graduate Studies and Research


62
Figure 14. Spectrophotometric growth curves of Planococcus citreus in
Trypticase Soy Broth incubated at 5, 20 and 35 C~


Table 8. Purification of an extracellular proteolytic enzyme from Planococcus citreus
Fraction
Volume
Total Activity3
(units x 10 )
Protein*5
(mg/ml)
Specific Activity0
(units/mg total protein)
Purification
Recovery
(%)
Cell-free broth
900
113.3
44.52
29.45
100.0
70% (N4) S04
precipitation
350
102.7
/
19.60
52.39
1.78x
78
Sephacryl
S-200 Superfine
115
65.3
1.42
461.65
15.67x
50
DEAE-Sephadex^
A-50
90
64.0
0.32
780.37
26.50x
49
a
^Determined using gelatin as substrate
Average of duplicate samples
Total activity/Total protein = specific activity (units of activity/mg protein)


Table 1. Chemical and physical tests available to measure shrimp quality
Test
Parameter Measured
Reference
acid-soluble orthophosphate
trichloroacetic acid and soluble
orthophosphate
adenosine triphosphate and degradation of adenine dinucleotides
its degradation products to to hypoxanthine
hypoxanthine
alcoholic tumeric solution
changes in percent transmission of
a yellow tumeric shrimp solution
amino-nitrogen
changes in amine nitrogen content
ammonia
ammonia content
B-vitamin content
cathecol ferric chloride
dimethylamine
direct microscopic counts
fluorescamine
free fatty acids
glycogen
content of B-complex vitamins
change in percent transmission of
shrimp filtrate mixed with cathecol
ferric chloride
degradation of trimethyl amine oxide
by enzyme (TMO) to DMA
actively and non-actively
metabolizing bacteria
changes in free amine fractions
percent of free fatty acids
glycogen content
14
64
75
14,19,62,93
75,143
15
85
31
109
107
31
14,93


LIST OF FIGURES (continued)
FIGURE
Page
13
Enzyme activity at 35 C for 15 min (pH 8) of the cell-free
broth of Planococcus citreus grown in Plate Count Broth,
Nutrient Broth and Trypticase Soy Broth for up to 96 hrs ..
59
14
Spectrophotometric growth curves of Planococcus citreus in
Trypticase Soy Broth incubated at 5, 20 and 35 C
62
15
Separation of the proteins present in the 55-70% ammonium
sulfate fraction using Sephacryl S-200
70
16
£
Ion-exchange chromatography using DEAE-Sephadex A-50 of
the pooled active fractions obtained in the molecular sieve
chromatography purification step
72
17
Acrylamide gel electrophoresis of the purified enzyme of
Planococcus citreus
74
18
Acrylamide gel electrophoresis of increasing concentration
of the purified extracellular enzyme of Planococcus citreus
75
19
Calibration curve used for the molecular weight estimation
of Planococcus citreus proteolytic enzyme using Sephacryl
S-200 column chromatography
78
20
Calibration curve used for the molecular weight estimation
of Planococcus citreus proteolytic enzyme using acrylamide
gel electrophoresis
79
21
Effect of ionic strength on the activity of the extracell
ular proteolytic enzyme of Planococcus citreus
81
22
Optimum pH of the extracellular proteolytic enzyme of
Planococcus citreus
83
23
Temperature optimum of the extracellular proteolytic enzyme
of Planococcus citreus
85
24
Thermal stability of the enzymes in the cell-free broths of
Planococcus citreus grown at 5 and 35 C and of the
purified enzyme
86
25
Effect of sodium chloride concentration on enzyme activity
89
26
Effect of sodium bisulfite concentration on enzyme activity
91
27
Effect of enzyme concentration on enzvme activitv
92
28
Effect of gelatin concentration on the reaction rate of the
Planococcus citreus extracellular enzvme
94
x


Figure 5. Exitation (curve A) and fluorescence (curve B) spectrum for the reaction of a shrimp protein-
trichloroacetic acid (TCA) filtrate with fluorescamine pH 8.


l/[s]mg/ml of Shrimp Protein
Figure 31. Lineweaver-Burlc plot of the Planococcus citreus extracellular enzyme illustrating V x
K values using shrimp protein as substrate.3
a
Transformation of the data presented in Figure 30.
and


LITERATURE REVIEW
In 1978, the shrimping industry was the most valuable fishery in
the United States (8). However, the quality of shrimp often falls short
of that expected by the consumer. Approximately 15-20% of the shrimp
landed is eventually lost due to quality deterioration. This deteriora
tion of shrimp quality is usually attributed to rapid bacterial enzymatic
changes of the fresh shrimp resulting from mishandling and/or inadequate
processing. These changes, along with the chemical and physical methods
for measuring shrimp quality, are discussed as a basis for the
investigations presented in this dissertation.
Shrimp Spoilage
Quality changes in shrimp during storage on ice can lead to major
economic losses in the. shrimp industry. Mechanical damage, bacterial
contamination and enzymatic activity may combine to cause undesirable
changes in the composition and quality of shrimp (20,34,38,39,43,45,53,
54,77,78,79,89,94,108,117,153,154).
The loss of acceptability of shrimp may be triggered by several
factors: 1) shrimp muscle enzymes, 2) direct microbial activity, 3)
microbial enzymes and/or 4) a combination of these factors. Defects
which may occur as a result of such reactions are formation of malodor
ous substances, flavor deterioration, toughness, mushiness, juiciness,
dryness and discoloration (116).
Proteolytic enzymes play an important role in the spoilage of shrimp
by degrading muscle proteins and polypeptides, forming amino acids which
3


Ill
Figure 34. Growth of Planococcus citreus in Trypticase Soy Broth
with and without shrimp protein at 20 C.


57
rapid growth of P. citreus. The optical density after 96 hours of incu
bation at 20 C was .314, .200 and .072 for TSB, PCB and NB, respectively
Figure 12 shows a similar trend; however, P. citreus growth was measured
by the Aerobic Plate Count technique (6). Again, we can observe that
the _P. citreus log count per ml increases slightly during the first
12 hours of incubation at 20 C. After 96 hrs of incubation, the _P.
citreus log count for TSB, PCB and NB was 6.02, 5.10 and 4.17, respec
tively. Consequently, TSB allowed for the "optimum" growth of _P.
citreus when grown at 20 C.
Nutritional components present in the growth medium are of utmost
importance for gram-positive microorganisms which are generally more
fastidious in its nutrient requirements than gram-negative bacteria (29)
Realizing these growth requirements of gram-positive microorganisms, the
results from this section are not surprising. TSB contains tryptone,
soytone, dextrose, sodium chloride and dipotassium phosphate (16). This
combination of nutrients provide an adequate nitrogen, carbohydrate,
vitamin and overall nutrient supply for the growth of _P. citreus. In
contrast, PCB and NB are not as nutritionally complex.
Figure 13 shows the enzyme activity of the cell-free broth of P.
citreus cells grown in TSB, NB and PCB for 96 hrs at 20 C. An active
extracellular enzyme fraction was produced by P. citreus in all three
media. However, after 96 hrs of growth, the amount of enzyme produced
by this organism in TSB is greater than that produced when grown in PCB
of NB. The enzyme activity after 96 hrs of incubation of the cell-free
broth of _P. citreus grown in TSB, PCB and NB was 323, 270 and 200 units
of activity, respectively. However, if the cells are harvested at
approximately midlog phase, the difference in the amount of enzyme


Figure 8. Increase in fluorescence intensity using gelatin as substrate and various amounts of the cell-
free broth after incubation at 35 C for 1 hr.


74
Figure 17. Acrylamide gel electrophoresis of the purified enzyme of
Planococcus citreus.


24
fluorescamine technique with the standard Lowry procedure (97) for the
monitoring of protein in a column effluent. The volumes used for fluo
rescamine assay were 10 to 20 percent of those used in the Lowry method,
and smaller amounts could have been used (140). Background interference
was negligible with the automated fluorescence method, and significant
peaks not discernible by the Lowry procedure were observed.
Due to the many advantages of the relatively new fluorometric tech
nique, it was used to measure the proteolytic enzyme activity of P.
citreus. The ability of this organism to grow on shrimp as well as to
hydrolyze various protein preparations promoted investigations to iso
late, purify and characterize the extracellular enzyme(s) produced by
P. citreus.


29
In order to determine the reaction spectrum of our working enzyme
solution when gelatin and shrimp protein were used as substrates, the
excitation and emission (fluorescent) wavelengths were scanned (48).
Figure 4 shows the excitation (curve A) and fluorescence (curve B)
spectra for TCA filtrates of the gelatin substrate. The excitation
spectrum has a maximum of 360 nm and a secondary peak at 390 nm. The
secondary peak at 390 nm was chosen because it results in minimal zero
time blank fluorescence values. The fluorescence emission maximum with
the excitation wavelength at 390 nm was at 475 nm. Figure 5 shows the
excitation (curve A) and fluorescence (curve B) emission spectra for TCA
filtrates of the shrimp protein substrate. The excitation spectrum has
a maximum peak at 375 nm and a secondary peak at 390 nm. Again, the
secondary peak was chosen. The fluorescence emission maximum with the
excitation wavelength at 390 nm was at 490 nm.
Efficacy of 5% Trichloroacetic Acid (TCA)
In order to determine the efficacy of 10 ml of 5% trichloroacetic
acid (TCA) in terminating the enzyme-substrate reaction, 5 ml aliquots
of substrate (gelatin) were incubated with 1 ml of cell-free broth and
10 ml of 57o TCA for 0, 10, 15, 30, 45 and 60 min at 35 C. A positive
control was done by incubating the enzyme-substrate mixture at 35 C for
0, 10, 15, 30, 45 and 60 min before adding the TCA. After the incubation
period, the positive control was terminated by adding 10 ml of 5% TCA.
t
Data in Figure 6 shows that 10 ml of 5% TCA were adequate for inhibiting
the enzyme substrate reaction effectively since there was no increase in
fluorescence intensity. The fluorescence intensity, observed when TCA
is immediately reacted with the enzyme and substrate, represents the
background fluorescence of the assay mixture.


RESULTS AND DISCUSSION
\
The ability of Planococcus citreus to grow in shrimp during ice
storage raised the question as to whether this organism could contribute
to the spoilage of shrimp. Various studies (3,4,5) have indicated that
this organism may contribute to the spoilage of this valuable marine
/
resource. In order to more clearly understand the contribution this
organism makes to the degradation of shrimp, an investigation was under
taken to study the extracellular proteolytic enzyme(s) produced by this
organism.
Proteolytic Activity of Cellular Fractions
The proteolytic activity of cellular fractions of _P. citreus cells
grown in Trypticase Soy Broth (TSB) was investigated in order to deter
mine the distribution of the enzyme activity in the isolated fractions.
In addition to the cell-free broth (extracellular fraction), whole cells,
washings of the whole cells, soluble intracellular and the cellular
particulate fraction were examined. Table 5 shows the total activity
(units of activity), protein content (mg/ml), specific activity (units
of activity/mg of total protein) and distribution of activity (%) for
all the fractions tested using both gelatin and shrimp protein sub
strates. The extracellular fraction showed the highest specific activ
ity, 29.450 units of activity/mg of protein and 27.540 units of activ
ity/mg of protein towards gelatin and shrimp protein, respectively.
This represented 95.9 and 95.8% of the total activity present in all of
the fractions towards gelatin and shrimp protein, respectively.
52


127
100. Mallory, L. M., B. Austin and R. R. Colwell. 1977. Numerical
taxonomy and ecology of oligotiogalic bacteria isolated from the
estaurine environment. Can. J. Microbiol. 23:733-750.
101. McCamam, M. W. and E. Robins. 1962. Fluorometric method for the
determination of phenylalanine in serum. J. Lab. Clin. Med. 59:
885-890.
102. Mendenhall, W. 1975. Introduction to Probability and Statistics.
4th Ed. Duxbery Press, North Seltuate, MA.
103. Migula, W. 1894. Uber ein neues System der Bakterein. Arb.
Bakt. Inst. Karlsruhe 1:235-238.
104. Migula, W. 1900. System det Baktenein. Gustav Fischer, Jeua.
105. Mills, G. L. and J. M. Wilkins. 1958. The isolation and proper
ties of a protease form Proteus vulgaris. Biochem. Biophys. Acta
30:63-70.
106. Miyata, K., K. Maejima, K. Tomoda and Masao Isona. 1970. Serratia
protease. Part I. Purification and general properties of the
enzyme. Agr. Biol. Chem. 34:310-318.
107. Moore, A. B. and R. R. Eitenmiller. 1980. Shrimp quality:
Biological and technological relationships. University of Georgia,
Research Bulletin No. 253, Athens, GA.
108. Nickelson, R. and C. Vanderzant. 1976. Bacteriology of shrimp.
Proc. of the First Annual Tropical and Subtropical Fisheries
Technological Conference, Corpus Christi, TX 1:254-270.
109. Nickelson, R., J. Hosch and L. E. Wyatt. 1975. A direct micro
scopic count procedure for the rapid estimation of bacterial
numbers of green-headless shrimp. J. Milk Food Technol. 38:76-77.
110. Noreau, J. and G. R. Drapeau. 1979. Isolation and properties of
the protease from the wild-type and mutant strains of Pseudomonas
fragi. J. Bacteriol. 140:911-916.
111. Novak, A. F., E. A. Fieger and M. E. Bailey. 1956. Rapid proce
dures for approximation of bacterial counts in shrimp and oysters.
Food Technol. 10:66-67.
112. Oeding, P. 1971. Serological investigations of Planococcus
strains. Int. J. Symp. Bacteriol. 21:323-325.
113. Pacaud, M. and J. Uriel. 1971. Isolation and some properties of
a proteolytic enzyme from Escherichia coli (Protease I). Eur. J.
Biochem. 23:435-442.
114. Pacaud, M. and C. Richard. 1975. Protease II from Escherichia
coli. J. Biol. Chem. 250:7771-7779.


11
new method using fluorescamine primarily detected only the non-protein,
non-ammonia, small molecular weight amines in shrimp homogenates. They
proposed that fluorescamine analysis could be useful in determining
changes in the free amine fractions. The shrimp industry still depends
on visual observation, smell and bacteriological testing for evaluating
overall shrimp quality, whereas, the Food and Drug Administration (FDA)
uses decomposition, filth and odor for the evaluation of shrimp quality.
Characteristics of Planococcus citreus
Koburger et al. (90) noted the presence of a high percentage of
gram-positive organisms following iced storage of rock shrimp (Sicyonia
brevirostris). These organisms comprised up to 68% of the isolates. Of
these 40% were Planococcus citreus, an aerobic gram-positive motile
coccus of marine origin producing an orange or yellow pigment. Informa
tion describing the isolation and characteristics of this organism is
limited. _P. citreus was previously named Micrococcus citreus (27). The
8th Edition of the Bergey's Manual of Determinative Bacteriology (29)
does have a description of the organism; however, it is limited in scope.
Cook in 1970 (45) and previous researchers working with shrimp placed
all aerobic gram-positive to gram-variable coccoid shaped bacteria in
the genus Micrococcus. In addition, Cook (45) noted that many of these
organisms isolated from shrimp were pigmented orange or yellow and were
motile. According to Bergey's manual (29), the only genus in the family
Micrococcaceae that is pigmented, either yellow or orange, and motile,
is Planococcus. This change in the taxonomic status of this organism
and the difficulty of demonstrating motility are probably the reasons
why Planococcus has not been reported in previous studies.
The taxonomic status of Planococcus citreus has changed markedly
through the years. In 1894 and again in 1900, Migula (103,104) made a


ISOLATION, PURIFICATION AND CHARACTERIZATION OF
AN EXTRACELLULAR PROTEOLYTIC ENZYME OF Planococcus citreus
BY
RICARDO J. ALVAREZ
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1981

A mis queridos padres:
Gracias por la ayuda brindada, el amor,
el apoyo moral y la vision de avanzar en la vida.
Con todo mi amor

ACKNOWLEDGMENTS
The author expresses his deepest gratitude to Dr. J. A. Koburger,
his major advisor, for his exceptional patience, guidance and encourage
ment throughout the course of this work. The author also wishes to
acknowledge the advice, support and all the help given by Drs. R. H.
Schmidt, W. S. Otwell, J. L. Oblinger and G. Bitton as members of his
supervisory committee. Thanks go to Dean J. L. Fry for his under
standing and support in delicate times.
Special appreciation is extended to Dr. J. R. Kirk for procuring a
much needed assistantship for the first two years of this research, to
Margie Summers for her beautiful graphic work, Beth Beville, Diane Dobsha,
Beth Johnsen and Mike Pyle for their patience in typing sections of this
dissertation. A very .special thank you goes to Melissa Michaels for the
typing of the final copy of this dissertation.
In addition, the author expresses thanks to Sam May for his help
and support around the laboratory and to Suzanne Davidson, Bridget
Walker and Janet Eastridge for their aid when needed. Thanks go to Dr.
L. D. Ingram for his constructive comments.
Recognition is also given to the faculty, staff and secretaries of
the Food Science and Human Nutrition Department for their cooperation
and to all fellow graduate students who shared with the author the years
at the University of Florida.
iii

Finally, he is deeply grateful to Mary Brannigan for her love,
patience and devotion, providing sentimental support and help through
out all phases of his course work. He thanks her for her understanding
and provision of many reasons to pursue all achievements in life.
iv

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
LIST OF TABLES vii
LIST OF FIGURES ix
ABSTRACT xii
INTRODUCTION 1
LITERATURE REVIEW 3
Shrimp Spoilage 3
Microbiological Characteristics of Shrimp 4
Measurement of Shrimp Spoilage
Characteristics of Planococcus citreus 11
Proteolytic Enzymes 17
Measuring Proteolytic Activity 19
MATERIALS AND METHODS 25
Planococcus citreus Cultures 25
Determination of Proteolytic Activity 25
Efficacy of 5% Trichloroacetic Acid (TCA) 29
Substrate Characteristics 33
Determination of Enzyme-Substrate Mixture Reaction Time 33
Growth Medium and Enzyme Production 36
Optimization of Enzyme Activity to Growth and Cell Number ... 36
Effect of Incubation Temperature on Enzyme Production and
Activity 38
Purification of the Extracellular Enzyme(s) 39
Ammonium Sulfate Precipitation 39
Molecular Sieve Chromatography 39
Ion-Exchange Chromatography 40
Confirmation of Enzyme Purity 42
Characterization of the Proteolytic Enzyme(s) 43
Molecular Weight Determination 43
Determination of the Purified Enzyme-Substrate Mixture
Reaction Time 43
Effect of Ionic Strength on Enzyme Activity 44
Determination of Optimum pH 44
Determination of Optimum Temperature 46
Thermal Stability 46
Effect of Sodium Chloride Concentration 47
v

TABLE OF CONTENTS (continued)
Page
Effect of Sodium Bisulfite Concentration 47
Effect of Enzyme Concentration 47
Effect of Substrate Concentration 47
Effect of Metal Ions on Enzyme Activity 48
Effect of Various Reagents on Enzyme Activity 48
Dipeptidase Activity 49
Enzyme Induction Studies 49
RESULTS AND DISCUSSION 52
Proteolytic Activity of Cellular Fractions 52
Growth Medium and Enzyme Production 55
Effect of Incubation Temperature on Enzyme Production and
Activity 60
Purification of Extracellular Enzyme(s) 66
Purity of the Extracellular Proteolytic Enzyme 71
Characterization of the Extracellular Proteolytic Enzyme .... 77
Molecular Weight Determination 77
Effect of Ionic Strength on Enzyme Activity 80
Optimum pH Determination 82
Optimum Temperature Determination 84
Thermal Stability 84
Effect of Sodium Chloride Concentration 87
Effect of Sodium Bisulfite Concentration 90
Effect of Enzyme Concentration 90
Effect of Substrate Concentration 93
Effect of Metal Ions on Enzyme Activity 98
Effect of Various Reagents on Enzyme Activity 101
Dipeptidase Activity 104
Enzyme Classification 104
Enzyme Induction Studies 104
SUMMARY AND CONCLUSIONS 115
LITERATURE CITED 120
BIOGRAPHICAL SKETCH
131

LIST OF TABLES
TABLE Page
1 Chemical and physical tests available to measure shrimp
quality 8
2 Hydrolysis of various protein sources by selected strains of
Planococcus citreus at 25 C 15
3 Proximate composition of the shrimp protein preparation .... 34
4 Composition of yeast carbon base medium 50
5 Proteolytic activity at 35 C for 15 min (pH 8) of cellular
fractions obtained from Planococcus citreus grown in
Trypticase Soy Broth (TSB) using gelatin and shrimp protein
as substrates 53
6 Units of enzyme activity per cell per hour (m) of Planococcus
citreus grown in Trypticase Soy Broth (TSB), Plate Count
Broth (PCB) and Nutrient Broth (NB) at mid-log phase 61
7 Enzyme activity measured at 5, 20 and 35 C (pH 8) of the
cell-free broths of Planococcus citreus grown in Trypticase
Soy Broth (TSB) at 5, 20 and 35 C for 108, 72 and 36 hrs,
respectively 64
8 Purification of an extracellular proteolytic enzyme from
Planococcus citreus 67
9Proteolytic activity at 35 C for 15 min (pH 8) of various
ammonium sulfate fractions of the cell-free broth of
Planococcus citreus 68
10 Effect of various metal ions on the activity (assayed at 35
C for 10 min (pH 8)) of the Planococcus citreus extracellu
lar enzyme 100
11 Effect of various reagents on the activity (assayed at 35 C
for 10 min (pH 8)) of the Planococcus citreus extracellular
enzyme 102
12 Dipeptidase activity of the Planococcus citreus extracellular
enzyme 105
vii

LIST OF TABLES (continued)
TABLE Page
13 Units of enzyme activity per cell per hour (m) of Planococcus
citreus grown in yeast carbon base supplemented with shrimp
protein and/or yeast extract at midlog phase Ill
14 Units of enzyme activity per cell per hour (m) of Planococcus
citreus grown in various media
vii

LIST OF FIGURES
FIGURE Page
1 Photomicrograph of Planococcus citreus cells showing
morphology and flagellation 14
2 Comparison of the Fluorescamine technique and the Lowry
procedure for determining protein concentration 23
3 Effect of pH adjustment of gelatin-trichloroacetic acid
(TCA) filtrates on fluorescence intensity 28
4 Excitation (curve A) and fluorescence (curve B) spectrum
for the reaction of a gelatin-trichloroacetic acid (TCA)
filtrate with fluorescamine at pH 8 30
5 Excitation (curve A) and fluorescence (curve B) spectrum
for the reaction of a shrimp protein-trichloroacetic acid
(TCA) filtrate with fluorescamine at pH 8 31
6 Efficacy of trichloroacetic acid (TCA) in terminating the
enzyme-substrate reaction 32
7 Increase in fluorescence intensity using the shrimp protein
preparation as substrate after incubation with cell-free
broth for 1 hr at 35 C 35
8 Increase in fluorescence intensity using gelatin as
substrate and various amounts of cell-free broth after
incubation at 35 C for 1 hr 37
9 Outline of steps for the purification of the extracellular
protease(s) of Planococcus citreus 41
10 Increase in fluorescence intensity following incubation of
gelatin and shrimp protein substrate with purified enzyme
for up to 1 hr at 35 C 45
11 Spectrophotometric growth curves of Planococcus citreus in
Plate Count Broth, Nutrient broth and Trypticase Soy Broth
at 20 C 56
12Aerobic plate counts of Planococcus citreus incubated in
Plate Count Broth, Nutrient Broth and Trypticase Soy Broth
at 20 C for 96 hrs
58

LIST OF FIGURES (continued)
FIGURE
Page
13
Enzyme activity at 35 C for 15 min (pH 8) of the cell-free
broth of Planococcus citreus grown in Plate Count Broth,
Nutrient Broth and Trypticase Soy Broth for up to 96 hrs ..
59
14
Spectrophotometric growth curves of Planococcus citreus in
Trypticase Soy Broth incubated at 5, 20 and 35 C
62
15
Separation of the proteins present in the 55-70% ammonium
sulfate fraction using Sephacryl S-200
70
16
£
Ion-exchange chromatography using DEAE-Sephadex A-50 of
the pooled active fractions obtained in the molecular sieve
chromatography purification step
72
17
Acrylamide gel electrophoresis of the purified enzyme of
Planococcus citreus
74
18
Acrylamide gel electrophoresis of increasing concentration
of the purified extracellular enzyme of Planococcus citreus
75
19
Calibration curve used for the molecular weight estimation
of Planococcus citreus proteolytic enzyme using Sephacryl
S-200 column chromatography
78
20
Calibration curve used for the molecular weight estimation
of Planococcus citreus proteolytic enzyme using acrylamide
gel electrophoresis
79
21
Effect of ionic strength on the activity of the extracell
ular proteolytic enzyme of Planococcus citreus
81
22
Optimum pH of the extracellular proteolytic enzyme of
Planococcus citreus
83
23
Temperature optimum of the extracellular proteolytic enzyme
of Planococcus citreus
85
24
Thermal stability of the enzymes in the cell-free broths of
Planococcus citreus grown at 5 and 35 C and of the
purified enzyme
86
25
Effect of sodium chloride concentration on enzyme activity
89
26
Effect of sodium bisulfite concentration on enzyme activity
91
27
Effect of enzyme concentration on enzvme activitv
92
28
Effect of gelatin concentration on the reaction rate of the
Planococcus citreus extracellular enzvme
94
x

LIST OF FIGURES (continued)
FIGURE Page
29 Lineweaver-Burk plot of the Planococcus citreus extracell
ular enzyme illustrating V and K values using gelatin
, .' max m 66 qr
as substrate ?->
30 Effect of shrimp protein concentration on the reaction rate
of the Planococcus citreus extracellular enzyme 96
31 Lineweaver-Burk plot of the Planococcus citreus extracell
ular enzyme illustrating V and K values using shrimp
. max m 07
protein as substrate 7/
32 Growth of Planococcus^ citreus in Yeast Carbon Base supple
mented with shrimp protein and/or yeast extract at 20 C ... 1-07
33 Proteolytic enzyme activity of the cell-free broth of
Planococcus citreus grown in Yeast Carbon Base supplemented
with shrimp protein and/or yeast extract 108
34 Growth of Planococcus citreus in Trypticase Soy Broth with
and without shrimp protein at 20 C HI
35 Proteolytic activity of the cell-free broth of Planococcus
citreus grown in Trypticase Soy Broth with and without
shrimp protein 112

Abstract of Dissertation Presented to the Graduate
Council of the University of Florida in Partial Fulfillment
of the Requirements for the Degree of Doctor of Philosophy
ISOLATION, PURIFICATION AND CHARACTERIZATION OF
AN EXTRACELLULAR PROTEOLYTIC ENZYME OF Planococcus citreus
By
Ricardo J. Alvarez
March 1981
Chairman: J. A, Koburger
Major Department: Food Science and Human Nutrition
Planococcus citreus is a gram-positive marine bacterium commonly
found in fresh and iced shrimp. Various studies have indicated that it
may contribute to spoilage of this valuable marine resource. In order
to understand the contribution of this organism to the degradation of
shrimp as well as other proteins, an investigation was undertaken to
study the extracellular proteolytic enzyme(s) of this organism. Results
indicated that the major portion (>95.0%) of the proteolytic activity
resided in the extracellular fraction.
Under the conditions tested, maximum extracellular enzyme produc
tion occurred in Trypticase Soy Broth (TSB) as observed by the highest
m value (units of enzyme activity per cell per hour). In addition, the
cell-free broth obtained from _P. citreus cells grown at 5 C for 108 hrs,
20 C for 72 hrs and 35 C for 36 hrs exhibited enzyme activity towards
shrimp protein at all three enzyme-substrate incubation temperatures (5,
20 and 35 C).
xii

P. citreus was grown in Trypticase Soy Broth at 20 C for 72 hrs.
Centrifugation, ammonium sulfate precipitation, Sephacryl S-200 Super-
£
fine molecular sieve chromatography, DEAE-Sephadex A-50 ion exchange
chromatography and acrylamide gel electrophoresis were used to purify
the extracellular enzyme(s). The enzyme was purified 26.50 fold (using
the fluorometric technique for activity measurement), and recovery of
the enzyme was above 49%. Gelatin and shrimp protein were used as sub
strates throughout the study. The molecular weight of the purified
protease was approximately 29,000 as measured by Sephacryl S-200 column
chromatography and acrylamide gel electrophoresis.
Maximum activity of the enzyme was at pH 8 and 35 C. Ionic
strengths of above 0.83 (0.75 M NaCl) decreased the activity of the
extracellular enzyme. Heat treatment at 65 C for 15 min destroyed the
activity of the purified enzyme. However, 1.0% of the residual enzyme
activity was still present in the cell-free broth of _P. citreus grown at
35 C for 36 hrs. In contrast, 15 min at 75 C were necessary to reduce
99.0% the activity of the enzymes in the cell-free broth of _P. citreus
grown at 5 C for 108 hrs. When shrimp protein was used as substrate,
sodium chloride concentrations of 0.0-0.5% increased enzyme activity,
while concentrations of 0.5-1.5% decreased enzyme activity. However,
when gelatin substrate was used, NaCl concentrations of 0.0-1.5% had
no effect on enzyme activity. The activity of the purified enzyme
decreased as the concentration of sodium bisulfite increased. Michaelis-
Menten kinetics were followed when gelatin and shrimp protein preparation
were used as substrates. The apparent values for gelatin and shrimp
protein were 0.98 mg/ml and 0.33 mg/ml, respectively. The apparent
V values were 666.67 and 431.03 units of activity for gelatin
xiii
I

and shrimp protein, respectively. Ferric chloride, mercuric chloride,
potassium chloride, ethylene diaminetetraacetic acid, citric acid,
cysteine, p-mercaptoethanol, potassium permanganate and formaldehyde
partially inactivated the enzyme. Calcium chloride increased the
activity of the extracellular proteolytic enzyme. Zinc chloride, p-
dioxane, manganese chloride and magnesium chloride had no effect on the
activity of the enzyme. The proteolytic enzyme exhibited peptidase
activity on various commercial synthetic dipeptides. The extracellular
proteolytic enzyme produced by J?. citreus was apparently not induced by
the presence of shrimp protein in the medium of growth. Enzyme produc
tion appeared to be related to the extent of growth of _P. citreus in the
medium.
xiv

INTRODUCTION
Quality deterioration and subsequent spoilage of shrimp during
storage are caused primarily by activities of indigenous tissue enzymes
and microbial enzymes (69). Various researchers (17,156) believe that
bacterial action plays a more important role than autolytic enzyme
release in causing spoilage of seafoods. During growth of the bacteria,
proteolysis of shrimp proteins and free amino acid formation by microbial
action has been observed. Enzymatic deamination and decarboxylation of
these amino acids from shrimp protein occur, resulting in the formation
of malodorous compounds (116).
Various types of bacteria have been reported to be present on
freshly caught shrimp. Numerous studies (3,42,43,45,116,142) have shown
the changes undergone by the bacterial flora of shrimp as the storage
period increases. Recent research (1,2,3,90) has noted the presence of
a gram-positive organism, Planococcus citreus, during shrimp storage.
The organism is described as a motile gram-positive coccus found in the
marine environment, capable of growing over a pH range of 7-10 between
5-35 C in broth containing 0.5-12% sodium chloride (NaCl), and the orga
nism is capable of hydrolyzing gelatin, cottonseed, soy and shrimp
protein.
The potential of _P. citreus as a "spoiler" of shrimp was shown by
the increase in pH and the rapid increase in the total volatile nitrogen/
amino acid-nitrogen ratio (TVN/AA-N) and trimethyl-amine nitrogen (TMN)
following growth of this organism on shrimp (4). The proteolytic
1

2
activity of this organism was further demonstrated by the decrease in
percent total extractable protein (percent TEP) in shrimp during storage
at 5 C (4,5) which had been inoculated with _P. citreus.
The proteolytic activity exhibited by this organism deserves addi
tional research in order to better understand the contribution of _P.
citreus to the degradation of shrimp protein. A study was therefore
undertaken to study the enzyme(s) responsible for protein degradation.
The optimum medium and stage in the growth cycle of _P. citreus were
determined for maximum extracellular enzyme(s) production. The effect
of incubation temperature (5, 20 and 35 C) on the growth of _P. citreus
and proteolytic enzyme production was also investigated. Purification
of the extracellular enzyme(s) was achieved by precipitation and chro
matographic techniques. Homogeneity of the enzyme was evaluated by gel
electrophoresis and chromatographic techniques. Optimum pH and tempera
ture, ionic strength effect, thermal stability, molecular weight, sodium
chloride effect, sodium bisulfite effect, enzyme concentration, substrate
concentration, and the effect of metal ions and other reagents were
investigated. In addition, the potential of the _P. citreus enzyme(s) to
degrade dipeptides and the possible effect of shrimp protein in the
growth medium inducing the extracellular enzyme(s) were studied.
Results obtained from this investigation indicate that _P. citreus,
while growing on shrimp, may contribute to the overall decrease in shrimp
quality during iced or refrigerated storage. In addition, information
about the characteristics of the enzyme(s) produced by P. citreus will
be introduced

LITERATURE REVIEW
In 1978, the shrimping industry was the most valuable fishery in
the United States (8). However, the quality of shrimp often falls short
of that expected by the consumer. Approximately 15-20% of the shrimp
landed is eventually lost due to quality deterioration. This deteriora
tion of shrimp quality is usually attributed to rapid bacterial enzymatic
changes of the fresh shrimp resulting from mishandling and/or inadequate
processing. These changes, along with the chemical and physical methods
for measuring shrimp quality, are discussed as a basis for the
investigations presented in this dissertation.
Shrimp Spoilage
Quality changes in shrimp during storage on ice can lead to major
economic losses in the. shrimp industry. Mechanical damage, bacterial
contamination and enzymatic activity may combine to cause undesirable
changes in the composition and quality of shrimp (20,34,38,39,43,45,53,
54,77,78,79,89,94,108,117,153,154).
The loss of acceptability of shrimp may be triggered by several
factors: 1) shrimp muscle enzymes, 2) direct microbial activity, 3)
microbial enzymes and/or 4) a combination of these factors. Defects
which may occur as a result of such reactions are formation of malodor
ous substances, flavor deterioration, toughness, mushiness, juiciness,
dryness and discoloration (116).
Proteolytic enzymes play an important role in the spoilage of shrimp
by degrading muscle proteins and polypeptides, forming amino acids which
3

4
enrich the natural substrates and are thus available for the growth of
microorganisms. Enzymatic deamination and decarboxylation of amino acids
may also occur rapidly, resulting in the formation of spoilage products.
Pedraja (116) observed that from the moment a shrimp is taken out of the
water, its free amino acid pool is affected to some extent by osmoregula
tion and also by the struggle during catching. Therefore, the onset of
enzymatic and bacterial actions will vary according to the factors
affecting the substrates available in shrimp muscle.
Another factor that can induce shrimp spoilage is mechanical damage.
Handling shrimp on the boats results in mechanical damage to the muscle,
which will accelerate microbial invasion. The expressible fluid with its
protein and amino acid content serves as an excellent medium for growth
and reproduction of invading microorganisms (116).
Microbiological Characteristics of Shrimp
The muscle tissue of freshly caught shrimp is generally regarded as
sterile (26); however, work by Lightner (97) showed bacteria in the gut,
gills and between muscle bundles of brown shrimp. Reports on the number
2
of bacteria found on freshly caught shrimp range from 2.5 x 10 to 2.0 x
10^ organisms per gram (org/g) with Gulf coast shrimp averaging 1.0 x 10^
org/g, whereas bay shrimp averaged 1.0 x 10^ org/g (31,34,39,43,78,142).
Work completed in our laboratory has shown that fresh shrimp from the
Gulf of Mexico had bacterial counts ranging from 4.0 x 10^ org/g to 2.0
x 10^ org/g, while shrimp from the Atlantic coast had bacterial counts
ranging from 4.5 x 105 to 3.6 x 106 org/g (1).
Various kinds of bacteria have been reported on freshly caught
shrimp. Initially, the microbial flora is a mixture of organisms from
both the marine and terrestial environment. In the early 1950s, Campbell

5
and Williams (31) and Williams et al. (154) isolated species of Achromo-
bacter, Bacillus, Micrococcus, Flavobacterium and Pseudomonas from Gulf
coast shrimp. Vanderzant et al. (142) reported that the flora of shrimp
from the Gulf of Mexico consisted of coryneforms, Achromobacter, Flavo
bacterium and Bacillus. In Pacific shrimp, Acinetobacter-Moraxella spe
cies were predominant (80). Lee and Pfeifer (94) reported that the flora
of Pacific shrimp (Pandalus jordani) consisted of Moraxe11a, Pseudomonas,
Acinetobacter, Arthrobacter and Flavobacterium-Cytophaga species. Cann
/
(32) and Cann et al. (33) found that coryneform organisms were predomi
nant in the bacterial flora of scampi, Nephrops norvegicus, with strains
of Achromobacter-Acinetobacter group and Pseudomonas, Cytophaga and
Micrococcus species also present. Koburger et al. (90) reported that
the Flavobacterium-Cytophaga group represented the majority of the
organisms of fresh rock shrimp (Sicyonia brevirostris), and Alvarez (1)
and Alvarez and Koburger (3) reported that Flavobacterium and Pseudomonas
were the predominant groups isolated from Penaeus shrimp from the East
and West coasts of Florida.
When shrimp are stored in ice, the number and kinds of bacteria
shift to a predominantly psychrotrophic flora (130). Psychrotrophs are
described as organisms having an optimal growth temperature of about 20
C. A comparatively longer storage life of iced shrimp from tropical
waters has been reported by Carrol et al. (34). Cann et al. (33) in
their review on tropical shrimp indicated that penaeid shrimp from the
Gulf of Thailand remained in acceptable condition for 12-16 days on ice,
whereas nontropical shrimp, such as Pandalus and Nephrops species, were
totally spoiled after 8-10 days. They attributed this difference to the
bacterial flora; the mesophilic flora on tropical shrimp are not active

6
at ice temperatures and little spoilage occurs until the psychrotrophic
flora develops. Cann et al. (33) stated that the amount of spoilage may
be related to the degree to which psychrotrophic strains are introduced
with the ice. Consequently, the rate of increase in bacterial growth
depends on the initial number of bacteria, handling on deck, and amount
and quality of ice used. Shewan (130) demonstrated that the action of
many psychrotrophic organisms resulted in rapid fish spoilage. The
principal organisms he mentioned were Pseudomonas, Aeromonas, Vibrio,
Moraxella, coryneforms and Flavobacterium. Castell and Mappleback (35)
concluded that Flavobacterium was among the most important of the fish-
spoilage bacteria. Flavobacterium is a frequently encountered bacterium
on fresh shrimp flesh.
*\
The bacterial flora of shrimp undergoes marked changes as the stor
age period increases. Campbell and Williams (31) showed Bacillus, Micro
coccus and Flavobacterium made up over 50% of the flora initially,
whereas the Achromobacter-Pseudomonas group accounted for 98% of the
flora after 16 days of iced storage. In a study on the bacterial spoil
age patterns of headless brown shrimp, Cook (45) noted that there was
only one consistent change in the bacterial types growing initially or
during the period of die-off. As the bacterial count began to rise,
Pseudomonas species became the predominant organism, accounting for 80-
100% of the bacterial types isolated. Vanderzant et al. (142) reported
that the predominant bacterial flora of fresh shrimp consisted of
coryneforms and that following storage Pseudomonas species predominated.
Cobb et al. (43) indicated that typical spoilage organisms of the genus
Pseudomonas are not usually found in freshly caught shrimp. It is not
until the shrimp are exposed to handling on board the vessel that this
organism becomes apparent.

