Characterization and comparison of plant and crustacean polyphenol oxidases

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Title:
Characterization and comparison of plant and crustacean polyphenol oxidases kinetics and inhibition by chemical methods
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xii, 216 leaves : ill., photos ; 29 cm.
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Chen, Jon-shang, 1958-
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Oxidases -- Analysis   ( lcsh )
Crustacea -- Analysis   ( lcsh )
Plants -- Analysis   ( lcsh )
Food Science and Human Nutrition thesis Ph. D
Dissertations, Academic -- Food Science and Human Nutrition -- UF
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bibliography   ( marcgt )
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Thesis:
Thesis (Ph. D.)--University of Florida, 1991.
Bibliography:
Includes bibliographical references (leaves 199-215).
Statement of Responsibility:
by Jon-Shang Chen.
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Typescript.
General Note:
Vita.

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University of Florida
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Full Text










CHARACTERIZATION AND COMPARISON OF PLANT AND CRUSTACEAN
POLYPHENOL OXIDASES: KINETICS AND INHIBITION BY CHEMICAL METHODS

















By

JON-SHANG CHEN


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

UNIVERSITY OF FLORIDA

1991


UNIVERSITY OF FLORIDA !! .PI~ES














ACKNOWLEDGEMENTS


I wish to express my sincere appreciation to Drs. Marty R. Marshall

(the chairman of the graduate supervisory committee) and Cheng-I Wei (the

cochairman) for their scientific guidance and financial support during my

graduate study. I would also like to extend my gratitude to the other

members of my graduate committee, Dr. James F. Preston, Dr. Murat 0.

Balaban, and Dr. Steven Otwell for their advice and assistance in

improving the depth of this research work. My special thanks are extended

to my wife, Shaw-Lien Hsia, for her invaluable support and encouragement.















TABLE OF CONTENTS


ACKNOWLEDGEMENTS. . . ... ....... .ii

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

LIST OF FIGURES . . ... ... .. .viii

ABSTRACT. . . ... . xii

INTRODUCTION. . . ... . 1

LITERATURE REVIEW . . . 4

Polyphenol Oxidase . . 4
Enzymatic Browning (Melanosis) . . 6
Inhibition/Prevention of Melanosis . ... 9
Kojic Acid. . . . 12
Supercritical Fluid (SCF) Treatment . 12

CHARACTERIZATION AND COMPARISON OF CRUSTACEAN AND PLANT POLYPHENOL
OXIDASES: KINETICS AND SOME PROPERTIES. . ... 16

Introduction . . .. 16
Materials and Methods . . 17
Extraction and Purification of Lobster Polyphenol Oxidase
(PPO) . . . 18
Extraction and Purification of Potato PPO . .. 19
Extraction and Purification of Apple PPO . .. 20
Extraction and Purification of White Shrimp and Grass Prawn
PPO . . . 21
Protein Quantitation and Enzyme Purity Determination. .. .. 23
PPO Activity Determination . .. 23
pH Optima and Stability of Lobster PPO . ... 24
Activation Energy and Thermostability of Lobster PPO. .. .. 24
Molecular Weight Determination of Lobster PPO ... 25
Enzyme Kinetics Study . .... 26
Results and Discussion . . .. 26
Effect of pH on Lobster PPO Activity . ... 26
Effect of pH on Lobster PPO Stability . .. 31
Effect of Temperature on Lobster PPO Activity and Stability 31
Activation Energy (Ea) of Lobster PPO . .. 39
Molecular Weight and Isoform Determination of Lobster PPO 40
Enzyme Kinetics of Lobster PPO. . ... 43









Conclusion. . .. .. . .51

STRUCTURAL COMPARISON OF CRUSTACEAN, PLANT, AND MUSHROOM POLYPHENOL
OXIDASES . . . ... 52

Introduction. . . . ... 52
Materials and Methods . . 53
Extraction of Mushroom, Potato, Lobster, and Shrimp PPO 53
Purification of Mushroom, Potato, Lobster, and Shrimp PPO 54
Protein Quantitation and Enzyme Purity Determination. .. .. 54
Enzyme Activity Assay . . .. 55
Anti-lobster PPO Antibody Production and Purification 55
Molecular Weight Determination of Anti-lobster PPO Antibody 56
Antibody Titer Determination by Enzyme-linked Immunosorbent
Assay (ELISA) ................ .. 57
Analysis of Antigenic Properties of PPO .. ... 58
Immunoblotting . . .. 58
Spectropolarimetric Analysis of PPO . 59
Results and Discussion. . . ... 60
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE) Profile of PPO . . .. 60
Antibody Production and Molecular Weight Determination. 63
Antibody Titer Determination . .... .63
Immunological Characteristics of PPO. . ... 70
Spectropolarimetric Analysis of PPO . .. 75
Conclusion. . . . .. 89