7
Alvarez and Koburger (3) reported that the numbers of Moraxella,
Vibrio/Aeromonas and Planococcus species isolated from Penaeus shrimp
remained relatively constant throughout 10 days of ice storage. However,
Flavobacterium isolates increased until the fifth day, then decreased
rapidly. Pseudomonas species showed the opposite trend. They decreased
until the fifth day, then increased rapidly. Other workers have observed
the presence of Flavobacterium in raw shrimp (31,80,90,94,142,143) and
have noted this decrease in numbers during ice storage with a subsequent
increase in Pseudomonas species. Cook (45) was unable to produce typical
spoilage when shrimp were inoculated with Flavobacterium species, indi
cating that they are probably an inert group of organisms found in
shrimp. In contrast, Pseudomonas species have been implicated as the
organisms primarily responsible for the spoilage of marine products
stored in ice (108,130).
Measurement of Shrimp Spoilage
Numerous methods for determining shrimp quality have been developed;
however, due to the complexity, time involved and inconsistent results of
many of these methods, only a few are routinely used by the industry and
then, only for internal quality control. In many of these chemical
tests, results can vary with the age of the shrimp, size, species, area
of catch and handling conditions. Many of the tests only indicate the
onset of spoilage (31,109). Table 1 lists the chemical and physical
tests that have been used to measure shrimp quality. Total volatile
nitrogen/amino acid-nitrogen (TVN/AA-N) ratio (40,41,42,43,64,75) is the
chemical test that shows the best correlation with organoleptic quality
measurements of shrimp. Moore and Eitenmiller (107) compared various
methods for measuring shrimp quality. They observed that a relatively

Table 1. Chemical and physical tests available to measure shrimp quality
Test
Parameter Measured
Reference
acid-soluble orthophosphate
trichloroacetic acid and soluble
orthophosphate
adenosine triphosphate and degradation of adenine dinucleotides
its degradation products to to hypoxanthine
hypoxanthine
alcoholic tumeric solution
changes in percent transmission of
a yellow tumeric shrimp solution
amino-nitrogen
changes in amine nitrogen content
ammonia
ammonia content
B-vitamin content
cathecol ferric chloride
dimethylamine
direct microscopic counts
fluorescamine
free fatty acids
glycogen
content of B-complex vitamins
change in percent transmission of
shrimp filtrate mixed with cathecol
ferric chloride
degradation of trimethyl amine oxide
by enzyme (TMO) to DMA
actively and non-actively
metabolizing bacteria
changes in free amine fractions
percent of free fatty acids
glycogen content
14
64
75
14,19,62,93
75,143
15
85
31
109
107
31
14,93

Table 1. (continued)
Test
hydrogen sulfide
hydration capacity
inosine monophosphate
indole
iodine titration
lactic acid
methylene blue reductase
peroxide number
PH
phenol red test paper
photoelectric reflection number
picric acid
skatole
Parameter Measured
Reference
measure of H^S presence 31
hydration of water insoluble protein
14,129,141
degradation of adenine dinucleotides
to IMP
132
utilization of tryptophane by
bacteria and its conversion
to indole
31,64,75,93
presence of iodine
14,64,75
lactic acid content
14
reduction of methylene blue by
bacteria
111
determines peroxide oxygen which
has formed at the double bonds in
unsaturated fatty acids
31
hydrogen ion concentration
14,19,68,69,85,
93,141
changes in pH
86
changes in light transmission of
shrimp extract
64
turbidity of shrimp filtrates with
picric acid
19
production of skatole
93

Table 1. (continued)
Test
Parameter Measured Reference
total fat
fat content 93
total nitrogen
ammonia content 93
trimethylamine nitrogen
degradation of trimethylamine oxide 14,19,31,44,64,67,
by enzyme (TMO) to TMA 75,85
total volatile nitrogen
volatile nitrogen compounds (ammonia) 64,75
tyrosine
free tyrosine levels 67
ultraviolet light-change
in fluorescence
ultraviolet absorption of 93
shrimp extracts
volatile acids
volatile acidic compounds 19,31,46,62,68,69
volatile reducing substances
measure of volatile nitrogen 62
compounds
volatile nitrogen
volatile nitrogen containing 85
compounds (ammonia)

11
new method using fluorescamine primarily detected only the non-protein,
non-ammonia, small molecular weight amines in shrimp homogenates. They
proposed that fluorescamine analysis could be useful in determining
changes in the free amine fractions. The shrimp industry still depends
on visual observation, smell and bacteriological testing for evaluating
overall shrimp quality, whereas, the Food and Drug Administration (FDA)
uses decomposition, filth and odor for the evaluation of shrimp quality.
Characteristics of Planococcus citreus
Koburger et al. (90) noted the presence of a high percentage of
gram-positive organisms following iced storage of rock shrimp (Sicyonia
brevirostris). These organisms comprised up to 68% of the isolates. Of
these 40% were Planococcus citreus, an aerobic gram-positive motile
coccus of marine origin producing an orange or yellow pigment. Informa
tion describing the isolation and characteristics of this organism is
limited. _P. citreus was previously named Micrococcus citreus (27). The
8th Edition of the Bergey's Manual of Determinative Bacteriology (29)
does have a description of the organism; however, it is limited in scope.
Cook in 1970 (45) and previous researchers working with shrimp placed
all aerobic gram-positive to gram-variable coccoid shaped bacteria in
the genus Micrococcus. In addition, Cook (45) noted that many of these
organisms isolated from shrimp were pigmented orange or yellow and were
motile. According to Bergey's manual (29), the only genus in the family
Micrococcaceae that is pigmented, either yellow or orange, and motile,
is Planococcus. This change in the taxonomic status of this organism
and the difficulty of demonstrating motility are probably the reasons
why Planococcus has not been reported in previous studies.
The taxonomic status of Planococcus citreus has changed markedly
through the years. In 1894 and again in 1900, Migula (103,104) made a

12
recommendation that flagellated cocci be included either in the genus
Planococcus or Planosarcina. This suggestion was accepted by only a few
authors, e.g., Krasil'nikov in 1949 (92). The majority of the authors
have included the flagellated cocci in the genus Micrococcus (22,83),
mainly because these cocci could only be differentiated from the other
members of the genus by their motility. Most authors have considered
motility to be a minor characteristic for the recognition of a new genus.
The findings of Bohacek et al. (23,24) that the flagellated cocci differ
considerably in the guanosine-cytosine (GC) content of their deoxyribo
nucleic acid (DNA) from other cocci shed new light on their taxonomic
position. It was proposed by Bohacek et al. (23) to include the flagel
lated cocci with a GC content ranging from 40-50% in the genus Piano-
coccus. In 1970, Kocur et al. (91) revised and outlined the genus
Planococcus. However, according to Index Bergeyana (86), the Planococcus
genus includes nine species (P. agilis, _P. casei, _P. citreus, _P. citro-
agilis, _P. europeans, P. loffleii, _P. luteus, _P. ochrolencus and J?.
roseus). Kocur et al. (91) evaluated the strains available in culture
and proposed that seven belong to one species, Planococcus citreus.
Although the remaining two species were closely related, he refrained
from giving a precise designation and labeled them only as Planococcus
species.
Schleifer and Kandler (126) found that the strains studied by
Bohacek et al. (23,24) and Kocur et al. (91) were uniform with respect
to the type of murein present in their cell walls and similar to that
of members of the genera Micrococcus and Staphylococcus. However,
serological investigation of _P. citreus by Oeding in 1971 (112) revealed
no antigenic relationship to staphylococci or micrococci.

13
Thirkell and Summerfield (137,138) studied the effect of varying
the sea salt concentration on the chemical composition of a purified
membrane fraction of _P. citreus. They concluded that the concentration
of salt in the medium affected the amount of membrane in the cell. Salt
concentrations above or below the normal 3% of sea water reduced the
amount of membrane material present. In addition, varying salt concen
tration had no significant effect on the amount of total neutral lipid,
glycolipid or phospholipid in the _P. citreus membrane preparations. But
a significant effect was observed on the amount of individual neutral
lipid or phospholipid classes present and on the number of individual
glycolipid components detected.
Our attention was directed toward this organism when, during a study
of the normal flora of rock shrimp (Sicyonia brevirostris), _P. citreus
was consistently isolated and found to increase in numbers during iced
storage (90). In this study, 68% of the isolates recovered were gram
positive cocci, with _F. citreus increasing from 10% of the isolates on
the fresh rock shrimp to 40% on the ice stored rock shrimp. In recent
work (1,2,3), _P. citreus has been found to be an important member of the
normal flora of Penaeus shrimp.
Alvarez and Koburger (5) described _P. citreus as a motile gram
positive coccus found in the marine environment, capable of growing over
a range of pH 7-10, 5-35 C, in broth containing 0.5-12% sodium chloride
(NaCl) and capable of hydrolyzing gelatin, cottonseed, soy and more
importantly to seafood microbiologists, shrimp protein. Figure 1 shows
a photomicrograph of citreus illustrating its morphology and flagella
tion. Table 2 shows the capabilities of this organism to hydrolyze
various protein sources.

14
Figure 1. Photomicrograph of Planococcus citreus cells showing morphology
and flagellation (6) (magnification 950X).

15
Table 2. Hydrolysis of various protein sources by selected strains of
Planococcus citreus at 25 C (4) (modification of Frazier (72)).
2
Protein Source
Isolate
i-H
G
H
4-
CT3
rH
0)
2
Whey
2
Cottonseed
iH
G
CO
cd
u
CO
&
6
H
Vj
x:
cn
m
rC
CO
H
2
Peanut
2
Corn germ
CM
>%
O
C/3
Hog blood
isolate^
2
Barley
A 17
+5
+
+
-
+
-
-
-
+
+
-
E 4
+
+
+
-
+
-
-
-
+
-
-
E 1
+
+
+
-
+
-
-
-
+
+
-
E 7
+
+
+
-
+
-
-
-
+
+
-
F 9
+
-
+
-
+
-
-
-
+
+
-
F 15
+
-
+
-
+
-
-
-
+
-
-
F 18
+
-
+
-
+
-
-
-
+
-
-
KS-1
+
-
+
-
+
-
-
-
+
-
-
KS-2
+
+
+
-
+
-
-
-
+
-
-
KS-3
+
-
+
-
+
-
-
-
+
-
-
KS-4
+
+
+
-
+
-
-
-
+
-
-
CS-1
+
+
+
-
+
-
-
-
+
-
-
From Difco Laboratories, Detroit, MI.
Protein isolates obtained from Southern Utilization Research and
Development Division, New Orleans, LA.
*Fresh samples were diluted 1:10 with 0.05 M phosphate buffer pH 7 and
ground in a Waring blender, dialyzed overnight with 10 volumes of the
same buffer (5 C) and lyophilized.
F. W. Knapp, Food Science and Human Nutrition Department, University
of Florida, Gainesville, FL.
'+ = hydrolysis; = no hydrolysis.

16
The reports (4,5) by Alvarez and Koburger outline some observations
on the distribution of _P. citreus in the marine environment. Of the 35
samples of marine origin examined for _P. citreus, only 5 yielded this
organism. Four were shrimp samples and the fifth was a stuffed flounder
sample that had been prepared in a plant that processed predominantly
shrimp. One of the shrimp samples from which Planococcus was isolated
had been in frozen storage for over six years. Fresh seafood (trout,
sheephead, mackerel, crab and oysters) as well as Gulf Coast waters and
sediments from the vicinity of Suwannee, Florida, were also examined for
_P. citreus without success. However, in more recent studies performed
by Mallory et al. (100), _P. citreus was isolated from estuarine areas of
Chesapeake Bay in low numbers.
Since the isolation of gram-positive organisms from iced seafood is
uncommon, Alvarez and Koburger (5) studied the contribution of _P. citreus
to the spoilage of Penaeus shrimp. They utilized gamma irradiation (600
Krads) to lower the number of bacteria in raw. shrimp and then inoculated
3
a portion of the shrimp with 5 x 10 _P. citreus cells per gram of shrimp
in order to study the changes produced by this organism. _P. citreus
3
counts increased in the inoculated shrimp from 5 x 10 bacteria/gram at
g
0 day to 1.9 x 10 bacteria/gram at the 16th day. The potential of _P.
citreus as a "spoiler" of shrimp was shown by an increase in pH and the
rapid increase in total volatile nitrogen/amino acid-nitrogen ratio (TVN/
AA-N) and trimethyl-amine nitrogen (TMN) content. In 1973, Cobb et al.
(42) reported a high correlation between total volatile nitrogen/amino
acid-nitrogen ratio (TVN/AA-N) and quality of shrimp. Later work (41)
suggested that the TVN/AA-N ratio and the logarithm of bacterial counts
increased at approximately the same rate after the initial lag phase of

17
bacterial growth and that a TVN/AA-N ratio of 1.3 indicated a limited
shelf life of the shrimp. Alvarez and Koburger (4,5) showed that _P.
citreus is capable of increasing the TVN/AA-N ratio at a similar rate as
the control sample (natural flora of shrimp). Thus, if the TVN/AA-N
ratio is an index of shrimp quality, _P. citreus is capable of shortening
the shelf life of shrimp. The proteolytic activity of this organism was
demonstrated by a significant decrease in percent extractable protein
(% TEP) in the early days of storage. Maximal percent TEP decrease was
observed between the 4th and the 12th day of storage of shrimp at 5 C
(5).
Proteolytic Enzymes
Enzymes are proteins with highly specific catalytic activities. As
catalysts, enzymes have the following properties: 1) they are effective
in small concentrations; 2) they remain unchanged in the reaction; 3) if
present in small concentrations relative to the substrate, they speed
attainment of equilibrium as reflected by increases in the rate constants
V
K. and K (A + B t > C + D, where A + B = reacting substances, C + D
K-1
= products of the A + B enzyme catalyzed reaction, = rate constant of
the forward reaction, K = rate constant of the reverse reaction).
However, an enzyme does not change the ratio K,/K = K (95).
1 1 eq
Most living organisms possess the ability to degrade proteins to
more readily absorbed substances. Such attacks on the peptide bond are
made possible by the presence of proteolytic enzymes. Although proteo
lytic enzymes from animal sources have been studied for more than a
century by both physiologists and biochemists, it was the work of Berg-
mann and Fruton (18) which led to a more complete understanding of the
mode of action of these enzymes. Their work established conclusively

18
that these enzymes exert a specificity toward the amino acids involved
in the peptide bonds which they attack. Bergmann and his students are
also responsible for the presently accepted classification of proteolytic
enzymes: They proposed that these enzymes be grouped into two classes
endopeptidases and exopeptidasesdepending upon whether they hydrolyzed
peptide bonds remote from, or near to, the end of the peptide chains of
their natural substrates. The former class includes such enzymes as
pepsin, trypsin and chymotrypsin, while the latter class contains the
dipeptidases and the amino and carboxy peptidases.
Proteinases in bacteria may be either intracellular or extracellular
depending upon whether they exert their activity within the cell or
whether they are excreted from the cell to attack proteins in the envi
ronment (10,58). Also, enzymes may be classified according to their
location in, on or around the cell: a) cell-bound: 1) truly intracell
ular, 2) surface-bound; and b) extracellular (58). Extracellular enzymes
are those enzymes which exist in the medium around the cell, having
originated from the cell without any alteration to cell structure greater
than that compatible with the cell's normal processes of growth and
reproduction. This distinction is not always clear and in some instances
it is entirely possible that autolysis of cells has permitted the escape
of intracellular enzymes into the culture filtrate. This is particularly
true when high proteolytic activity is dependent upon prolonged
incubation of the culture (74).
In 1964, the International Union of Biochemistry (54) recommended a
scheme for numbering enzymes, which is currently used for the classifi
cation of enzymes. Enzymes are divided into groups on the basis of the
type of reaction catalyzed, and this, together with the name(s) of the

19
substrate(s), provides a basis for naming individual enzymes. Each
enzyme number contains four elements; the first element (1 through 6)
shows to which of the 6 main groups of enzymes the particular enzyme
belongs (the six main groups are made on the basis of the general chemi
cal reaction catalyzed); the second and third elements show the subclass
and sub-subclass, respectively, thus defining the type of reaction; and
the fourth element is the serial number of the enzyme within its sub-sub-
class. Enzymes can be divided into six main groups: oxidoreductases,
transferases, hydrolases, lyases, isomerases and ligases.
Active extracellular proteinases are produced by numerous species of
Clostridium, Proteus, Bacillus, Pseudomonas, Micrococcus, Streptococcus,
Escherichia, Cytophaga and Staphylococcus (11,12,36,58,59,65,70,84,87,
105,110,113,114,118,136,139,155,157).
The continued study of these bacterial enzymes is important for at
least two reasons: (a) proteolysis by microorganisms plays an important
role in the biogeochemical cycles (74) and is responsible for numerous
environmental interrelationships; (b) the purification and the elucida
tion of their bond specificities are certain to lead to the discovery of
new enzymes with new properties not previously known.
Measuring Proteolytic Activity
Many methods are available for measuring proteolytic activity. Some
are based on the measurement of increase in protein (or nitrogen) solu
bility in the supernatant after centrifugation of the reaction mixture.
The most frequently cited method for measuring protein in solution is
that of Lowry et al. (98) in which the tyrosinetryptophan groups of
proteins in solution, or precipitated with acid, are reacted with alka
line Folin-phenol reagent after an alkaline copper treatment (71) to

20
produce a blue color that is measured in a spectrophotometer. Other
methods record proteolysis as the increase in ultraviolet absorption at
280 nm or the increase in absorbance (660 nm) of the tyrosine-tryptophan
filtrate after trichloroacetic acid (TCA) precipitation of the undigested
protein reacted with diluted (2:1) phenol reagent solution (9).
Schwabe (127) described a method which permited the assay of the
proteolytic enzyme activity on hemoglobin utilizing the fluorescamine
technique. The assay is about 100 times more sensitive than the Lowry
method, much faster and less complicated. He observed that the two main
obstacles for the successful use of fluorescamine in his assay system
were (1) the high blank produced by the reaction of e-amino groups of
the protein and (2) the fluorescent quenching effect of the hemoglobin.
The high blank of the hemoglobin he substantially suppressed by a chemi
cal modification, i.e., succinylation. Hemoglobin is usually used as a
2% solution of which only 10 pi are pipetted into 2 ml of phosphate buf
fer used for the reaction. He observed that the enzyme activity as mea
sured by the fluorescamine method remained linear throughout thirty min
utes while the Lowry method indicated a definite slowing of the reaction
beginning at about ten minutes. This was due to the fact that fluores
camine detects an increase in free amino groups while the Lowry reagent
as well as the direct measurement of absorption at 280 nm depends on the
production of tyrosine or tryptophan containing peptides. A possible
explanation for this discrepancy is that the enzyme in its initial
attack on the hemoglobin molecule releases large peptides which are TCA
soluble and that subsequent enzyme action further degrades these large
peptides without significantly increasing the number of TCA-soluble
fragments containing tyrosine or tryptophan moieties. A reagent
depending upon primary amine groups is not subject to this error (125).

21
Fluorescamine is a new reagent for the detection of amino acids,
peptides, proteins and primary amines in the picomole range (18,133,140).
Its reaction with amines is almost instantaneous at room temperature in
aqueous media. The products are highly fluorescent, whereas the reagent
and its degradation products are nonfluorescent.
McCaman and Robins (101) introduced a fluorometric method now widely
used for assay of serum phenylalanine which is based on the interaction
of ninhydrin and peptides. Samejima et al. (124,125) found that it was
the phenylacetaldehyde formed on interaction with ninhydrin which com
bined with additional ninhydrin and peptide or any other primary amine
to yield highly fluorescent products. The structure of these products
was subsequently elucidated by Weigele et al. (145), who then synthesized
a novel reagent (145). This reagent 4-phenylspiro (furan-2(3H),I'-
phthalan) 3,3'-dione (fluorescamine) reacts directly with primary amines
to form highly fluorescent products.
Several factors make fluorescamine suitable for assaying primary
amines, including amino acids, peptides and proteins. At pH 8-9, the
reaction with primary amines proceeds at room temperature (140) within a
fraction of a second. Excess reagent is concomitantly destroyed within
several seconds (140). Fluorescamine, as well as its hydrolysis prod
ucts, is nonfluorescent. Studies with small peptides have shown that
the reaction goes to near completion (about 80% to 95% of theoretical
yield) even when fluorescamine is not present in excess. The following
is an example of the reaction of fluorescamine with an amine group
illustrating the product formed (fluorophor) and the rate of the reaction
(100-500 msec). In addition, the reaction of water with fluorescamine
with the formation of a nonfluorescent product is also shown.

22
(nonfluorescent)
Hydrolysis products
(nonfluorescent)
Primary amines are first buffered to an appropriate pH (7-8), and
then fluorescamine, dissolved in a water miscible, nonhydroxylic solvent
such as acetone or dioxane, is added. The reaction is complete, and in
less than a minute excess reagent is destroyed. The resulting fluores
cence is proportional to the amine concentration, and the fluorophors
are stable over several hours. The above properties lend themselves
well to automation (123). It should be noted that fluorescamine does
not react with proline or hydroxyproline, which are not primary amines.
This disadvantage can be overcome by introducing an appropriate inter
mediate step to convert these amino acids to primary amines (63,146).
An additional advantage of the fluoresamine assay is that comparatively
little fluorescence is developed with ammonia. Therefore, ammonia does
not interfere with an analysis to the extent that it does in the colori
metric ninhydrin procedure. Figure 2 shows a comparison of the

OPTICAL DENSITY RELATIVE FLUORESCENCE
23
Figure 2. Comparison between the Fluorescamine technique and the Lowry procedure
(l^O) for determining protein concentration. a
Chromatography of a partially purified enzyme of guinea pig neuro-
physin monitored by the fluorescamine and the Lowry procedure.

24
fluorescamine technique with the standard Lowry procedure (97) for the
monitoring of protein in a column effluent. The volumes used for fluo
rescamine assay were 10 to 20 percent of those used in the Lowry method,
and smaller amounts could have been used (140). Background interference
was negligible with the automated fluorescence method, and significant
peaks not discernible by the Lowry procedure were observed.
Due to the many advantages of the relatively new fluorometric tech
nique, it was used to measure the proteolytic enzyme activity of P.
citreus. The ability of this organism to grow on shrimp as well as to
hydrolyze various protein preparations promoted investigations to iso
late, purify and characterize the extracellular enzyme(s) produced by
P. citreus.

MATERIALS AND METHODS
Unless otherwise specified, Difco (55,56) or Baltimore Biological
Laboratories (BBL) (16) products were used for all microbiological
analyses. Serial dilutions used Butterfields Phosphate buffer and fol
lowed the procedures outlined in the Compendium of Methods for the
Microbiological Examination of Foods (6). All chemicals used were
reagent grade meeting American Chemical Society specifications. All
media and glassware were autoclaved for 15 min at 121 C unless label
directions specified otherwise.
Planococcus citreus Cultures
The culture of _P. citreus used in this study, A-17, was isolated
from rock shrimp (Sicyonia brevirostris) (90). The culture chosen was
able to grow well in shrimp during iced storage and showed strong pro
teolytic activity toward various protein preparations. The isolate used
for the study was grown on Plate Count Agar slants (Difco) with 0.5%
sodium chloride (NaCl) added and incubated at 20 C for 72 hrs (4).
Appropriate dilutions in buffer were made to obtain a concentration of
3
approximately 5 x 10 organisms per ml. The A-17 isolate used was
capable of hydrolyzing gelatin, whey, cottonseed, soy, hog blood and
shrimp protein preparations (4). It also grew well in 0.5% to 16% NaCl
and pH 7.0 to 10.9.
Determination of Proteolytic Activity
A modified fluorescamine fluorescent (fluorometric) technique (129)
was used to measure enzyme activity. Fluorescamine is capable of
25

26
detection of amino acids, peptides, proteins and primary amines in the
picomole range (140).
P. citreus cells were grown in various media throughout the study.
After incubation, the cultures were centrifuged in a RC-5 Superspeed
Refrigerated Centrifuge (Sorval, Dupont Co. Instruments, Newtown, CT) at
a force of 20,000 x g for 30 min. The supernatant (cell-free broth) was
used for further investigations involving extracellular enzymes. The
cell pellet was washed twice with 0.05 M phosphate buffer (pH 8). The
whole cells were then resuspended with 10-20 ml of the same buffer,
transferred to a dry ice chilled Eaton pressure cell (60) and allowed to
freeze under dry ice for 3 hrs. The frozen microbial cells were disinte
grated using the Eaton pressure cell at a constant pressure of 7.03 x 10^
kg/m2 on a Carver hydraulic laboratory press (F. S. Carver, Inc., Summit,
NJ). The ruptured cell extract was fractionated into intracellular solu
ble and particulate fractions by centrifugation at a force of 12,000 x g
for 15 min. The particulate fraction was resuspended in 10 ml of 0.05 M
phosphate buffer prior to enzyme activity determinations of all frac
tions. Five milliliters of the substrate (gelatin or shrimp protein)
were reacted with 1 ml of each of the above fractions for 15 min at 35 C.
The reactions were terminated by adding 10 ml of 5% TCA. Zero time
blanks were prepared by adding the trichloroacetic acid (TCA, Fisher
Scientific Co., Fairlanes, NJ) before the incubation period (see latter
part of this section).
One milliliter of the cell-free broth or 100 yl of the purified
enzyme was reacted with 5 ml of substrate (gelatin or shrimp protein) for
the appropriate reaction time (to be determined) at 35 C, pH 8. The
enzyme-substrate reaction was stopped by precipitating the mixture with

27
10 ml of 5% TCA. After 5 to 10 min, to allow the proteins to settle,
the solution was filtered through Whatman #1 filter paper. Two hundred
microliters of the TCA filtrate were transferred to a 13 x 100 mm test
tube (Dispo culture tubes, Scientific Products, McGraw Park, IL) and the
volume brought to 1.5 ml with 0.5 M sodium phosphate buffer, pH 8.
0
While the test tube was vigorously mixed in a Vortex Mixture (Scientific
Products, Evanston, NY), 0.5 ml of fluorescamine in dioxane (30 mg/100
ml, Eastman Kodak Corp., Rochester, NY) was rapidly added to the buffered
protein solution. A model 204-A Fluorescence Spectrophotometer (Perkin
Elmer Corp., Norwalk, CT) was used to measure fluorescence intensity.
Zero time blanks were prepared by adding 10 ml of 5% TCA after adding the
enzyme and prior incubation of the mixture. This blank represented the
background activity present in the mixture at zero time. Zero time
fluorescence reading was subtracted from the reading of the substrate-
enzyme mixture after the appropriate incubation time.
Total enzyme activity was expressed as the change in 0.1 fluores
cence units of the TCA filtrate per milliliter of enzyme per minute.
Specific activity was expressed as the units of total enzyme activity/mg
of protein present (units of activity/mg of protein).
Previous research involving the use of the fluorescamine technique
(47,127,140) indicated that pH affected fluorescence intensity. Buffers
of pH from 2 to 10 (see buffers described on pg. 44) were used to deter
mine the effect of varying the pH of the buffer on fluorescence intens
ity. TCA filtrates (0.2 ml) were reacted with 1.3 ml of the various
buffers (pH 2-pH 10) before addition of the fluorescamine reagent.
Figure 3 indicates that addition of pH 8 buffer resulted in the highest
fluorescence itensity. Consequently, pH 8 buffer was used for the
remainder of the research.

23
Figure 3. Effect of pH adjustment of gelatin-trichloroacetic acid (TCA).filtrates
vfiaorescence intensity.

29
In order to determine the reaction spectrum of our working enzyme
solution when gelatin and shrimp protein were used as substrates, the
excitation and emission (fluorescent) wavelengths were scanned (48).
Figure 4 shows the excitation (curve A) and fluorescence (curve B)
spectra for TCA filtrates of the gelatin substrate. The excitation
spectrum has a maximum of 360 nm and a secondary peak at 390 nm. The
secondary peak at 390 nm was chosen because it results in minimal zero
time blank fluorescence values. The fluorescence emission maximum with
the excitation wavelength at 390 nm was at 475 nm. Figure 5 shows the
excitation (curve A) and fluorescence (curve B) emission spectra for TCA
filtrates of the shrimp protein substrate. The excitation spectrum has
a maximum peak at 375 nm and a secondary peak at 390 nm. Again, the
secondary peak was chosen. The fluorescence emission maximum with the
excitation wavelength at 390 nm was at 490 nm.
Efficacy of 5% Trichloroacetic Acid (TCA)
In order to determine the efficacy of 10 ml of 5% trichloroacetic
acid (TCA) in terminating the enzyme-substrate reaction, 5 ml aliquots
of substrate (gelatin) were incubated with 1 ml of cell-free broth and
10 ml of 57o TCA for 0, 10, 15, 30, 45 and 60 min at 35 C. A positive
control was done by incubating the enzyme-substrate mixture at 35 C for
0, 10, 15, 30, 45 and 60 min before adding the TCA. After the incubation
period, the positive control was terminated by adding 10 ml of 5% TCA.
t
Data in Figure 6 shows that 10 ml of 5% TCA were adequate for inhibiting
the enzyme substrate reaction effectively since there was no increase in
fluorescence intensity. The fluorescence intensity, observed when TCA
is immediately reacted with the enzyme and substrate, represents the
background fluorescence of the assay mixture.

Figure 4. Exitation (curve A) and fluorescence (curve B) spectrum for the reaction of a gelatin-trichloro
acetic acid (TCA) filtrate with fluorescamine at pH 8.

Figure 5. Exitation (curve A) and fluorescence (curve B) spectrum for the reaction of a shrimp protein-
trichloroacetic acid (TCA) filtrate with fluorescamine pH 8.

Figure 6. Efficacy of trichloroacetic acid (TCA) in terminating the enzyme-substrate reaction.

33
Substrate Characteristics
Two substrates were used throughout the study: gelatin (Difco)
(1.2 mg/ml) and a shrimp protein preparation (0.6 mg/ml). Higher concen
trations of the shrimp protein preparation were not used because of
solubility problems in the buffer systems used. The shrimp protein prep
aration was prepared as follows: fresh raw shrimp meat ground in a War
ing blender with 0.05 M phosphate buffer (pH 7, 1:10 dilution), dialyzed
overnight with four changes of the same buffer at 5 C and lyophylized for
preservation (Virtis Freeze Dryer, Gardiner, NY). Protein, fat, moisture
and ash were determined for the shrimp protein preparation. Protein was
determined by the AOAC standard micro-Kjeldahl method (13). Crude fat
was determined by a modification of the AOAC method (13) using the Gold-
fisch solvent chamber. Approximately 2 grams of sample were extracted
overnight with petroleum ether. Moisture was determined in a vacuum
oven at 70 C for 12 hrs. Ashing was done in a muffle furnace at 600 C
for 8 hrs. Table 3 shows that the shrimp protein preparation consisted
of 77.44% protein, 5.40% fat, 8.95% moisture, 6.50% ash and 1.71%
carbohydrate (calculated by difference).
Determination of Enzyme-Substrate Mixture Reaction Time
Five milliliters of substrate and an aliquot of cell-free broth were
incubated at 35 C for 0, 5, 10, 15, 20, 30 and 60 min in order to deter
mine the time course of enzyme activity and apparent optimum reaction
time. In experiments involving gelatin, 0.5, 1.0 and 2.0 ml of cell-free
broth were used while 1.0 ml of cell-free broth was used with the shrimp
protein substrate. An incubation time of 15 min was an appropriate
enzyme-substrate contact reaction time when shrimp were used as substrate
(Figure 7). In addition, when gelatin was used as a substrate and

34
Table 3. Proximate composition of the shrimp protein preparation.3
Percent (%)
Protein
77.44
Fat
5.40
Moisture
8.95
Ash
6.50
Carbohydrate^*
1.71
£
Average of two determinations
^Calculated by difference

A FLUORESCENCE
35
Figure 7. Increase in fluorescence intensity using the shrimp protein
preparation as substrate after incubation with cell-free
broth for up to 1 hr at 35 C.

36
various amounts of cell-free broth were reacted with gelatin, a 15 min
reaction time was also a sufficient substrate reaction time (Figure 8).
This reaction time was used for the remainder of the study.
Growth Medium and Enzyme Production
Various media were used to determine growth rates and production of
extracellular enzyme(s) by _P. citreus. Three hundred milliliters of
Plate Count Broth (PCB) + 0.5% NaCl, Nutrient Broth (NB) + 0.5% NaCl and
Trypticase Soy Broth (TSB) were used to grow the organism. Incubation
was at 20 C for up to 96 hrs. Samples were drawn at 0, 12, 24, 48, 72
and 96 hrs. All samples were assayed for growth by measuring optical
density at 600 nm in a Spectronic-20 Spectrophotometer (Bausch and Lomb,
Rochester, NY) and by plating in Plate Count Agar (PCA) with incubation
at 20 C for 5 days. Cultures were centrifuged in a RC-5 Superspeed
Refrigerated Centrifuge at a force of 20,000 x g for 30 min. The sedi
ment was discarded. One milliliter of the cell-free broth was assayed
for enzyme activity with gelatin substrate. _P. citreus growth and
enzyme analyses were done three times and each time in duplicate.
Optimization of Enzyme Activity to Growth and Cell Number
The optimum time for cell harvesting along the logarithmic section
of the growth curve (approximately midlog) of the organism was selected.
The logarithm of the cell count was plotted against the incubation time.
The specific growth rate (k) of each medium was calculated using the
equation In B = In B + k (where B = bacterial count at time x, B =
x o x o
bacterial count at time 0 (both in midlog growth phase), k = specific
growth rate, t = time, hrs). The units of enzyme activity per cell per
hour (m) were then calculated using the formula (58,124): M M = £
t ok
(Bx BQ) (where Mt = enzyme activity at cell number B M = enzyme

Figure 8. Increase in fluorescence intensity using gelatin as substrate and various amounts of the cell-
free broth after incubation at 35 C for 1 hr.

38
activity at cell number B k = specific growth rate). This formula was
o
din k'
obtained through the integration of the following equation: = m Bq e
(where dm/dt = change in enzyme activity over time). The calculated m's
were compared for the three media used. A test to observe any difference
between the values for the units of enzyme activity per cell per hour (m)
in each medium was designed using the Statistical Analysis System (SAS)
program package (15) for analysis of variance. A completely randomized
design (102,132) was used in that the major source of error to be
considered was due to the nutritional differences between media.
Effect of Incubation Temperature on Enzyme Production and Activity
The effect of incubation temperature (5, 20 and 35 C) on the growth
of _P. citreus and its ability to produce an active extracellular enzyme
was investigated. Five-hundred-milliliter Erlenmeyer flasks containing
3
100 ml of medium were inoculated with approximately 5 x 10 P_. citreus
and incubated at the three temperatures. All samples were assayed for
growth by measuring optical density at 600 nm in a Spectronic-20 spectro
photometer and for proteolytic activity using the fluorometric technique.
For the 35 C grown cells, samples were drawn at 0, 6, 12, 24, 48, 72 and
96 hrs. For the 20 C grown cells, samples were drawn at 0, 12, 24, 48,
72, 96 and 120 hrs. Finally, for the 5 C grown cells, samples were drawn
at 0, 24, 48, 72, 96, 120 and 144 hrs. After determining the midlog
phase of growth for _P. citreus at each temperature, _P. citreus cells were
then harvested at this stage. Enzyme activity determinations were done
using the cell-free broth obtained from growing the organism at the three
temperatures until midlog phase. Five milliliters of the shrimp protein
preparation were incubated with 1 ml of each cell-free broth at 5 C for
60 min, 20 C for 30 min and 35 C for 15 min. Analyses were done three
times and each time in duplicate.

39
In addition, direct microscopic observations of the cells grown at
5, 20 and 35 C were conducted. Any morphological change due to growth
temperature was observed and recorded.
Purification of the Extracellular Enzyme(s)
Planococcus citreus was grown in the selected medium at 20 C until
midlog phase. The cells were then centrifuged at a force of 20,000 x g
for 30 min. This cell-free broth was used in the purification procedure.
Ammonium Sulfate Precipitation
Fractional precipitation of the enzyme(s) in the cell-free broth was
accomplished with 0-55%, 55-70% and 70-100% ammonium sulfate saturation
(Mallinckrodt, Inc., Paris, KY). The required amount of ammonium sulfate
was added with stirring until dissolved (88). The mixture was allowed to
equilibrate for 1 hr at 4 C and centrifuged at a force of 20,000 x g for
20 min. The precipitate was resuspended with 10 ml of Butterfield's
phosphate buffer at pH 8 and dialyzed for 16 hrs (dialysis tubing #24,
Scientific Products, McGraw Park, IL) against 500 ml of 0.05 M phosphate
buffer pH 8 (108,153). Each ammonium sulfate fraction was then assayed
for proteolytic activity using gelatin as substrate.
Molecular Sieve Chromatography
Sephacryl S-200 Superfine (Pharmacia Fine Chemicals, Uppsala,
Sweden), a high resolution chromatographic medium for gel filtration was
used to separate the enzyme solution according to molecular weight after
the ammonium sulfate precipitation step. A 30 x 2.5 cm column was packed
R R
with Sephacryl S-200 Superfine gel and a Pharmacia peristaltic pump
(p-3) was used to pack the column at a speed of 40 ml/cm/hr (120). The
enzyme solution was eluted using reverse flow at a speed of 30 ml/cm/hr.
Five milliliter fractions were collected in each tube with 0.02 M phos-

40
phate buffer pH 7 as the eluting agent using a Gilson Fraction collector
(Model FC-220K Fractionator, Gilson Medical Electronics, Inc., Middle-
town, WI). Enzyme activity of each fraction collected was then measured
using gelatin as substrate. The protein present in the fractions was
determined by following absorbance at 280 nm using a Beckman Model 25
spectrophotometer (Beckman Instruments, Inc., Fullerton, CA).
Ion-Exchange Chromatography
The active fractions recovered from the gel filtration step were
pooled and further separated by ion-exchange chromatography using DEAE-
Sephadex A-50 (Pharmacia Fine Chemicals, Uppsala, Sweden). The column
was prepared following the procedures given by Pharmacia Fine Chemicals
(119). A 40 x 2.5 cm column was prepared and the protein eluted with
0.02 M phosphate buffer with a linear gradient of tris 0.01 M NaCl-tris
0.15 M NaCl (47) at a rate of 25 ml/hr (1.5 reading in the peristaltic
pump) and collected in 10 ml fractions. Two hundred fifty milliliters
of 0.01 M and 0.15 M NaCl solutions were placed in each vessel for the
linear gradient. The protein present in the fractions was followed by
reading the absorbance at 280 nm using a Beckman Model 25 spectrophotom
eter. Enzyme activity of each fraction was measured with gelatin as the
substrate. All column studies were duplicated.
The protein content of each fraction eluted using the Sephacryl
S-200 Superfine and the DEAE-Sephadex^ A-50 was also analyzed by the
Lowry (98) method for protein with Bovine Serum Albumin (Sigma Chemical
Co., St. Louis, MO) as the standard. Protein content is expressed as
mg/ml. Figure 9 summarizes the steps followed in the purification of
the extracellular proteolytic enzyme(s) of P. citreus.

41
STERILE
TRYPTICASE SOY BROTH
Figure 9: Outline of steps for the purification of the extracellular
protease(s) of Planococcus citreus.

42
Confirmation of Enzyme Purity
A modification of the Weber and Osborn (144) method for sodium
dodecyl sulfate-poly acrylamide gel (SDS-PAG) gel electrophoresis was
used. A Buchler 3-1500 electrophoresis apparatus (Buchler Instruments
Corp., Fort Lee, NJ) was used to evaluate the purity of the isolated
extracellular enzyme.
A 10% acrylamide:BIS, 30:0.8 gel was prepared and allowed to poly
merize for 2 hrs. A sample of the purified enzyme was diluted 1:1 with
the sample buffer. The sample buffer consisted of 0.01 M sodium phos
phate (pH 7), 10% sodium dodecyl sulfate, 0.1% dithiothrietol, 10% glyc
erol and 0.001% bromocresol blue. The protein solutions were placed onto
the gels (50 yg protein/gel, 100 yg/gel, 150 yg/gel and 200 yg/gel) and
were layered carefully with electrode buffer (pH 8.3) to the top of each
tube. The lower electrode chamber was then 2/3 filled with electrode
buffer. The tubes in the apparatus were then lowered into the electrode
chamber. The upper chamber was filled with water to approximately 1 inch
over the tube top. The water jacket was connected and the electrode
wires from the power source were also connected. A constant current of
1-1.5 mAmps/gel was applied until the marker dye band just exited from
the gels (approximately 3 hrs). The gels were immediately removed from
their tubes. The gels were fixed overnight in a fresh 50% TCA solution.
The fixed gels were then stained 1-2 hrs with 0.1% Coomassie brilliant
blue solution made up fresh in 50% TCA at 37 C in a water bath. The gels
were further diffusion-destained by repeated washings in 7% acetic acid
(17-72 hrs). Gels were then stored in 7% acetic acid (82).

43
Characterization of the Proteolytic Enzyme(s)
Molecular Weight Determination
Two methods (7,21,150) were used to estimate the molecular weight
of the enzyme(s).
A 2.5 x 30 cm column packed with Sephacryl S-200 (Pharmacia Fine
Chemicals, Uppsala, Sweden) was used and the following standards applied:
Ribonuclease A (13,000 MW), Trypsin (23,500 MW), Pepsin (45,000 MW),
Bovine Serum Albumin (70,000 MW) and Aldolase (158,000 MW) (Pharmacia
Fine Chemicals, Piscataway, NJ) following the procedures suggested by
Pharmacia Fine Chemicals (116). The value of each protein sample was
calculated and plotted against the corresponding molecular weight
V V
6 O
(K = 7T~ where V = column void volume, V = elution volumes and
av Vt V o e
t o
= total column volume). Protein was monitored at 280 nm using a
Beckman Model 25 spectrophotometer (Beckman Instruments, Inc., Fullerton,
CA).
Bio-Rad Low Molecular Weight Protein Standards (10,000-100,000) for
SDS Gel Electrophoresis were also used for molecular weight determination
D
using SDSPAG gel electrophoresis. The instructions outlined by Bio-Rad
(Bio-Rad Laboratories, Richmond, CA) were followed (21). The proteins
included were Phosphorylase B, Bovine Serum Albumin, Ovalbumin, Carbonic
Anhydrase, Soybean Trypsin Inhibitor, Lysozyme and the purified enzyme.
The motility of the enzyme was then compared to the relative motility
(Rm) of the standards.
Determination of the Purified Enzyme-Substrate Mixture Reaction Time
Five milliliters of substrate (gelatin or shrimp protein) and 100 yl
of the purified enzyme were incubated at 35 C for 0, 5, 10, 15, 30 and 60
min in order to determine the apparent optimum reaction time.

44
Enzyme activity was measured using the Fluorescamine technique. Figure
10 shows that 10 min was the optimum reaction time for the purified
enzyme-substrate (gelatin or shrimp protein) reaction mixture. This
optimum reaction time was used for the remainder of the characterization
of the extracellular proteolytic enzyme.
Effect of Ionic Strength on Enzyme Activity
The effect of ionic strength on enzyme activity was investigated.
Gelatin (1.2 mg/ml) was dissolved in the following solutions of sodium
chloride (NaCl): 0.05 M (y 0.13), 0.08 M (y = 0.16), 0.18 M (y =
0.26), 0.25 M (y = 0.35), 0.34 M (y 0.42), 0.51 M (y = 0.59), 0.75 M
(y = 0.83), 1.00 M (y = 1.08) and 1.5 M (y = 1.58). The NaCl was dis
solved in 0.05 M phosphate buffer (pH 8). Five milliliters of this mix
ture were reacted with 100 ul of the purified enzyme and incubated at
35 C for the selected reaction time (10 min).
Determination of Optimum pH
Buffers of varying pH from pH 2 to pH 10 were used to determine the
optimum pH for the proteolytic activity of the enzyme(s). The following
buffers were used:
Ionic Strength
pH 2
0.1 M citric acid
0.25
3
47.0 ml of 0.1 M citric acid + 3.5 ml of
0.1 M sodium citrate
0.35
4
33.0 ml of 0.1 M citric acid + 17.0 ml of
0.1 M sodium citrate
0.45
5
20.5 ml of 0.1 M citric acid + 29.5 ml of
0.1 M sodium citrate
0.45
6
88 ml of 0.2 M monobasic sodium phosphate +
12.5 ml dibasic sodium phosphate
0.45
7
39.0 ml of 0.2 M monobasic sodium phosphate +
61.0 ml of 0.2 M dibasic sodium phosphate
0.35

A FLUORESCENCE
45
INCUBATION TIME AT 35 C (min)
Figure 10. Increase in fluorescence intensity following incubation of
gelatin and shrimp protein substrate with purified enzyme
for up to 1 hr at 35 C.

46
Ionic Strength
8 5.3 ml of 0.2 M monobasic sodium phosphate 0.25
+ 95.0 ml of 0.2 M dibasic sodium phosphate
9 50 ml of 0.2 M glycine + 8.8 ml of 0.2 M NaOH 0.22
10 50 ml of 0.2 M glycine + 32.0 M NaOH 0.20
Gelatin (1.2 mg/ml) or shrimp protein (0.6 mg/ml) were dissolved in
the various buffers. Any pH adjustments due to the addition of the sub
strates were done using 10 mM HC1 or 10 mM NaOH. Five milliliters of
this mixture were reacted with 100 pi of the purified enzyme and incu
bated at 35 C for the selected reaction time (10 min).
Determination of Optimum Temperature
Five milliliters of gelatin or shrimp protein substrate and 100 pi
of the purified enzyme were incubated at 5, 10, 20, 35, 45, 55 and 65 C
for 10 min at the optimum pH determined in the previous section.
Thermal Stability
_P. citreus was incubated at 5 and 35 C in 300 ml of Trypticase Soy
Broth (TSB). Cell-free broths obtained at midlog phase, 108 and 36 hrs
for the 5 and 35 C grown cells, respectively, were used in this study.
Five milliliters of the cell-free broths were incubated at 35, 45, 55,
65, 75 and 85 C for 15 min. The heat treated cell-free broths solutions
were rapidly cooled (87), and their activity was assayed at 35 C for 15
min using gelatin as substrate. The residual activities at each solution
were compared to the activity observed when the cell-free broths were
incubated with the substrate at 35 C for 15 min.
In addition, 1 ml of the purified enzyme was also incubated at 35,
45, 55, 65, 75 and 85 C for 10 min. The heat treated purified enzyme
solution was cooled, and its activity assayed at 35 C for 10 min using

47
gelatin as substrate. The residual activities of each solution were
compared to the activity observed when the purified enzyme was incubated
with gelatin at 35 C for 10 min.
Effect of Sodium Chloride Concentration
Various concentrations of NaCl were tested for their effect on
enzyme activity. Concentrations of 0.00, 0.25, 0.50, 0.75, 1.00, 1.25
and 1.50% were used. NaCl was dissolved in 0.05 M phosphate buffer pH 8.
The shrimp protein (0.6 mg/ml) and gelatin (1.2 mg/ml) were dissolved in
the NaCl solutions. Five milliliters of the NaCl solutions were incu
bated with 100 yl of the purified enzyme at 35 C for 10 min.
Effect of Sodium Bisulfite Concentration
Various concentrations of sodium bisulfite (NaHSO^) were tested for
their effect on enzyme activity. Concentrations of 0.0, 0.5, 1.0, 2.0
and 3.0% were tested. NaHSO^ (J. T. Baker Chemical Co., Phillipsburg,
NJ) was dissolved in 0.05 M phosphate buffer pH 8. The shrimp substrate
was dissolved in these NaHSO^ solutions (0.6 mg/ml). Five milliliters
of the NaHSO^ solutions were incubated with 100 Pi of the purified
enzyme at 35 C for 10 min.
Effect of Enzyme Concentration
Various quantities of enzyme (from 0 to 200 Pi) were tested to
observe the effect of enzyme concentration on enzyme activity. Five mil
liliters of substrate (gelatin or shrimp protein) were incubated with 0,
50, 75, 100 and 200 pi of enzyme at 35 C for 10 min.
Effect of Substrate Concentration
The enzyme was incubated with various concentrations of gelatin and
shrimp protein in order to determine substrate saturation conditions.
For the gelatin substrate, 0.00, 0.15, 0.30, 0.45, 0.60 and 1.20 mg/ml

48
were tested. However, for the shrimp substrate, 0.000, 0.075, 0.100,
0.125, 0.150, 0.300 and 0.600 mg/ml were tested. Five milliliters of
each substrate solution were reacted with 100 yl of the purified enzyme
at 35 C for 10 min. From these data, Lineweaver-Burk plots were derived,
and K and V values for each substrate were extrapolated from these
m max
plots (95,152).
Effect of Metal Ions on Enzyme Activity
Calcium chloride (10, 20 mM), ferric chloride (1, 20 mM), magnesium
chloride (10, 20 mM), mercurous chloride (1, 20 mM), zinc chloride (10,
20 mM), manganese chloride (10, 20 mM) and potassium chloride (5, 20 mM)
were tested for their effect on enzyme activity (all metals were dis
solved in 0.05 tris-HCl buffer). For the control, a buffer with no
metal ions added was used (76,87). Five milliliters of substrate (gela
tin) 100 yl of enzyme and 1 ml of the metal ion buffer solution were
reacted for 10 min at 35 C. The fluorometer reading of the control
sample was compared to^ the reading of the metal ion samples.
Effect of Various Reagents on Enzyme Activity
Ethylene diaminetetraacetic acid (EDTA) (10, 20 mM), citric acid
(10, 20 mM), formaldehyde (1, 20 mM), potassium cyanide (KCN) (1, 20 mM),
potassium permanganate (KMnO^) (1, 20 mM), cysteine (1, 20 mM), 2-
mercaptoethanol (1, 20 mM), p-dioxane (10, 20 mM) and trichloroacetic
acid (TCA) (5, 10%) were tested for their effect on the proteolytic
activity of the J?. citreus enzyme (76,87). All reagents were dissolved
and/or mixed with 0.05 M tris-HCl buffer. Five milliliters of substrate
(gelatin), 100 yl of enzyme and 1 ml of the appropriate reagent buffer
solution were reacted at 35 C for 10 min. A control with no reagent
added was used and the fluorometer reading from the various reagents was
compared to the control.

49
Dipeptidase Activity
The potential of the P. citreus enzyme to degrade peptides was
investigated. DL-leucylglycine, DL-leucyl-DL-alanine, glycyl-DL-leucine,
DL-alanylglycine and L-leucyl-l-tryptrophan (Sigma Chemical Co., St.
Louis, MO) were used in this study. Fifty milligrams of each dipeptide
were dissolved in 50 ml of phosphate buffer, pH 8. Five milliliters of
the dipeptide solutions were incubated with 100 pi of the purified
enzyme at 35 C for 10 min. The reaction was terminated by adding 10 ml
of 5% TCA. Zero time blanks were done by adding the TCA to the enzyme-
peptide mixture before the incubation period.
Enzyme Induction Studies
_P. citreus was grown in various media in order to determine if the
extracellular proteolytic enzyme produced by this organism is induced by
shrimp protein. Three-hundred milliliters of the following were used:
(1) Yeast Carbon Base (YCB) (control)
(2) YCB + 1.0% Shrimp Protein
(3) YCB + 0.1% Yeast Extract
(4) YCB + 0.1% Yeast Extract + 1.0% Shrimp Protein
Table 4 shows the composition of the Yeast Carbon Base medium (YCB).
_P. citreus growth and enzyme activity were analyzed at 0, 24, 48, 72
and 96 hrs following incubation at 20 C. Cell numbers were determined
by pour plating into Trypticase Soy Agar (TSA) with incubation at 20 C
for 5 days. Five milliliters of the shrimp substrate were incubated
with 1 ml of the cell-free broth from each culture for 15 min at 35 C.
The reaction was terminated by adding 10 ml of 5% TCA. Zero time blanks
were done by adding the TCA to the cell-free broth-substrate mixture
before the incubation period. This study was done twice in duplicate.

50
Table 4. Composition of yeast carbon base medium (56).
Formula in Grams per Liter of Distilled Water
Boric Acid
0.500
mg
Copper Sulfate
0.040
Potassium Iodide
0.100
Ferric Chloride
0.200
Manganese Sulfate
0.400
Sodium Molybdate
0.200
Zinc Sulfate
0.400
Biotin
0.002
mg
Calcium Pantothenate
0.400
Folic Acid
0.002
Inositol
2.000
Niacin
0.400
p-Aminobenzoic Acid
0.200
Pyridoxine
0.400
Riboflavin
0.200
Thiamine HC1
0.400
L-Histidine HC1
0.001
g*
DL-Methionine
0.002
DL-Tryptophan
0.002
Potassium Phosphate
1.000
g
Magnesium Sulfate
0.500
Sodium Chloride
0.100
Calcium Chloride
0.100
Dextrose
10.000
Final pH of the base adjusted to 7.5

51
The data was analyzed in a similar manner as the Growth Medium and
Enzyme Activity data. Again, a comparison of the m's for each medium
used (m = units of enzyme activity/cell/hr) was attempted using the SAS
program package for analysis of variance (15).
The Duncan's New Multiple-Range Test (pg. 187-190 (132)) was used to
compare any difference in the calculated means of the data obtained after
analysis of variance in the "Optimization of enzyme activity to growth
and cell number" section (pgs. 36 and 38), "Optimum pH determination"
section (pg. 82), "Optimum temperature determination" section (pg. 84)
and "Enzyme induction study" section (pg. 105-115). The Duncan's New
Multiple-Range Test was done using the SAS program package (15).

RESULTS AND DISCUSSION
\
The ability of Planococcus citreus to grow in shrimp during ice
storage raised the question as to whether this organism could contribute
to the spoilage of shrimp. Various studies (3,4,5) have indicated that
this organism may contribute to the spoilage of this valuable marine
/
resource. In order to more clearly understand the contribution this
organism makes to the degradation of shrimp, an investigation was under
taken to study the extracellular proteolytic enzyme(s) produced by this
organism.
Proteolytic Activity of Cellular Fractions
The proteolytic activity of cellular fractions of _P. citreus cells
grown in Trypticase Soy Broth (TSB) was investigated in order to deter
mine the distribution of the enzyme activity in the isolated fractions.
In addition to the cell-free broth (extracellular fraction), whole cells,
washings of the whole cells, soluble intracellular and the cellular
particulate fraction were examined. Table 5 shows the total activity
(units of activity), protein content (mg/ml), specific activity (units
of activity/mg of total protein) and distribution of activity (%) for
all the fractions tested using both gelatin and shrimp protein sub
strates. The extracellular fraction showed the highest specific activ
ity, 29.450 units of activity/mg of protein and 27.540 units of activ
ity/mg of protein towards gelatin and shrimp protein, respectively.
This represented 95.9 and 95.8% of the total activity present in all of
the fractions towards gelatin and shrimp protein, respectively.
52

Table 5. Proteolytic activity at 35 C for 15 min (pH 8) of cellular fractions obtained from
Planococcus citreus grown in Trypticase Soy Broth (TSB) using gelatin and shrimp as
substrates.
Fractions
Total Enzyme
Activity (units)
Protein3
(mg/ml)
Specific Activity*3
(units/mg total protein)
Distribution of
Activity (%)
Gelatin
Shrimp
Gelatin
Shrimp
Gelatin
Shrimp
Whole cells
386.7
395.6
23.40
0.136
0.139
0.4
0.5
Extracellular
131,100.0
122,590.0
44.50
29.450
27.540
95.0
95.8
1st washing
251.1
253.3
6.26
0.401
0.406
1.3
1.4
2nd washing
163.3
133.3
3.26
0.500
0.408
1.6
1.4
Intracellular
101.1
81.1
10.33
0.098
0.079
0.4
0.3
Particulates
171.1
192.2
11.26
0.152
0.171
0.6
0.6
^Average of duplicate samples
Total activity/Total protein = specific activity (units of activity/mg protein)

54
The whole cell fraction (cell bound fraction), both whole cell washings
(loosely bound to cell wall fraction) and the particulate fraction
exhibited low specific activity towards both high molecular weight sub
strates. The intracellular soluble fraction (the soluble fraction after
the differential centrifugation of ruptured cells) exhibited the lowest
specific activity when gelatin and shrimp protein were used as substrates
(Table 5). These results show that the major portion (>95.0%) of the
active enzyme towards these two high molecular weight substrates resides
in the extracellular fraction.
Most microorganisms can synthesize various enzymes within their
cell structure. Each enzyme system may have its own unique characteris
tics, and these characteristics will vary depending on the enzyme, the
substrate and conditions during the enzyme-substrate reaction. In addi
tion, the location of the proteinase(s) within the bacterial cell may
vary markedly between microorganisms. Various researchers (53,115,135,
139) have studied the location of particular bacterial proteinases
within the cell and how this location relates to the function of the
enzyme. Thomas et al. (139), using gentle procedures for cell fraction
ation, suggested two criteria for the location of a proteinase produced
by Streptococcus lactis. The two criteria they suggested were: 1)
intact cells (whole cells) possessed substantial proteinase activity
when incubated with a high molecular weight substrate; 2) most of the
cell-bound proteinase activity was released during spheroplast formation.
The solubilized cell wall, plasma membrane and cytoplasm fractions con
tained 84%, 0% and 16% activity, respectively, of the total proteinase
activity with casein as substrate (139). In the results presented in
this dissertation, the whole cell and cellular particulate fractions of

55
the P. cltreus cells showed little enzyme activity towards gelatin and
shrimp protein (both high molecular weight substrates).
Thomas et al. (139) also concluded that the cell wall proteinase
may serve a similar nutritional role in nature as the surface-bound pro-
teinases discussed by Payne and Gilvarg (115) and Sussman and Gilvarg
(135). Gilvarg and his co-workers stated that surface-bound protein
ase (s) appear to serve a nutritional role by hydrolyzing proteins to
amino acids or peptides that are small enough to enter the cell. In
turn, Payne, Sussman and Gilvarg (115,135) also suggested that the
intracellular peptidases could further hydrolyze the peptides formed and
release their constituent amino acids, thus, permitting the utilization
of the protein substrate for growth. In Table 5, we can observe that
certain _P. citreus fractions (whole cells, intracellular and cellular
particulate) had substantial amounts of protein present. Perhaps some
of the protein present in these fractions include other enzymes (i.e.,
peptidases) that can utilize the peptides produced by the action of the
extracellular protease(s) that later may enter the _P. citreus cell. In
this manner, _P. citreus cells could fully utilize the protein available
(i.e., shrimp protein as well as other proteins) for their growth.
Growth Medium and Enzyme Production
Trypticase Soy Broth (TSB), Plate Count Broth (PCB) + 0.5% NaCl and
Nutrient Broth (NB) + 0.5% NaCl were used to determine growth rates and
production of extracellular proteolytic enzyme(s) by _P. citreus. Figure
11 shows the growth of _P. citreus, as measured by the increase in optical
density (600 nm), in the three media used. In all three media, P.
citreus exhibited a 12 hr lag phase in which an increase in optical den
sity was not evident. After this lag period, TSB supported the most

OPTICAL DENSITY (600 nm)
56
INCUBATION TIME AT 20C(hr)
Figure 11. Spectrophotometric growth curves of Planococcus citreus in
Plate Count Broth, Nutrient Broth and Trypticase Soy Broth
at 20 C.

57
rapid growth of P. citreus. The optical density after 96 hours of incu
bation at 20 C was .314, .200 and .072 for TSB, PCB and NB, respectively
Figure 12 shows a similar trend; however, P. citreus growth was measured
by the Aerobic Plate Count technique (6). Again, we can observe that
the _P. citreus log count per ml increases slightly during the first
12 hours of incubation at 20 C. After 96 hrs of incubation, the _P.
citreus log count for TSB, PCB and NB was 6.02, 5.10 and 4.17, respec
tively. Consequently, TSB allowed for the "optimum" growth of _P.
citreus when grown at 20 C.
Nutritional components present in the growth medium are of utmost
importance for gram-positive microorganisms which are generally more
fastidious in its nutrient requirements than gram-negative bacteria (29)
Realizing these growth requirements of gram-positive microorganisms, the
results from this section are not surprising. TSB contains tryptone,
soytone, dextrose, sodium chloride and dipotassium phosphate (16). This
combination of nutrients provide an adequate nitrogen, carbohydrate,
vitamin and overall nutrient supply for the growth of _P. citreus. In
contrast, PCB and NB are not as nutritionally complex.
Figure 13 shows the enzyme activity of the cell-free broth of P.
citreus cells grown in TSB, NB and PCB for 96 hrs at 20 C. An active
extracellular enzyme fraction was produced by P. citreus in all three
media. However, after 96 hrs of growth, the amount of enzyme produced
by this organism in TSB is greater than that produced when grown in PCB
of NB. The enzyme activity after 96 hrs of incubation of the cell-free
broth of _P. citreus grown in TSB, PCB and NB was 323, 270 and 200 units
of activity, respectively. However, if the cells are harvested at
approximately midlog phase, the difference in the amount of enzyme

LOG PLANOCOCCUS CITREUS/ml
58
INCUBATION TIME AT 20 C (hr)
Figure 12. Aerobic plate counts of Planococcus citreus incubated in
Plate Count Broth, Nutrient Broth and Trypticase Soy Broth
at 20 C for 96 hrs.

ENZYME ACTIVITY (xIO1)
59
INCUBATION TIME AT 20C (hr)
igure 13. Enzyme activity at 35 C for 15 min (pH 8) of the cell-free broth
of Planococcus citreus cells grown in Plate Count Broth, Nutrient
Broth and Trypticase Soy Broth for up to 96 hrs.

60
produced (as measured by enzyme activity) is more clearly observed.
After 48 hrs of incubation, the enzyme activity for the _P. citreus grown
in TSB, PCB and NB was 200, 100 and 85.5 units of activity, respectively.
Under the conditions tested, _P. citreus exhibited maximum enzyme produc
tion when grown in TSB.
The units of activity per cell per hr (m) was calculated for each
medium used and the results are shown in Table 6. The average m value
for TSB, PCB and NB was 168.50, 105.67 and 59.32, respectively. These
data show that the amount of enzyme produced by actively growing _P.
citreus cells (midlog phase) in TSB is higher than that produced when
_P. citreus is grown in PCB or NB. The analysis of the data supports
this observation. A significant difference (a = 0.05 level) was
observed between the m values of TSB, PCB and NB (Table 6).
Thus, after evaluating the results from this section, TSB was cho
sen as the best medium for _P. citreus growth and enzyme production and
was used for the remainder of the study. The combination of nutrients
in TSB allowed for the rapid growth of _P. citreus and by doing so, per
mitted the production of more extracellular proteolytic enzyme. In
addition to the combination of nutrients in TSB, the presence of 0.25%
dextrose may play a role in extracellular enzyme production. Dextrose
has been suggested as a possible inducer of a variety of enzymes (57),
although this effect was not specifically tested in these experiments.
Effect of Incubation Temperature on Enzyme Production and Activity
The ability of _P. citreus to produce an active extracellular enzyme
at 5, 20 and 35 C was investigated in order to determine the ability of
the extracellular enzyme(s) to affect shrimp protein at refrigeration
(5 C) or iced temperatures. Figure 14 shows the increase in optical

61
Table 6. Units of enzyme activity per cell per hour (m) of Planococcus
citreus grown in Trypticase Soy Broth (TSB), Plate Count
Broth (PCB) and Nutrient Broth (NB) at midlog phase.
Medium
mean m value^
TSB
148.50a
PCB
105.67b
NB
59.32C
'
Cells were grown at 20 C and enzyme activity was measured at 35 C
2for 15 min (pH 8).
average of 6 observations
Means followed by the same letter do not differ significantly at the
a = 0.05 (r from Anova table 0.984)

62
Figure 14. Spectrophotometric growth curves of Planococcus citreus in
Trypticase Soy Broth incubated at 5, 20 and 35 C~

63
density of _P. citreus cultures grown at 5 C for 144 hrs, 20 C for 120
hrs and 35 C for 96 hrs. After different time intervals for growth
adaptation, _P. citreus grew at all three temperatures. Direct micro
scopic observation of _P. citreus cells growing at the three temperatures
revealed a difference in cell arrangements. When cells were grown at 35
C, the predominant morphology present was clusters of gram-positive cocci.
At this temperature, the cells are rapidly growing and dividing, thus,
possibly accounting for the observed predominance of clusters. At 20 C
a mixture of tetrads, pairs and single cells were observed which is the
predominant morphology exhibited by this organism, as described in
Bergey's Manual of Determinative Bacteriology (29). At 5 C the predomi
nant cell arrangement observed was single cells. This particular morpho
logical structure might predominate because of the slow metabolic rate
at this temperature, although these rates were not investigated. Indi
vidual cells have more surface area for the uptake of nutrients. At 5 C,
the movement of nutrients within the cell is slow. Consequently, the
increase in surface area is particularly desirable from a nutritional
standpoint.
Table 7 illustrates the enzyme activity of the cell-free broth of
_P. citreus grown at 5 C for 108 hrs, 20 C for 72 hrs and 35 C for 36
hrs (midlog at each temperature) and incubated with shrimp protein at 5,
20 and 35 C for 60, 30 and 15 min, respectively. _P. citreus produced an
active extracellular enzyme(s) when grown at all temperatures. In addi
tion, the cell-free broth obtained from the three temperatures of growth
exhibited activity at all three enzyme-substrate incubation temperatures
(5, 20 and 35 C). As the temperature of growth increased from 5 to 35
C, the enzyme activity increased at a similar rate at the three enzyme-

Table 7. Enzyme activity measured at 5, 20 and 35 C (pH 8) of the cell-free broths of Planococcus
citreus grown in Trypticase Soy Broth (TSB) at 5, 20 and 35 C for 108, 72 and 36 hrs,
respectively.
Enzyme-Substrate
Incubation Temperature (C)
Temperature of Growth (C)
5
20
35
5
24.18
+
l.lla
58.18 2.08
116.17
8.40
20
25.65
/
+
1.20
72.30 2.04
139.67
7.88
35
28.56

1.35
98.55 3.10
178.93
7.68
Average of 6 observations standard deviation

65
substrate incubation temperatures. A higher _P. citreus count was
observed at 35 C and the production of extracellular enzyme(s) was also
higher at all three enzyme-substrate incubation temperatures. This
indicates that the amount of enzyme produced by _P. citreus is related to
the amount of growth of the organism in the medium. In addition, as the
enzyme-substrate incubation temperature increased from 5 to 35 C, the
enzyme activity of the cell-free broths increased. Although enzyme
activity is present at the lower temperatures, the data presented indi
cate that the optimum temperature of the extracellular protease system
may be close to 35 C. Consequently, the results indicate that _P. citreus
can indeed produce an active extracellular enzyme(s) capable of utilizing
the protein in shrimp when shrimp is stored at refrigerated or iced
temperatures.
The effect of refrigeration temperatures on enzyme activity has
been studied (50,51). In most of the research, the majority of the
enzymes studied lost activity when incubated at low temperatures. Stud
ies have shown that lactic streptococci characteristically produced less
acid after storage at refrigerated temperatures. Such stored cells also
show a diminished residual proteinase activity (49,50,51,52,149). The
researchers stated that after storage at 3 C, the enzyme showed gross
structural alterations with a concomitant loss of activity. Gel filtra
tion and sedimentation velocity data indicate that inactivation of the
enzyme was a result of aggregation to higher molecular weight forms (50).
However, several investigators (49,52,131) previously suggested that
storage inactivation of enzymes may be caused by induced conformational
or structural changes. Scutton and Utter (128) and Havir et al. (81)
observed that inactivation of various enzymes by low temperature storage

66
was due to dissociation of the molecules into subunits. The inactivated
enzymes could be reactivated by warming to room temperature.
Purification of the Extracellular Enzyme(s)
Planococcus citreus was grown in Trypticase Soy Broth (TSB) at 20
C for 72 hrs. The cell-free broth was used in the isolation of the
extracellular enzyme(s) of this organism. The cell-free broth had a
total activity of 1.31 x 10^ units of activity (total enzyme activity =
change in 0.1 fluorescence units of the TCA filtrate per milliliter of
enzyme per minute), 44.52 mg/ml of protein and a specific activity of
29.45 units of activity/mg protein (Table 8). The cell-free broth was
then fractionated with 0-55%, 55-70% and 70-100% ammonium sulfate
((nh4)2so4).
After overnight dialysis (16 hrs) in phosphate buffer pH 8, the
activity of the 0-55%, 55-70% and 70-100% ammonium sulfate precipitates
was measured. Table 9 shows the proteolytic activity of the various
fractions examined. The specific activity of each fraction was 4.59,
52.39 and 3.99 units of activity/mg of protein for the 0-55%, 55-70% and
70-100% fractions, respectively. Eighty-six percent of the activity was
present in the 55-70% fraction. This is compared to 7.5 and 6.5% for
the 0-55% and 70-100% fractions, respectively (Table 9). Ammonium sul
fate precipitation is a common method used to precipitate proteins for
their purification. As the ammonium sulfate concentration is raised
from zero, the solubility of a given protein at first usually increases
but then the "salting-in" effect comes to an end and as the salt concen
tration is raised to higher values a "salting-out" effect is observed
and the protein becomes progressively less soluble (65). The major por
tion of the extracellular proteolytic enzyme(s) of P. citreus was salted

Table 8. Purification of an extracellular proteolytic enzyme from Planococcus citreus
Fraction
Volume
Total Activity3
(units x 10 )
Protein*5
(mg/ml)
Specific Activity0
(units/mg total protein)
Purification
Recovery
(%)
Cell-free broth
900
113.3
44.52
29.45
100.0
70% (N4) S04
precipitation
350
102.7
/
19.60
52.39
1.78x
78
Sephacryl
S-200 Superfine
115
65.3
1.42
461.65
15.67x
50
DEAE-Sephadex^
A-50
90
64.0
0.32
780.37
26.50x
49
a
^Determined using gelatin as substrate
Average of duplicate samples
Total activity/Total protein = specific activity (units of activity/mg protein)

Table 9. Proteolytic activity at 35 C for 15 min (pH 8) of various ammonium sulfate
fractions of the cell-free broth of Planococcus citreus.
Fraction
Total Activity3
(units x 10 )
Protein^
(mg/ml)
Specific Activity0
(units/mg total protein)
Distribution of
Activity (%)
0-55%
8.31
18.07
4.59
7.5
55-70%
102.70
19.60
52.39
86.0
70-100%
5.00
12.50
3.99
6.5
g
^Determined using gelatin as substrate
^Average of duplicate samples
Total activity/Total protein = specific activity (units of activity/mg protein)

69
out between 55-70% ammonium sulfate saturation. Table 9 shows that 86%
of the activity towards gelatin is observed in this fraction. Table 8
shows that the activity of the 55-70% ammonium sulfate fraction was 1.78
times greater in specific activity than the cell-free broth. A 78%
recovery of the extracellular enzyme(s) was achieved in this step of
the enzyme purification.
£
Sephacryl S-200 Superfine, a high resolution chromatographic
medium for gel filtration of proteins, nucleic acids, polysaccharides
and biopolymers (120), was used to separate the enzyme(s) present in the
55-70% ammonium sulfate fraction according to molecular weight. Figure
15 shows that four protein peaks were recovered after the elution of the
£
enzyme fraction through the Sephacryl S-200 column. However, when the
proteolytic activity was measured, the majority of the activity was pre
sent in protein peak C (third peak in Figure 15). Peak C had a specific
activity of 651.0 units. The enzyme(s) was purified 15.67 times and 50%
of the enzyme was recovered in this step (Table 8). The fractions com
prising peak C were pooled for further purification. The percent recov
ery of the extracellular proteolytic enzyme of _P. citreus after molecular
sieve chromatography using Sephacryl S-200 Superfine was within the
range of most of the enzymes recovered when the more traditional
£
Sephadex gels have been used (70,113,114,136).
The pooled fractions of peak C were further rechromatographed using
DEAE-Sephadex A-50 (functional group -C^H^N+iC^H^)A-50 gels are
usually used for low and medium molecular weight proteins (up to
200,000). Ion exchange chromatography may be defined as the reversible
exchange of ions in solution with ions electrostatically bound to an
insoluble support medium. The ion exchanger is the inert support medium

ABSORBANCE (280 nm
R
Figure 15. Separation of proteins present in the 55-70 % ammonium sulfate fraction using Sephacryl
S-200. a
cl
30 x 2.5 cm column, eluted with 0.02 M phosphate buffer pH 7.
I
ENZYME ACTIVITY (xIO

71
to which is covalently bound positive (in the case of the anionic
exchanger) or negative (in the case of a cation exchanger) functional
groups (48). A sodium chloride (NaCl) gradient (range of ionic strength,
y = 0.11 0.23) was used with the ion exchange column to elute the pro
tein components. A gradient is a physical method of constantly changing
the salt concentration of a solution that is being passed through the
column creating a constant and linear increase in concentration (48).
Figure 16 shows one major peak after ion exchange of the pooled active
fractions from peak C. The isolated peak exhibited a specific activity
of 780.37 units (Table 8). The proteolytic enzyme was purified 26.50
times and 49% recovery was achieved (Table 8). Fractions 17 to 21
(Figure 16) were pooled for future characterization.
Purity of the Extracellular Proteolytic Enzyme
Many methods can be used to establish the purity of an enzyme prep
aration. However, the best indication of purity of an enzyme prepara
tion is by the consistent failure to detect heterogeneity when several
analytical techniques are used (i.e., a single peak in chromatographic
systems, a single band on electrophoresis, a single band after isoelec
tric focusing and/or one component in solubility or precipitation tests).
However, the final criterion for purity is the demonstration of a unique
amino acid sequence (61,65) but this is rarely done in order to
demonstrate purity.
The recovery of the isolated peak (Figure 16) as a single entity
with homogeneous activity after DEAE-Sephadex A-50 ion-exchange chroma-
tography was the first indication that the major extracellular
proteolytic activity of _P. citreus was isolated in a purified form.

Figure 16. Ion-exchange chromatography using DEAE-Sephadex^ A-50 of the pooled active fractions obtained
in the molecular sieve chromatography step.
a40 x 2.5 column, eluted with 0.02 M phosphate buffer pH 7 with a linear
gradient of 0.01 M NaCl- tris 0.15 M NaCl at a rate of 25 ml/hr.

73
According to Cooper (48) electrophoretic techniques have become
principal tools for characterizing macromolecules and for assaying their
purity. Figure 17 shows a single band after SDS-PAG electrophoresis
using 50 pi of the purified enzyme. A single homogeneous band is indic
ative of the presence of only one enzyme, i.e., the purity of the extra
cellular enzyme of _P. citreus. In addition, as an additional test for
purity, increasing amounts of the purified extracellular enzyme were
added to the gels. Enzyme concentrations of 50 yg protein/gel, 100 yg/
gel, 150 yg/gel and 200 yg/gel were used. Figure 18 shows that a single
band is recovered after SDS-PAG electrophoresis of each protein fraction.
Thus, these results add to the evidence indicating the purity of the
extracellular proteolytic enzyme of _P. citreus. Consequently, an extra
cellular proteolytic enzyme produced by _P. citreus was purified 26.50
times using the procedures outlined previously with 49.0% of the enzyme
being recovered (Table 8). The specific activity of the enzyme was
780.37 units of activity/mg protein.
Schwabe (127) reported the use of the fluorescamine reagent to mea
sure proteolytic enzyme activity of cathepsin enzymes using hemoglobin
a substrate. He stated that while the fluorescamine reagent has been
used successfully for quantitative amino acid analysis, protein and pep
tide determination, it has also beneficial applications in enzymology.
In addition, Schwabe (127) compared the fluorometric technique with the
Lowry method (98). He concluded that the fluorometric method was 100
times more sensitive than the Lowry method, much faster and less com
plicated. The fluorometric technique proved to be an efficient method
for the measurement of proteolytic enzyme activity.