INHIBITION MECHANISM OF KOJIC ACID ON SOME PLANT AND CRUSTACEAN
POLYPHENOL OXIDASES . . 90

Introduction . . . 90
Materials and Methods . . 91
Extraction and Purification of Potato PPO . .. 92
DEAE-cellulose Chromatography . .. 93
Gel Filtration . . .. 93
Extraction and Purification of Apple PPO . .. 94
Hydroxylapatite Chromatography . ... 94
Extraction and Purification of Grass Prawn PPO ... 95
Extraction and Purification of Lobster PPO ... 96
Extraction and Purification of white shrimp PPO .. 97
Protein Quantitation and Enzyme Purity Determination. ... 98
Enzyme Activity Assay . . .. 98
Effect of Kojic Acid on Enzyme Activity . 99
Enzyme Kinetics Study . . 100
Effect of Pre-incubation Temperature on PPO Inhibition by
Kojic Acid . . 102
Effect of Kojic Acid on the Hydroxylation Capability of PPO 103
Effect of Kojic Acid on Oxygen Uptake by PPO Reaction 103
Effect of Kojic Acid on Reduction of Cupric Copper .... 104
Effect of Kojic Acid on Quinone Products. . 104
Enzymatic Activities of Kojic Acid-treated PPO ... 105
Statistical Analysis . . ... 106









Results and Discussion. . . ... 107
Effect of Kojic Acid on Mushroom PPO Activity ... 107
Effect of Kojic Acid on Potato PPO Activity ... 107
Effect of Kojic Acid on Apple PPO Activity. .. 114
Effect of Kojic acid on Crustacean PPO Activity ...... 117
Enzyme Kinetics . . .. .. 117
Effect of Pre-incubation Temperature on PPO Inhibition by
Kojic Acid. . . .. 129
Effect of Kojic Acid on the Hydroxylation Capability of PPO 129
Effect of Kojic Acid on Oxygen Uptake by PPO Reaction 131
Effect of Kojic Acid on Reduction of Cupric Copper. .131
Effect of Kojic Acid on Quinone Products. . ... 135
Enzymatic Activities of Kojic Acid-treated PPO. .. 140
Conclusion. . . ... ....... 140

EFFECT OF CARBON DIOXIDE ON THE INACTIVATION OF PLANT AND
CRUSTACEAN POLYPHENOL OXIDASES. . ... 143

Introduction. . . ... ....... 143
Materials and Methods . . 144
Extraction and Purification of Lobster, Shrimp, and Potato
PPO . .. ... .144
Protein Quantitation and Enzyme Purity Determination. ... 145
Enzyme Activity Assay .......... .. 146
Effect of Carbon Dioxide (1 atm) on PPO Activity. ...... 146
pH Control Study. . ... 147
Effect of High Pressure (58 atm) Carbon Dioxide on PPO
Activity. . . 147
Kinetics of PPO Inactivation. . ... 150
Nondenaturing Polyacrylamide Gel Electrophoresis of Carbon
Dioxide (1 atm)-treated PPO .. . ..... 151
Mass Balance of High Pressure Carbon Dioxide-treated and
Nontreated PPO. . . .. 151
Polyacrylamide Gel Isoelectric Focusing of Carbon Dioxide-
treated PPO . .. ... .152
Spectropolarimetric Analysis of PPO . .. 153
Study of Restoration of Carbon Dioxide-treated PPO Activity 153
Results and Discussion .................. .. 154
Effect of Carbon Dioxide (1 atm) on PPO Activity. ... 154
Effect of Nitrogen on PPO Activity. . ... 157
Effect of High Pressure Carbon Dioxide on PPO Activity. 159
Kinetics of PPO Inactivation . .. 166
Polyacrylamide Gel Electrophoresis of Carbon Dioxide (1 atm)-
treated PPO . . 168
Mass Balance of High Pressure Carbon Dioxide-treated and
Nontreated PPO . . 168
Polyacrylamide Gel Isoelectric Focusing of Carbon Dioxide-
treated PPO . . 172
Spectropolarimetric Analysis of High Pressure Carbon Dioxide-
treated PPO . . 172
Restorative Ability of Carbon Dioxide-treated PPO Activity. 186
Conclusion . . . 189









CONCLUSIONS . . . 197

REFERENCE LIST..................... ..... .199

BIOGRAPHICAL SKETCH .. .. .. . 216














LIST OF TABLES


TABLE PAGE

1 Comparison of Kinetic Properties of Polyphenol Oxidase (PPO)
from Various Sources . . .. 30

2 Comparison of Polyphenol Oxidase (PPO) Activity between
Florida Spiny Lobster and Western Australian Lobster 49
3 Secondary Structure Estimates of Various Polyphenol Oxidases
(PPOs) from Far UV Circular Dichroic Spectra ... 88
4 Inhibitory Mechanism of Kojic Acid on Polyphenol Oxidase
Obtained from Various Sources. . ... 124

5 Effect of Different Pre-incubation Temperatures on the
Inhibition of Various Polyphenol Oxidases (PPOs) by Kojic
Acid . . .. ... .130

6 Inhibitory Effect of Kojic Acid on the Consumption of Oxygen
by Polyphenol Oxidase. . . ... 134

7 Enzymatic Activity of Kojic Acid-treated Polyphenol Oxidase
Following Kojic Acid Removal . .... 141

8 Effect of Nitrogen on Florida Spiny Lobster Polyphenol Oxidase
Activity . . ... .158

9 Kinetic Parameters of Florida Spiny Lobster Polyphenol Oxidase
Inactivation by Carbon Dioxide (1 atm) at Various
Temperatures . . ... ...... 167

10 Mass Balance of Protein Contents from High Pressure CO2-
treated and Nontreated PPO . . 171

11 Secondary Structure Estimates of Nontreated Control and High
Pressure CO -treated Florida Spiny Lobster, Brown Shrimp,
and Potato Polyphenol Oxidases (PPOs) from Far UV Circular
Dichroic Spectra . .. .. 185















LIST OF FIGURES


FIGURE PAGE

1 Mechanism of Polyphenol Oxidase-catalyzed Reaction .. 8

2 Structure of Kojic Acid (5-hydroxy-2-hydroxymethyl-y-
pyrone). . . ... ...... .14
3 Effect of pH on the Activity of Polyphenol Oxidase (PPO)
Isolated from Florida Spiny Lobster and Western
Australian Lobster . .... 28

4 Effect of pH on the Stability of Polyphenol Oxidase (PPO)
Isolated from Florida Spiny Lobster and Western
Australian Lobster . . 33

5 Effect of Temperature on the Activity of Polyphenol Oxidase
(PPO) Isolated from Florida Spiny Lobster and Western
Australian Lobster . . .. 35

6 Effect of Temperature on the Stability of Polyphenol
Oxidase (PPO) Isolated from Florida Spiny Lobster and
Western Australian Lobster . .. 38

7 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE, 7.5% Gel) Profile of Polyphenol Oxidase from
Florida Spiny Lobster (FSL) and Western Australian
Lobster (WAL). . . 42

8 Double Reciprocal Plots for the Oxidation of DL-DOPA and
Catechol by Florida Spiny Lobster Polyphenol Oxidase
(PPO). . . . 45

9 Double Reciprocal Plots for the Oxidation of DL-DOPA and
Catechol by Western Australian Lobster Polyphenol
Oxidase (PPO). . . ... .. 47

10 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE, 7.5% Gel) Profile of Polyphenol Oxidase from
Mushroom, Potato, Florida Spiny Lobster, White Shrimp,
and Brown Shrimp . .... 62

11 Profile of Anti-lobster PPO Antibody Production in
Immunized Hen during the Immunization Period .. 65


viii









12 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE, 7.5% Gel) Profile of Anti-lobster PPO
Antibody . . .. 67

13 The Titer Determination of Antibody against Florida Spiny
Lobster PPO versus Mushroom, Potato, Florida Spiny
Lobster, White Shrimp, and Brown Shrimp PPO. .. 69

14 Analysis of Antigenic Properties of Purified Mushroom,
Potato, Florida Spiny Lobster, White Shrimp, and Brown
Shrimp PPO by Competitive ELISA. . ... 72

15 Profile of Protein Standard (SDS-6H), Purified Mushroom,
Potato, Florida Spiny Lobster, White Shrimp, and Brown
Shrimp PPO, and Crude Lobster PPO Preparation on a
Nitrocellulose Membrane. . ... 74

16 Determination of the Specific Reactivity of Anti-lobster
PPO Antibody with Crude Lobster PPO Preparation,
Purified Mushroom, Potato, Florida Spiny Lobster, White
Shrimp, and Brown Shrimp PPO as well as SDS-6H Protein
Standard as Analyzed by Immunoblotting Technique 77

17 The far UV Circular Dichroic Spectra of Mushroom PPO 79

18 The far UV Circular Dichroic Spectra of Potato PPO 81

19 The far UV Circular Dichroic Spectra of Florida Spiny
Lobster PPO. . .. .. 83

20 The far UV Circular Dichroic Spectra of White Shrimp PPO 85

21 The far UV Circular Dichroic Spectra of Brown Shrimp PPO 87

22 Effect of Concentration-related Inhibitory Effect of Kojic
Acid on Mushroom Tyrosinase (PPO) Activity on DL-P-3,4-
dihydroxyphenylalanine (DL-DOPA) .... .... 109

23 The Concentration-related Inhibitory Effect of Kojic Acid
on the Oxidation of DL-DOPA and L-Tyrosine by Mushroom
PPO. . . .111

24 The Concentration-related Inhibitory Effect of Kojic Acid
on the Oxidation of Chlorogenic Acid and Catechol by
Potato PPO . . .. 113

25 The Concentration-related Inhibitory Effect of Kojic Acid
on the Oxidation of 4-Methylcatechol and Chlorogenic
Acid by Apple PPO. . . ... 116









26 The Concentration-related Inhibitory Effect of Kojic Acid
on the Oxidation of L-DOPA and Catechol by White Shrimp
PPO. . . ... . 119
27 The Concentration-related Inhibitory Effect of Kojic Acid
on the Oxidation of DL-DOPA and Catechol by Grass Prawn
PPO. . . ... ....... 121

28 The Concentration-related Inhibitory Effect of Kojic Acid
on the Oxidation of DL-DOPA and Catechol by Lobster PPO. 123

29 Effect of Kojic Acid on the Hydroxylation of Monohydroxy-
phenol by Mushroom Tyrosinase (PPO). . ... 133
30 Effect of Kojic Acid on Reduction of Cu2+ to Cu+ in a
Model System . . 137

31 Change in Absorption Spectra of Dopaquinone due to the
Addition of Kojic Acid. (a) Dopaquinone Produced from
the Action of Mushroom PPO on DL-DOPA; and
(b) Dopaquinone Plus Kojic Acid. . ... 139