74
Figure 17. Acrylamide gel electrophoresis of the purified enzyme of
Planococcus citreus.

75
50 yg/gel 100 yg/gel 150 yg/gel 200 yg/gel
Figure 18. Acrylamide gel electrophoresis of increasing concentrations
of the purified extracellular enzyme of Planococcus citreus.

76
With the presence of the relatively new Fluorometric technique,
that appears to be more sensitive and reproducible than the traditional
methods available for measuring proteolytic enzyme activity, the results
of various previous research with extracellular enzymes (36,78,84,88,
136) using the Anson method (9) could have possibly resulted in higher
recoveries and higher measurable total enzyme activity. The following
investigators are some of those who used the Anson method to study the
various enzymes. Tarrant et al. (136) working with Pseudomonas fragi in
pig muscle isolated an extracellular proteolytic enzyme with only 18%
recovery after partial purification. Husein and McDonald (84) character
ized an extracellular proteinase from Micrococcus freudenreichii using
casein as substrate with 23% recovery after partial purification.
Christison and Martin (36) isolated and preliminarily characterized an
extracellular protease of Cytophaga spp. using casein, hemoglobin and
£
azocoll as substrates. After chromatography with DEAE-Cellulose only
26% of the enzyme was recovered. Khan et al. (88), looking at the
extracellular proteases of Mucor pusillus, isolated and characterized
two fractions. However, after DEAE-Sephadex A-50 only 29.3% of the
milk-clotting fraction was recovered and 47.0% of the fraction with pro
tease activity toward hemoglobin was recovered. Gnosspelius (76) puri
fied an extracellular protease from Myxococcus virescens using phosphate
precipitation, gel exclusion and ion exchange chromatography. Only
20.1% was recovered after the chromatographic step. In the work reported
U
in this dissertation, following DEAE-Sephadex A-50, 49.0% of the extra
cellular enzyme of _P. citreus was recovered when the Fluorometric method
was used to measure proteolytic activity.

77
Characterization of the Extracellular Proteolytic Enzyme
The fractions collected (17-21) from peak B (Figure 16) were pooled
and used for the characterization of the extracellular enzyme of _P.
citreus.
Molecular Weight Determination
Two methods were used to determine the molecular weight of the
enzyme, column chromatography (Sephacryl S-200 Superfine) and acrylamide
gel electrophoresis. Standards ranging from a molecular weight of 10,000
to 200,000 were used to determine the molecular weight of the _P. citreus
enzyme. Using both techniques, the molecular weight of the extracellular
enzyme of this organism was approximately 29,000. Figures 19 and 20 show
£
the molecular weight determination using Sephacryl S-200 and acrylamide
gel electrophoresis, respectively. Different standards were used in each
case to assure that the molecular weight was estimated correctly.
A search of the literature was done in order to compare the molecu
lar weight of the extracellular proteolytic enzyme of J?. citreus with
extracellular proteases from other microorganisms. Pacaud and Uriel
(113) estimated the molecular weight of a protease from Escherichia coli
using electrophoresis on polyacrylamide gels and sucrose-density gradient
centrifugation to be about 43,000. Four years later, Pacaud and Richaud
V
(114) estimated the molecular weight of a second protease of E. coli
using gel filtration and SDA-acrylamide gels to be 58,000. Drapeau et
al. (59) estimated the molecular weight of an extracellular protease of
Staphylococcus aureus to be approximately 12,000 using sedimentation
equilibrium and gel electrophoresis studies. Arvidson et al. (12)
reported the molecular weight of an extracellular (alkaline protease)
enzyme from _S. aureus to be approximately 12,500. Later, he reported

78
Figure 19. Calibration curve for the molecular weight estimation o.
Planococcus citreus proteolytic enzyme using Sephacryl
S-200 column chromatography.
q
40 x 2.5 column, eluted with 0.02 M phosphate
buffer at a rate of 15 ml/hr.

MOLECULAR WEIGHT
Figure 20. Calibration curve for the molecular weight estimation of Planococcus citreus proteolytic
enzyme using acrylamide gel electrophoresis.
al0 % acrylamide: Bis 30:0.8 gel, 1-1.5 mAMP/ gel for 3 hrs.

80
(11) the molecular weight of a EDTA-sensitive S.. aureus protease as
28,000. Recently, Hoshida et al. (137) estimated the molecular weight
of a proteolytic enzyme from Bacillus sphaericus to be about 26,000.
Gnosspelius (76) working with an extracellular enzyme of Myxococcus
virescens reported its molecular weight as 26,000. Thus, the apparent
molecular weight of the extracellular enzyme of _P. citreus (MW 29,000)
is within the range of other extracellular proteolytic enzymes reported
in the literature.
Effect of Ionic Strength on Enzyme Activity
The effect of salts on the solubility of proteins is well known.
The solubility is usually a function of the ionic strength. In condi
tions of high ionic strength, the ions attract around themselves the
polarizable water molecules, making less water available for the pro
teins since, at high salt concentrations, the number of charged groups
contributed by the salts is enormous compared with those of the proteins.
Consequently, the solubility of the proteins decreases (152). In addi
tion, any change in the charges of an enzyme may cause various transfor
mations in structure or active site configuration that could affect its
activity towards the substrate. Figure 21 shows that ionic strengths (p)
of 0.15-0.83 did not alter the attraction of the _P. citreus extracellular
enzyme towards gelatin substrate. However, as the ionic strength was
increased the activity of the enzyme decreased. An ionic strength of
1.60 (1.5 M NaCl) caused a decrease in enzyme activity of approximately
60%. Thus, if ionic strengths above 0.83 (0.75 M NaCl) are used they
may cause a change in solubility of the enzyme, charged groups, confor
mation of the enzyme, active site stability and/or active site availa
bility to the substrate. Gnosspelius (76) stated that variations in the

ENZYME ACTIVITY (x I01)
IONIC STRENGTH (/l)
Figure 21. Effect of ionic strenght on the activity of the extracellular proteolytic enzyme of
Planococcus citreus.
aActivity assayed at 35 C for 10 min, pH 8.

82
ionic strength did not signficantly influence the activity of Myxococcus
virescens when casein was used as the substrate. However, the actual
data for this observation were not presented in the literature.
In that the activity of the proteolytic enzyme was not affected by
ionic strengths of p=0.83 or lower, the buffers shown in page 44,
(Determination of Optimum pH) were considered acceptable and were used
for the determination of the optimum pH of the _P. citreus enzyme.
Optimum pH Determination
The pH optimum of an enzyme is dependent upon a number of experi
mental parameters. Changes in pH may cause changes in the ionization
of prototropic groups (groups capable of ionization) in the active site
of an enzyme. These prototropic groups in the active site may be
involved in maintaining the proper configuration of the site, in binding
a substrate to enzyme and/or in transformation of substrate to products
(133). However, there is usually a zone of maximum ion stability in
which enzyme activity is maximal. Enzyme inactivation also increases on
the acid and alkaline sides of this maximum activity zone. Observing
Figure 22, enzyme activity was maximum at pH 8 when both gelatin and
shrimp protein were used as substrates. The activity dropped as the pH
became more acidic or alkaline. Although not statistically significant,
a slightly higher activity was evident at the alkaline pH's (9 and 10)
when shrimp protein was used as substrate.
The majority of the bacterial enzymes studied have shown maximum
proteolytic activity at neutral pH's (57,65,151). The enzyme isolated
in this study resembles the bacterial proteolytic enzyme from Proteus
vulgaris (105), Bacillus sphaericus (155), Staphylococcus aureus (11,12),
Serratia marcescens (106) and Pseudomonas spp. (87) in that they all

ENZYME ACTIVITY (x 10')
83
pH
Figure 22. Optimum pH of the extracellular proteolytic enzyme of
Planococcus citreus.a
a
Gelatin and shrimp protein substrate incubated
at 35 C for 10 min, pH 8.

84
require a slightly alkaline pH for optimum activity. Considerable
activity is present at neutral pH's; the pH of freshly caught shrimp is
around neutrality. During shrimp storage, the pH of shrimp will
increase (5).
Optimum Temperature Determination
Changes in temperature may affect enzymatic reactions in a number
of ways. Some of these effects may include: a) stability of the enzyme;
b) affinity of enzymes for activators and inhibitors; c) ionization of
prototropic groups; d) enzyme-substrate affinity; and e) velocity of
breakdown of enzyme-substrate complex (131). The optimum temperature of
the _P. citreus extracellular enzyme when both shrimp protein and gelatin
were used as substrates was 35 C (Figure 23). Although not statistically
signficant, a slightly higher enzyme activity was observed at the lower
temperatures (5 and 10 C) using shrimp protein as substrate as compared
to gelatin. However, at the higher temperatures (45 and 55 C) the
reverse was evident. Thus, as previously observed with the cell-free
broth (enzyme crude extract), the purified enzyme of _P. citreus can
exhibit activity at the temperatures of refrigerated shrimp.
Thermal Stability
The cell-free broths obtained from _P. citreus cells grown in Trypti-
case Soy Broth (TSB) at 5 and 35 C for 108 and 36 hrs, respectively, and
the _P. citreus purified enzyme were incubated at 35, 45, 55, 65, 75 and
85 C for 15 min in order to study the various temperatures effects on
stability. The enzyme activity of the cell-free broths and purified
enzyme remaining after the various heat treatments was assayed using
gelatin as the substrate and compared to the activity observed with the
unheated cell-free broths and purified enzyme. Figure 24 shows the

ENZYME ACTIVITY (xIO1)
Si
Figure 23. Temperature optimum of the extracellular proteolytic enzyme of Planococcus citreus.
Activity assayed at 35 C for 10 min, pH 8.

TEMPERATURE (C)
Figure 24. Thermal stability of the enzymes in the cell-free broths of Planococcus citreus grown at
5 and 35 C and of the purified enzyme.
aCell-free broths incubated for 15 min at each temperature and activity assayed
at 35 C for 15 min (pH 8) and purified enzyme incubated for 10 min and activity
assayed at 35 C for 10 min (pH 8).

87
thermal stablity of the cell-free broth of JP. citreus cells grown at 5
and 35 C and of the purified enzyme. In general, as the temperature
increased, the activity of all three fractions decreased. After 15 min
of incubation at 65 C, all the activity was lost in the purified enzyme
fraction and only 1% was left in the 35 C cell-free broth fraction.
However, 28% of the activity still remained in the 5 C cell-free broth
fraction. After 15 min at 75 C, 1% of the activity of the 5 C cell-free
broth fraction still remained. Perhaps the enzymes in the cell-free
broth of _P. citreus grown and stored at 5 C have undergone a structural
change (50,51) or have a slightly different structure than the enzyme
produced at 35 C. This change could result in an enzyme conformation
with an active site that is more protected from increased temperatures.
Usually, an enzyme is more stable to temperature changes in an
intact tissue or in an homogenate where its structure is protected by
the presence of other colloidal material (i.e. proteins, carbohydrates,
etc.) than it is in a purified form (147,151). However, in general,
those enzymes which have molecular weights ranging from 12,000 to 50,000
are composed of single polypeptide chains and having disulfide bonds are
usually more resistant to heat treatment. The larger the enzyme and the
more complex its structure the more susceptible it is to increases in
temperature (151). Figure 24 shows that the cell-free broths are more
stable to heat than the purified enzyme.
Effect of Sodium Chloride Concentration
The effect of increasing sodium chloride (NaCl) concentration on
enzyme activity was examined. Concentrations of 0-1.50% NaCl were
investigated. When shrimp protein was used as the substrate, the activ
ity of the extracellular enzyme increased until 0.50 g/100 ml NaCl (0.5%)

88
was reached, then the activity started decreasing. However, when gelatin
was used as the substrate, salt concentration (0-1.50%) had no apparent
effect on the activity of the enzyme (Figure 25). The concentrations of
the NaCl solutions used in this study, 0-0.26 M NaCl, have an ionic
strength of p=0.15-0.34 and are not within the ionic strength range that
resulted in decreased enzyme activity (Figure 21).
The effect observed when shrimp protein is used as substrate is
probably due to an initial increase in solubility of the substrate due
to the increase in salt concentration. Structurally, gelatin is a small
protein when compared to shrimp protein. Perhaps the increase in solu
bility allowed an easier enzyme-substrate interaction, thus, accounting
for the initial increase in enzyme activity.
The effect of higher concentrations of NaCl on enzyme activity was
not investigated. However, Figure 21 illustrates that an ionic strength
of 0.83 (0.75 M NaCl) or above resulted in decreased enzyme activity.
Reversible inactivation of the enzyme and substrate effects due to higher
NaCl concentrations were not investigated.
Arvidson and coworkers (11,12) showed that the activity of both
extracellular proteases I and II from Staphylococcus aureus (neutral and
alkaline protease, respectively) was reduced by concentrations of 0.5 M
NaCl or above. Gnosspelius (76) stated that an NaCl concentration of 0.2
M in the assay mixture had no effect on the activity of the Myxococcus
virescens extracellular enzyme. However, higher NaCl concentrations
decreased the proteolytic activity.
During the storage of Penaeus shrimp on ice, the salt concentration
will decrease due to the leaching of the salt as the ice melts or perco
lates through the shrimp. As the NaCl concentration decreases in shrimp,
the activity of the J?. citreus enzyme will be enhanced.

ENZYME ACTIVITY (x I01)
Figure 25. Effect of sodium chloride (NaCl) concentration on enzyme activity.3
Activity assayed at 35 C for 10 min (pH 8).

90
Effects of Sodium Bisulfite Concentration
To control black spotting in shrimp (148), sodium bisulfite (NaHSO^)
is used to inhibit enzymatic oxidation of both tyrosine and dihydroxy-
phenylalanine thereby preventing darkening of the shell (66,96). Since
1956, agencies such as the former Florida State Department of Conserva
tion (30), now the Department of Natural Resources, have recommended
dipping shrimp in a 1.25% sodium bisulfite solution for 1 min to control
black spot development. Therefore, the effect of sodium bisulfite con
centration (0 to 3%) on the activity of the extracellular proteolytic
enzyme of _P. citreus was investigated. Figure 26 shows that as the con
centration of sodium bisulfite increases, the activity of the enzyme
decreases. When sodium bisulfite dissociates in water, it may affect
enzyme activity by reducing disulfite (-S=S-) linkages (57). The activ
ity of the enzyme in the presence of 1.25% sodium bisulfite was approxi
mately 240 units of activity. Thus, with the addition of 1.25% sodium
bisulfite, approximately 47% of the activity of the proteolytic enzyme
was lost. However, as the concentration of sodium bisulfite decreases
(leaches out in the melt water) (148) the activity of the enzyme should
be less affected.
Effect of Enzyme Concentration
Rates of enzyme-catalyzed reactions are directly dependent on
enzyme concentration (151). The effect of enzyme concentration (0 to
200 yl) on the activity of the _P. citreus enzyme when gelatin and shrimp
protein were used as substrates was investigated. By observing Figure
27, the enzyme preparations used for characterization followed a linear
relationship with increasing levels of enzyme. According to Dixon and
Webb (57) when the plot passes through the origin, inhibitors are usually

ENZYME ACTIVITY (x 10')
igure 6. Effect of sodium bisulfite (NaHSO^) concentration on enzyme activity.3
a .
Activity assayed at 35 C for 10 min (pH 8).

ENZYME ACTIVITY (xIO1)
92
ENZYME CONCENTRATION (/j.\)
Figure 27. Effect of enzyme concentration on enzyme activity.
Increasing enzyme concentrations were incubated with the
substrates at 35 C for 10 min (pH 8).

93
absent from the preparation. Thus, looking at Figure 27, we can observe
that inhibitors were not present in the preparation.
Effect of Substrate Concentration
Substrate concentration is one of the most important factors which
determine the velocity of enzyme reactions. Figures 28 and 30 illustrate
the effect of substrate concentration on the velocity of the reaction
when gelatin (0 to 1.2 mg/ml) and shrimp protein (0 to 0.6 mg/ml) sub
strates, respectively, were used. Both enzyme-substrate reactions fol
lowed Michaelis-Menten kinetics. That is, the enzyme E first combines
with the substrate S to form the enzyme substrate complex ES; the latter
then breaks down in a second step to form the free enzyme E and the
K1
product P: E + S E + P. Figures 28 and 30 follow the traditional
TT
Michaelis-Menten shape curve (95). Enzyme kinetic calculations (95,152)
were done in order to add to the information about the _P. citreus extra
cellular enzyme and to determine substrate saturation conditions.
The K values (Michaelis-Menten constant) calculated in this study
m
are apparent K^'s. Whole protein substrates (either gelatin or shrimp
protein) rather than specific synthetic amide substrates were used.
True values are calculated using specific substrates and having a
definite knowledge of the enzyme's active site (57). Additional
research is required to demonstrate the active site of the P. citreus
extracellular enzyme.
Apparent and V values were calculated by transforming the
data in Figures 28 and 30. Doublereciprocal plots (Lineweaver-Burk
plots) were done and they are shown in Figures 29 and 31 for gelatin
and shrimp protein substrates, respectively. The apparent K values
m
for the gelatin and shrimp protein substrates were 0.98 mg/ml and 0.33

ENZYME ACTIVITY (xIO1)
94
GELATIN CONCENTRATION (mg/ml)
Figure 28. Effect of gelatin concentration on the reaction rate of the
Planococcus citreus extracellular enzyme.3
g
Increasing gelatin concentrations were incubated with
the enzyme at 35 C for 10 min (pH 3).

Transformation of data presented in Figure 29.

ENZYME ACTIVITY ( xI01)
50
Figure 30.
Effect of shrimp protein concentration on the reaction rate of the Planococcus citreus
extracellular enzyme.
Increasing shrimp protein concentrations were incubated with the enzyme at
35 C for 10 min (pH 8).
vO
CT

l/[s]mg/ml of Shrimp Protein
Figure 31. Lineweaver-Burlc plot of the Planococcus citreus extracellular enzyme illustrating V x
K values using shrimp protein as substrate.3
a
Transformation of the data presented in Figure 30.
and

98
mg/ml, respectively. The apparent V values for gelatin and shrimp
protein were 666.67 and 431.03 units of activity, respectively. Since
K is defined as the rate of the disappearance of the enzyme-substrate
m
(ES) complex to the appearance of the ES complex, it appears that
gelatin has a higher affinity for the enzyme than shrimp protein.
Consequently, is lower with shrimp protein as substrate. This is
shown in Figures 29 and 31. _P. citreus extracellular proteolytic enzyme
can utilize and degrade gelatin at a more rapid rate than shrimp protein.
Various factors could be responsible for observing a higher V^ax when
gelatin is used as substrate. Gelatin, with a molecular weight of 90,000
(152), is a simple protein when compared to shrimp protein. Four amino
acids comprise 70% of the total amino acid composition. Glycine and
alanine add to approximately 50% of the amino acids present in gelatin
(152). Possibly, the relative simplicity of gelatin makes this protein
more available to the action of the _P. citreus extracellular enzyme. In
addition, gelatin showed higher solubility than shrimp protein in the
buffer system used. Perhaps, this increased solubility allowed for an
easier enzyme-substrate interaction. However, shrimp protein has a more
complex primary structure, and as it was prepared in this study, it is
probably a mixture of proteins. Eighteen amino acids comprise approxi
mately 60% of the total amino acid composition (53). The composition of
the shrimp protein preparation probably makes it a more complex substrate
for the .P. citreus extracellular enzyme. Consequently, shrimp protein
is not as easily available for the reaction with the enzyme.
Effect of Metal Ions on Enzyme Activity
The effect of various metal ions on the activity of the extracellu
lar proteolytic enzyme of _P. citreus was investigated. The effect of

99
two concentrations of each metal ion is shown in Table 10. Calcium
chloride (CaCl2) increased the activity of the proteolytic enzyme, while
ferric chloride (FeCl^), mercuric chloride (HgCl^ and potassium chloride
(KC1) all suppressed the activity of the enzyme to some extent. Zinc
chloride (ZnCl2), magnesium chloride (MgCl^) and manganese chloride
(MnCl2) had little effect on enzyme activity.
Divalent cations, except CaCl^, had no appreciable effect on the
activity of the extracellular enzyme. The ionic radius of divalent cat
ions is such that one of their primary functions is to coordinate the sub
strate to the active site (37). Mg and Ca, the alkaline earth cations,
participate in the formation of the catalytically active conformation.
Monovalent ions are usually involved in transport mechanisms in the
cell (37), thus, usually not as an integral part of the active center.
Possibly, K ions could be displacing a required ion resulting in the
suppression of enzyme activity. In contrast, Figure 25 showed that NaCl
concentrations had no effect on enzyme activity when gelatin was used as
the substrate. This apparent effect of K+ but not of Na+, possibly
related to the larger ionic radius of K+ ions, should be the subject
of additional investigations.
Salts of heavy metals, such as silver, copper, mercury and lead,
react rapidly and at low concentrations with sulfhydryl groups; however,
they also react with other groups including the imidazole, carboxyl and
peptide groups (151). At high concentrations, heavy metals can inhibit
by neutralizing charges on the protein and/or by forming cross linkages
between protein molecules (57). Iron salts have been found to activate
some enzymes but suppress others (57). Some metal ions can activate
enzymes by: 1) becoming an integral part of the active site; 2) linking

Table 10. Effect of various metal ions on the activity (assayed at 35 C for 10 min (pH 8))
of the Planococcus citreus extracellular enzyme.
Metal Ion
Concentration
(mM)
Enzyme Activity
% Residual Activity
b
none (control)
458.37

2.08
100.0
CaCl
10
492.89

1.57
107.5
z
20
553.61
/

2.10
120.7
FeCl
1
153.61

2.97
33.5
J
20
105.33

2.87
23.0
MgCl
10
460.16

2.77
100.4
4
20
470.11

2.85
102.6
HgCl,
1
285.71

2.41
62.3
20
243.11

1.44
53.0
MnCl.
10
430.17

0.75
93.8
4
20
408.16

1.89
89.1
ZnCl
10
449.95

2.24
98.2
4
20
435.00

1.89
94.9
KC1
5
287.72

2.10
62.8
20
247.67

6.37
54.0
^Average of 6 observations
Compared to the control sample
100

101
the enzyme with the substrate; 3) changing the equilibrium constant of
enzyme reaction; 4) changing surface charge of enzyme; 5) removing
inhibitors; and 6) inducing a more active enzyme conformation (151).
Kato et al. (87) reported that calcium chloride (CaCl^) and magne
sium chloride (MgCip activated the enzyme of a marine-psychrotrophic
bacterium (Pseudomonas spp.) and mercuric chloride (HgC^) and ferric
sulfate (FeSO^) suppressed the enzyme. Arvidson (11) reported that
magnesium chloride (MgCl^) zinc chloride (ZnC^) and calcium chloride
(CaCip activated a protease from Staphylococcus aureus. However,
Pacaud and Uriel (112) stated that calcium chloride (CaC^), manganese
chloride (MnCl^) and ferric chloride (FeC^) activated an enzyme from
Escherichia coli but magnesium chloride (MgCl^), zinc chloride (ZnCl^)
and mercuric chloride (HgC^) had no effect on the activity. The
results of this study indicate the diversity of effects ions can have
on the activity of the enzyme produced by _P. citreus.
Effect of Various Reagents on Enzyme Activity
Various reagents were tested to observe their effect on the activity
of the extracellular proteolytic enzyme of _P. citreus. None of the
reagents tested activated the enzyme (Table 11). Dioxane, one of the
reagents used in the enzyme activity assay, had no apparent effect on
the activity of the enzyme at either concentration examined (10 and 20
mM). The percent residual activity observed was 98.9 and 98.6%, respec
tively. The results observed with trichloroacetic acid (TCA, 5 and 10%)
indicate that both concentrations can terminate the activity of the
enzyme (2.0 and 0.0% residual activity, respectively). These results
are comparable to those observed in Figure 6.

Table 11. Effect of
(pH 8))of
various reagents
the Planococcus
on the enzyme activity (assayed
citreus extracellular enzyme.
at 35 C for 10 min
Reagent
Concentration
Enzyme Activity
% Residual Activity^
None (control)
449.95
5.59
100.00
EDTA
10 mM
87.72
2.64
19.50
20 mM
58.50
2.33
13.00
Citric Acid
10 mM
150.72
3.85
33.50
20 mM
129.72
6.30
28.70
Formaldehyde
1 mM
286.33
2.59
63.60
20 mM
239.56
2.83
53.20
KCN
1 mM
389.89
2.86
86.70
20 mM
363.45
3.50
80.80
KMnO.
1 mM
9.28
1.56
2.10
20 mM
0.00
0.00
0.00
TCA
5%
8.87
1.75
2.00
10%
3.05
0.93
0.68
Cysteine
1 mM
209.83
4.21
46.60
20 mM
181.95
6.50
40.40
p-mercaptoethanol
1 mM
284.17
6.23
63.20
20 mM
247.00
3.28
54.90
p-Dioxane
10 mM
445.17
3.40
98.90
20 mM
443.45
3.00
98.60
2
^Average of 6 observations standard deviation
Compared to the control sample
102

103
The enzyme was inactivated by citric acid and EDTA, which act as
metal ion chelators. Considerable activity was lost by the enzyme when
cysteine and p-mercaptoethanol were added. These reagents reduce and
interchange with sulfhydryl groups (SH groups) in proteins resulting in
a possible reorganization of the enzyme structure (98). In addition,
cysteine can also bind and remove metals (57,155). Possibly, these two
compounds might be binding a required trace metal resulting in
the observed suppression of enzyme activity. The enzyme lost practically
all the activity when 1 mM potassium permanganate (KMnO^) was added
(2.10% residual activity) and all the activity when 20 mM KMnO^ was
added. When 1 mM or 20 mM of formaldehyde was added, 63.6 and 53.2%
residual activity, respectively, was observed. These results are compa
rable to those observed when p-mercaptoethanol was added. Formaldehyde
can react with SH groups forming methylene bridges between amino acids
and amide groups. However, it can also link a-amino acids (73), possibly
making them unavailable for the reaction with the Fluoram reagent.
Potassium cyanide (KCN) affected the activity of the enzyme to a small
degree. Cyanide groups are known to combine with cofactors (metal ions)
in substrates of enzymes when a C=0 group is involved (37). Thus, CN
is usually a carbonyl group inhibitor.
Numerous researchers (12,36,87,113,119,157) have reported on the
effect of various reagents on extracellular proteases. Kato et al. (87)
reported that KMnO^, EDTA and citrate inactivated a Pseudomonas spp.
protease while KCN and p-Dioxane did not affect the activity of the
enzyme. Yoshida et al. (157), Christison and Martin (36) and Arvidson
(12) stated that EDTA and citrate inhibited the proteolytic enzymes from
Bacillus sphaericus, Cytophaga spp. and Staphylococcus aureus. However,

104
a protease from Escherichia coli (113) was not inhibited by EDTA,
cysteine nor p-mercaptoethanol.
The results of this study indicate that the _P. citreus enzyme may
contain a metal cofactor and possibly sulfhydryl groups. Additional
work should be conducted to confirm these findings.
Dipeptidase Activity
Table 12 shows the activity of the purified _P. citreus extracellular
enzyme towards five synthetic dipeptides. Although lower when compared
to the activity on the whole protein substrates, enzyme activity was
observed in all five peptides. The highest activity was observed with
DL-alanylglycine (47.67 units of activity). In order to make predictions
on the specificity of this enzyme, additional peptides should be
investigated. Consequently, the extracellular enzyme of _P. citreus can
utilize and degrade dipeptides to their constituent amino acids.
Enzyme Classification
According to the International Union of Biochemistry scheme (54)
for numbering enzymes, the _P. citreus proteolytic enzyme would be class
ified as: 3.4.1 (acting on peptide bonds, an a-amino-acyl-peptide
hydrolase). The data presented in this study point to the possibility
of having an aminopeptidase enzyme; however, further studies with
synthetic peptides are necessary for the complete classification of the
_P. citreus enzyme. In addition, studies need to be conducted to deter
mine if the _P. citreus enzyme exhibits endo or exopeptidase activity.
Enzyme Induction Studies
Various media were used in order to determine if the extracellular
proteolytic enzyme produced by _P. citreus was induced by shrimp protein.
Induction is the complete de novo synthesis of enzyme molecules in the

105
Table 12. Dipeptidase activity of the Planococcus citreus extracellular
enzyme.
Dipeptide
Enzyme Activity
a
DL-leucylglycine
DL-leucyl-DL-alanine
L-leucyl-L-Tryptophane
glycyl-DL-leucine
DL-alanylglycine
11.67 0.89
22.94 0.44
32.61 1.98
11.56 0.18
47.67 2.48
a
Average of 6 observations standard deviation

106
presence of a specific substrate (28,57,73). A substantial number of
bacterial exo-enzymes appear to be induced by their substrate or closely
related compounds (122) while others are continuously being synthesized
by the microorganisms during their growth. According to Pollock (121),
enzyme induction does not introduce a new pattern of protein structure
into the cell. Whether constituent or induced, and whatever inducer is
used, the enzyme formed appears to be identical (121).
Yeast Carbon Base (YCB), a minimal substrate level medium, was used
in this study (see Table 4) and was fortified with shrimp protein and/or
yeast extract. In addition, a study with Trypticase Soy Broth (TSB)
fortified with shrimp protein was also done.
Figure 32 shows the growth of _P. citreus in the various media fol
lowing 96 hrs of incubation at 20 C. Overall, _P. citreus grew poorly in
all four media tested. About a 1.15 log increase in _P. citreus cell
number was observed in YCB, a 1.20 log increase in YCB + 1.0% shrimp
protein (YCBS), a 1.40. log increase in YCB + 0.1% yeast extract (YCBY)
and a 1.60 log increase in YCB + 1.0% shrimp protein + 0.1% yeast extract
(YCBSY). Therefore, as the nutrients in the growth medium increased,
improved growth of _P. citreus was observed.
The proteolytic activity of the cell-free broth of _P. citreus grown
in Yeast Carbon Base supplemented with shrimp protein and/or yeast
extract is shown in Figure 33. After 96 hrs at 20 C, the proteolytic
enzyme activity of the cell-free broths from each medium was 141, 114,
90 and 77.5 units for YCBSY, YCBY, YCBS and YCB, respectively. Thus, as
observed with the _P. citreus growth data (Figure 32), as the nutritional
composition of the growth medium increased, the amount of enzyme produced
also increased (as measured by an increase in total enzyme activity).

LOG PLANOCOCCUS CITREUS/ml
107
Figure 32. Growth of Planococcus citreus in Yeast Carbon Base
supplemented with shrimp protein and/or yeast extract
at 20 C.

ENZYME ACTIVITY
108
Figure 33. Proteolytic enzyme activity of the cell-free broth of Planococcus
citreus grown in Yeast Carbon Base supplemented with shrimp
protein and/or yeast extract.

109
The units of enzyme activity per cell per hr (m) was calculated for
each medium used. Table 13 shows that the average m value for YCB, YCBS,
YCBY and YCBSY was 4.77, 7.62, 27.82 and 37.12, respectively. These data
agree with the results in Figures 32 and 33. As the nutrients in the
growth medium increased, more enzyme was produced per _P. citreus cell.
Thus, it seems that the nutritional composition of the medium of growth,
not the mere presence of shrimp protein, influences enzyme production.
No significant difference (a=0.05 level) was found between YCB (control)
and YCB with shrimp protein added (Table 13). The results presented
indicate that the enzyme produced by citreus is constantly being pro
duced as long as there is cell growth. Consequently, the enzyme is
constituent.
The results shown in these induction studies reflect the production
of an enzyme by _P. citreus in a minimal medium in which the maximum
growth attained was 1.6 logs after 96 hrs of incubation at 20 C. Figure
34 shows the growth of _P. citreus cells in Trypticase Soy Broth (TSB)
with and without shrimp protein added. TSB was previously chosen as the
best medium for J?. citreus growth and enzyme production (see Growth
Medium and Enzyme Production section). _P. citreus log count increased
approximately 3.75 and 3.80 logs in TSB and TSB + 1.0% shrimp protein,
respectively. Thus, _P. citreus grew equally as well as in media with
or without shrimp protein added.
Figure 35 shows the proteolytic activity of the cell-free broth of
_P. citreus cells grown in TSB with and without shrimp protein. After
96 hrs at 20 C, the proteolytic enzyme activity of the cell-free broths
was 297 and 295 units of activity for TSB and TSB + 1.0% shrimp protein,
respectively. Thus, these data support the previous results in showing

no
Table 13. Units of enzyme activity per cell per hr (m) of Planococcus
citreus grown in Yeast Carbon Base supplemented with shrimp
protein and/or yeast extract at midlog phase.
Medium Mean m
Yeast Carbon Base (YSB) 4.77a
Yeast Carbon Base + 1.0% shrimp protein (YCBS) 7.62a
Yeast Carbon Base + 0.1% yeast extract (YCBY) 27.82^
Yeast Carbon Base + 1.0% shrimp protein + 0.1%
yeast extract (YCBSY) 37.12C
average of 6 observations
Means followed by the same letter do not differ significantly at the
a=0.05 level (r from Anova table 0.978)

Ill
Figure 34. Growth of Planococcus citreus in Trypticase Soy Broth
with and without shrimp protein at 20 C.

ENZYME ACTIVITY (x I01)
112
INCUBATION TIME AT 20C (hr)
Figure 35. Proteolytic enzyme activity of the cell-free broth of
Planococcus citreus grown in Trypticase Soy Broth with
and without shrimp protein.

113
that _P. citreus can produce an extracellular enzyme(s) in the presence
or absence of shrimp protein in the growth medium. The units of enzyme
activity per cell per hr (m) of the enzyme produced in TSB and TSB + 1.0%
shrimp protein were 147.04 and 146.58, respectively. These values are
not significantly different (a=0.05 level). However, when compared to
the m values in Table 13, both of these m values are significantly
different (a=0.05 level).
Consequently, these data appear to indicate that the extracellular
proteolytic enzyme produced by _P. citreus is not induced by the presence
of shrimp protein in the growth medium. The enzyme is produced in mini
mal media and its activity per cell per hr increases as the nutrient
composition in the medium increases. P. citreus produces the extracel
lular enzyme even in the presence of surplus nutrients. Table 14 illus
trates these findings more clearly. There appears to be no effect, e.g.,
gene repression, by any of the factors present in the various media used.
Consequently, the production of the extracellular proteolytic enzyme by
_P. citreus appears to be related more to growth of the organism than to
the presence of any specific nutrient.

114
Table 14. Units of enzyme activity per cell per hr (m) of Planococcus
citreus grown in various media.
Medium
Mean m value^
Trypticase Soy Broth (TSB)
147.68a
Trypticase Soy Broth + 1.0% shrimp protein
146.58a
Plate Count Broth (PCB)
105.67b
Nutrient Broth (NB)
59.32C
Yeast Carbon Base + 1.0% shrimp protein + 0.1%
yeast extract (YCBSY)
37.lld
Yeast Carbon Base + 0.1% yeast extract (YCBY)
27.82
Yeast Carbon Base + 1.0% shrimp protein (YCBS)
7.62f
Yeast Carbon Base (YCB)
4.77f
1Average of 10 observations for TSB, 4 for TSB + 1.0% shrimp protein
and 6 for the other media used
Means followed £y the same letter do not differ significantly at the
a=0.05 level (r from Anova table 0.994)

SUMMARY AND CONCLUSIONS
An extracellular proteolytic enzyme from a marine organism, Piano-
coccus citreus, was isolated, purified and characterized. Ammonium sul-
fate precipitation, Sephacryl S-200 Superfine chromatography and DEAE-
£
Sephadex A-50 ion exchange chromatography were used to purify the
enzyme. A single band present after acrylamide gel electrophoresis, as
well as chromatography, showed the purity of the extracellular proteo
lytic enzyme. In addition, the fluorometric technique proved to be an
efficient, fast and economical (less enzyme is required) method for the
measurement of enzyme activity.
Each fraction of the _P. citreus cells studied (extracellular, whole
cells, washings of whole cells, soluble intracellular and cellular par
ticulate) exhibited proteolytic activity. However, the major portion,
greater than 95.0%, of the active enzyme towards the high molecular
weight substrates (gelatin and shrimp protein) was recovered in the
extracellular fraction.
Trypticase Soy Broth (TSB), a highly nutritious medium, proved to
be the best medium for culturing _P. citreus cells and for the production
of the extracellular proteolytic enzyme. A highly significant difference
(a=0.05 level) was observed between the units of enzyme activity produced
per cell per hr (m) by _P. citreus grown in Trypticase Soy Broth, Plate
Count Broth and Nutrient Broth.
The following properties were characteristic of the P. citreus
extracellular proteolytic enzyme:
115

116
1) The cell-free broth obtained from _P. citreus cells grown at 5 C
for 108 hrs, 20 C for 72 hrs and 35 C for 36 hrs exhibited enzyme activ
ity towards shrimp protein at all three enzyme-substrate incubation
temperatures (5, 20 and 35 C). Thus, _P. citreus when grown at 5 C pro
duces an extracellular enzyme capable of utilizing the protein in shrimp
stored either at refrigeration or higher temperatures.
2) The major portion of the extracellular proteolytic enzyme of _P.
citreus was recovered at an ammonium sulfate concentration between 55-70%
saturation. Eighty-six percent of the total activity was recovered in
this fraction.
3) Using the fluorometric method for activity measurements, the
protease was purified 26.50 fold with a recovery of approximately 49%.
The specific activity of the purified enzyme was 780.37 (units of
activity/mg of protein).
4) Purity of the enzyme was demonstrated by the presence of a
single band after acrylamide gel electrophoresis using various protein
concentrations as well as by the presence of a single peak with homo
geneous activity after ion-exchange chromatography.
5) The molecular weight of the _P. citreus enzyme was approximately
29,000 according to column chromatography using Sephacryl S-200 and
acrylamide gel electrophoresis.
6) Ionic strengths of 0.15-0.83 had no effect on the activity of
the extracellular enzyme.
7) pH optimum of the proteolytic enzyme was 8. Activity of the
enzyme decreased as the pH deviated from this optimum.
8) The optimum temperature for the JP. citreus enzyme was 35 C;
however, activity was observed at 5 C.