32 Apparatus Used for Studying Polyphenol Oxidase (PPO)
Inactivation by High Pressure CO . .. 149
33 Effect of Carbon Dioxide (1 atm) on the Change in pH and
Enzyme Activity of Florida Spiny Lobster PPO Heated at
330 (a), 380 (b), or 430C (c). . ... 156
34 Effect of High Pressure (58 atm) Carbon Dioxide on the
Change in pH and Enzyme Activity of Florida Spiny
Lobster PPO heated at 43C . .. 161
35 Effect of High Pressure (58 atm) Carbon Dioxide on the
Change in pH and Enzyme Activity of Brown Shrimp PPO
heated at 43C . . 163
36 Effect of High Pressure (58 atm) Carbon Dioxide on the
Change in pH and Enzyme Activity of Potato PPO heated at
430C . . 165

37 Nondenaturing Polyacrylamide Gel Electrophoresis (PAGE,
7.5% Gel) Profile of Carbon Dioxide (1 atm)-treated
Florida Spiny Lobster PPO . 170
38 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
(SDS-PAGE, 7.5% Gel) Profile of High Pressure (58 atm)
Carbon Dioxide-treated Florida Spiny Lobster PPO 174









39 Polyacrylamide Gel Isoelectric Focusing (IEF, 5% Gel)
Profile of Carbon Dioxide (1 atm)-treated Florida Spiny
Lobster PPO . ..... 176

40 Polyacrylamide Gel Isoelectric Focusing (IEF, 5% Gel)
Profile of High Pressure (58 atm) Carbon Dioxide-treated
Florida Spiny Lobster PPO. . 178

41 Comparison of Far UV Circular Dichroic Spectra of
Nontreated Control and High Pressure (58 atm) Carbon
Dioxide-treated Florida Spiny Lobster PPO. .. 180

42 Comparison of Far UV Circular Dichroic Spectra of
Nontreated Control and High Pressure (58 atm) Carbon
Dioxide-treated Brown Shrimp PPO . .... 182

43 Comparison of Far UV Circular Dichroic Spectra of
Nontreated Control and High Pressure (58 atm) Carbon
Dioxide-treated Potato PPO . .... 184

44 The Restorative Ability of Carbon Dioxide (1 atm)-treated
Florida Spiny Lobster PPO Activity and the Pertinent
Environmental pH Changes during Frozen-storage .. 188

45 The Restorative Ability of High Pressure (58 atm) Carbon
Dioxide-treated Florida Spiny Lobster PPO Activity and
the Pertinent Environmental pH Changes during Frozen-
storage. . ... ..... 191

46 The Restorative Ability of High Pressure (58 atm) Carbon
Dioxide-treated Brown Shrimp PPO Activity and the
Pertinent Environmental pH Changes during Frozen-storage 193

47 The Restorative Ability of High Pressure (58 atm) Carbon
Dioxide-treated Potato PPO Activity and the Pertinent
Environmental pH Changes during Frozen-storage .195














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

CHARACTERIZATION AND COMPARISON OF PLANT AND CRUSTACEAN
POLYPHENOL OXIDASES: KINETICS AND INHIBITION BY CHEMICAL METHODS

By

JON-SHANG CHEN

December, 1991

Chairman: M.R. Marshall
Cochair: C.I. Wei
Major Department: Food Science and Human Nutrition

The characterization and comparison of polyphenol oxidase (PPO, E.C.

1.14.18.1) from crustaceans (lobster and shrimp) and plants were made.

PPO from various plant and crustacean sources varied with respect to

molecular weight, pH and temperature effects on activity and stability,

substrate specificity, and other kinetic parameters. Differences in

conformational structure among PPOs from mushroom, potato, lobster, white

shrimp, and brown shrimp were investigated using spectropolarimetric and

immunological methods. Varied secondary structures were observed among

these PPOs, although they were shown to possess similar antigenic

determinants.

Kojic acid [5-hydroxy-2-(hydroxymethyl)---pyrone], a fungal

metabolite, exhibited competitive and/or mixed types of inhibition on the

oxidation of various phenolic substrates (L-tyrosine, DL-P-3,4-dihydroxy

phenylalanine, 4-methylcatechol, catechol, and chlorogenic acid) by

mushroom, potato, apple, and crustacean (lobster, grass prawn, and white
xii









shrimp) PPOs. In addition, kojic acid was capable of reducing o-quinones

back to diphenols and prevented melanin formation.

The inactivation of lobster, brown shrimp, and potato PPO by CO2 was

studied. When exposed to CO2 (1 atm) at 330, 380, or 430C, lobster PPO

showed a decline in enzyme activity with treatment time. Studies on

inactivation kinetics revealed that PPO was more labile to CO2 and heat

than to heat alone. The use of polyacrylamide gel electrophoresis showed

that there were no differences in protein patterns and isoelectric

profiles between the CO2 (1 atm)-treated and untreated PPO. When lobster,

brown shrimp, and potato PPOs were subjected to high pressure (58 atm) CO2

at 430C, the inactivation of these PPOs followed trends similar to the

atmospheric CO, experiments. Crustacean PPOs, however, were more

susceptible to inactivation by high pressure CO2 than by atmospheric CO,.

Differences in the secondary structures between the high pressure CO2-

treated and the nontreated PPO were evident by spectropolarimetric

analysis.














INTRODUCTION


Undesirable enzymatic browning causing the discoloration or
formation of black spots melanosiss) by polyphenol oxidase (E.C.