117
9) After 15 min of incubation of the purified enzyme at 65 C, no
activity was observed and only 1.0% activity remained in the cell-free
broth of _P. citreus grown at 35 C for 36 hrs. However, after 15 min at
75 C, 1.0% activity still remained in the cell-free broth of _P. citreus
cells grown at 5 C for 108 hrs. Results indicated that the enzyme sys
tem in the crude preparations (cell-free broths) was less affected by
temperature changes than the purified enzyme. In addition, the enzyme
system produced by _P. citreus grown at 5 C was more stable to changes in
temperature than the 35 C crude enzyme preparation. Perhaps the enzymes
present in the 5 C crude extract have an enzyme configuration that better
protects the active site from temperature changes.
However, when shrimp is processed (boiled, canned or broiled), the
enzyme should be easily inactivated.
10) When shrimp protein was used as substrate, the activity of the
enzyme increased as the sodium chloride (NaCl) concentration increased
up to 0.5% NaCl. Enzyme activity decreased with higher concentrations
of NaCl (0.5-1.5%). When gelatin was used as the substrate, NaCl con
centrations (1-1.5%) had no effect on enzyme activity. The increase in
NaCl concentration up to 0.5% might have caused an increase in shrimp
protein solubility, thus, making shrimp protein more available for the
_P. citreus enzyme.
11) As the concentration of sodium bisulfite (NaHSO^) was increased
from 0 to 3%, the activity of the protease decreased (first-order reac
tion). Approximately 47% of the activity was lost when 1.25% sodium
bisulfite was present in the medium.
12) As the concentration of enzyme increased (0-200 yl), the rate
of the reaction increased when gelatin and shrimp protein were used as
substrates.

118
13) Michaelis-Menten kinetics were followed when gelatin and
shrimp protein were used a substrates.
14) The apparent K values for gelatin and shrimp protein were
m
0.98 mg/ml and 0.33 mg/ml, respectively. The apparent V values were
max
666.67 and 431.03 (units of activity), respectively. This indicates
that the _P. citreus extracellular enzyme can degrade gelatin faster
than shrimp protein.
15) FeCl^, HgC^ and KC1 inhibited the enzyme to some extent,
while CaC^ activated the extracellular enzyme. ZnC^, MgC^ and
MnCl^ had no appreciable effect on the activity of the proteolytic
+3 +2
enzyme. The repressing effect of Fe and Hg on the activity of
this extracellular enzyme may indicate that the enzyme contains
sulfhydryl groups. p-Dioxane had no effect on the activity of the
proteolytic enzyme. EDTA, citric acid, cysteine, p-mercaptoethanol,
potassium permangate and formaldehyde inactivated the enzyme to dif
ferent degrees indicating that the extracellular enzyme was affected by
metal chelators. These results indicate that the enzyme may contain a
metal ion as cofactor and possibly sulfhydryl groups.
16) The proteolytic enzyme of _P. citreus exhibited activity against
the following dipeptides: DL-leucylglycine, DL-leucyl-DL-alanine, L-
leucyl-L-tryptophane, glycyl-DL-leucine and DL-alanylglycine. The high
est activity was observed with DL-alanylglycine.
17) Preliminary classification of the enzyme shows that it is an
a-amino-acy1-peptide hydrolase (3.4.1). Additional studies with syn
thetic peptides are necessary for complete classification.
18) The extracellular proteolytic enzyme produced by P. citreus
was not induced by the presence of shrimp protein in the growth medium.

119
The enzyme appears to be produced continuously during growth of the
organism.
Results obtained under the conditions of these investigations indi
cate that Planococcus citreus produces an extracellular enzyme which is
active at refrigerated temperatures and capable of degrading shrimp pro
tein. In addition, this enzyme is capable of cleaving dipeptides to
their constituent amino acids. Consequently, the production of this
enzyme by _P. citreus while growing on shrimp may contribute to the
overall decrease in shrimp quality during iced or refrigerated storage.
The function of the extracellular enzyme appears to be one of
supplying nitrogenous compounds to the cell. _P. citreus does not
actively utilize carbohydrates for growth, rather its metabolism is
directed towards the utilization of proteins. In a nutrient limited
marine environment, it appears advantageous from an evolutionary stand
point to produce a single extracellular enzyme of broad specificity.
This dependence upon nitrogen compounds by _P. citreus could be one
reason for the unique ecological association that exits between P.
citreus and shrimp in nature.

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BIOGRAPHICAL SKETCH
Ricardo Javier Alvarez was born on November 4, 1954, in Santiago,
Chile. In June, 1972, he graduated from Colegio San Ignacio de Loyola
in San Juan, Puerto Rico. He attended the University of South Florida,
and in June, 1976, he received his Bachelor of Science in microbiology.
He enrolled as a graduate student in the Food Science and Human Nutri
tion Department, University of Florida, in January, 1977. He received
his Master of Science degree in June, 1978. He anticipates receiving
his Ph.D. degree in food science and human nutrition with a minor in
environmental engineering in March, 1981.
131

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Dr. J. L. Oblinger
Associate Professor of
and Human Nutrition
Food Science
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Dr.k'G. Bitton
Associate Professor of Environmental
Engineering Sciences

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Dr. R. H. Schmidt
Associate Professor of Food Science
and Human Nutrition
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Assistant Professor of Food Science
and Human Nutrition
This dissertation was submitted to the Graduate Faculty of the College
of Agriculture and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
March, 1981
Dean for Graduate Studies and Research



63
density of _P. citreus cultures grown at 5 C for 144 hrs, 20 C for 120
hrs and 35 C for 96 hrs. After different time intervals for growth
adaptation, _P. citreus grew at all three temperatures. Direct micro
scopic observation of _P. citreus cells growing at the three temperatures
revealed a difference in cell arrangements. When cells were grown at 35
C, the predominant morphology present was clusters of gram-positive cocci.
At this temperature, the cells are rapidly growing and dividing, thus,
possibly accounting for the observed predominance of clusters. At 20 C
a mixture of tetrads, pairs and single cells were observed which is the
predominant morphology exhibited by this organism, as described in
Bergey's Manual of Determinative Bacteriology (29). At 5 C the predomi
nant cell arrangement observed was single cells. This particular morpho
logical structure might predominate because of the slow metabolic rate
at this temperature, although these rates were not investigated. Indi
vidual cells have more surface area for the uptake of nutrients. At 5 C,
the movement of nutrients within the cell is slow. Consequently, the
increase in surface area is particularly desirable from a nutritional
standpoint.
Table 7 illustrates the enzyme activity of the cell-free broth of
_P. citreus grown at 5 C for 108 hrs, 20 C for 72 hrs and 35 C for 36
hrs (midlog at each temperature) and incubated with shrimp protein at 5,
20 and 35 C for 60, 30 and 15 min, respectively. _P. citreus produced an
active extracellular enzyme(s) when grown at all temperatures. In addi
tion, the cell-free broth obtained from the three temperatures of growth
exhibited activity at all three enzyme-substrate incubation temperatures
(5, 20 and 35 C). As the temperature of growth increased from 5 to 35
C, the enzyme activity increased at a similar rate at the three enzyme-


ENZYME ACTIVITY (x I01)
Figure 25. Effect of sodium chloride (NaCl) concentration on enzyme activity.3
Activity assayed at 35 C for 10 min (pH 8).


103
The enzyme was inactivated by citric acid and EDTA, which act as
metal ion chelators. Considerable activity was lost by the enzyme when
cysteine and p-mercaptoethanol were added. These reagents reduce and
interchange with sulfhydryl groups (SH groups) in proteins resulting in
a possible reorganization of the enzyme structure (98). In addition,
cysteine can also bind and remove metals (57,155). Possibly, these two
compounds might be binding a required trace metal resulting in
the observed suppression of enzyme activity. The enzyme lost practically
all the activity when 1 mM potassium permanganate (KMnO^) was added
(2.10% residual activity) and all the activity when 20 mM KMnO^ was
added. When 1 mM or 20 mM of formaldehyde was added, 63.6 and 53.2%
residual activity, respectively, was observed. These results are compa
rable to those observed when p-mercaptoethanol was added. Formaldehyde
can react with SH groups forming methylene bridges between amino acids
and amide groups. However, it can also link a-amino acids (73), possibly
making them unavailable for the reaction with the Fluoram reagent.
Potassium cyanide (KCN) affected the activity of the enzyme to a small
degree. Cyanide groups are known to combine with cofactors (metal ions)
in substrates of enzymes when a C=0 group is involved (37). Thus, CN
is usually a carbonyl group inhibitor.
Numerous researchers (12,36,87,113,119,157) have reported on the
effect of various reagents on extracellular proteases. Kato et al. (87)
reported that KMnO^, EDTA and citrate inactivated a Pseudomonas spp.
protease while KCN and p-Dioxane did not affect the activity of the
enzyme. Yoshida et al. (157), Christison and Martin (36) and Arvidson
(12) stated that EDTA and citrate inhibited the proteolytic enzymes from
Bacillus sphaericus, Cytophaga spp. and Staphylococcus aureus. However,


ENZYME ACTIVITY (xIO1)
59
INCUBATION TIME AT 20C (hr)
igure 13. Enzyme activity at 35 C for 15 min (pH 8) of the cell-free broth
of Planococcus citreus cells grown in Plate Count Broth, Nutrient
Broth and Trypticase Soy Broth for up to 96 hrs.


ABSORBANCE (280 nm
R
Figure 15. Separation of proteins present in the 55-70 % ammonium sulfate fraction using Sephacryl
S-200. a
cl
30 x 2.5 cm column, eluted with 0.02 M phosphate buffer pH 7.
I
ENZYME ACTIVITY (xIO


43
Characterization of the Proteolytic Enzyme(s)
Molecular Weight Determination
Two methods (7,21,150) were used to estimate the molecular weight
of the enzyme(s).
A 2.5 x 30 cm column packed with Sephacryl S-200 (Pharmacia Fine
Chemicals, Uppsala, Sweden) was used and the following standards applied:
Ribonuclease A (13,000 MW), Trypsin (23,500 MW), Pepsin (45,000 MW),
Bovine Serum Albumin (70,000 MW) and Aldolase (158,000 MW) (Pharmacia
Fine Chemicals, Piscataway, NJ) following the procedures suggested by
Pharmacia Fine Chemicals (116). The value of each protein sample was
calculated and plotted against the corresponding molecular weight
V V
6 O
(K = 7T~ where V = column void volume, V = elution volumes and
av Vt V o e
t o
= total column volume). Protein was monitored at 280 nm using a
Beckman Model 25 spectrophotometer (Beckman Instruments, Inc., Fullerton,
CA).
Bio-Rad Low Molecular Weight Protein Standards (10,000-100,000) for
SDS Gel Electrophoresis were also used for molecular weight determination
D
using SDSPAG gel electrophoresis. The instructions outlined by Bio-Rad
(Bio-Rad Laboratories, Richmond, CA) were followed (21). The proteins
included were Phosphorylase B, Bovine Serum Albumin, Ovalbumin, Carbonic
Anhydrase, Soybean Trypsin Inhibitor, Lysozyme and the purified enzyme.
The motility of the enzyme was then compared to the relative motility
(Rm) of the standards.
Determination of the Purified Enzyme-Substrate Mixture Reaction Time
Five milliliters of substrate (gelatin or shrimp protein) and 100 yl
of the purified enzyme were incubated at 35 C for 0, 5, 10, 15, 30 and 60
min in order to determine the apparent optimum reaction time.


OPTICAL DENSITY RELATIVE FLUORESCENCE
23
Figure 2. Comparison between the Fluorescamine technique and the Lowry procedure
(l^O) for determining protein concentration. a
Chromatography of a partially purified enzyme of guinea pig neuro-
physin monitored by the fluorescamine and the Lowry procedure.


87
thermal stablity of the cell-free broth of JP. citreus cells grown at 5
and 35 C and of the purified enzyme. In general, as the temperature
increased, the activity of all three fractions decreased. After 15 min
of incubation at 65 C, all the activity was lost in the purified enzyme
fraction and only 1% was left in the 35 C cell-free broth fraction.
However, 28% of the activity still remained in the 5 C cell-free broth
fraction. After 15 min at 75 C, 1% of the activity of the 5 C cell-free
broth fraction still remained. Perhaps the enzymes in the cell-free
broth of _P. citreus grown and stored at 5 C have undergone a structural
change (50,51) or have a slightly different structure than the enzyme
produced at 35 C. This change could result in an enzyme conformation
with an active site that is more protected from increased temperatures.
Usually, an enzyme is more stable to temperature changes in an
intact tissue or in an homogenate where its structure is protected by
the presence of other colloidal material (i.e. proteins, carbohydrates,
etc.) than it is in a purified form (147,151). However, in general,
those enzymes which have molecular weights ranging from 12,000 to 50,000
are composed of single polypeptide chains and having disulfide bonds are
usually more resistant to heat treatment. The larger the enzyme and the
more complex its structure the more susceptible it is to increases in
temperature (151). Figure 24 shows that the cell-free broths are more
stable to heat than the purified enzyme.
Effect of Sodium Chloride Concentration
The effect of increasing sodium chloride (NaCl) concentration on
enzyme activity was examined. Concentrations of 0-1.50% NaCl were
investigated. When shrimp protein was used as the substrate, the activ
ity of the extracellular enzyme increased until 0.50 g/100 ml NaCl (0.5%)


17
bacterial growth and that a TVN/AA-N ratio of 1.3 indicated a limited
shelf life of the shrimp. Alvarez and Koburger (4,5) showed that _P.
citreus is capable of increasing the TVN/AA-N ratio at a similar rate as
the control sample (natural flora of shrimp). Thus, if the TVN/AA-N
ratio is an index of shrimp quality, _P. citreus is capable of shortening
the shelf life of shrimp. The proteolytic activity of this organism was
demonstrated by a significant decrease in percent extractable protein
(% TEP) in the early days of storage. Maximal percent TEP decrease was
observed between the 4th and the 12th day of storage of shrimp at 5 C
(5).
Proteolytic Enzymes
Enzymes are proteins with highly specific catalytic activities. As
catalysts, enzymes have the following properties: 1) they are effective
in small concentrations; 2) they remain unchanged in the reaction; 3) if
present in small concentrations relative to the substrate, they speed
attainment of equilibrium as reflected by increases in the rate constants
V
K. and K (A + B t > C + D, where A + B = reacting substances, C + D
K-1
= products of the A + B enzyme catalyzed reaction, = rate constant of
the forward reaction, K = rate constant of the reverse reaction).
However, an enzyme does not change the ratio K,/K = K (95).
1 1 eq
Most living organisms possess the ability to degrade proteins to
more readily absorbed substances. Such attacks on the peptide bond are
made possible by the presence of proteolytic enzymes. Although proteo
lytic enzymes from animal sources have been studied for more than a
century by both physiologists and biochemists, it was the work of Berg-
mann and Fruton (18) which led to a more complete understanding of the
mode of action of these enzymes. Their work established conclusively


60
produced (as measured by enzyme activity) is more clearly observed.
After 48 hrs of incubation, the enzyme activity for the _P. citreus grown
in TSB, PCB and NB was 200, 100 and 85.5 units of activity, respectively.
Under the conditions tested, _P. citreus exhibited maximum enzyme produc
tion when grown in TSB.
The units of activity per cell per hr (m) was calculated for each
medium used and the results are shown in Table 6. The average m value
for TSB, PCB and NB was 168.50, 105.67 and 59.32, respectively. These
data show that the amount of enzyme produced by actively growing _P.
citreus cells (midlog phase) in TSB is higher than that produced when
_P. citreus is grown in PCB or NB. The analysis of the data supports
this observation. A significant difference (a = 0.05 level) was
observed between the m values of TSB, PCB and NB (Table 6).
Thus, after evaluating the results from this section, TSB was cho
sen as the best medium for _P. citreus growth and enzyme production and
was used for the remainder of the study. The combination of nutrients
in TSB allowed for the rapid growth of _P. citreus and by doing so, per
mitted the production of more extracellular proteolytic enzyme. In
addition to the combination of nutrients in TSB, the presence of 0.25%
dextrose may play a role in extracellular enzyme production. Dextrose
has been suggested as a possible inducer of a variety of enzymes (57),
although this effect was not specifically tested in these experiments.
Effect of Incubation Temperature on Enzyme Production and Activity
The ability of _P. citreus to produce an active extracellular enzyme
at 5, 20 and 35 C was investigated in order to determine the ability of
the extracellular enzyme(s) to affect shrimp protein at refrigeration
(5 C) or iced temperatures. Figure 14 shows the increase in optical


126
85. Ilyengar, J. R., K. Viscneswariak, M. N. Moryani and D. S. Bhatia.
1960. Assessment of the progressive spoilage of ice-stored shrimp.
J. Fish. Res. Brd. Can. 17:475-485.
86. Index Bergeyana. 1966. Eds. R. E. Buchanan, J. G. Holt and Erwin
F. Tessel. The Williams and Wilkins Co., Baltimore, MD.
87. Kato, N., T. Nagasana, S. Adachi, Y. Tani and K. Ogata. 1972.
Purification and properties of proteases from a marine-psychro-
philic bacterium. Agr. Biol. Chem. 36:1185-1192.
88. Khan, M. R., J. A. Blain and J. E. D. Patterson. 1979. Extra
cellular proteases of Mucor pusillus. Appl. Environ. Microbiol.
37:719-724.
89. Koburger, J. A., R. F., Matthews and W. E. McCullough. 1972. Some
observations on the heading of Penaeus shrimp. Proc. Gulf and
Caribbean Fish. Inst. 26:144-148.
90. Koburger, J. A., A. R. Norden and G. M. Kempler. 1975. The
microbial flora of rock shrimp Sicyonia brevirostris. J. Milk
Food Technol. 38:747-749.
91. Kocur, M., Z. Pacova, W. Hodgkiss and T. Martinec. 1970. The
taxonomic status of the genus Planococcus Migula 1894. Int'l.
J. of System. Bacteriol. 20:241-248.
92. Krasil'nikov, N. A. 1949. Opredelitel bakteriji aktinomycetov.
Moskva.
93. Lane, C. E. and B. E. Whittaker. 1954. The use of ultraviolet
("black") light for determining quality of iced shrimp. Proc.
Gulf and Caribbean Fish. Inst. 6:13-18.
94. Lee, J. S. and K. K. Pfeifer. 1977. Microbial characteristics of
Pacific shrimp (Pandalus Jordan). Appl. Environ. Microbiol. 33:
853-859.
95. Lehninger, A. L. 1975. Biochemistry. 2nd Ed. Worth Publishers,
Inc., New York, NY.
96. Lerner, A. B. and T. B. Fitzpatrick. 1950. Biochemistry of
melanin formation. Physiol. Rev. 30:91-126.
97. Lightner, D. V. 1974. Normal postmortem changes in the brown
shrimp, Penaeus aztecus. Fish. Bull. 72:223-225.
98. Lowry, D. H., N. J. Rosenbrough, A. L. Farr and R. J. Randall.
1951. Protein measurement with the Folin phenol reagent. J.
Biol. Chem. 193:265-275.
99. Madsen, H. B. 1963. Mercaptide forming agents. In: Metabolic
Inhibitors. Vol. I. Eds. R. M. Hochster and J. H. Quaste^
Academic Press, Inc., New York, NY.


ISOLATION, PURIFICATION AND CHARACTERIZATION OF
AN EXTRACELLULAR PROTEOLYTIC ENZYME OF Planococcus citreus
BY
RICARDO J. ALVAREZ
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1981


44
Enzyme activity was measured using the Fluorescamine technique. Figure
10 shows that 10 min was the optimum reaction time for the purified
enzyme-substrate (gelatin or shrimp protein) reaction mixture. This
optimum reaction time was used for the remainder of the characterization
of the extracellular proteolytic enzyme.
Effect of Ionic Strength on Enzyme Activity
The effect of ionic strength on enzyme activity was investigated.
Gelatin (1.2 mg/ml) was dissolved in the following solutions of sodium
chloride (NaCl): 0.05 M (y 0.13), 0.08 M (y = 0.16), 0.18 M (y =
0.26), 0.25 M (y = 0.35), 0.34 M (y 0.42), 0.51 M (y = 0.59), 0.75 M
(y = 0.83), 1.00 M (y = 1.08) and 1.5 M (y = 1.58). The NaCl was dis
solved in 0.05 M phosphate buffer (pH 8). Five milliliters of this mix
ture were reacted with 100 ul of the purified enzyme and incubated at
35 C for the selected reaction time (10 min).
Determination of Optimum pH
Buffers of varying pH from pH 2 to pH 10 were used to determine the
optimum pH for the proteolytic activity of the enzyme(s). The following
buffers were used:
Ionic Strength
pH 2
0.1 M citric acid
0.25
3
47.0 ml of 0.1 M citric acid + 3.5 ml of
0.1 M sodium citrate
0.35
4
33.0 ml of 0.1 M citric acid + 17.0 ml of
0.1 M sodium citrate
0.45
5
20.5 ml of 0.1 M citric acid + 29.5 ml of
0.1 M sodium citrate
0.45
6
88 ml of 0.2 M monobasic sodium phosphate +
12.5 ml dibasic sodium phosphate
0.45
7
39.0 ml of 0.2 M monobasic sodium phosphate +
61.0 ml of 0.2 M dibasic sodium phosphate
0.35


34
Table 3. Proximate composition of the shrimp protein preparation.3
Percent (%)
Protein
77.44
Fat
5.40
Moisture
8.95
Ash
6.50
Carbohydrate^*
1.71
£
Average of two determinations
^Calculated by difference


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Dr. R. H. Schmidt
Associate Professor of Food Science
and Human Nutrition
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Assistant Professor of Food Science
and Human Nutrition
This dissertation was submitted to the Graduate Faculty of the College
of Agriculture and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
March, 1981
Dean for Graduate Studies and Research


84
require a slightly alkaline pH for optimum activity. Considerable
activity is present at neutral pH's; the pH of freshly caught shrimp is
around neutrality. During shrimp storage, the pH of shrimp will
increase (5).
Optimum Temperature Determination
Changes in temperature may affect enzymatic reactions in a number
of ways. Some of these effects may include: a) stability of the enzyme;
b) affinity of enzymes for activators and inhibitors; c) ionization of
prototropic groups; d) enzyme-substrate affinity; and e) velocity of
breakdown of enzyme-substrate complex (131). The optimum temperature of
the _P. citreus extracellular enzyme when both shrimp protein and gelatin
were used as substrates was 35 C (Figure 23). Although not statistically
signficant, a slightly higher enzyme activity was observed at the lower
temperatures (5 and 10 C) using shrimp protein as substrate as compared
to gelatin. However, at the higher temperatures (45 and 55 C) the
reverse was evident. Thus, as previously observed with the cell-free
broth (enzyme crude extract), the purified enzyme of _P. citreus can
exhibit activity at the temperatures of refrigerated shrimp.
Thermal Stability
The cell-free broths obtained from _P. citreus cells grown in Trypti-
case Soy Broth (TSB) at 5 and 35 C for 108 and 36 hrs, respectively, and
the _P. citreus purified enzyme were incubated at 35, 45, 55, 65, 75 and
85 C for 15 min in order to study the various temperatures effects on
stability. The enzyme activity of the cell-free broths and purified
enzyme remaining after the various heat treatments was assayed using
gelatin as the substrate and compared to the activity observed with the
unheated cell-free broths and purified enzyme. Figure 24 shows the


A FLUORESCENCE
45
INCUBATION TIME AT 35 C (min)
Figure 10. Increase in fluorescence intensity following incubation of
gelatin and shrimp protein substrate with purified enzyme
for up to 1 hr at 35 C.


Table 1. (continued)
Test
hydrogen sulfide
hydration capacity
inosine monophosphate
indole
iodine titration
lactic acid
methylene blue reductase
peroxide number
PH
phenol red test paper
photoelectric reflection number
picric acid
skatole
Parameter Measured
Reference
measure of H^S presence 31
hydration of water insoluble protein
14,129,141
degradation of adenine dinucleotides
to IMP
132
utilization of tryptophane by
bacteria and its conversion
to indole
31,64,75,93
presence of iodine
14,64,75
lactic acid content
14
reduction of methylene blue by
bacteria
111
determines peroxide oxygen which
has formed at the double bonds in
unsaturated fatty acids
31
hydrogen ion concentration
14,19,68,69,85,
93,141
changes in pH
86
changes in light transmission of
shrimp extract
64
turbidity of shrimp filtrates with
picric acid
19
production of skatole
93


ENZYME ACTIVITY (xIO1)
94
GELATIN CONCENTRATION (mg/ml)
Figure 28. Effect of gelatin concentration on the reaction rate of the
Planococcus citreus extracellular enzyme.3
g
Increasing gelatin concentrations were incubated with
the enzyme at 35 C for 10 min (pH 3).


no
Table 13. Units of enzyme activity per cell per hr (m) of Planococcus
citreus grown in Yeast Carbon Base supplemented with shrimp
protein and/or yeast extract at midlog phase.
Medium Mean m
Yeast Carbon Base (YSB) 4.77a
Yeast Carbon Base + 1.0% shrimp protein (YCBS) 7.62a
Yeast Carbon Base + 0.1% yeast extract (YCBY) 27.82^
Yeast Carbon Base + 1.0% shrimp protein + 0.1%
yeast extract (YCBSY) 37.12C
average of 6 observations
Means followed by the same letter do not differ significantly at the
a=0.05 level (r from Anova table 0.978)


Table 1. (continued)
Test
Parameter Measured Reference
total fat
fat content 93
total nitrogen
ammonia content 93
trimethylamine nitrogen
degradation of trimethylamine oxide 14,19,31,44,64,67,
by enzyme (TMO) to TMA 75,85
total volatile nitrogen
volatile nitrogen compounds (ammonia) 64,75
tyrosine
free tyrosine levels 67
ultraviolet light-change
in fluorescence
ultraviolet absorption of 93
shrimp extracts
volatile acids
volatile acidic compounds 19,31,46,62,68,69
volatile reducing substances
measure of volatile nitrogen 62
compounds
volatile nitrogen
volatile nitrogen containing 85
compounds (ammonia)


119
The enzyme appears to be produced continuously during growth of the
organism.
Results obtained under the conditions of these investigations indi
cate that Planococcus citreus produces an extracellular enzyme which is
active at refrigerated temperatures and capable of degrading shrimp pro
tein. In addition, this enzyme is capable of cleaving dipeptides to
their constituent amino acids. Consequently, the production of this
enzyme by _P. citreus while growing on shrimp may contribute to the
overall decrease in shrimp quality during iced or refrigerated storage.
The function of the extracellular enzyme appears to be one of
supplying nitrogenous compounds to the cell. _P. citreus does not
actively utilize carbohydrates for growth, rather its metabolism is
directed towards the utilization of proteins. In a nutrient limited
marine environment, it appears advantageous from an evolutionary stand
point to produce a single extracellular enzyme of broad specificity.
This dependence upon nitrogen compounds by _P. citreus could be one
reason for the unique ecological association that exits between P.
citreus and shrimp in nature.


LOG PLANOCOCCUS CITREUS/ml
107
Figure 32. Growth of Planococcus citreus in Yeast Carbon Base
supplemented with shrimp protein and/or yeast extract
at 20 C.


39
In addition, direct microscopic observations of the cells grown at
5, 20 and 35 C were conducted. Any morphological change due to growth
temperature was observed and recorded.
Purification of the Extracellular Enzyme(s)
Planococcus citreus was grown in the selected medium at 20 C until
midlog phase. The cells were then centrifuged at a force of 20,000 x g
for 30 min. This cell-free broth was used in the purification procedure.
Ammonium Sulfate Precipitation
Fractional precipitation of the enzyme(s) in the cell-free broth was
accomplished with 0-55%, 55-70% and 70-100% ammonium sulfate saturation
(Mallinckrodt, Inc., Paris, KY). The required amount of ammonium sulfate
was added with stirring until dissolved (88). The mixture was allowed to
equilibrate for 1 hr at 4 C and centrifuged at a force of 20,000 x g for
20 min. The precipitate was resuspended with 10 ml of Butterfield's
phosphate buffer at pH 8 and dialyzed for 16 hrs (dialysis tubing #24,
Scientific Products, McGraw Park, IL) against 500 ml of 0.05 M phosphate
buffer pH 8 (108,153). Each ammonium sulfate fraction was then assayed
for proteolytic activity using gelatin as substrate.
Molecular Sieve Chromatography
Sephacryl S-200 Superfine (Pharmacia Fine Chemicals, Uppsala,
Sweden), a high resolution chromatographic medium for gel filtration was
used to separate the enzyme solution according to molecular weight after
the ammonium sulfate precipitation step. A 30 x 2.5 cm column was packed
R R
with Sephacryl S-200 Superfine gel and a Pharmacia peristaltic pump
(p-3) was used to pack the column at a speed of 40 ml/cm/hr (120). The
enzyme solution was eluted using reverse flow at a speed of 30 ml/cm/hr.
Five milliliter fractions were collected in each tube with 0.02 M phos-


Figure 16. Ion-exchange chromatography using DEAE-Sephadex^ A-50 of the pooled active fractions obtained
in the molecular sieve chromatography step.
a40 x 2.5 column, eluted with 0.02 M phosphate buffer pH 7 with a linear
gradient of 0.01 M NaCl- tris 0.15 M NaCl at a rate of 25 ml/hr.


ACKNOWLEDGMENTS
The author expresses his deepest gratitude to Dr. J. A. Koburger,
his major advisor, for his exceptional patience, guidance and encourage
ment throughout the course of this work. The author also wishes to
acknowledge the advice, support and all the help given by Drs. R. H.
Schmidt, W. S. Otwell, J. L. Oblinger and G. Bitton as members of his
supervisory committee. Thanks go to Dean J. L. Fry for his under
standing and support in delicate times.
Special appreciation is extended to Dr. J. R. Kirk for procuring a
much needed assistantship for the first two years of this research, to
Margie Summers for her beautiful graphic work, Beth Beville, Diane Dobsha,
Beth Johnsen and Mike Pyle for their patience in typing sections of this
dissertation. A very .special thank you goes to Melissa Michaels for the
typing of the final copy of this dissertation.
In addition, the author expresses thanks to Sam May for his help
and support around the laboratory and to Suzanne Davidson, Bridget
Walker and Janet Eastridge for their aid when needed. Thanks go to Dr.
L. D. Ingram for his constructive comments.
Recognition is also given to the faculty, staff and secretaries of
the Food Science and Human Nutrition Department for their cooperation
and to all fellow graduate students who shared with the author the years
at the University of Florida.
iii


99
two concentrations of each metal ion is shown in Table 10. Calcium
chloride (CaCl2) increased the activity of the proteolytic enzyme, while
ferric chloride (FeCl^), mercuric chloride (HgCl^ and potassium chloride
(KC1) all suppressed the activity of the enzyme to some extent. Zinc
chloride (ZnCl2), magnesium chloride (MgCl^) and manganese chloride
(MnCl2) had little effect on enzyme activity.
Divalent cations, except CaCl^, had no appreciable effect on the
activity of the extracellular enzyme. The ionic radius of divalent cat
ions is such that one of their primary functions is to coordinate the sub
strate to the active site (37). Mg and Ca, the alkaline earth cations,
participate in the formation of the catalytically active conformation.
Monovalent ions are usually involved in transport mechanisms in the
cell (37), thus, usually not as an integral part of the active center.
Possibly, K ions could be displacing a required ion resulting in the
suppression of enzyme activity. In contrast, Figure 25 showed that NaCl
concentrations had no effect on enzyme activity when gelatin was used as
the substrate. This apparent effect of K+ but not of Na+, possibly
related to the larger ionic radius of K+ ions, should be the subject
of additional investigations.
Salts of heavy metals, such as silver, copper, mercury and lead,
react rapidly and at low concentrations with sulfhydryl groups; however,
they also react with other groups including the imidazole, carboxyl and
peptide groups (151). At high concentrations, heavy metals can inhibit
by neutralizing charges on the protein and/or by forming cross linkages
between protein molecules (57). Iron salts have been found to activate
some enzymes but suppress others (57). Some metal ions can activate
enzymes by: 1) becoming an integral part of the active site; 2) linking


109
The units of enzyme activity per cell per hr (m) was calculated for
each medium used. Table 13 shows that the average m value for YCB, YCBS,
YCBY and YCBSY was 4.77, 7.62, 27.82 and 37.12, respectively. These data
agree with the results in Figures 32 and 33. As the nutrients in the
growth medium increased, more enzyme was produced per _P. citreus cell.
Thus, it seems that the nutritional composition of the medium of growth,
not the mere presence of shrimp protein, influences enzyme production.
No significant difference (a=0.05 level) was found between YCB (control)
and YCB with shrimp protein added (Table 13). The results presented
indicate that the enzyme produced by citreus is constantly being pro
duced as long as there is cell growth. Consequently, the enzyme is
constituent.
The results shown in these induction studies reflect the production
of an enzyme by _P. citreus in a minimal medium in which the maximum
growth attained was 1.6 logs after 96 hrs of incubation at 20 C. Figure
34 shows the growth of _P. citreus cells in Trypticase Soy Broth (TSB)
with and without shrimp protein added. TSB was previously chosen as the
best medium for J?. citreus growth and enzyme production (see Growth
Medium and Enzyme Production section). _P. citreus log count increased
approximately 3.75 and 3.80 logs in TSB and TSB + 1.0% shrimp protein,
respectively. Thus, _P. citreus grew equally as well as in media with
or without shrimp protein added.
Figure 35 shows the proteolytic activity of the cell-free broth of
_P. citreus cells grown in TSB with and without shrimp protein. After
96 hrs at 20 C, the proteolytic enzyme activity of the cell-free broths
was 297 and 295 units of activity for TSB and TSB + 1.0% shrimp protein,
respectively. Thus, these data support the previous results in showing


26
detection of amino acids, peptides, proteins and primary amines in the
picomole range (140).
P. citreus cells were grown in various media throughout the study.
After incubation, the cultures were centrifuged in a RC-5 Superspeed
Refrigerated Centrifuge (Sorval, Dupont Co. Instruments, Newtown, CT) at
a force of 20,000 x g for 30 min. The supernatant (cell-free broth) was
used for further investigations involving extracellular enzymes. The
cell pellet was washed twice with 0.05 M phosphate buffer (pH 8). The
whole cells were then resuspended with 10-20 ml of the same buffer,
transferred to a dry ice chilled Eaton pressure cell (60) and allowed to
freeze under dry ice for 3 hrs. The frozen microbial cells were disinte
grated using the Eaton pressure cell at a constant pressure of 7.03 x 10^
kg/m2 on a Carver hydraulic laboratory press (F. S. Carver, Inc., Summit,
NJ). The ruptured cell extract was fractionated into intracellular solu
ble and particulate fractions by centrifugation at a force of 12,000 x g
for 15 min. The particulate fraction was resuspended in 10 ml of 0.05 M
phosphate buffer prior to enzyme activity determinations of all frac
tions. Five milliliters of the substrate (gelatin or shrimp protein)
were reacted with 1 ml of each of the above fractions for 15 min at 35 C.
The reactions were terminated by adding 10 ml of 5% TCA. Zero time
blanks were prepared by adding the trichloroacetic acid (TCA, Fisher
Scientific Co., Fairlanes, NJ) before the incubation period (see latter
part of this section).
One milliliter of the cell-free broth or 100 yl of the purified
enzyme was reacted with 5 ml of substrate (gelatin or shrimp protein) for
the appropriate reaction time (to be determined) at 35 C, pH 8. The
enzyme-substrate reaction was stopped by precipitating the mixture with


Figure 4. Exitation (curve A) and fluorescence (curve B) spectrum for the reaction of a gelatin-trichloro
acetic acid (TCA) filtrate with fluorescamine at pH 8.


36
various amounts of cell-free broth were reacted with gelatin, a 15 min
reaction time was also a sufficient substrate reaction time (Figure 8).
This reaction time was used for the remainder of the study.
Growth Medium and Enzyme Production
Various media were used to determine growth rates and production of
extracellular enzyme(s) by _P. citreus. Three hundred milliliters of
Plate Count Broth (PCB) + 0.5% NaCl, Nutrient Broth (NB) + 0.5% NaCl and
Trypticase Soy Broth (TSB) were used to grow the organism. Incubation
was at 20 C for up to 96 hrs. Samples were drawn at 0, 12, 24, 48, 72
and 96 hrs. All samples were assayed for growth by measuring optical
density at 600 nm in a Spectronic-20 Spectrophotometer (Bausch and Lomb,
Rochester, NY) and by plating in Plate Count Agar (PCA) with incubation
at 20 C for 5 days. Cultures were centrifuged in a RC-5 Superspeed
Refrigerated Centrifuge at a force of 20,000 x g for 30 min. The sedi
ment was discarded. One milliliter of the cell-free broth was assayed
for enzyme activity with gelatin substrate. _P. citreus growth and
enzyme analyses were done three times and each time in duplicate.
Optimization of Enzyme Activity to Growth and Cell Number
The optimum time for cell harvesting along the logarithmic section
of the growth curve (approximately midlog) of the organism was selected.
The logarithm of the cell count was plotted against the incubation time.
The specific growth rate (k) of each medium was calculated using the
equation In B = In B + k (where B = bacterial count at time x, B =
x o x o
bacterial count at time 0 (both in midlog growth phase), k = specific
growth rate, t = time, hrs). The units of enzyme activity per cell per
hour (m) were then calculated using the formula (58,124): M M = £
t ok
(Bx BQ) (where Mt = enzyme activity at cell number B M = enzyme


13
Thirkell and Summerfield (137,138) studied the effect of varying
the sea salt concentration on the chemical composition of a purified
membrane fraction of _P. citreus. They concluded that the concentration
of salt in the medium affected the amount of membrane in the cell. Salt
concentrations above or below the normal 3% of sea water reduced the
amount of membrane material present. In addition, varying salt concen
tration had no significant effect on the amount of total neutral lipid,
glycolipid or phospholipid in the _P. citreus membrane preparations. But
a significant effect was observed on the amount of individual neutral
lipid or phospholipid classes present and on the number of individual
glycolipid components detected.
Our attention was directed toward this organism when, during a study
of the normal flora of rock shrimp (Sicyonia brevirostris), _P. citreus
was consistently isolated and found to increase in numbers during iced
storage (90). In this study, 68% of the isolates recovered were gram
positive cocci, with _F. citreus increasing from 10% of the isolates on
the fresh rock shrimp to 40% on the ice stored rock shrimp. In recent
work (1,2,3), _P. citreus has been found to be an important member of the
normal flora of Penaeus shrimp.
Alvarez and Koburger (5) described _P. citreus as a motile gram
positive coccus found in the marine environment, capable of growing over
a range of pH 7-10, 5-35 C, in broth containing 0.5-12% sodium chloride
(NaCl) and capable of hydrolyzing gelatin, cottonseed, soy and more
importantly to seafood microbiologists, shrimp protein. Figure 1 shows
a photomicrograph of citreus illustrating its morphology and flagella
tion. Table 2 shows the capabilities of this organism to hydrolyze
various protein sources.