1.14.18.1; PPO) on the surface of many vegetable, fruit, and seafood

(crustacean) products has been of great concern to food scientists. For

food processors, the formation of melanin pigments not only imparts the

problems in sensory attributes and, hence, the marketability of the

product, but often lower its nutritive value as well (Synge, 1975).

Kinetic properties have been studied for PPO from various sources of
vegetables and fruits (Schwimmer, 1981) and more recently from crustaceans

(Ferrer et al., 1989a; Rolle et al., 1991; Simpson et al., 1987, 1988a).

However, little comparative biochemical information exists between PPO

from plants and crustaceans. A preliminary study conducted in this

laboratory revealed that Western Australian lobster (Panulirus cygnus) was

far less susceptible to melanosis during storage at refrigeration

temperature than Florida spiny lobster (Panulirus argus) maintained under

the same conditions. It is speculated that the susceptibility to

melanosis could be attributed to the difference in PPO activity between

these two lobster species. Thus, the first objective of this study was to

characterize and determine the kinetic properties of PPO enzymes from

these two lobsters as well as from other crustacean and plant sources.

Many chemical and physical methods have been studied for their

effectiveness on the inhibition of enzymatic browning. Browning may be

1









2

prevented not only by inactivating the enzyme, but also by eliminating

substrate (02 or polyphenol) necessary for the reaction, or by reacting

with the products of the enzyme reaction thus preventing the formation of

the quinones necessary for the preceding non-enzymatic steps. Although

some chemicals have been shown to inhibit PPO activities, their use in

food processing is restricted by many concerns such as toxicity,

wholesomeness, effect on taste, flavor, texture, etc. (Vamos-Vigyazo,

1981). Kojic acid (5-hydroxy-2-hydroxymethyl---pyrone), a fungal

metabolite produced by many species of Aspergillus and Penicillium

(Kinosita and Shikata, 1964), has been reported for its inhibitory effect

on mushroom PPO (Saruno et al., 1979). Kojic acid mixed with ascorbic

acid and citric acid constitutes a Japanese commercial product which is

used as a tyrosinase inhibitor in foods. Since only limited information

was available on the inhibitory activity and mechanism of kojic acid on

PPO, the second objective of this study was to investigate the inhibitory

activity of this compound on crustacean (Florida spiny lobster, white

shrimp, and grass prawn) mushroom, and plant (potato and apple) PPO and to

elaborate the mechanisms of inhibition.

In past years, treatment in an atmosphere modified with carbon

dioxide has been used as an application for retarding enzyme activity, to

preserve food quality and to extend the shelf-life of food products.

Recently, inactivation of peroxidase, PPO, pectinesterase, a-amylase,

glucose oxidase, lipase, or catalase by supercritical fluid using CO2 as

the solvent has been reported by many workers (Arreola, 1990; Christianson

et al., 1984; Taniguchi et al., 1987; Zemel, 1989). However, information

concerning the inhibitory effect and the inhibition mechanism of CO2 on








3

plant and crustacean PPO was limited. Thus, the third objective of this

study was to investigate the effect of CO2 (atmospheric and high pressure)

on the inhibition of lobster, brown shrimp, and potato PPO.














LITERATURE REVIEW


Polyphenol Oxidase


Polyphenol oxidase (PPO) (E.C. 1.14.18.1.), also known as

tyrosinase, polyphenolase, phenolase, catechol oxidase, cresolase, and

catecholase, is widely distributed in nature (Schwimmer, 1981). In

addition to its general occurrence in plants, it can also be found in

microorganisms, especially fungi, and in some animal tissue (Brown,

1967). PPO plays an important role in the resistance of plants to

microbial or viral infections and probably to adverse climate (Vamos-

Vigyazo, 1981). The vast literature dealing with the role of the PPO-

polyphenol system in plant pathology has been extensively reviewed

(Bonner, 1957; Farkas and Kiraly, 1962). A simplified explanation of this

role in the resistance of plants to infections is that the quinones formed

upon the action of the enzyme undergo secondary polymerization reactions

yielding dark, insoluble polymers; the tissues impregnated with these

polymers act as barriers to prevent further spreading of the infection.

This is considered by some authors to be the main function of the enzyme

(Macrae and Duggleby, 1968). However, plants resistant to adverse

climatic conditions have, in general, higher PPO activities than

susceptible varieties (Khrushcheva and Krehin, 1965).

In addition to the involvement in phenolic compounds biosynthesis,

the PPO enzymes also indirectly participate in auxin biosynthesis. The











primary products of its action on o-diphenols, the o-quinones, react with

tryptophan to form indole acetic acid via indolepyruvic acid (Gordon and

Paleg, 1961). Thus, PPO together with auxin degrading enzyme

(peroxidase), might play a role in plant growth regulation (Vamos-Vigyazo,

1981). The quinones formed upon PPO action may also participate in

reactions similar to those leading to nonenzymatic browning and

humification and thus contribute to producing organic matter of soil

(Synge, 1975).

PPO from insects and crustaceans plays an important role in the
sclerotization during molting (Andersen, 1971; Brunet, 1980; Cobb, 1977;

Ferrer et al., 1989a; Summers, 1967; Vinayakam and Nellaiappan, 1987). In

this process, PPO oxidizes diphenols to quinones, which interact with

certain side groups on adjacent proteins, thus linking them together

(Stevenson, 1985). N-Acetyldopamine (Andersen, 1979) and 3,4-

dihydroxybenzoic acid (Pryor et al., 1962) have been identified as the

cross-linking agents in crustacean and cockroach, respectively. Another

function closely related to plant systems was PPO's involvement in the

process of wound repair and calcification of the cuticle (Stevenson,

1985).

Most PPO's are copper-containing enzymes which catalyze two entirely
different reactions: (a) the hydroxylation of monophenols to the

corresponding o-dihydroxy compounds, which is called cresolase activity,

and (b) the oxidation of o-dihydroxy phenols to o-quinones, which is

termed catechol activity. The activity of cresolase involves three steps

which can be represented by the following equation (Mason, 1956):

Protein-Cu2+-02 + monophenol Protein-Cu2+ + o-quinone + H20









6
The protein-copper-oxygen complex is formed by combining one molecule of

oxygen with the protein to which two adjacent cuprous atoms are attached.

Catecholase activity involves the oxidation of 2 molecules of o-diphenols

to 2 molecules of o-quinones, resulting in the reduction of one molecule

of oxygen to two molecules of water. The sequence for the PPO-catalyzed

reaction proposed by Mason (1957) is shown in Figure 1. The enzyme-oxygen

complex serves as the hydroxylating or dehydroxylating intermediate, and

(Cu)n represents the actual charge designation of the copper at the active
site. The overall reaction involves the use of one molecule of oxygen;

one atom of which goes into the formation of diphenol, and the other is

reduced to water. Substrate specificity varies considerably for PPO from

various sources (Aurand and Woods, 1973).


Enzymatic Browning (Melanosis)

Food scientists' primary interest in PPO is in the enzymatic

browning phenomenon and its effect on food quality. o-Quinones, the

primary products of PPO oxidative reaction can (a) interact with each

other to form high molecular weight polymers, (b) form macromolecular

complexes with amino acids or proteins, and (c) oxidize compounds of lower

oxidation-reduction potentials (Mathew and Parpia, 1971). Enzymatic

browning is a desirable process for the manufacture of black tea, sultana

grape, and ground coffee bean (De Amorim and Silva, 1968; Grncarevic and
Hawker, 1971; Takeo, 1966).

Unfavorable darkening on the surface of many fruits, vegetables, and
seafood products is primarily due to the indirect consequence of PPO

oxidizing phenols to orthoquinones, which in turn rapidly polymerize to


















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u-, +- r
e "as



Cr +J X








8


cr

00
C



I +
I 0


xc
+ 0
S+:3
0" CL )0
< X, o



00 cI 6 0

00





t-
*L_
0 w





+ I
C =: .1

0+ ZI )


co









9

form brown pigments or melanins (Joslyn and Ponting, 1951). Enzymatic

browning of fruits, vegetables, and seafood products (mainly shrimp and

lobster) due to PPO activity has been extensively studied (Ferrer et al.,

1989a; Flurkey and Jen, 1978; Harel et al., 1966; Koburger et al., 1985;

Macrae and Duggleby, 1968; Madero and Finne, 1982; Mapson et al.,1963;

Mosel and Hermann, 1974; Palmer, 1963; Patil and Zucker, 1965; Savagaon

and Sreenivasan, 1978; Sciancalepore and Longone, 1984; Simpson et al.,

1987, 1988a; Walker, 1962, 1964; Weurman and Swain, 1955). Although not

harmful to consumers, it is the formation of melanins on the surface of

these products by PPO that decreases their market value, making them

unappealing and the perception of spoilage (Alford and Fieger, 1952;

Bailey and Fieger, 1954; Bailey et al., 1960a, 1960b; Faulkner et al.,

1954; Ogawa et al., 1984).

Inhibition/Prevention of Melanosis


Undesirable enzymatic browning occurring in agricultural products

causes an enormous economical loss for food industries. Thus, in order to

reduce this loss, the occurrence of melanosis on food products must be

prevented or inhibited. Inhibition of PPO and prevention of enzymatic

browning are often treated as one and the same subject. Browning can be

prevented by inactivating PPO, by eliminating one of the substrates

necessary for the reaction (02 and the polyphenols), or by reacting the

products of PPO metabolism which in turn inhibit the formation of the

colored compounds produced in the secondary, non-enzymatic reaction steps.

As PPO is a metalloprotein with copper as the prosthetic group (Bailey et

al., 1960a; Bendall and Gregory, 1966; Mayer, 1962; Smith and Kruger,









10

1962), the activity of this enzyme can be inhibited by metal-chelating

agents. The effects of cyanide, carbon monoxide, sodium-diethyl-dithio-

carbamate (DIECA), mercaptobenzothiazole, dimercaptopropanol, potassium

methyl xanthate, fluoride, azide, borate, benzoic acid, small peptides,

polyvinylpyrrolidone (PVP), 2,3-naphthalenediol, ascorbic acid, and

dichlorodifluoromethane on plant PPO activity have been extensively

studied; these chemical inhibitors act primarily on the enzyme (Bedrosian

et al., 1959; Harel et al., 1967; Jones et al., 1965; Mayer et al., 1964;

Palmer and Roberts, 1967; Pierpoint, 1966; Pifferi et al., 1974; Robb et

al., 1966; Taeufel and Voigt, 1964; Walker, 1964, 1975, 1976; Walker and

Wilson, 1975; Yasunobu and Norris, 1957).