47
gelatin as substrate. The residual activities of each solution were
compared to the activity observed when the purified enzyme was incubated
with gelatin at 35 C for 10 min.
Effect of Sodium Chloride Concentration
Various concentrations of NaCl were tested for their effect on
enzyme activity. Concentrations of 0.00, 0.25, 0.50, 0.75, 1.00, 1.25
and 1.50% were used. NaCl was dissolved in 0.05 M phosphate buffer pH 8.
The shrimp protein (0.6 mg/ml) and gelatin (1.2 mg/ml) were dissolved in
the NaCl solutions. Five milliliters of the NaCl solutions were incu
bated with 100 yl of the purified enzyme at 35 C for 10 min.
Effect of Sodium Bisulfite Concentration
Various concentrations of sodium bisulfite (NaHSO^) were tested for
their effect on enzyme activity. Concentrations of 0.0, 0.5, 1.0, 2.0
and 3.0% were tested. NaHSO^ (J. T. Baker Chemical Co., Phillipsburg,
NJ) was dissolved in 0.05 M phosphate buffer pH 8. The shrimp substrate
was dissolved in these NaHSO^ solutions (0.6 mg/ml). Five milliliters
of the NaHSO^ solutions were incubated with 100 Pi of the purified
enzyme at 35 C for 10 min.
Effect of Enzyme Concentration
Various quantities of enzyme (from 0 to 200 Pi) were tested to
observe the effect of enzyme concentration on enzyme activity. Five mil
liliters of substrate (gelatin or shrimp protein) were incubated with 0,
50, 75, 100 and 200 pi of enzyme at 35 C for 10 min.
Effect of Substrate Concentration
The enzyme was incubated with various concentrations of gelatin and
shrimp protein in order to determine substrate saturation conditions.
For the gelatin substrate, 0.00, 0.15, 0.30, 0.45, 0.60 and 1.20 mg/ml


OPTICAL DENSITY (600 nm)
56
INCUBATION TIME AT 20C(hr)
Figure 11. Spectrophotometric growth curves of Planococcus citreus in
Plate Count Broth, Nutrient Broth and Trypticase Soy Broth
at 20 C.


SUMMARY AND CONCLUSIONS
An extracellular proteolytic enzyme from a marine organism, Piano-
coccus citreus, was isolated, purified and characterized. Ammonium sul-
fate precipitation, Sephacryl S-200 Superfine chromatography and DEAE-
£
Sephadex A-50 ion exchange chromatography were used to purify the
enzyme. A single band present after acrylamide gel electrophoresis, as
well as chromatography, showed the purity of the extracellular proteo
lytic enzyme. In addition, the fluorometric technique proved to be an
efficient, fast and economical (less enzyme is required) method for the
measurement of enzyme activity.
Each fraction of the _P. citreus cells studied (extracellular, whole
cells, washings of whole cells, soluble intracellular and cellular par
ticulate) exhibited proteolytic activity. However, the major portion,
greater than 95.0%, of the active enzyme towards the high molecular
weight substrates (gelatin and shrimp protein) was recovered in the
extracellular fraction.
Trypticase Soy Broth (TSB), a highly nutritious medium, proved to
be the best medium for culturing _P. citreus cells and for the production
of the extracellular proteolytic enzyme. A highly significant difference
(a=0.05 level) was observed between the units of enzyme activity produced
per cell per hr (m) by _P. citreus grown in Trypticase Soy Broth, Plate
Count Broth and Nutrient Broth.
The following properties were characteristic of the P. citreus
extracellular proteolytic enzyme:
115


50
Table 4. Composition of yeast carbon base medium (56).
Formula in Grams per Liter of Distilled Water
Boric Acid
0.500
mg
Copper Sulfate
0.040
Potassium Iodide
0.100
Ferric Chloride
0.200
Manganese Sulfate
0.400
Sodium Molybdate
0.200
Zinc Sulfate
0.400
Biotin
0.002
mg
Calcium Pantothenate
0.400
Folic Acid
0.002
Inositol
2.000
Niacin
0.400
p-Aminobenzoic Acid
0.200
Pyridoxine
0.400
Riboflavin
0.200
Thiamine HC1
0.400
L-Histidine HC1
0.001
g*
DL-Methionine
0.002
DL-Tryptophan
0.002
Potassium Phosphate
1.000
g
Magnesium Sulfate
0.500
Sodium Chloride
0.100
Calcium Chloride
0.100
Dextrose
10.000
Final pH of the base adjusted to 7.5


82
ionic strength did not signficantly influence the activity of Myxococcus
virescens when casein was used as the substrate. However, the actual
data for this observation were not presented in the literature.
In that the activity of the proteolytic enzyme was not affected by
ionic strengths of p=0.83 or lower, the buffers shown in page 44,
(Determination of Optimum pH) were considered acceptable and were used
for the determination of the optimum pH of the _P. citreus enzyme.
Optimum pH Determination
The pH optimum of an enzyme is dependent upon a number of experi
mental parameters. Changes in pH may cause changes in the ionization
of prototropic groups (groups capable of ionization) in the active site
of an enzyme. These prototropic groups in the active site may be
involved in maintaining the proper configuration of the site, in binding
a substrate to enzyme and/or in transformation of substrate to products
(133). However, there is usually a zone of maximum ion stability in
which enzyme activity is maximal. Enzyme inactivation also increases on
the acid and alkaline sides of this maximum activity zone. Observing
Figure 22, enzyme activity was maximum at pH 8 when both gelatin and
shrimp protein were used as substrates. The activity dropped as the pH
became more acidic or alkaline. Although not statistically significant,
a slightly higher activity was evident at the alkaline pH's (9 and 10)
when shrimp protein was used as substrate.
The majority of the bacterial enzymes studied have shown maximum
proteolytic activity at neutral pH's (57,65,151). The enzyme isolated
in this study resembles the bacterial proteolytic enzyme from Proteus
vulgaris (105), Bacillus sphaericus (155), Staphylococcus aureus (11,12),
Serratia marcescens (106) and Pseudomonas spp. (87) in that they all


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Table 5. Proteolytic activity at 35 C for 15 min (pH 8) of cellular fractions obtained from
Planococcus citreus grown in Trypticase Soy Broth (TSB) using gelatin and shrimp as
substrates.
Fractions
Total Enzyme
Activity (units)
Protein3
(mg/ml)
Specific Activity*3
(units/mg total protein)
Distribution of
Activity (%)
Gelatin
Shrimp
Gelatin
Shrimp
Gelatin
Shrimp
Whole cells
386.7
395.6
23.40
0.136
0.139
0.4
0.5
Extracellular
131,100.0
122,590.0
44.50
29.450
27.540
95.0
95.8
1st washing
251.1
253.3
6.26
0.401
0.406
1.3
1.4
2nd washing
163.3
133.3
3.26
0.500
0.408
1.6
1.4
Intracellular
101.1
81.1
10.33
0.098
0.079
0.4
0.3
Particulates
171.1
192.2
11.26
0.152
0.171
0.6
0.6
^Average of duplicate samples
Total activity/Total protein = specific activity (units of activity/mg protein)


Table 9. Proteolytic activity at 35 C for 15 min (pH 8) of various ammonium sulfate
fractions of the cell-free broth of Planococcus citreus.
Fraction
Total Activity3
(units x 10 )
Protein^
(mg/ml)
Specific Activity0
(units/mg total protein)
Distribution of
Activity (%)
0-55%
8.31
18.07
4.59
7.5
55-70%
102.70
19.60
52.39
86.0
70-100%
5.00
12.50
3.99
6.5
g
^Determined using gelatin as substrate
^Average of duplicate samples
Total activity/Total protein = specific activity (units of activity/mg protein)


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Dr. J. L. Oblinger
Associate Professor of
and Human Nutrition
Food Science
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Dr.k'G. Bitton
Associate Professor of Environmental
Engineering Sciences


33
Substrate Characteristics
Two substrates were used throughout the study: gelatin (Difco)
(1.2 mg/ml) and a shrimp protein preparation (0.6 mg/ml). Higher concen
trations of the shrimp protein preparation were not used because of
solubility problems in the buffer systems used. The shrimp protein prep
aration was prepared as follows: fresh raw shrimp meat ground in a War
ing blender with 0.05 M phosphate buffer (pH 7, 1:10 dilution), dialyzed
overnight with four changes of the same buffer at 5 C and lyophylized for
preservation (Virtis Freeze Dryer, Gardiner, NY). Protein, fat, moisture
and ash were determined for the shrimp protein preparation. Protein was
determined by the AOAC standard micro-Kjeldahl method (13). Crude fat
was determined by a modification of the AOAC method (13) using the Gold-
fisch solvent chamber. Approximately 2 grams of sample were extracted
overnight with petroleum ether. Moisture was determined in a vacuum
oven at 70 C for 12 hrs. Ashing was done in a muffle furnace at 600 C
for 8 hrs. Table 3 shows that the shrimp protein preparation consisted
of 77.44% protein, 5.40% fat, 8.95% moisture, 6.50% ash and 1.71%
carbohydrate (calculated by difference).
Determination of Enzyme-Substrate Mixture Reaction Time
Five milliliters of substrate and an aliquot of cell-free broth were
incubated at 35 C for 0, 5, 10, 15, 20, 30 and 60 min in order to deter
mine the time course of enzyme activity and apparent optimum reaction
time. In experiments involving gelatin, 0.5, 1.0 and 2.0 ml of cell-free
broth were used while 1.0 ml of cell-free broth was used with the shrimp
protein substrate. An incubation time of 15 min was an appropriate
enzyme-substrate contact reaction time when shrimp were used as substrate
(Figure 7). In addition, when gelatin was used as a substrate and


41
STERILE
TRYPTICASE SOY BROTH
Figure 9: Outline of steps for the purification of the extracellular
protease(s) of Planococcus citreus.


118
13) Michaelis-Menten kinetics were followed when gelatin and
shrimp protein were used a substrates.
14) The apparent K values for gelatin and shrimp protein were
m
0.98 mg/ml and 0.33 mg/ml, respectively. The apparent V values were
max
666.67 and 431.03 (units of activity), respectively. This indicates
that the _P. citreus extracellular enzyme can degrade gelatin faster
than shrimp protein.
15) FeCl^, HgC^ and KC1 inhibited the enzyme to some extent,
while CaC^ activated the extracellular enzyme. ZnC^, MgC^ and
MnCl^ had no appreciable effect on the activity of the proteolytic
+3 +2
enzyme. The repressing effect of Fe and Hg on the activity of
this extracellular enzyme may indicate that the enzyme contains
sulfhydryl groups. p-Dioxane had no effect on the activity of the
proteolytic enzyme. EDTA, citric acid, cysteine, p-mercaptoethanol,
potassium permangate and formaldehyde inactivated the enzyme to dif
ferent degrees indicating that the extracellular enzyme was affected by
metal chelators. These results indicate that the enzyme may contain a
metal ion as cofactor and possibly sulfhydryl groups.
16) The proteolytic enzyme of _P. citreus exhibited activity against
the following dipeptides: DL-leucylglycine, DL-leucyl-DL-alanine, L-
leucyl-L-tryptophane, glycyl-DL-leucine and DL-alanylglycine. The high
est activity was observed with DL-alanylglycine.
17) Preliminary classification of the enzyme shows that it is an
a-amino-acy1-peptide hydrolase (3.4.1). Additional studies with syn
thetic peptides are necessary for complete classification.
18) The extracellular proteolytic enzyme produced by P. citreus
was not induced by the presence of shrimp protein in the growth medium.


ENZYME ACTIVITY (x I01)
112
INCUBATION TIME AT 20C (hr)
Figure 35. Proteolytic enzyme activity of the cell-free broth of
Planococcus citreus grown in Trypticase Soy Broth with
and without shrimp protein.


TABLE OF CONTENTS (continued)
Page
Effect of Sodium Bisulfite Concentration 47
Effect of Enzyme Concentration 47
Effect of Substrate Concentration 47
Effect of Metal Ions on Enzyme Activity 48
Effect of Various Reagents on Enzyme Activity 48
Dipeptidase Activity 49
Enzyme Induction Studies 49
RESULTS AND DISCUSSION 52
Proteolytic Activity of Cellular Fractions 52
Growth Medium and Enzyme Production 55
Effect of Incubation Temperature on Enzyme Production and
Activity 60
Purification of Extracellular Enzyme(s) 66
Purity of the Extracellular Proteolytic Enzyme 71
Characterization of the Extracellular Proteolytic Enzyme .... 77
Molecular Weight Determination 77
Effect of Ionic Strength on Enzyme Activity 80
Optimum pH Determination 82
Optimum Temperature Determination 84
Thermal Stability 84
Effect of Sodium Chloride Concentration 87
Effect of Sodium Bisulfite Concentration 90
Effect of Enzyme Concentration 90
Effect of Substrate Concentration 93
Effect of Metal Ions on Enzyme Activity 98
Effect of Various Reagents on Enzyme Activity 101
Dipeptidase Activity 104
Enzyme Classification 104
Enzyme Induction Studies 104
SUMMARY AND CONCLUSIONS 115
LITERATURE CITED 120
BIOGRAPHICAL SKETCH
131


122
27. Breed, R. S., E. G. D. Murray and N. R. Smith. 1957. Bergey's
Manual of Determinative Bacteriology. 7th Ed. The Williams and
Wilkins Co., Baltimore, MD.
28. Brock, T. D. Biology of Microorganisms. 2nd Ed. Prentice-Hall,
Inc., Englewood Cliffs, NJ.
29. Buchanan, R. E. and N. E. Gibbons. 1974. Bergey*s Manual of
Determinative Bacteriology. 8th Ed. The Williams and Wilkins
Co., Baltimore, MD.
30. Camber, C. I., M. H. Vance and J. E. Alexander. 1956. How to use
sodium bisulfite to control "black spot" on shrimp. Fla. State
Bd. of Conservation. The Miami Laboratory, University of Miami
Special Services Bulletin No. 12.
31. Campbell, L. L. and 0. B. Williams. 1952. The bacteriology of
Gulf Coast shrimp. IV. Bacteriological, chemical and organoleptic
changes in ice storage. Food Technol. 6:125-126.
32. Cann, 0. C. 1973. Bacteriological aspects of tropical shrimp.
FDA Conference on Fishery Products, Tokyo, Japan.
33. Cann, 0. C., G. Hobbs, B. R. Wilson and R. W. Horsley. 1971. The
bacteriology of 'scampi' (Nephrops norvegicus). I. Detailed
investigation of the bacterial flora of freshly caught samples.
J. Fd. Technol. 6:153-161.
34. Carrol, B. J., G. B. Reese and B. G. Ward. 1968. Microbiological
study of iced shrimp: Excerpts from the 1965 iced-shrimp sympo
sium. Bureau of Commercial Fisheries. Circular 284.
35. Castell, C. H. and E. G. Mappleback. 1952. The importance of
Flavobacterium in fish spoilage. J. Fish. Res. Brd. Can. 9:
148-154.
36. Christison, J. and S. M. Martin. 1971. Isolation and preliminary
characterization of an extracellular protease of Cytophaga spp.
Can. J. Microbiol. 17:1207-1216.
37. Clark, W. D. 1963. Inhibition of amino acid decarboxylases. In:
Metabolic Inhibitors. Vol. I. Eds. R. M. Hochster and J. H.
Quaste. Academic Press, Inc., New York, NY.
38. Clark, E. D. and L. MacNaughton. 1917. Shrimp: Handling, trans
portation, and uses. U. S. Department of Agriculture, Bulletin
No. 538, Washington, DC.
. Cobb, B. F. III. 1976. Biochemistry and physiology of shrimp
effect on use as food. Proc. of the First Annual Tropical and
Subtropical Fisheries Technological Conference, Corpus Christi,
TX 1:142-150.
39


LIST OF FIGURES
FIGURE Page
1 Photomicrograph of Planococcus citreus cells showing
morphology and flagellation 14
2 Comparison of the Fluorescamine technique and the Lowry
procedure for determining protein concentration 23
3 Effect of pH adjustment of gelatin-trichloroacetic acid
(TCA) filtrates on fluorescence intensity 28
4 Excitation (curve A) and fluorescence (curve B) spectrum
for the reaction of a gelatin-trichloroacetic acid (TCA)
filtrate with fluorescamine at pH 8 30
5 Excitation (curve A) and fluorescence (curve B) spectrum
for the reaction of a shrimp protein-trichloroacetic acid
(TCA) filtrate with fluorescamine at pH 8 31
6 Efficacy of trichloroacetic acid (TCA) in terminating the
enzyme-substrate reaction 32
7 Increase in fluorescence intensity using the shrimp protein
preparation as substrate after incubation with cell-free
broth for 1 hr at 35 C 35
8 Increase in fluorescence intensity using gelatin as
substrate and various amounts of cell-free broth after
incubation at 35 C for 1 hr 37
9 Outline of steps for the purification of the extracellular
protease(s) of Planococcus citreus 41
10 Increase in fluorescence intensity following incubation of
gelatin and shrimp protein substrate with purified enzyme
for up to 1 hr at 35 C 45
11 Spectrophotometric growth curves of Planococcus citreus in
Plate Count Broth, Nutrient broth and Trypticase Soy Broth
at 20 C 56
12Aerobic plate counts of Planococcus citreus incubated in
Plate Count Broth, Nutrient Broth and Trypticase Soy Broth
at 20 C for 96 hrs
58


51
The data was analyzed in a similar manner as the Growth Medium and
Enzyme Activity data. Again, a comparison of the m's for each medium
used (m = units of enzyme activity/cell/hr) was attempted using the SAS
program package for analysis of variance (15).
The Duncan's New Multiple-Range Test (pg. 187-190 (132)) was used to
compare any difference in the calculated means of the data obtained after
analysis of variance in the "Optimization of enzyme activity to growth
and cell number" section (pgs. 36 and 38), "Optimum pH determination"
section (pg. 82), "Optimum temperature determination" section (pg. 84)
and "Enzyme induction study" section (pg. 105-115). The Duncan's New
Multiple-Range Test was done using the SAS program package (15).


88
was reached, then the activity started decreasing. However, when gelatin
was used as the substrate, salt concentration (0-1.50%) had no apparent
effect on the activity of the enzyme (Figure 25). The concentrations of
the NaCl solutions used in this study, 0-0.26 M NaCl, have an ionic
strength of p=0.15-0.34 and are not within the ionic strength range that
resulted in decreased enzyme activity (Figure 21).
The effect observed when shrimp protein is used as substrate is
probably due to an initial increase in solubility of the substrate due
to the increase in salt concentration. Structurally, gelatin is a small
protein when compared to shrimp protein. Perhaps the increase in solu
bility allowed an easier enzyme-substrate interaction, thus, accounting
for the initial increase in enzyme activity.
The effect of higher concentrations of NaCl on enzyme activity was
not investigated. However, Figure 21 illustrates that an ionic strength
of 0.83 (0.75 M NaCl) or above resulted in decreased enzyme activity.
Reversible inactivation of the enzyme and substrate effects due to higher
NaCl concentrations were not investigated.
Arvidson and coworkers (11,12) showed that the activity of both
extracellular proteases I and II from Staphylococcus aureus (neutral and
alkaline protease, respectively) was reduced by concentrations of 0.5 M
NaCl or above. Gnosspelius (76) stated that an NaCl concentration of 0.2
M in the assay mixture had no effect on the activity of the Myxococcus
virescens extracellular enzyme. However, higher NaCl concentrations
decreased the proteolytic activity.
During the storage of Penaeus shrimp on ice, the salt concentration
will decrease due to the leaching of the salt as the ice melts or perco
lates through the shrimp. As the NaCl concentration decreases in shrimp,
the activity of the J?. citreus enzyme will be enhanced.


38
activity at cell number B k = specific growth rate). This formula was
o
din k'
obtained through the integration of the following equation: = m Bq e
(where dm/dt = change in enzyme activity over time). The calculated m's
were compared for the three media used. A test to observe any difference
between the values for the units of enzyme activity per cell per hour (m)
in each medium was designed using the Statistical Analysis System (SAS)
program package (15) for analysis of variance. A completely randomized
design (102,132) was used in that the major source of error to be
considered was due to the nutritional differences between media.
Effect of Incubation Temperature on Enzyme Production and Activity
The effect of incubation temperature (5, 20 and 35 C) on the growth
of _P. citreus and its ability to produce an active extracellular enzyme
was investigated. Five-hundred-milliliter Erlenmeyer flasks containing
3
100 ml of medium were inoculated with approximately 5 x 10 P_. citreus
and incubated at the three temperatures. All samples were assayed for
growth by measuring optical density at 600 nm in a Spectronic-20 spectro
photometer and for proteolytic activity using the fluorometric technique.
For the 35 C grown cells, samples were drawn at 0, 6, 12, 24, 48, 72 and
96 hrs. For the 20 C grown cells, samples were drawn at 0, 12, 24, 48,
72, 96 and 120 hrs. Finally, for the 5 C grown cells, samples were drawn
at 0, 24, 48, 72, 96, 120 and 144 hrs. After determining the midlog
phase of growth for _P. citreus at each temperature, _P. citreus cells were
then harvested at this stage. Enzyme activity determinations were done
using the cell-free broth obtained from growing the organism at the three
temperatures until midlog phase. Five milliliters of the shrimp protein
preparation were incubated with 1 ml of each cell-free broth at 5 C for
60 min, 20 C for 30 min and 35 C for 15 min. Analyses were done three
times and each time in duplicate.


46
Ionic Strength
8 5.3 ml of 0.2 M monobasic sodium phosphate 0.25
+ 95.0 ml of 0.2 M dibasic sodium phosphate
9 50 ml of 0.2 M glycine + 8.8 ml of 0.2 M NaOH 0.22
10 50 ml of 0.2 M glycine + 32.0 M NaOH 0.20
Gelatin (1.2 mg/ml) or shrimp protein (0.6 mg/ml) were dissolved in
the various buffers. Any pH adjustments due to the addition of the sub
strates were done using 10 mM HC1 or 10 mM NaOH. Five milliliters of
this mixture were reacted with 100 pi of the purified enzyme and incu
bated at 35 C for the selected reaction time (10 min).
Determination of Optimum Temperature
Five milliliters of gelatin or shrimp protein substrate and 100 pi
of the purified enzyme were incubated at 5, 10, 20, 35, 45, 55 and 65 C
for 10 min at the optimum pH determined in the previous section.
Thermal Stability
_P. citreus was incubated at 5 and 35 C in 300 ml of Trypticase Soy
Broth (TSB). Cell-free broths obtained at midlog phase, 108 and 36 hrs
for the 5 and 35 C grown cells, respectively, were used in this study.
Five milliliters of the cell-free broths were incubated at 35, 45, 55,
65, 75 and 85 C for 15 min. The heat treated cell-free broths solutions
were rapidly cooled (87), and their activity was assayed at 35 C for 15
min using gelatin as substrate. The residual activities at each solution
were compared to the activity observed when the cell-free broths were
incubated with the substrate at 35 C for 15 min.
In addition, 1 ml of the purified enzyme was also incubated at 35,
45, 55, 65, 75 and 85 C for 10 min. The heat treated purified enzyme
solution was cooled, and its activity assayed at 35 C for 10 min using


P. citreus was grown in Trypticase Soy Broth at 20 C for 72 hrs.
Centrifugation, ammonium sulfate precipitation, Sephacryl S-200 Super-
£
fine molecular sieve chromatography, DEAE-Sephadex A-50 ion exchange
chromatography and acrylamide gel electrophoresis were used to purify
the extracellular enzyme(s). The enzyme was purified 26.50 fold (using
the fluorometric technique for activity measurement), and recovery of
the enzyme was above 49%. Gelatin and shrimp protein were used as sub
strates throughout the study. The molecular weight of the purified
protease was approximately 29,000 as measured by Sephacryl S-200 column
chromatography and acrylamide gel electrophoresis.
Maximum activity of the enzyme was at pH 8 and 35 C. Ionic
strengths of above 0.83 (0.75 M NaCl) decreased the activity of the
extracellular enzyme. Heat treatment at 65 C for 15 min destroyed the
activity of the purified enzyme. However, 1.0% of the residual enzyme
activity was still present in the cell-free broth of _P. citreus grown at
35 C for 36 hrs. In contrast, 15 min at 75 C were necessary to reduce
99.0% the activity of the enzymes in the cell-free broth of _P. citreus
grown at 5 C for 108 hrs. When shrimp protein was used as substrate,
sodium chloride concentrations of 0.0-0.5% increased enzyme activity,
while concentrations of 0.5-1.5% decreased enzyme activity. However,
when gelatin substrate was used, NaCl concentrations of 0.0-1.5% had
no effect on enzyme activity. The activity of the purified enzyme
decreased as the concentration of sodium bisulfite increased. Michaelis-
Menten kinetics were followed when gelatin and shrimp protein preparation
were used as substrates. The apparent values for gelatin and shrimp
protein were 0.98 mg/ml and 0.33 mg/ml, respectively. The apparent
V values were 666.67 and 431.03 units of activity for gelatin
xiii
I


Table 7. Enzyme activity measured at 5, 20 and 35 C (pH 8) of the cell-free broths of Planococcus
citreus grown in Trypticase Soy Broth (TSB) at 5, 20 and 35 C for 108, 72 and 36 hrs,
respectively.
Enzyme-Substrate
Incubation Temperature (C)
Temperature of Growth (C)
5
20
35
5
24.18
+
l.lla
58.18 2.08
116.17
8.40
20
25.65
/
+
1.20
72.30 2.04
139.67
7.88
35
28.56

1.35
98.55 3.10
178.93
7.68
Average of 6 observations standard deviation


65
substrate incubation temperatures. A higher _P. citreus count was
observed at 35 C and the production of extracellular enzyme(s) was also
higher at all three enzyme-substrate incubation temperatures. This
indicates that the amount of enzyme produced by _P. citreus is related to
the amount of growth of the organism in the medium. In addition, as the
enzyme-substrate incubation temperature increased from 5 to 35 C, the
enzyme activity of the cell-free broths increased. Although enzyme
activity is present at the lower temperatures, the data presented indi
cate that the optimum temperature of the extracellular protease system
may be close to 35 C. Consequently, the results indicate that _P. citreus
can indeed produce an active extracellular enzyme(s) capable of utilizing
the protein in shrimp when shrimp is stored at refrigerated or iced
temperatures.
The effect of refrigeration temperatures on enzyme activity has
been studied (50,51). In most of the research, the majority of the
enzymes studied lost activity when incubated at low temperatures. Stud
ies have shown that lactic streptococci characteristically produced less
acid after storage at refrigerated temperatures. Such stored cells also
show a diminished residual proteinase activity (49,50,51,52,149). The
researchers stated that after storage at 3 C, the enzyme showed gross
structural alterations with a concomitant loss of activity. Gel filtra
tion and sedimentation velocity data indicate that inactivation of the
enzyme was a result of aggregation to higher molecular weight forms (50).
However, several investigators (49,52,131) previously suggested that
storage inactivation of enzymes may be caused by induced conformational
or structural changes. Scutton and Utter (128) and Havir et al. (81)
observed that inactivation of various enzymes by low temperature storage


90
Effects of Sodium Bisulfite Concentration
To control black spotting in shrimp (148), sodium bisulfite (NaHSO^)
is used to inhibit enzymatic oxidation of both tyrosine and dihydroxy-
phenylalanine thereby preventing darkening of the shell (66,96). Since
1956, agencies such as the former Florida State Department of Conserva
tion (30), now the Department of Natural Resources, have recommended
dipping shrimp in a 1.25% sodium bisulfite solution for 1 min to control
black spot development. Therefore, the effect of sodium bisulfite con
centration (0 to 3%) on the activity of the extracellular proteolytic
enzyme of _P. citreus was investigated. Figure 26 shows that as the con
centration of sodium bisulfite increases, the activity of the enzyme
decreases. When sodium bisulfite dissociates in water, it may affect
enzyme activity by reducing disulfite (-S=S-) linkages (57). The activ
ity of the enzyme in the presence of 1.25% sodium bisulfite was approxi
mately 240 units of activity. Thus, with the addition of 1.25% sodium
bisulfite, approximately 47% of the activity of the proteolytic enzyme
was lost. However, as the concentration of sodium bisulfite decreases
(leaches out in the melt water) (148) the activity of the enzyme should
be less affected.
Effect of Enzyme Concentration
Rates of enzyme-catalyzed reactions are directly dependent on
enzyme concentration (151). The effect of enzyme concentration (0 to
200 yl) on the activity of the _P. citreus enzyme when gelatin and shrimp
protein were used as substrates was investigated. By observing Figure
27, the enzyme preparations used for characterization followed a linear
relationship with increasing levels of enzyme. According to Dixon and
Webb (57) when the plot passes through the origin, inhibitors are usually


76
With the presence of the relatively new Fluorometric technique,
that appears to be more sensitive and reproducible than the traditional
methods available for measuring proteolytic enzyme activity, the results
of various previous research with extracellular enzymes (36,78,84,88,
136) using the Anson method (9) could have possibly resulted in higher
recoveries and higher measurable total enzyme activity. The following
investigators are some of those who used the Anson method to study the
various enzymes. Tarrant et al. (136) working with Pseudomonas fragi in
pig muscle isolated an extracellular proteolytic enzyme with only 18%
recovery after partial purification. Husein and McDonald (84) character
ized an extracellular proteinase from Micrococcus freudenreichii using
casein as substrate with 23% recovery after partial purification.
Christison and Martin (36) isolated and preliminarily characterized an
extracellular protease of Cytophaga spp. using casein, hemoglobin and
£
azocoll as substrates. After chromatography with DEAE-Cellulose only
26% of the enzyme was recovered. Khan et al. (88), looking at the
extracellular proteases of Mucor pusillus, isolated and characterized
two fractions. However, after DEAE-Sephadex A-50 only 29.3% of the
milk-clotting fraction was recovered and 47.0% of the fraction with pro
tease activity toward hemoglobin was recovered. Gnosspelius (76) puri
fied an extracellular protease from Myxococcus virescens using phosphate
precipitation, gel exclusion and ion exchange chromatography. Only
20.1% was recovered after the chromatographic step. In the work reported
U
in this dissertation, following DEAE-Sephadex A-50, 49.0% of the extra
cellular enzyme of _P. citreus was recovered when the Fluorometric method
was used to measure proteolytic activity.


18
that these enzymes exert a specificity toward the amino acids involved
in the peptide bonds which they attack. Bergmann and his students are
also responsible for the presently accepted classification of proteolytic
enzymes: They proposed that these enzymes be grouped into two classes
endopeptidases and exopeptidasesdepending upon whether they hydrolyzed
peptide bonds remote from, or near to, the end of the peptide chains of
their natural substrates. The former class includes such enzymes as
pepsin, trypsin and chymotrypsin, while the latter class contains the
dipeptidases and the amino and carboxy peptidases.
Proteinases in bacteria may be either intracellular or extracellular
depending upon whether they exert their activity within the cell or
whether they are excreted from the cell to attack proteins in the envi
ronment (10,58). Also, enzymes may be classified according to their
location in, on or around the cell: a) cell-bound: 1) truly intracell
ular, 2) surface-bound; and b) extracellular (58). Extracellular enzymes
are those enzymes which exist in the medium around the cell, having
originated from the cell without any alteration to cell structure greater
than that compatible with the cell's normal processes of growth and
reproduction. This distinction is not always clear and in some instances
it is entirely possible that autolysis of cells has permitted the escape
of intracellular enzymes into the culture filtrate. This is particularly
true when high proteolytic activity is dependent upon prolonged
incubation of the culture (74).
In 1964, the International Union of Biochemistry (54) recommended a
scheme for numbering enzymes, which is currently used for the classifi
cation of enzymes. Enzymes are divided into groups on the basis of the
type of reaction catalyzed, and this, together with the name(s) of the


105
Table 12. Dipeptidase activity of the Planococcus citreus extracellular
enzyme.
Dipeptide
Enzyme Activity
a
DL-leucylglycine
DL-leucyl-DL-alanine
L-leucyl-L-Tryptophane
glycyl-DL-leucine
DL-alanylglycine
11.67 0.89
22.94 0.44
32.61 1.98
11.56 0.18
47.67 2.48
a
Average of 6 observations standard deviation


LOG PLANOCOCCUS CITREUS/ml
58
INCUBATION TIME AT 20 C (hr)
Figure 12. Aerobic plate counts of Planococcus citreus incubated in
Plate Count Broth, Nutrient Broth and Trypticase Soy Broth
at 20 C for 96 hrs.


16
The reports (4,5) by Alvarez and Koburger outline some observations
on the distribution of _P. citreus in the marine environment. Of the 35
samples of marine origin examined for _P. citreus, only 5 yielded this
organism. Four were shrimp samples and the fifth was a stuffed flounder
sample that had been prepared in a plant that processed predominantly
shrimp. One of the shrimp samples from which Planococcus was isolated
had been in frozen storage for over six years. Fresh seafood (trout,
sheephead, mackerel, crab and oysters) as well as Gulf Coast waters and
sediments from the vicinity of Suwannee, Florida, were also examined for
_P. citreus without success. However, in more recent studies performed
by Mallory et al. (100), _P. citreus was isolated from estuarine areas of
Chesapeake Bay in low numbers.
Since the isolation of gram-positive organisms from iced seafood is
uncommon, Alvarez and Koburger (5) studied the contribution of _P. citreus
to the spoilage of Penaeus shrimp. They utilized gamma irradiation (600
Krads) to lower the number of bacteria in raw. shrimp and then inoculated
3
a portion of the shrimp with 5 x 10 _P. citreus cells per gram of shrimp
in order to study the changes produced by this organism. _P. citreus
3
counts increased in the inoculated shrimp from 5 x 10 bacteria/gram at
g
0 day to 1.9 x 10 bacteria/gram at the 16th day. The potential of _P.
citreus as a "spoiler" of shrimp was shown by an increase in pH and the
rapid increase in total volatile nitrogen/amino acid-nitrogen ratio (TVN/
AA-N) and trimethyl-amine nitrogen (TMN) content. In 1973, Cobb et al.
(42) reported a high correlation between total volatile nitrogen/amino
acid-nitrogen ratio (TVN/AA-N) and quality of shrimp. Later work (41)
suggested that the TVN/AA-N ratio and the logarithm of bacterial counts
increased at approximately the same rate after the initial lag phase of


123
40. Cobb, B. F. and C. Vanderzant. 1971. Biochemical changes in
shrimp inoculated with Pseudomonas, Bacillus and coryneform
bacterium. J. Milk Food Technol. 34:533-540.
41. Cobb, B. F. and C. Vanderzant. 1975. Development of a chemical
test for shrimp quality. J. Food Sci. 40:121-124.
42. Cobb, B. F., I. Alaniz and C. A. Thompson. 1973. Biochemical and
microbial studies on shrimp: Volatile nitrogen and amino nitrogen
analyses. J. Food Sci. 38:431-436.
43. Cobb, B. F., C. Vanderzant, C. A. Thompson and C. S. Custer. 1973.
Chemical characteristics, bacterial counts, and potential shelf-
life of shrimp of various locations on the northwestern Gulf of
Mexico. J. Milk Food Technol. 36:463-468.
44. Conway, E. J. 1958. Microdiffusion Analysis and Volumetric Error,
p. 199. The Macmillan Company, New York, NY.
45. Cook, D. W. 1970. A study of bacterial spoilage patterns in iced
Penaeus shrimp. Microbiology Section, Gulf Coast Research Labora
tory, Ocean Springs, MS.
46. Cook, D. W. and R. E. Bowman. 1972. Retardation of shrimp spoil
age with ethylene diaminetetraacetic acid. J. Miss. Acad. Sci.
17:38-43.
47. Cooper, T. G. 1977. The Tools of Biochemistry. John Wiley and
Sons, Inc., New York, NY.
48. Coppola, E. D., A. F. Wickroski and J. G. Hanna. 1975. Fluoro-
metric determination of nitrite in cured meats. J. of the AOAC
58:469-473.
49. Cowman, R. A. and M. L. Speck. 1965. Activity of lactic strepto
cocci following storage at refrigeration temperaures. J. Dairy
Sci. 48:1441-1444.
50. Cowman, R. A. and M. L. Speck. 1969. Low temperature as an
environmental stress on microbial enzymes. Cryobiology 5:291-299.
51. Cowman, R. A. and M. L. Speck. 1975. Ultralow temperature storage
of lactic streptococci. J. Dairy Sci. 58:1531-1532.
52. Cowman, R. A., H. E. Swaisgood and M. L. Speck. 1967. Proteinase
enzyme system of lactic streptococci. II. Role of membrane pro
teinase in cellular function. J. Bacterid. 94:942-948.
53. Dabrowski, T., E. Kolakowski and B. Karnicka. 1969. Chemical
composition of shrimp flesh (Parapenaeus spp.) and its nutritive
value. J. Fish. Res. Brd. Can. 26:2969-2974.


Figure 6. Efficacy of trichloroacetic acid (TCA) in terminating the enzyme-substrate reaction.