Other compounds acting as reducing agents which reduce o-quinones to

diphenols or react with plant PPO substrates to lessen the extent of

enzymatic browning are ascorbic acid, SO2, sodium sulfite, sodium

bisulfite, sodium metabisulfite, potassium metabisulfite, cysteine,

2-mercaptoethanol, glutathione, benzenesulphinic acid, DIECA, Na-ethyl

xanthate, and PVP (Cash et al., 1976; Embs and Markakis, 1965; Feinberg et

al., 1976; Haisman, 1974; Hope, 1961; Markakis and Embs, 1966; Mapson,

1965; Mapson and Wager, 1961; Muneta, 1966; Muneta and Walradt, 1968;

Muneta and Wang, 1977; Ponting, 1960; Ponting and Jackson, 1972;

Sayavedra-Soto and Montgomery, 1986; Singh and Ahlawati, 1974; Taeufel and

Voigt, 1964; Vamos-Vigyazo, 1981; Walker, 1964). For the prevention of

melanosis in crustaceans, numerous agents including ascorbic acid and

sodium ascorbate, ascorbate and citrate, L-tyrosine, L-methionine, citric

acid, L-cysteine, sodium diethyl dithiocarbamate, sodium tripolyphosphate

(STP), sodium sulfite, and sodium bisulfite have been broadly studied









11

(Antony and Nair, 1975; Bailey and Fieger, 1954; Faulkner et al., 1954;

Ferrer et al., 1989b; Fink, 1988; Madero, 1982; Madero and Finne, 1982).

Many methods are available for inhibiting/inactivating PPO. The

application of these methods or chemicals for inhibition in the food

industry, however, is limited. Problems related to off-flavor, off-odor,

toxicity, and economic feasibility all affect the application of chemicals

(Eskin et al., 1971). Sulfiting agents listed as Generally Recognized As

Safe (GRAS) by the Food and Drug Administration (FDA) since 1959 (FDA,

1984) have been widely used to prevent melanosis of agricultural and

seafood products. However, due to the recent health concern and questions

regarding the safety of sulfiting agents, the FDA currently proposed

revoking the GRAS status of these additives for use on fruits and

vegetables intended to be served or sold fresh or raw to consumers. The

FDA also requires label declaration of these agents added to food or as an

ingredient whenever they are present in the finished product at a level

equal to or higher than 10 ppm as SO2 (FDA, 1985). Furthermore, the FDA

requires that sulfite-treated shrimp products having residue levels

greater than 10 ppm must bear a label stating the presence of these

additives (FDA, 1985).

The maximum allowable residual level of sulfite on shrimp in the raw

edible portion is 100 ppm as SO2 (Marshall et al., 1984), which is not

hazardous to the majority of the population. After exposure to these
additives even at this level, however, people still suffer adverse

reactions ranging from mild to severe symptoms (Lecos, 1986). Therefore,

the search for an alternative which can inhibit melanosis but does not

cause adverse reactions becomes a necessity.











Kojic Acid

The inhibitory effect of some tyrosine-3-hydroxylase inhibitors of

microbial origins such as aquayamycin, chrothiomycin, and oudenone on PPO

activity has been studied (Ayukawa et al., 1968, 1969; Nagatsu et al.,

1968; Sezaki et al., 1968; Umezawa et al., 1970). Kojic acid (5-hydroxy-

2-hydroxymethyl-7-pyrone) (Figure 2) produced in the culture filtrate of

Aspergillus albus was shown in vitro to inhibit the activity of mushroom

tyrosinase using DOPA and tyrosine as substrates (Saruno et al., 1979).

At low concentration, kojic acid was found to inhibit the in vitro

oxidation of a number of D-amino acids, L-phenylalanine and a few related

compounds (Bajpai et al., 1981). Production of kojic acid, generally

considered as a secondary metabolite from carbohydrate metabolism under

aerobic conditions, has been extensively studied (Bajpai et al., 1981,

1982a, 1982b; Morton et al., 1945; Saruno et al., 1979). Although kojic

acid can be considered a potential candidate to inhibit melanosis, the use

of this chemical, depends not only on its production but also its

potential toxicity to humans.


Supercritical fluid (SCF) Treatment

Intensive study of supercritical fluids for extraction of food

components has mostly concentrated on decaffeination, deodorization, aroma

recovery, oil recovery, oil refining, and fractionation (Bulley et al.,

1984; Christianson et al., 1984; Friedrich and Pryde, 1984; Lee et al.,

1986; Maddocks and Gibson, 1977; Peter and Brunner, 1978; Snyder et al.,

1984; Schultz et al., 1967; Stahl et al., 1978, 1980, 1984; Taniguchi et

al., 1985; Vollbrecht, 1982; Zosel, 1979, 1981). Modified atmospheres






























C
0
O
S.-



I






I


0-
X
O








.0




Lf



0


'-a
u

u





*4-

U





l..