42
Confirmation of Enzyme Purity
A modification of the Weber and Osborn (144) method for sodium
dodecyl sulfate-poly acrylamide gel (SDS-PAG) gel electrophoresis was
used. A Buchler 3-1500 electrophoresis apparatus (Buchler Instruments
Corp., Fort Lee, NJ) was used to evaluate the purity of the isolated
extracellular enzyme.
A 10% acrylamide:BIS, 30:0.8 gel was prepared and allowed to poly
merize for 2 hrs. A sample of the purified enzyme was diluted 1:1 with
the sample buffer. The sample buffer consisted of 0.01 M sodium phos
phate (pH 7), 10% sodium dodecyl sulfate, 0.1% dithiothrietol, 10% glyc
erol and 0.001% bromocresol blue. The protein solutions were placed onto
the gels (50 yg protein/gel, 100 yg/gel, 150 yg/gel and 200 yg/gel) and
were layered carefully with electrode buffer (pH 8.3) to the top of each
tube. The lower electrode chamber was then 2/3 filled with electrode
buffer. The tubes in the apparatus were then lowered into the electrode
chamber. The upper chamber was filled with water to approximately 1 inch
over the tube top. The water jacket was connected and the electrode
wires from the power source were also connected. A constant current of
1-1.5 mAmps/gel was applied until the marker dye band just exited from
the gels (approximately 3 hrs). The gels were immediately removed from
their tubes. The gels were fixed overnight in a fresh 50% TCA solution.
The fixed gels were then stained 1-2 hrs with 0.1% Coomassie brilliant
blue solution made up fresh in 50% TCA at 37 C in a water bath. The gels
were further diffusion-destained by repeated washings in 7% acetic acid
(17-72 hrs). Gels were then stored in 7% acetic acid (82).


49
Dipeptidase Activity
The potential of the P. citreus enzyme to degrade peptides was
investigated. DL-leucylglycine, DL-leucyl-DL-alanine, glycyl-DL-leucine,
DL-alanylglycine and L-leucyl-l-tryptrophan (Sigma Chemical Co., St.
Louis, MO) were used in this study. Fifty milligrams of each dipeptide
were dissolved in 50 ml of phosphate buffer, pH 8. Five milliliters of
the dipeptide solutions were incubated with 100 pi of the purified
enzyme at 35 C for 10 min. The reaction was terminated by adding 10 ml
of 5% TCA. Zero time blanks were done by adding the TCA to the enzyme-
peptide mixture before the incubation period.
Enzyme Induction Studies
_P. citreus was grown in various media in order to determine if the
extracellular proteolytic enzyme produced by this organism is induced by
shrimp protein. Three-hundred milliliters of the following were used:
(1) Yeast Carbon Base (YCB) (control)
(2) YCB + 1.0% Shrimp Protein
(3) YCB + 0.1% Yeast Extract
(4) YCB + 0.1% Yeast Extract + 1.0% Shrimp Protein
Table 4 shows the composition of the Yeast Carbon Base medium (YCB).
_P. citreus growth and enzyme activity were analyzed at 0, 24, 48, 72
and 96 hrs following incubation at 20 C. Cell numbers were determined
by pour plating into Trypticase Soy Agar (TSA) with incubation at 20 C
for 5 days. Five milliliters of the shrimp substrate were incubated
with 1 ml of the cell-free broth from each culture for 15 min at 35 C.
The reaction was terminated by adding 10 ml of 5% TCA. Zero time blanks
were done by adding the TCA to the cell-free broth-substrate mixture
before the incubation period. This study was done twice in duplicate.


22
(nonfluorescent)
Hydrolysis products
(nonfluorescent)
Primary amines are first buffered to an appropriate pH (7-8), and
then fluorescamine, dissolved in a water miscible, nonhydroxylic solvent
such as acetone or dioxane, is added. The reaction is complete, and in
less than a minute excess reagent is destroyed. The resulting fluores
cence is proportional to the amine concentration, and the fluorophors
are stable over several hours. The above properties lend themselves
well to automation (123). It should be noted that fluorescamine does
not react with proline or hydroxyproline, which are not primary amines.
This disadvantage can be overcome by introducing an appropriate inter
mediate step to convert these amino acids to primary amines (63,146).
An additional advantage of the fluoresamine assay is that comparatively
little fluorescence is developed with ammonia. Therefore, ammonia does
not interfere with an analysis to the extent that it does in the colori
metric ninhydrin procedure. Figure 2 shows a comparison of the


5
and Williams (31) and Williams et al. (154) isolated species of Achromo-
bacter, Bacillus, Micrococcus, Flavobacterium and Pseudomonas from Gulf
coast shrimp. Vanderzant et al. (142) reported that the flora of shrimp
from the Gulf of Mexico consisted of coryneforms, Achromobacter, Flavo
bacterium and Bacillus. In Pacific shrimp, Acinetobacter-Moraxella spe
cies were predominant (80). Lee and Pfeifer (94) reported that the flora
of Pacific shrimp (Pandalus jordani) consisted of Moraxe11a, Pseudomonas,
Acinetobacter, Arthrobacter and Flavobacterium-Cytophaga species. Cann
/
(32) and Cann et al. (33) found that coryneform organisms were predomi
nant in the bacterial flora of scampi, Nephrops norvegicus, with strains
of Achromobacter-Acinetobacter group and Pseudomonas, Cytophaga and
Micrococcus species also present. Koburger et al. (90) reported that
the Flavobacterium-Cytophaga group represented the majority of the
organisms of fresh rock shrimp (Sicyonia brevirostris), and Alvarez (1)
and Alvarez and Koburger (3) reported that Flavobacterium and Pseudomonas
were the predominant groups isolated from Penaeus shrimp from the East
and West coasts of Florida.
When shrimp are stored in ice, the number and kinds of bacteria
shift to a predominantly psychrotrophic flora (130). Psychrotrophs are
described as organisms having an optimal growth temperature of about 20
C. A comparatively longer storage life of iced shrimp from tropical
waters has been reported by Carrol et al. (34). Cann et al. (33) in
their review on tropical shrimp indicated that penaeid shrimp from the
Gulf of Thailand remained in acceptable condition for 12-16 days on ice,
whereas nontropical shrimp, such as Pandalus and Nephrops species, were
totally spoiled after 8-10 days. They attributed this difference to the
bacterial flora; the mesophilic flora on tropical shrimp are not active


124
54. Damn, H. C. Ed. 1966. The Handbook of Biochemistry and
Biophysics. The World Publishing Company, New York, NY.
55. Difco Manual of Dehydrated Cultures, Media and Reagents for Micro
biological and Clinical Laboratory Procedures. 1974. 9th Ed.
Difco Laboratories, Detroit, MI.
56. Difco Supplementary Literature. 1962. Difco Laboratories,
Detroit, MI.
57. Dixon, M and E. C. Webb. 1958. Enzymes. Academic Press, Inc.,
New York, NY.
58. Doelle, H. W. 1975. Bacterial Metabolism. 2nd Ed. Academic
Press, Inc., New York, NY.
59. Drapeau, G. R., Y. Boily and J. Houmard. 1972. Purification and
properties of an extracellular protease of Staphylococcus aureus.
J. Biol. Chem. 247:6720-6726.
60. Eaton, N. R. 1962. New press for disruption of microorganisms.
J. Bacteriol. 83:1359-1360.
61. Elmore, D. T. 1968. Peptides and Proteins. University Press,
Cambridge, MA.
62. Farber, L. and M. Ferro. 1956. Volatile reducing substances
(VRS) and volatile nitrogen compounds in relation to spoilage in
canned fish. Food Technol. 10:303-304.
63. Felix, A. M. and G. Terkelsen. 1973. Total fluorometric amino
acid analysis using fluorescamine. Arch. Biochem. Biophys. 157:
1777-1782.
64. Fellers, C. R., M. Gagnon and R. Khatchikian. 1957. Biochemical
and bacteriological methods for determining shrimp quality. Proc.
Gulf Caribbean Fish. Inst. 9th Annual Session 23-26.
65. Ferdinand, W. 1969. The Enzyme Molecule. John Wiley and Sons,
Inc., New York, NY.
66. Fieger, E. A. 1951. Cause and prevention of black spot on shrimp.
IQ Refrig. 120:49-50.
67. Fieger, E. A. and J. J. Friloux. 1954. A comparison of objective
tests for quality of Gulf shrimp. Food Technol. 8:35-38.
68. Flick, G. J. and R. T. Lovell. 1972. Postmortem biochemical
changes in muscle of Gulf shrimp, Penaeus aztecus. J. Food Sci.
37:609-611.
69. Flores, S. C. and D. L. Crawford. 1973. Postmortem quality
changes in iced Pacific shrimp (Pandalus jordan). J. Food Sci.
38:575-579.


7
Alvarez and Koburger (3) reported that the numbers of Moraxella,
Vibrio/Aeromonas and Planococcus species isolated from Penaeus shrimp
remained relatively constant throughout 10 days of ice storage. However,
Flavobacterium isolates increased until the fifth day, then decreased
rapidly. Pseudomonas species showed the opposite trend. They decreased
until the fifth day, then increased rapidly. Other workers have observed
the presence of Flavobacterium in raw shrimp (31,80,90,94,142,143) and
have noted this decrease in numbers during ice storage with a subsequent
increase in Pseudomonas species. Cook (45) was unable to produce typical
spoilage when shrimp were inoculated with Flavobacterium species, indi
cating that they are probably an inert group of organisms found in
shrimp. In contrast, Pseudomonas species have been implicated as the
organisms primarily responsible for the spoilage of marine products
stored in ice (108,130).
Measurement of Shrimp Spoilage
Numerous methods for determining shrimp quality have been developed;
however, due to the complexity, time involved and inconsistent results of
many of these methods, only a few are routinely used by the industry and
then, only for internal quality control. In many of these chemical
tests, results can vary with the age of the shrimp, size, species, area
of catch and handling conditions. Many of the tests only indicate the
onset of spoilage (31,109). Table 1 lists the chemical and physical
tests that have been used to measure shrimp quality. Total volatile
nitrogen/amino acid-nitrogen (TVN/AA-N) ratio (40,41,42,43,64,75) is the
chemical test that shows the best correlation with organoleptic quality
measurements of shrimp. Moore and Eitenmiller (107) compared various
methods for measuring shrimp quality. They observed that a relatively


6
at ice temperatures and little spoilage occurs until the psychrotrophic
flora develops. Cann et al. (33) stated that the amount of spoilage may
be related to the degree to which psychrotrophic strains are introduced
with the ice. Consequently, the rate of increase in bacterial growth
depends on the initial number of bacteria, handling on deck, and amount
and quality of ice used. Shewan (130) demonstrated that the action of
many psychrotrophic organisms resulted in rapid fish spoilage. The
principal organisms he mentioned were Pseudomonas, Aeromonas, Vibrio,
Moraxella, coryneforms and Flavobacterium. Castell and Mappleback (35)
concluded that Flavobacterium was among the most important of the fish-
spoilage bacteria. Flavobacterium is a frequently encountered bacterium
on fresh shrimp flesh.
*\
The bacterial flora of shrimp undergoes marked changes as the stor
age period increases. Campbell and Williams (31) showed Bacillus, Micro
coccus and Flavobacterium made up over 50% of the flora initially,
whereas the Achromobacter-Pseudomonas group accounted for 98% of the
flora after 16 days of iced storage. In a study on the bacterial spoil
age patterns of headless brown shrimp, Cook (45) noted that there was
only one consistent change in the bacterial types growing initially or
during the period of die-off. As the bacterial count began to rise,
Pseudomonas species became the predominant organism, accounting for 80-
100% of the bacterial types isolated. Vanderzant et al. (142) reported
that the predominant bacterial flora of fresh shrimp consisted of
coryneforms and that following storage Pseudomonas species predominated.
Cobb et al. (43) indicated that typical spoilage organisms of the genus
Pseudomonas are not usually found in freshly caught shrimp. It is not
until the shrimp are exposed to handling on board the vessel that this
organism becomes apparent.


ENZYME ACTIVITY ( xI01)
50
Figure 30.
Effect of shrimp protein concentration on the reaction rate of the Planococcus citreus
extracellular enzyme.
Increasing shrimp protein concentrations were incubated with the enzyme at
35 C for 10 min (pH 8).
vO
CT


ENZYME ACTIVITY (x 10')
igure 6. Effect of sodium bisulfite (NaHSO^) concentration on enzyme activity.3
a .
Activity assayed at 35 C for 10 min (pH 8).


116
1) The cell-free broth obtained from _P. citreus cells grown at 5 C
for 108 hrs, 20 C for 72 hrs and 35 C for 36 hrs exhibited enzyme activ
ity towards shrimp protein at all three enzyme-substrate incubation
temperatures (5, 20 and 35 C). Thus, _P. citreus when grown at 5 C pro
duces an extracellular enzyme capable of utilizing the protein in shrimp
stored either at refrigeration or higher temperatures.
2) The major portion of the extracellular proteolytic enzyme of _P.
citreus was recovered at an ammonium sulfate concentration between 55-70%
saturation. Eighty-six percent of the total activity was recovered in
this fraction.
3) Using the fluorometric method for activity measurements, the
protease was purified 26.50 fold with a recovery of approximately 49%.
The specific activity of the purified enzyme was 780.37 (units of
activity/mg of protein).
4) Purity of the enzyme was demonstrated by the presence of a
single band after acrylamide gel electrophoresis using various protein
concentrations as well as by the presence of a single peak with homo
geneous activity after ion-exchange chromatography.
5) The molecular weight of the _P. citreus enzyme was approximately
29,000 according to column chromatography using Sephacryl S-200 and
acrylamide gel electrophoresis.
6) Ionic strengths of 0.15-0.83 had no effect on the activity of
the extracellular enzyme.
7) pH optimum of the proteolytic enzyme was 8. Activity of the
enzyme decreased as the pH deviated from this optimum.
8) The optimum temperature for the JP. citreus enzyme was 35 C;
however, activity was observed at 5 C.


98
mg/ml, respectively. The apparent V values for gelatin and shrimp
protein were 666.67 and 431.03 units of activity, respectively. Since
K is defined as the rate of the disappearance of the enzyme-substrate
m
(ES) complex to the appearance of the ES complex, it appears that
gelatin has a higher affinity for the enzyme than shrimp protein.
Consequently, is lower with shrimp protein as substrate. This is
shown in Figures 29 and 31. _P. citreus extracellular proteolytic enzyme
can utilize and degrade gelatin at a more rapid rate than shrimp protein.
Various factors could be responsible for observing a higher V^ax when
gelatin is used as substrate. Gelatin, with a molecular weight of 90,000
(152), is a simple protein when compared to shrimp protein. Four amino
acids comprise 70% of the total amino acid composition. Glycine and
alanine add to approximately 50% of the amino acids present in gelatin
(152). Possibly, the relative simplicity of gelatin makes this protein
more available to the action of the _P. citreus extracellular enzyme. In
addition, gelatin showed higher solubility than shrimp protein in the
buffer system used. Perhaps, this increased solubility allowed for an
easier enzyme-substrate interaction. However, shrimp protein has a more
complex primary structure, and as it was prepared in this study, it is
probably a mixture of proteins. Eighteen amino acids comprise approxi
mately 60% of the total amino acid composition (53). The composition of
the shrimp protein preparation probably makes it a more complex substrate
for the .P. citreus extracellular enzyme. Consequently, shrimp protein
is not as easily available for the reaction with the enzyme.
Effect of Metal Ions on Enzyme Activity
The effect of various metal ions on the activity of the extracellu
lar proteolytic enzyme of _P. citreus was investigated. The effect of


Transformation of data presented in Figure 29.


ENZYME ACTIVITY (x I01)
IONIC STRENGTH (/l)
Figure 21. Effect of ionic strenght on the activity of the extracellular proteolytic enzyme of
Planococcus citreus.
aActivity assayed at 35 C for 10 min, pH 8.


77
Characterization of the Extracellular Proteolytic Enzyme
The fractions collected (17-21) from peak B (Figure 16) were pooled
and used for the characterization of the extracellular enzyme of _P.
citreus.
Molecular Weight Determination
Two methods were used to determine the molecular weight of the
enzyme, column chromatography (Sephacryl S-200 Superfine) and acrylamide
gel electrophoresis. Standards ranging from a molecular weight of 10,000
to 200,000 were used to determine the molecular weight of the _P. citreus
enzyme. Using both techniques, the molecular weight of the extracellular
enzyme of this organism was approximately 29,000. Figures 19 and 20 show
£
the molecular weight determination using Sephacryl S-200 and acrylamide
gel electrophoresis, respectively. Different standards were used in each
case to assure that the molecular weight was estimated correctly.
A search of the literature was done in order to compare the molecu
lar weight of the extracellular proteolytic enzyme of J?. citreus with
extracellular proteases from other microorganisms. Pacaud and Uriel
(113) estimated the molecular weight of a protease from Escherichia coli
using electrophoresis on polyacrylamide gels and sucrose-density gradient
centrifugation to be about 43,000. Four years later, Pacaud and Richaud
V
(114) estimated the molecular weight of a second protease of E. coli
using gel filtration and SDA-acrylamide gels to be 58,000. Drapeau et
al. (59) estimated the molecular weight of an extracellular protease of
Staphylococcus aureus to be approximately 12,000 using sedimentation
equilibrium and gel electrophoresis studies. Arvidson et al. (12)
reported the molecular weight of an extracellular (alkaline protease)
enzyme from _S. aureus to be approximately 12,500. Later, he reported


113
that _P. citreus can produce an extracellular enzyme(s) in the presence
or absence of shrimp protein in the growth medium. The units of enzyme
activity per cell per hr (m) of the enzyme produced in TSB and TSB + 1.0%
shrimp protein were 147.04 and 146.58, respectively. These values are
not significantly different (a=0.05 level). However, when compared to
the m values in Table 13, both of these m values are significantly
different (a=0.05 level).
Consequently, these data appear to indicate that the extracellular
proteolytic enzyme produced by _P. citreus is not induced by the presence
of shrimp protein in the growth medium. The enzyme is produced in mini
mal media and its activity per cell per hr increases as the nutrient
composition in the medium increases. P. citreus produces the extracel
lular enzyme even in the presence of surplus nutrients. Table 14 illus
trates these findings more clearly. There appears to be no effect, e.g.,
gene repression, by any of the factors present in the various media used.
Consequently, the production of the extracellular proteolytic enzyme by
_P. citreus appears to be related more to growth of the organism than to
the presence of any specific nutrient.


117
9) After 15 min of incubation of the purified enzyme at 65 C, no
activity was observed and only 1.0% activity remained in the cell-free
broth of _P. citreus grown at 35 C for 36 hrs. However, after 15 min at
75 C, 1.0% activity still remained in the cell-free broth of _P. citreus
cells grown at 5 C for 108 hrs. Results indicated that the enzyme sys
tem in the crude preparations (cell-free broths) was less affected by
temperature changes than the purified enzyme. In addition, the enzyme
system produced by _P. citreus grown at 5 C was more stable to changes in
temperature than the 35 C crude enzyme preparation. Perhaps the enzymes
present in the 5 C crude extract have an enzyme configuration that better
protects the active site from temperature changes.
However, when shrimp is processed (boiled, canned or broiled), the
enzyme should be easily inactivated.
10) When shrimp protein was used as substrate, the activity of the
enzyme increased as the sodium chloride (NaCl) concentration increased
up to 0.5% NaCl. Enzyme activity decreased with higher concentrations
of NaCl (0.5-1.5%). When gelatin was used as the substrate, NaCl con
centrations (1-1.5%) had no effect on enzyme activity. The increase in
NaCl concentration up to 0.5% might have caused an increase in shrimp
protein solubility, thus, making shrimp protein more available for the
_P. citreus enzyme.
11) As the concentration of sodium bisulfite (NaHSO^) was increased
from 0 to 3%, the activity of the protease decreased (first-order reac
tion). Approximately 47% of the activity was lost when 1.25% sodium
bisulfite was present in the medium.
12) As the concentration of enzyme increased (0-200 yl), the rate
of the reaction increased when gelatin and shrimp protein were used as
substrates.


Abstract of Dissertation Presented to the Graduate
Council of the University of Florida in Partial Fulfillment
of the Requirements for the Degree of Doctor of Philosophy
ISOLATION, PURIFICATION AND CHARACTERIZATION OF
AN EXTRACELLULAR PROTEOLYTIC ENZYME OF Planococcus citreus
By
Ricardo J. Alvarez
March 1981
Chairman: J. A, Koburger
Major Department: Food Science and Human Nutrition
Planococcus citreus is a gram-positive marine bacterium commonly
found in fresh and iced shrimp. Various studies have indicated that it
may contribute to spoilage of this valuable marine resource. In order
to understand the contribution of this organism to the degradation of
shrimp as well as other proteins, an investigation was undertaken to
study the extracellular proteolytic enzyme(s) of this organism. Results
indicated that the major portion (>95.0%) of the proteolytic activity
resided in the extracellular fraction.
Under the conditions tested, maximum extracellular enzyme produc
tion occurred in Trypticase Soy Broth (TSB) as observed by the highest
m value (units of enzyme activity per cell per hour). In addition, the
cell-free broth obtained from _P. citreus cells grown at 5 C for 108 hrs,
20 C for 72 hrs and 35 C for 36 hrs exhibited enzyme activity towards
shrimp protein at all three enzyme-substrate incubation temperatures (5,
20 and 35 C).
xii


128
115. Payne, J. W. and C. Gilvarg. 1971. Peptide transport. Adv. in
Enzymol. 35:187-244.
116. Pedraja, R. R. 1970. Change in composition of shrimp and other
marine animals during processing. Food Technol. 24:1355-1360.
117. Peterson, A. C. and M. F. Gunderson. 1960. Some characteristics
of proteolytic enzymes from Pseudomonas fluorescens. Appl.
Microbiol. 8:98-103.
118. Peplow, A. J., J. A. Koburger and H. Appledorf. Effect of ice
storage on the total weight, proximate composition and mineral
content of shrimp. Proc. of the Third Annual Tropical and Sub
tropical Fisheries Technological Conference, New Orleans, LA
3:92-102.
119. Pharmacia Fine Chemicals. 1975. Sephadex Ion Exchangers: A Guide
to Ion Exchange Chromatography. Uppsala, Sweden.
120. Pharmacia Fine Chemicals. 1976. Sephacryl S-200 Superfine: For
High Performance Gel Filtration. Uppsala, Sweden.
121. Pollock, M. R. 1960. Induced formations of enzymes. In: The
Enzymes. Vol. I, 2nd Ed. Eds. P. D. Boger, H. Hardy and K.
Myrback. Academic Press, Inc., New York, NY.
122. Pollock, M. R. 1962. Exoenzymes. In: The Bacteria: Volume IV:
The Physiology of Growth. Eds. I. C. Gunsalus and R. Y. Stainer.
Academic Press, Inc., New York, NY.
123. Preston, K. R. 1976. An automated fluorometric assay for pro
teolytic activity in wheat. Cereal Chem. 52:451-458.
124. Samejima, K., W. Dairman and S. Udenfriend. 1971. Condensation
of ninhydrin with aldehydes and primary amines to yield highly
fluorescent ternary compounds. 1. Studies of the mechanisms of
the reaction and some characteristics of the condensation products.
Anal. Biochem. 42:222-236.
125. Samejima, K., W. Dairman, J. Stone and S. Udenfriend. 1971. Con
densation of ninhydrin with aldehydes and primary amines to yield
highly fluorescent ternary compounds. 2. Application to the
detection and assay of peptides, amino acids, amines and amino
sugars. Anal. Biochem. 42:237-247.
126. Schleifer, K. H. and D. Kandler. 1970. Amino acid sequence of
the murein of Planococcus and other Micrococcaceae. J. Bacteriol.
103:387-392.
127. Schwabe, C. 1973. A fluorescent assay for proteolytic enzymes.
Anal. Biochem. 53:484-490.
128. Scrutton, M. C. and M. F. Utter. 1965. Pyruvate carboxylase.
III. Some physical and chemical properties of the highly
purified enzyme. J. Biol. Chem. 240:1-9.


LIST OF TABLES (continued)
TABLE Page
13 Units of enzyme activity per cell per hour (m) of Planococcus
citreus grown in yeast carbon base supplemented with shrimp
protein and/or yeast extract at midlog phase Ill
14 Units of enzyme activity per cell per hour (m) of Planococcus
citreus grown in various media
vii


129
129. Shelef, L. A. and J. M. Jay. 1971. Hydration capacity as an
index of shrimp microbial quality. J. Food Sci. 36:994-997.
130. Shewan, J. M. 1971. The microbiology of fish and fishery
products a progress report. J. Appl. Bacteriol. 34:299-315.
131. Shuster, C. W. and M. Doudoroff. 1962. A cold-sensitive D(-)-
beta-hydroxybutyric acid dehydrogenase from Rhodospirilium rubrum.
J. Biol. Chem. 237:603-607.
132. Steele, R. G. D. and J. H. Torrie. 1960. Principles and Proce
dures for Statisticians. McGraw-Hill Co., Inc., New York, NY.
133. Stein, S., P. Bohlen, J. Stone, W. Dairman and S. Udenfriend.
1973. Amino acid analysis with fluorescamine at the picomole
level. Arch. Biochem.- Biophys. 155:202-212.
134. Stone, F. E. 1971. Inosine monophosphate (IMP) and hypoxanthin
formation in three species of shrimp held on ice. J. Milk Food
Techno1. 34:354-356.
135. Sussman, A. J. and C. Gilvarg. 1971. Peptide transport and
metabolism in bacteria. Ann. Rev. Biochem. 40:397-408.
136. Tarrant, P. J. V., N. Jenkins, A. M. Pearson and T. R. Outson.
1973. Proteolytic enzyme preparation from Pseudomonas fragi:
Its action on pig muscle. Appl. Microbiol. 25:996-1005.
137. Thirkell, D. and M. Summerfield. 1977. The effect of varying
sea salt concentration in the growth medium on the chemical com
position of a purified membrane fraction from Planococcus citreus
Migula. Anton, van Leeuwenhoek 43:37-42.
138. Thirkell, D. and M. Summerfield. 1977. The membrane lipids of
Planococcus citreus Migula from cells grown in the presence of
three different concentrations of sea salt added to a basic
medium. Anton, van Leeuwenhoek 43:43-54.
139. Thomas, T. D., E. D. W. Jarvis and N. A. Skipper. 1974. Locali
zation of proteinase(s) near the cell surface of Streptococcus
lactis. J. Bacteriol. 118:329-333.
140. Udenfriend, S., S. Stein, P. Bohlen and W. Dairman. 1972.
Fluorescamine: A reagent for assay of amino acids, peptides,
proteins, and primary amines in the picomole range. Science
178:871-872.
141. Vanderzant, C. and R. Nickelson. 1971. Comparison of extract-
release volume, pH and agar Plate Count of shrimp. J. Milk
Food Technol. 34:115-118.
142. Vanderzant, C., E. Mroz and R. Nickelson. 1970. Microbial flora
of Gulf of Mexico and pond shrimp. J. Milk Food Technol. 33:346-
350.


55
the P. cltreus cells showed little enzyme activity towards gelatin and
shrimp protein (both high molecular weight substrates).
Thomas et al. (139) also concluded that the cell wall proteinase
may serve a similar nutritional role in nature as the surface-bound pro-
teinases discussed by Payne and Gilvarg (115) and Sussman and Gilvarg
(135). Gilvarg and his co-workers stated that surface-bound protein
ase (s) appear to serve a nutritional role by hydrolyzing proteins to
amino acids or peptides that are small enough to enter the cell. In
turn, Payne, Sussman and Gilvarg (115,135) also suggested that the
intracellular peptidases could further hydrolyze the peptides formed and
release their constituent amino acids, thus, permitting the utilization
of the protein substrate for growth. In Table 5, we can observe that
certain _P. citreus fractions (whole cells, intracellular and cellular
particulate) had substantial amounts of protein present. Perhaps some
of the protein present in these fractions include other enzymes (i.e.,
peptidases) that can utilize the peptides produced by the action of the
extracellular protease(s) that later may enter the _P. citreus cell. In
this manner, _P. citreus cells could fully utilize the protein available
(i.e., shrimp protein as well as other proteins) for their growth.
Growth Medium and Enzyme Production
Trypticase Soy Broth (TSB), Plate Count Broth (PCB) + 0.5% NaCl and
Nutrient Broth (NB) + 0.5% NaCl were used to determine growth rates and
production of extracellular proteolytic enzyme(s) by _P. citreus. Figure
11 shows the growth of _P. citreus, as measured by the increase in optical
density (600 nm), in the three media used. In all three media, P.
citreus exhibited a 12 hr lag phase in which an increase in optical den
sity was not evident. After this lag period, TSB supported the most


73
According to Cooper (48) electrophoretic techniques have become
principal tools for characterizing macromolecules and for assaying their
purity. Figure 17 shows a single band after SDS-PAG electrophoresis
using 50 pi of the purified enzyme. A single homogeneous band is indic
ative of the presence of only one enzyme, i.e., the purity of the extra
cellular enzyme of _P. citreus. In addition, as an additional test for
purity, increasing amounts of the purified extracellular enzyme were
added to the gels. Enzyme concentrations of 50 yg protein/gel, 100 yg/
gel, 150 yg/gel and 200 yg/gel were used. Figure 18 shows that a single
band is recovered after SDS-PAG electrophoresis of each protein fraction.
Thus, these results add to the evidence indicating the purity of the
extracellular proteolytic enzyme of _P. citreus. Consequently, an extra
cellular proteolytic enzyme produced by _P. citreus was purified 26.50
times using the procedures outlined previously with 49.0% of the enzyme
being recovered (Table 8). The specific activity of the enzyme was
780.37 units of activity/mg protein.
Schwabe (127) reported the use of the fluorescamine reagent to mea
sure proteolytic enzyme activity of cathepsin enzymes using hemoglobin
a substrate. He stated that while the fluorescamine reagent has been
used successfully for quantitative amino acid analysis, protein and pep
tide determination, it has also beneficial applications in enzymology.
In addition, Schwabe (127) compared the fluorometric technique with the
Lowry method (98). He concluded that the fluorometric method was 100
times more sensitive than the Lowry method, much faster and less com
plicated. The fluorometric technique proved to be an efficient method
for the measurement of proteolytic enzyme activity.


61
Table 6. Units of enzyme activity per cell per hour (m) of Planococcus
citreus grown in Trypticase Soy Broth (TSB), Plate Count
Broth (PCB) and Nutrient Broth (NB) at midlog phase.
Medium
mean m value^
TSB
148.50a
PCB
105.67b
NB
59.32C
'
Cells were grown at 20 C and enzyme activity was measured at 35 C
2for 15 min (pH 8).
average of 6 observations
Means followed by the same letter do not differ significantly at the
a = 0.05 (r from Anova table 0.984)


2
activity of this organism was further demonstrated by the decrease in
percent total extractable protein (percent TEP) in shrimp during storage
at 5 C (4,5) which had been inoculated with _P. citreus.
The proteolytic activity exhibited by this organism deserves addi
tional research in order to better understand the contribution of _P.
citreus to the degradation of shrimp protein. A study was therefore
undertaken to study the enzyme(s) responsible for protein degradation.
The optimum medium and stage in the growth cycle of _P. citreus were
determined for maximum extracellular enzyme(s) production. The effect
of incubation temperature (5, 20 and 35 C) on the growth of _P. citreus
and proteolytic enzyme production was also investigated. Purification
of the extracellular enzyme(s) was achieved by precipitation and chro
matographic techniques. Homogeneity of the enzyme was evaluated by gel
electrophoresis and chromatographic techniques. Optimum pH and tempera
ture, ionic strength effect, thermal stability, molecular weight, sodium
chloride effect, sodium bisulfite effect, enzyme concentration, substrate
concentration, and the effect of metal ions and other reagents were
investigated. In addition, the potential of the _P. citreus enzyme(s) to
degrade dipeptides and the possible effect of shrimp protein in the
growth medium inducing the extracellular enzyme(s) were studied.
Results obtained from this investigation indicate that _P. citreus,
while growing on shrimp, may contribute to the overall decrease in shrimp
quality during iced or refrigerated storage. In addition, information
about the characteristics of the enzyme(s) produced by P. citreus will
be introduced


INTRODUCTION
Quality deterioration and subsequent spoilage of shrimp during
storage are caused primarily by activities of indigenous tissue enzymes
and microbial enzymes (69). Various researchers (17,156) believe that
bacterial action plays a more important role than autolytic enzyme
release in causing spoilage of seafoods. During growth of the bacteria,
proteolysis of shrimp proteins and free amino acid formation by microbial
action has been observed. Enzymatic deamination and decarboxylation of
these amino acids from shrimp protein occur, resulting in the formation
of malodorous compounds (116).
Various types of bacteria have been reported to be present on
freshly caught shrimp. Numerous studies (3,42,43,45,116,142) have shown
the changes undergone by the bacterial flora of shrimp as the storage
period increases. Recent research (1,2,3,90) has noted the presence of
a gram-positive organism, Planococcus citreus, during shrimp storage.
The organism is described as a motile gram-positive coccus found in the
marine environment, capable of growing over a pH range of 7-10 between
5-35 C in broth containing 0.5-12% sodium chloride (NaCl), and the orga
nism is capable of hydrolyzing gelatin, cottonseed, soy and shrimp
protein.
The potential of _P. citreus as a "spoiler" of shrimp was shown by
the increase in pH and the rapid increase in the total volatile nitrogen/
amino acid-nitrogen ratio (TVN/AA-N) and trimethyl-amine nitrogen (TMN)
following growth of this organism on shrimp (4). The proteolytic
1


101
the enzyme with the substrate; 3) changing the equilibrium constant of
enzyme reaction; 4) changing surface charge of enzyme; 5) removing
inhibitors; and 6) inducing a more active enzyme conformation (151).
Kato et al. (87) reported that calcium chloride (CaCl^) and magne
sium chloride (MgCip activated the enzyme of a marine-psychrotrophic
bacterium (Pseudomonas spp.) and mercuric chloride (HgC^) and ferric
sulfate (FeSO^) suppressed the enzyme. Arvidson (11) reported that
magnesium chloride (MgCl^) zinc chloride (ZnC^) and calcium chloride
(CaCip activated a protease from Staphylococcus aureus. However,
Pacaud and Uriel (112) stated that calcium chloride (CaC^), manganese
chloride (MnCl^) and ferric chloride (FeC^) activated an enzyme from
Escherichia coli but magnesium chloride (MgCl^), zinc chloride (ZnCl^)
and mercuric chloride (HgC^) had no effect on the activity. The
results of this study indicate the diversity of effects ions can have
on the activity of the enzyme produced by _P. citreus.
Effect of Various Reagents on Enzyme Activity
Various reagents were tested to observe their effect on the activity
of the extracellular proteolytic enzyme of _P. citreus. None of the
reagents tested activated the enzyme (Table 11). Dioxane, one of the
reagents used in the enzyme activity assay, had no apparent effect on
the activity of the enzyme at either concentration examined (10 and 20
mM). The percent residual activity observed was 98.9 and 98.6%, respec
tively. The results observed with trichloroacetic acid (TCA, 5 and 10%)
indicate that both concentrations can terminate the activity of the
enzyme (2.0 and 0.0% residual activity, respectively). These results
are comparable to those observed in Figure 6.


75
50 yg/gel 100 yg/gel 150 yg/gel 200 yg/gel
Figure 18. Acrylamide gel electrophoresis of increasing concentrations
of the purified extracellular enzyme of Planococcus citreus.


78
Figure 19. Calibration curve for the molecular weight estimation o.
Planococcus citreus proteolytic enzyme using Sephacryl
S-200 column chromatography.
q
40 x 2.5 column, eluted with 0.02 M phosphate
buffer at a rate of 15 ml/hr.


27
10 ml of 5% TCA. After 5 to 10 min, to allow the proteins to settle,
the solution was filtered through Whatman #1 filter paper. Two hundred
microliters of the TCA filtrate were transferred to a 13 x 100 mm test
tube (Dispo culture tubes, Scientific Products, McGraw Park, IL) and the
volume brought to 1.5 ml with 0.5 M sodium phosphate buffer, pH 8.
0
While the test tube was vigorously mixed in a Vortex Mixture (Scientific
Products, Evanston, NY), 0.5 ml of fluorescamine in dioxane (30 mg/100
ml, Eastman Kodak Corp., Rochester, NY) was rapidly added to the buffered
protein solution. A model 204-A Fluorescence Spectrophotometer (Perkin
Elmer Corp., Norwalk, CT) was used to measure fluorescence intensity.
Zero time blanks were prepared by adding 10 ml of 5% TCA after adding the
enzyme and prior incubation of the mixture. This blank represented the
background activity present in the mixture at zero time. Zero time
fluorescence reading was subtracted from the reading of the substrate-
enzyme mixture after the appropriate incubation time.
Total enzyme activity was expressed as the change in 0.1 fluores
cence units of the TCA filtrate per milliliter of enzyme per minute.
Specific activity was expressed as the units of total enzyme activity/mg
of protein present (units of activity/mg of protein).
Previous research involving the use of the fluorescamine technique
(47,127,140) indicated that pH affected fluorescence intensity. Buffers
of pH from 2 to 10 (see buffers described on pg. 44) were used to deter
mine the effect of varying the pH of the buffer on fluorescence intens
ity. TCA filtrates (0.2 ml) were reacted with 1.3 ml of the various
buffers (pH 2-pH 10) before addition of the fluorescamine reagent.
Figure 3 indicates that addition of pH 8 buffer resulted in the highest
fluorescence itensity. Consequently, pH 8 buffer was used for the
remainder of the research.


4
enrich the natural substrates and are thus available for the growth of
microorganisms. Enzymatic deamination and decarboxylation of amino acids
may also occur rapidly, resulting in the formation of spoilage products.
Pedraja (116) observed that from the moment a shrimp is taken out of the
water, its free amino acid pool is affected to some extent by osmoregula
tion and also by the struggle during catching. Therefore, the onset of
enzymatic and bacterial actions will vary according to the factors
affecting the substrates available in shrimp muscle.
Another factor that can induce shrimp spoilage is mechanical damage.
Handling shrimp on the boats results in mechanical damage to the muscle,
which will accelerate microbial invasion. The expressible fluid with its
protein and amino acid content serves as an excellent medium for growth
and reproduction of invading microorganisms (116).
Microbiological Characteristics of Shrimp
The muscle tissue of freshly caught shrimp is generally regarded as
sterile (26); however, work by Lightner (97) showed bacteria in the gut,
gills and between muscle bundles of brown shrimp. Reports on the number
2
of bacteria found on freshly caught shrimp range from 2.5 x 10 to 2.0 x
10^ organisms per gram (org/g) with Gulf coast shrimp averaging 1.0 x 10^
org/g, whereas bay shrimp averaged 1.0 x 10^ org/g (31,34,39,43,78,142).
Work completed in our laboratory has shown that fresh shrimp from the
Gulf of Mexico had bacterial counts ranging from 4.0 x 10^ org/g to 2.0
x 10^ org/g, while shrimp from the Atlantic coast had bacterial counts
ranging from 4.5 x 105 to 3.6 x 106 org/g (1).
Various kinds of bacteria have been reported on freshly caught
shrimp. Initially, the microbial flora is a mixture of organisms from
both the marine and terrestial environment. In the early 1950s, Campbell


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
LIST OF TABLES vii
LIST OF FIGURES ix
ABSTRACT xii
INTRODUCTION 1
LITERATURE REVIEW 3
Shrimp Spoilage 3
Microbiological Characteristics of Shrimp 4
Measurement of Shrimp Spoilage
Characteristics of Planococcus citreus 11
Proteolytic Enzymes 17
Measuring Proteolytic Activity 19
MATERIALS AND METHODS 25
Planococcus citreus Cultures 25
Determination of Proteolytic Activity 25
Efficacy of 5% Trichloroacetic Acid (TCA) 29
Substrate Characteristics 33
Determination of Enzyme-Substrate Mixture Reaction Time 33
Growth Medium and Enzyme Production 36
Optimization of Enzyme Activity to Growth and Cell Number ... 36
Effect of Incubation Temperature on Enzyme Production and
Activity 38
Purification of the Extracellular Enzyme(s) 39
Ammonium Sulfate Precipitation 39
Molecular Sieve Chromatography 39
Ion-Exchange Chromatography 40
Confirmation of Enzyme Purity 42
Characterization of the Proteolytic Enzyme(s) 43
Molecular Weight Determination 43
Determination of the Purified Enzyme-Substrate Mixture
Reaction Time 43
Effect of Ionic Strength on Enzyme Activity 44
Determination of Optimum pH 44
Determination of Optimum Temperature 46
Thermal Stability 46
Effect of Sodium Chloride Concentration 47
v


114
Table 14. Units of enzyme activity per cell per hr (m) of Planococcus
citreus grown in various media.
Medium
Mean m value^
Trypticase Soy Broth (TSB)
147.68a
Trypticase Soy Broth + 1.0% shrimp protein
146.58a
Plate Count Broth (PCB)
105.67b
Nutrient Broth (NB)
59.32C
Yeast Carbon Base + 1.0% shrimp protein + 0.1%
yeast extract (YCBSY)
37.lld
Yeast Carbon Base + 0.1% yeast extract (YCBY)
27.82
Yeast Carbon Base + 1.0% shrimp protein (YCBS)
7.62f
Yeast Carbon Base (YCB)
4.77f
1Average of 10 observations for TSB, 4 for TSB + 1.0% shrimp protein
and 6 for the other media used
Means followed £y the same letter do not differ significantly at the
a=0.05 level (r from Anova table 0.994)


15
Table 2. Hydrolysis of various protein sources by selected strains of
Planococcus citreus at 25 C (4) (modification of Frazier (72)).
2
Protein Source
Isolate
i-H
G
H
4-
CT3
rH
0)
2
Whey
2
Cottonseed
iH
G
CO
cd
u
CO
&
6
H
Vj
x:
cn
m
rC
CO
H
2
Peanut
2
Corn germ
CM
>%
O
C/3
Hog blood
isolate^
2
Barley
A 17
+5
+
+
-
+
-
-
-
+
+
-
E 4
+
+
+
-
+
-
-
-
+
-
-
E 1
+
+
+
-
+
-
-
-
+
+
-
E 7
+
+
+
-
+
-
-
-
+
+
-
F 9
+
-
+
-
+
-
-
-
+
+
-
F 15
+
-
+
-
+
-
-
-
+
-
-
F 18
+
-
+
-
+
-
-
-
+
-
-
KS-1
+
-
+
-
+
-
-
-
+
-
-
KS-2
+
+
+
-
+
-
-
-
+
-
-
KS-3
+
-
+
-
+
-
-
-
+
-
-
KS-4
+
+
+
-
+
-
-
-
+
-
-
CS-1
+
+
+
-
+
-
-
-
+
-
-
From Difco Laboratories, Detroit, MI.
Protein isolates obtained from Southern Utilization Research and
Development Division, New Orleans, LA.
*Fresh samples were diluted 1:10 with 0.05 M phosphate buffer pH 7 and
ground in a Waring blender, dialyzed overnight with 10 volumes of the
same buffer (5 C) and lyophilized.
F. W. Knapp, Food Science and Human Nutrition Department, University
of Florida, Gainesville, FL.
'+ = hydrolysis; = no hydrolysis.


Table 11. Effect of
(pH 8))of
various reagents
the Planococcus
on the enzyme activity (assayed
citreus extracellular enzyme.
at 35 C for 10 min
Reagent
Concentration
Enzyme Activity
% Residual Activity^
None (control)
449.95
5.59
100.00
EDTA
10 mM
87.72
2.64
19.50
20 mM
58.50
2.33
13.00
Citric Acid
10 mM
150.72
3.85
33.50
20 mM
129.72
6.30
28.70
Formaldehyde
1 mM
286.33
2.59
63.60
20 mM
239.56
2.83
53.20
KCN
1 mM
389.89
2.86
86.70
20 mM
363.45
3.50
80.80
KMnO.
1 mM
9.28
1.56
2.10
20 mM
0.00
0.00
0.00
TCA
5%
8.87
1.75
2.00
10%
3.05
0.93
0.68
Cysteine
1 mM
209.83
4.21
46.60
20 mM
181.95
6.50
40.40
p-mercaptoethanol
1 mM
284.17
6.23
63.20
20 mM
247.00
3.28
54.90
p-Dioxane
10 mM
445.17
3.40
98.90
20 mM
443.45
3.00
98.60
2
^Average of 6 observations standard deviation
Compared to the control sample
102


125
70. Fogarty, W. M. and P. J. Griffin. 1973. Production and purifica
tion of the metalloprotease of Bacillus polymyxa. Appl. Microbiol.
26:185-190.
71. Folin, D. and V. Ciocalteau. 1927. On tyrosine and tryptophane
determinations in proteins. J. Biol. Chem. 73:627-630.
72. Frazier, W. C. 1926. A method for the detection of changes in
gelatin due to bacteria. J. Infectious Diseases 39:300-308.
73. Fruton, J. S. and S. Simmons. 1960. General Biochemistry. 2nd
Ed. John Wiley and Sons, Inc., New York, NY.
74. Fry, B. A. 1955. The Nitrogen Metabolism of Microorganisms.
John Wiley and Sons, Inc., New York, NY.
75. Gaguon, M. and C. R. Fellers. 1958. Biochemical methods for
determining shrimp quality. I. Study of analytical methods.
Food Technol. 12:340-343.
76. Gnosspelius, G. 1977. Purification and properties of an extra
cellular protease from Myxococcus virescens. J. Bacteriol. 132:
17-25.
77. Green, M. 1949. Bacteriology of shrimp. I. Introduction and
development of experimental procedures. Food Res. 14:365-371.
78. Green, M. 1949. Bacteriology of shrimp. II. Quantitative
studies of freshly caught and iced shrimp. Food Res. 14:372-383.
79. Green, M. 1949.. Bacteriology of shrimp. III. Quantitative
studies of frozen shrimp. Food Res. 14:384-394.
80. Harrison, J. M. and J. S. Lee. 1968. Microbial evaluation of
Pacific shrimp processing. Appl. Microbiol. 18:188-192.
81. Havir, E. A., H. Tamir, S. Ratner and R. C. Warner. 1965.
Biosynthesis of urea. XI. Preparaton and properties of
crystalline arginosuccinase. J. Biol. Chem. 240:3079-3088.
82. Hay, J. D., R. W. Currie and F. H. Wolfe. 1973. Polyacrylamide
disc gel electrophoresis of fresh and aged chicken muscle proteins
in sodium dodecylsulfate. J. Food Sci. 38:987-990.
83. Hucher, G. J. and R. S. Breed. 1957. In: Bergeys Manual of
Determinative Bacteriology. 7th Ed. Eds. R. S. Breed, E. G. D.
Murray and W. R. Smith. Bailliere, Tindall and Cox, London,
England.
84. Husain, I. and I. J. McDonald. 1958. Characteristics of an
extracellular proteinase from Micrococcus freudenreichii. Can.
J. Microbiol. 4:237-242.


19
substrate(s), provides a basis for naming individual enzymes. Each
enzyme number contains four elements; the first element (1 through 6)
shows to which of the 6 main groups of enzymes the particular enzyme
belongs (the six main groups are made on the basis of the general chemi
cal reaction catalyzed); the second and third elements show the subclass
and sub-subclass, respectively, thus defining the type of reaction; and
the fourth element is the serial number of the enzyme within its sub-sub-
class. Enzymes can be divided into six main groups: oxidoreductases,
transferases, hydrolases, lyases, isomerases and ligases.
Active extracellular proteinases are produced by numerous species of
Clostridium, Proteus, Bacillus, Pseudomonas, Micrococcus, Streptococcus,
Escherichia, Cytophaga and Staphylococcus (11,12,36,58,59,65,70,84,87,
105,110,113,114,118,136,139,155,157).
The continued study of these bacterial enzymes is important for at
least two reasons: (a) proteolysis by microorganisms plays an important
role in the biogeochemical cycles (74) and is responsible for numerous
environmental interrelationships; (b) the purification and the elucida
tion of their bond specificities are certain to lead to the discovery of
new enzymes with new properties not previously known.
Measuring Proteolytic Activity
Many methods are available for measuring proteolytic activity. Some
are based on the measurement of increase in protein (or nitrogen) solu
bility in the supernatant after centrifugation of the reaction mixture.
The most frequently cited method for measuring protein in solution is
that of Lowry et al. (98) in which the tyrosinetryptophan groups of
proteins in solution, or precipitated with acid, are reacted with alka
line Folin-phenol reagent after an alkaline copper treatment (71) to


20
produce a blue color that is measured in a spectrophotometer. Other
methods record proteolysis as the increase in ultraviolet absorption at
280 nm or the increase in absorbance (660 nm) of the tyrosine-tryptophan
filtrate after trichloroacetic acid (TCA) precipitation of the undigested
protein reacted with diluted (2:1) phenol reagent solution (9).
Schwabe (127) described a method which permited the assay of the
proteolytic enzyme activity on hemoglobin utilizing the fluorescamine
technique. The assay is about 100 times more sensitive than the Lowry
method, much faster and less complicated. He observed that the two main
obstacles for the successful use of fluorescamine in his assay system
were (1) the high blank produced by the reaction of e-amino groups of
the protein and (2) the fluorescent quenching effect of the hemoglobin.
The high blank of the hemoglobin he substantially suppressed by a chemi
cal modification, i.e., succinylation. Hemoglobin is usually used as a
2% solution of which only 10 pi are pipetted into 2 ml of phosphate buf
fer used for the reaction. He observed that the enzyme activity as mea
sured by the fluorescamine method remained linear throughout thirty min
utes while the Lowry method indicated a definite slowing of the reaction
beginning at about ten minutes. This was due to the fact that fluores
camine detects an increase in free amino groups while the Lowry reagent
as well as the direct measurement of absorption at 280 nm depends on the
production of tyrosine or tryptophan containing peptides. A possible
explanation for this discrepancy is that the enzyme in its initial
attack on the hemoglobin molecule releases large peptides which are TCA
soluble and that subsequent enzyme action further degrades these large
peptides without significantly increasing the number of TCA-soluble
fragments containing tyrosine or tryptophan moieties. A reagent
depending upon primary amine groups is not subject to this error (125).


71
to which is covalently bound positive (in the case of the anionic
exchanger) or negative (in the case of a cation exchanger) functional
groups (48). A sodium chloride (NaCl) gradient (range of ionic strength,
y = 0.11 0.23) was used with the ion exchange column to elute the pro
tein components. A gradient is a physical method of constantly changing
the salt concentration of a solution that is being passed through the
column creating a constant and linear increase in concentration (48).
Figure 16 shows one major peak after ion exchange of the pooled active
fractions from peak C. The isolated peak exhibited a specific activity
of 780.37 units (Table 8). The proteolytic enzyme was purified 26.50
times and 49% recovery was achieved (Table 8). Fractions 17 to 21
(Figure 16) were pooled for future characterization.
Purity of the Extracellular Proteolytic Enzyme
Many methods can be used to establish the purity of an enzyme prep
aration. However, the best indication of purity of an enzyme prepara
tion is by the consistent failure to detect heterogeneity when several
analytical techniques are used (i.e., a single peak in chromatographic
systems, a single band on electrophoresis, a single band after isoelec
tric focusing and/or one component in solubility or precipitation tests).
However, the final criterion for purity is the demonstration of a unique
amino acid sequence (61,65) but this is rarely done in order to
demonstrate purity.
The recovery of the isolated peak (Figure 16) as a single entity
with homogeneous activity after DEAE-Sephadex A-50 ion-exchange chroma-
tography was the first indication that the major extracellular
proteolytic activity of _P. citreus was isolated in a purified form.


ENZYME ACTIVITY
108
Figure 33. Proteolytic enzyme activity of the cell-free broth of Planococcus
citreus grown in Yeast Carbon Base supplemented with shrimp
protein and/or yeast extract.


80
(11) the molecular weight of a EDTA-sensitive S.. aureus protease as
28,000. Recently, Hoshida et al. (137) estimated the molecular weight
of a proteolytic enzyme from Bacillus sphaericus to be about 26,000.
Gnosspelius (76) working with an extracellular enzyme of Myxococcus
virescens reported its molecular weight as 26,000. Thus, the apparent
molecular weight of the extracellular enzyme of _P. citreus (MW 29,000)
is within the range of other extracellular proteolytic enzymes reported
in the literature.
Effect of Ionic Strength on Enzyme Activity
The effect of salts on the solubility of proteins is well known.
The solubility is usually a function of the ionic strength. In condi
tions of high ionic strength, the ions attract around themselves the
polarizable water molecules, making less water available for the pro
teins since, at high salt concentrations, the number of charged groups
contributed by the salts is enormous compared with those of the proteins.
Consequently, the solubility of the proteins decreases (152). In addi
tion, any change in the charges of an enzyme may cause various transfor
mations in structure or active site configuration that could affect its
activity towards the substrate. Figure 21 shows that ionic strengths (p)
of 0.15-0.83 did not alter the attraction of the _P. citreus extracellular
enzyme towards gelatin substrate. However, as the ionic strength was
increased the activity of the enzyme decreased. An ionic strength of
1.60 (1.5 M NaCl) caused a decrease in enzyme activity of approximately
60%. Thus, if ionic strengths above 0.83 (0.75 M NaCl) are used they
may cause a change in solubility of the enzyme, charged groups, confor
mation of the enzyme, active site stability and/or active site availa
bility to the substrate. Gnosspelius (76) stated that variations in the


and shrimp protein, respectively. Ferric chloride, mercuric chloride,
potassium chloride, ethylene diaminetetraacetic acid, citric acid,
cysteine, p-mercaptoethanol, potassium permanganate and formaldehyde
partially inactivated the enzyme. Calcium chloride increased the
activity of the extracellular proteolytic enzyme. Zinc chloride, p-
dioxane, manganese chloride and magnesium chloride had no effect on the
activity of the enzyme. The proteolytic enzyme exhibited peptidase
activity on various commercial synthetic dipeptides. The extracellular
proteolytic enzyme produced by J?. citreus was apparently not induced by
the presence of shrimp protein in the medium of growth. Enzyme produc
tion appeared to be related to the extent of growth of _P. citreus in the
medium.
xiv


Finally, he is deeply grateful to Mary Brannigan for her love,
patience and devotion, providing sentimental support and help through
out all phases of his course work. He thanks her for her understanding
and provision of many reasons to pursue all achievements in life.
iv


48
were tested. However, for the shrimp substrate, 0.000, 0.075, 0.100,
0.125, 0.150, 0.300 and 0.600 mg/ml were tested. Five milliliters of
each substrate solution were reacted with 100 yl of the purified enzyme
at 35 C for 10 min. From these data, Lineweaver-Burk plots were derived,
and K and V values for each substrate were extrapolated from these
m max
plots (95,152).
Effect of Metal Ions on Enzyme Activity
Calcium chloride (10, 20 mM), ferric chloride (1, 20 mM), magnesium
chloride (10, 20 mM), mercurous chloride (1, 20 mM), zinc chloride (10,
20 mM), manganese chloride (10, 20 mM) and potassium chloride (5, 20 mM)
were tested for their effect on enzyme activity (all metals were dis
solved in 0.05 tris-HCl buffer). For the control, a buffer with no
metal ions added was used (76,87). Five milliliters of substrate (gela
tin) 100 yl of enzyme and 1 ml of the metal ion buffer solution were
reacted for 10 min at 35 C. The fluorometer reading of the control
sample was compared to^ the reading of the metal ion samples.
Effect of Various Reagents on Enzyme Activity
Ethylene diaminetetraacetic acid (EDTA) (10, 20 mM), citric acid
(10, 20 mM), formaldehyde (1, 20 mM), potassium cyanide (KCN) (1, 20 mM),
potassium permanganate (KMnO^) (1, 20 mM), cysteine (1, 20 mM), 2-
mercaptoethanol (1, 20 mM), p-dioxane (10, 20 mM) and trichloroacetic
acid (TCA) (5, 10%) were tested for their effect on the proteolytic
activity of the J?. citreus enzyme (76,87). All reagents were dissolved
and/or mixed with 0.05 M tris-HCl buffer. Five milliliters of substrate
(gelatin), 100 yl of enzyme and 1 ml of the appropriate reagent buffer
solution were reacted at 35 C for 10 min. A control with no reagent
added was used and the fluorometer reading from the various reagents was
compared to the control.


A mis queridos padres:
Gracias por la ayuda brindada, el amor,
el apoyo moral y la vision de avanzar en la vida.
Con todo mi amor


ENZYME ACTIVITY (x 10')
83
pH
Figure 22. Optimum pH of the extracellular proteolytic enzyme of
Planococcus citreus.a
a
Gelatin and shrimp protein substrate incubated
at 35 C for 10 min, pH 8.


69
out between 55-70% ammonium sulfate saturation. Table 9 shows that 86%
of the activity towards gelatin is observed in this fraction. Table 8
shows that the activity of the 55-70% ammonium sulfate fraction was 1.78
times greater in specific activity than the cell-free broth. A 78%
recovery of the extracellular enzyme(s) was achieved in this step of
the enzyme purification.
£
Sephacryl S-200 Superfine, a high resolution chromatographic
medium for gel filtration of proteins, nucleic acids, polysaccharides
and biopolymers (120), was used to separate the enzyme(s) present in the
55-70% ammonium sulfate fraction according to molecular weight. Figure
15 shows that four protein peaks were recovered after the elution of the
£
enzyme fraction through the Sephacryl S-200 column. However, when the
proteolytic activity was measured, the majority of the activity was pre
sent in protein peak C (third peak in Figure 15). Peak C had a specific
activity of 651.0 units. The enzyme(s) was purified 15.67 times and 50%
of the enzyme was recovered in this step (Table 8). The fractions com
prising peak C were pooled for further purification. The percent recov
ery of the extracellular proteolytic enzyme of _P. citreus after molecular
sieve chromatography using Sephacryl S-200 Superfine was within the
range of most of the enzymes recovered when the more traditional
£
Sephadex gels have been used (70,113,114,136).
The pooled fractions of peak C were further rechromatographed using
DEAE-Sephadex A-50 (functional group -C^H^N+iC^H^)A-50 gels are
usually used for low and medium molecular weight proteins (up to
200,000). Ion exchange chromatography may be defined as the reversible
exchange of ions in solution with ions electrostatically bound to an
insoluble support medium. The ion exchanger is the inert support medium


ENZYME ACTIVITY (xIO1)
92
ENZYME CONCENTRATION (/j.\)
Figure 27. Effect of enzyme concentration on enzyme activity.
Increasing enzyme concentrations were incubated with the
substrates at 35 C for 10 min (pH 8).


21
Fluorescamine is a new reagent for the detection of amino acids,
peptides, proteins and primary amines in the picomole range (18,133,140).
Its reaction with amines is almost instantaneous at room temperature in
aqueous media. The products are highly fluorescent, whereas the reagent
and its degradation products are nonfluorescent.
McCaman and Robins (101) introduced a fluorometric method now widely
used for assay of serum phenylalanine which is based on the interaction
of ninhydrin and peptides. Samejima et al. (124,125) found that it was
the phenylacetaldehyde formed on interaction with ninhydrin which com
bined with additional ninhydrin and peptide or any other primary amine
to yield highly fluorescent products. The structure of these products
was subsequently elucidated by Weigele et al. (145), who then synthesized
a novel reagent (145). This reagent 4-phenylspiro (furan-2(3H),I'-
phthalan) 3,3'-dione (fluorescamine) reacts directly with primary amines
to form highly fluorescent products.
Several factors make fluorescamine suitable for assaying primary
amines, including amino acids, peptides and proteins. At pH 8-9, the
reaction with primary amines proceeds at room temperature (140) within a
fraction of a second. Excess reagent is concomitantly destroyed within
several seconds (140). Fluorescamine, as well as its hydrolysis prod
ucts, is nonfluorescent. Studies with small peptides have shown that
the reaction goes to near completion (about 80% to 95% of theoretical
yield) even when fluorescamine is not present in excess. The following
is an example of the reaction of fluorescamine with an amine group
illustrating the product formed (fluorophor) and the rate of the reaction
(100-500 msec). In addition, the reaction of water with fluorescamine
with the formation of a nonfluorescent product is also shown.


LITERATURE CITED
1. Alvarez, R. J. 1978. Effect of delayed heading on the microbial
and organoleptic characteristics of shrimp. Master's Thesis.
University of Florida, Gainesville, FL.
2. Alvarez, R. J. and J. A. Koburger. 1978. Microbial development in
shrimp as affected by delayed heading. Proc. of the Third Annual
Tropical and Subtropical Fisheries Technological Conference, New
Orleans, LA 3:102-110.-
3. Alvarez, R. J. and J. A. Koburger. 1979. Effect of delayed head
ing on some quality attributes of Penaeus shrimp. J. Food Prot.
42:407-409.
4. Alvarez, R. J. and J. A. Koburger. 1979. Planococcus citreus:
Its potential for shrimp spoilage. Proc. of the Fourth Annual
Tropical and Subtropical Fisheries Technological Conference, St.
Petersburg, FL 4:79-90.
5. Alvarez, R. J. and J. A. Koburger. 1980. Effect of Planococcus
citreus on selected quality indices of Penaeus shrimp. J. Food
Prot. (accepted for publication).
6. American Public Health Association. 1976. Compendium of Methods
for the Microbiological Examination of Foods. Ed. M. L. Speck.
APHA, Washington, DC.
7. Andrews, P. 1964. Estimation of the molecular weights of proteins
by Sephadex gel-filtration. Biochem. J. 91:222-223.
8. Anonymous. 1979. Shrimping '78. The Fish Boat. Aug. 1979,
p. 45-49.
9. Anson, M. L. 1938. The estimation of pepsin, trypsin, papain,
and cathepsin with hemoglobin. J. Gen. Physiol. 22:79-89.
10. Anstrop, K. 1978. Industrial approach to enzyme production. In:
Biotechnological Applications of Proteins and Enzymes. Eds. Z.
Bohak and N. Sharon. Academic Press, Inc., NY.
11. Arvidson, S. 1973. Studies on extracellular proteolytic enzymes
from Staphylococcus aureus. II. Isolation and characterization
of an EDTA-sensitive protease. Biochem. Biophys. Acta 303:96-105.
120


Table 10. Effect of various metal ions on the activity (assayed at 35 C for 10 min (pH 8))
of the Planococcus citreus extracellular enzyme.
Metal Ion
Concentration
(mM)
Enzyme Activity
% Residual Activity
b
none (control)
458.37

2.08
100.0
CaCl
10
492.89

1.57
107.5
z
20
553.61
/

2.10
120.7
FeCl
1
153.61

2.97
33.5
J
20
105.33

2.87
23.0
MgCl
10
460.16

2.77
100.4
4
20
470.11

2.85
102.6
HgCl,
1
285.71

2.41
62.3
20
243.11

1.44
53.0
MnCl.
10
430.17

0.75
93.8
4
20
408.16

1.89
89.1
ZnCl
10
449.95

2.24
98.2
4
20
435.00

1.89
94.9
KC1
5
287.72

2.10
62.8
20
247.67

6.37
54.0
^Average of 6 observations
Compared to the control sample
100


MOLECULAR WEIGHT
Figure 20. Calibration curve for the molecular weight estimation of Planococcus citreus proteolytic
enzyme using acrylamide gel electrophoresis.
al0 % acrylamide: Bis 30:0.8 gel, 1-1.5 mAMP/ gel for 3 hrs.


LIST OF TABLES
TABLE Page
1 Chemical and physical tests available to measure shrimp
quality 8
2 Hydrolysis of various protein sources by selected strains of
Planococcus citreus at 25 C 15
3 Proximate composition of the shrimp protein preparation .... 34
4 Composition of yeast carbon base medium 50
5 Proteolytic activity at 35 C for 15 min (pH 8) of cellular
fractions obtained from Planococcus citreus grown in
Trypticase Soy Broth (TSB) using gelatin and shrimp protein
as substrates 53
6 Units of enzyme activity per cell per hour (m) of Planococcus
citreus grown in Trypticase Soy Broth (TSB), Plate Count
Broth (PCB) and Nutrient Broth (NB) at mid-log phase 61
7 Enzyme activity measured at 5, 20 and 35 C (pH 8) of the
cell-free broths of Planococcus citreus grown in Trypticase
Soy Broth (TSB) at 5, 20 and 35 C for 108, 72 and 36 hrs,
respectively 64
8 Purification of an extracellular proteolytic enzyme from
Planococcus citreus 67
9Proteolytic activity at 35 C for 15 min (pH 8) of various
ammonium sulfate fractions of the cell-free broth of
Planococcus citreus 68
10 Effect of various metal ions on the activity (assayed at 35
C for 10 min (pH 8)) of the Planococcus citreus extracellu
lar enzyme 100
11 Effect of various reagents on the activity (assayed at 35 C
for 10 min (pH 8)) of the Planococcus citreus extracellular
enzyme 102
12 Dipeptidase activity of the Planococcus citreus extracellular
enzyme 105
vii


66
was due to dissociation of the molecules into subunits. The inactivated
enzymes could be reactivated by warming to room temperature.
Purification of the Extracellular Enzyme(s)
Planococcus citreus was grown in Trypticase Soy Broth (TSB) at 20
C for 72 hrs. The cell-free broth was used in the isolation of the
extracellular enzyme(s) of this organism. The cell-free broth had a
total activity of 1.31 x 10^ units of activity (total enzyme activity =
change in 0.1 fluorescence units of the TCA filtrate per milliliter of
enzyme per minute), 44.52 mg/ml of protein and a specific activity of
29.45 units of activity/mg protein (Table 8). The cell-free broth was
then fractionated with 0-55%, 55-70% and 70-100% ammonium sulfate
((nh4)2so4).
After overnight dialysis (16 hrs) in phosphate buffer pH 8, the
activity of the 0-55%, 55-70% and 70-100% ammonium sulfate precipitates
was measured. Table 9 shows the proteolytic activity of the various
fractions examined. The specific activity of each fraction was 4.59,
52.39 and 3.99 units of activity/mg of protein for the 0-55%, 55-70% and
70-100% fractions, respectively. Eighty-six percent of the activity was
present in the 55-70% fraction. This is compared to 7.5 and 6.5% for
the 0-55% and 70-100% fractions, respectively (Table 9). Ammonium sul
fate precipitation is a common method used to precipitate proteins for
their purification. As the ammonium sulfate concentration is raised
from zero, the solubility of a given protein at first usually increases
but then the "salting-in" effect comes to an end and as the salt concen
tration is raised to higher values a "salting-out" effect is observed
and the protein becomes progressively less soluble (65). The major por
tion of the extracellular proteolytic enzyme(s) of P. citreus was salted


BIOGRAPHICAL SKETCH
Ricardo Javier Alvarez was born on November 4, 1954, in Santiago,
Chile. In June, 1972, he graduated from Colegio San Ignacio de Loyola
in San Juan, Puerto Rico. He attended the University of South Florida,
and in June, 1976, he received his Bachelor of Science in microbiology.
He enrolled as a graduate student in the Food Science and Human Nutri
tion Department, University of Florida, in January, 1977. He received
his Master of Science degree in June, 1978. He anticipates receiving
his Ph.D. degree in food science and human nutrition with a minor in
environmental engineering in March, 1981.
131


104
a protease from Escherichia coli (113) was not inhibited by EDTA,
cysteine nor p-mercaptoethanol.
The results of this study indicate that the _P. citreus enzyme may
contain a metal cofactor and possibly sulfhydryl groups. Additional
work should be conducted to confirm these findings.
Dipeptidase Activity
Table 12 shows the activity of the purified _P. citreus extracellular
enzyme towards five synthetic dipeptides. Although lower when compared
to the activity on the whole protein substrates, enzyme activity was
observed in all five peptides. The highest activity was observed with
DL-alanylglycine (47.67 units of activity). In order to make predictions
on the specificity of this enzyme, additional peptides should be
investigated. Consequently, the extracellular enzyme of _P. citreus can
utilize and degrade dipeptides to their constituent amino acids.
Enzyme Classification
According to the International Union of Biochemistry scheme (54)
for numbering enzymes, the _P. citreus proteolytic enzyme would be class
ified as: 3.4.1 (acting on peptide bonds, an a-amino-acyl-peptide
hydrolase). The data presented in this study point to the possibility
of having an aminopeptidase enzyme; however, further studies with
synthetic peptides are necessary for the complete classification of the
_P. citreus enzyme. In addition, studies need to be conducted to deter
mine if the _P. citreus enzyme exhibits endo or exopeptidase activity.
Enzyme Induction Studies
Various media were used in order to determine if the extracellular
proteolytic enzyme produced by _P. citreus was induced by shrimp protein.
Induction is the complete de novo synthesis of enzyme molecules in the


93
absent from the preparation. Thus, looking at Figure 27, we can observe
that inhibitors were not present in the preparation.
Effect of Substrate Concentration
Substrate concentration is one of the most important factors which
determine the velocity of enzyme reactions. Figures 28 and 30 illustrate
the effect of substrate concentration on the velocity of the reaction
when gelatin (0 to 1.2 mg/ml) and shrimp protein (0 to 0.6 mg/ml) sub
strates, respectively, were used. Both enzyme-substrate reactions fol
lowed Michaelis-Menten kinetics. That is, the enzyme E first combines
with the substrate S to form the enzyme substrate complex ES; the latter
then breaks down in a second step to form the free enzyme E and the
K1
product P: E + S E + P. Figures 28 and 30 follow the traditional
TT
Michaelis-Menten shape curve (95). Enzyme kinetic calculations (95,152)
were done in order to add to the information about the _P. citreus extra
cellular enzyme and to determine substrate saturation conditions.
The K values (Michaelis-Menten constant) calculated in this study
m
are apparent K^'s. Whole protein substrates (either gelatin or shrimp
protein) rather than specific synthetic amide substrates were used.
True values are calculated using specific substrates and having a
definite knowledge of the enzyme's active site (57). Additional
research is required to demonstrate the active site of the P. citreus
extracellular enzyme.
Apparent and V values were calculated by transforming the
data in Figures 28 and 30. Doublereciprocal plots (Lineweaver-Burk
plots) were done and they are shown in Figures 29 and 31 for gelatin
and shrimp protein substrates, respectively. The apparent K values
m
for the gelatin and shrimp protein substrates were 0.98 mg/ml and 0.33


TEMPERATURE (C)
Figure 24. Thermal stability of the enzymes in the cell-free broths of Planococcus citreus grown at
5 and 35 C and of the purified enzyme.
aCell-free broths incubated for 15 min at each temperature and activity assayed
at 35 C for 15 min (pH 8) and purified enzyme incubated for 10 min and activity
assayed at 35 C for 10 min (pH 8).


ENZYME ACTIVITY (xIO1)
Si
Figure 23. Temperature optimum of the extracellular proteolytic enzyme of Planococcus citreus.
Activity assayed at 35 C for 10 min, pH 8.


MATERIALS AND METHODS
Unless otherwise specified, Difco (55,56) or Baltimore Biological
Laboratories (BBL) (16) products were used for all microbiological
analyses. Serial dilutions used Butterfields Phosphate buffer and fol
lowed the procedures outlined in the Compendium of Methods for the
Microbiological Examination of Foods (6). All chemicals used were
reagent grade meeting American Chemical Society specifications. All
media and glassware were autoclaved for 15 min at 121 C unless label
directions specified otherwise.
Planococcus citreus Cultures
The culture of _P. citreus used in this study, A-17, was isolated
from rock shrimp (Sicyonia brevirostris) (90). The culture chosen was
able to grow well in shrimp during iced storage and showed strong pro
teolytic activity toward various protein preparations. The isolate used
for the study was grown on Plate Count Agar slants (Difco) with 0.5%
sodium chloride (NaCl) added and incubated at 20 C for 72 hrs (4).
Appropriate dilutions in buffer were made to obtain a concentration of
3
approximately 5 x 10 organisms per ml. The A-17 isolate used was
capable of hydrolyzing gelatin, whey, cottonseed, soy, hog blood and
shrimp protein preparations (4). It also grew well in 0.5% to 16% NaCl
and pH 7.0 to 10.9.
Determination of Proteolytic Activity
A modified fluorescamine fluorescent (fluorometric) technique (129)
was used to measure enzyme activity. Fluorescamine is capable of
25


121
12. Arvidson, S., T. Holme and B. Lindholm. 1972. Studies on extra
cellular proteolytic enzymes from Staphylococcus aureus. I.
Purification and characterization of one neutral and one alkaline
protease. Biochem. Biophys. Acta 302:135-148.
13. Association of Official Analytical Chemists. 1970. In: Official
Methods of Analysis. 11th Ed. AOAC, Washington, DC.
14. Bailey, M. E., E. A. Fieger and A. F. Novak. 1956. Objective
tests applicable to quality studies of ice-stored shrimp. Food
Res. 21:611-620.
15. Barr, A., S. H. Goodnight, J. R. Sail and J. T. Helwig. 1979.
A Users Guide to SAS-1979. Sparks Press, Raleigh, NC.
16. BBL Manual of Products and Laboratory Procedures. 1969. 5th Ed.
BBL, Division of BioQuest, Cockeysville, MD.
17. Beatty, S. A. and N. E. Gibbons. 1936. The measurement of
spoilage in fish. J. Biol. Bd. of Canada 3:77-90.
18. Bergmann, M. and J. S. Fruton. 1941. Proteolytic enzymes. Ann.
Rev. of Biochem. 10:31-46.
19. Bethae, S. and M. E. Ambrose. 1962. Comparison of pH, trimethyl-
amine content and picric acid turbidity as indices of iced shrimp
quality. Comm. Fisheries Rev. 24:7-10.
20. Bieler, A. C., R. F. Matthews and J. A. Koburger. 1972. Rock
shrimp quality as influenced by handling procedures. Proc. Gulf
and Caribbean Fish. Inst. 25:56-61.
21. Bio-Rad Laboratories. 1978. Molecular Weight Protein Standards
for SDS Gel Electrophoresis: Instruction Manual. Bio-Rad Labs.,
Richmond, CA.
22. Blajevic, D. J. and G. M. Ederer. 1975. Principles of Biochemical
Tests in Diagnostic Microbiology. John Wiley and Sons, New York,
NY.
23. Bohacek, J., M. Kocur and T. Martinec. 1967. DNA base composition
and taxonomy of some micrococci. J. Gen. Microbiol. 46:369-376.
24. Bohacek, J., M. Kocur and T. Martinec. 1968. Deoxyribonucleic
acid composition of some marine and halophilic micrococci. J.
Appl. Bacteriol. 31:215-219.
25. Bohlen, P., S. Stein, W. Darnman and S. Udenfriend. 1973. Fluoro-
metric assay of proteins in the nanogram range. Arch. Biochem.
Biophys. 155:213-220.
26. Boyle, P. J. and R. Mitchell. 1978. Absence of microorganisms in
crustacean digestive tracks. Science 200:1157-1159.


23
Figure 3. Effect of pH adjustment of gelatin-trichloroacetic acid (TCA).filtrates
vfiaorescence intensity.


12
recommendation that flagellated cocci be included either in the genus
Planococcus or Planosarcina. This suggestion was accepted by only a few
authors, e.g., Krasil'nikov in 1949 (92). The majority of the authors
have included the flagellated cocci in the genus Micrococcus (22,83),
mainly because these cocci could only be differentiated from the other
members of the genus by their motility. Most authors have considered
motility to be a minor characteristic for the recognition of a new genus.
The findings of Bohacek et al. (23,24) that the flagellated cocci differ
considerably in the guanosine-cytosine (GC) content of their deoxyribo
nucleic acid (DNA) from other cocci shed new light on their taxonomic
position. It was proposed by Bohacek et al. (23) to include the flagel
lated cocci with a GC content ranging from 40-50% in the genus Piano-
coccus. In 1970, Kocur et al. (91) revised and outlined the genus
Planococcus. However, according to Index Bergeyana (86), the Planococcus
genus includes nine species (P. agilis, _P. casei, _P. citreus, _P. citro-
agilis, _P. europeans, P. loffleii, _P. luteus, _P. ochrolencus and J?.
roseus). Kocur et al. (91) evaluated the strains available in culture
and proposed that seven belong to one species, Planococcus citreus.
Although the remaining two species were closely related, he refrained
from giving a precise designation and labeled them only as Planococcus
species.
Schleifer and Kandler (126) found that the strains studied by
Bohacek et al. (23,24) and Kocur et al. (91) were uniform with respect
to the type of murein present in their cell walls and similar to that
of members of the genera Micrococcus and Staphylococcus. However,
serological investigation of _P. citreus by Oeding in 1971 (112) revealed
no antigenic relationship to staphylococci or micrococci.