0)














17
0
I
0


0


0

Q.
a)
E






0
0t






C'
I
-o

0
"0
--
I
LC)


O)


O
10


0
I:









15

(carbon dioxide or C02-saturated brines) have been used successfully to

preserve food quality and extend shelf-life (Barnett et al., 1978; Brown

et al., 1980; Bullard and Collins, 1978; Gee and Brown, 1987a, 1987b;

Lannelongue et al., 1982; Longard and Reiger, 1974; Shewan, 1950; Vernath

and Robe, 1979; Villemure et al., 1986; Woyewoda et al., 1984; Yokoseki et

al., 1956). Studies of seafood preservation using a modified atmosphere

of CO2 showed that the lower internal pH of the tissue was due to exposure

of the external CO2 present and resulted in a rapid acidification of the

internal cellular environment (Aickin and Thomas, 1975; Boron and DeWeer,

1976; Thomas, 1974; Thomas and Ellis, 1976; Turin and Warner, 1977).

Intracellular and extracellular acidification of tissue could produce an

antimicrobial effect and also influence many different enzymatic

activities (Parkin et al., 1981). SCF possesses physicochemical

properties intermediate between those of liquids and gases which enhance

its efficacy as solvents; the higher gas density gives good solvent power,

while the lower viscosity and higher diffusivity provide the SCF with

higher gas permeability (Rizvi et al., 1986). Carbon dioxide is used as

a solvent for SCF because it is nontoxic, nonflammable, inexpensive and

readily available (Hardardottir and Kinsella, 1988) and it has a

relatively low critical temperature (33.1C) and pressure (72 atm) (Rizvi

et al., 1986). Eldridge et al. (1986) reported that SCF produced minimal

detrimental effects on the functional properties of proteins. The

application of SCF using CO2 as a solvent in seafood processing could be

a benefit because it possesses characteristics of antimicrobial action,

lipid (cholesterol) reduction, and enzymatic inhibitory activities.














CHARACTERIZATION AND COMPARISON OF CRUSTACEAN AND PLANT POLYPHENOL
OXIDASES: KINETICS AND SOME PROPERTIES

Introduction


Polyphenol oxidase (PPO) (E.C. 1.14.18.1.), also known as

tyrosinase, polyphenolase, phenolase, catechol oxidase, cresolase, and

catecholase, is widely distributed in nature (Schwimmer, 1981). In

addition to its general occurrence in plants, it can also be found in

microorganisms, especially in fungi, and in some animal organs (Brown,

1967). Unfavorable darkening of many fruits and vegetables after cutting

is primarily due to the action of this enzyme. Enzymatic browning of

fruits and vegetables due to PPO activity has been widely reported

(Flurkey and Jen, 1978; Harel et al., 1966; Macrae and Duggleby, 1968;

Mapson et al., 1963; Mosel and Herrman, 1974; Palmer, 1963; Patil and

Zucker, 1965; Sciancalepore and Longone, 1984; Walker, 1962, 1964; Weurman

and Swain, 1955). However, the role of PPO in crustaceans is not well

documented considering the variation in susceptibility to melanosis.

PPO plays an important role in the sclerotization of insects and

crustaceans during the molting cycle (Andersen, 1971; Brunet, 1980;

Summers, 1967; Vinayakam and Nellaiappan, 1987). However, it is the

formation of melanins causing darkening on the surface of seafood products

due to PPO action which is of concern to the seafood industry. PPO

enzymes from various crustaceans have been partially characterized (Madero

and Finne, 1982; Nakagawa and Nagayama, 1981; Simpson et al., 1987,









17

1988a). Although kinetic properties have been studied for PPO from

various sources of vegetables and fruits (Schwimmer, 1981) and more

recently from crustaceans (Chen et al., 1991a; Ferrer et al., 1989a; Rolle

et al., 1991), little comparative biochemical information exists between

PPO from plants and crustaceans. A preliminary study conducted in this

laboratory revealed that Western Australian lobsters were far less

susceptible than the Florida spiny lobsters to melanosis during storage at

refrigeration temperature. A similar phenomenon was observed on pink

shrimp (Penaeus duorarum) in comparison to white shrimp (Penaeus

setiferus) (Simpson et al., 1988a). These workers characterized the PPO

properties and they attributed the difference in susceptibility to

melanosis to the varied physiological efficiency of the enzyme and the

distribution of phenolic substrates in the shrimp (Simpson et al., 1988a).

Koburger et al. (1985) also showed that South African lobster tails were

less susceptible to melanosis than Florida spiny lobster tails. Since the

water habitat of Western Australian lobster is colder than that of Florida

spiny lobster, it is speculated that the PPO activity for these two

species could be different. Thus, the objectives of this study were to

characterize the PPO from these two lobster species and to compare their

kinetic properties to PPOs from other plant and crustacean sources.


Materials and Methods

Fresh Florida spiny lobster (Panulirus argus) tails obtained from

the Whitney Marine Laboratory at Marineland, FL were transported in ice to

the laboratory and stored at -200C. Frozen Western Australian lobster

(Panulirus cygnus) tails were purchased from the Beaver Street Foods









18
(Jacksonville, FL); these lobsters were found to contain less than 5 ppm

sulfite background residue when randomly selected samples from each batch

were checked using the method of Simpson et al. (1988b). Mushroom

(Agaricus bispora) tyrosinase with an activity of 2,200 units/mg solid was

purchased from Sigma Chemical Co. (St. Louis, MO). Russet potatoes and Red

Delicious apples were purchased from a local supermarket. White shrimp

(Penaeus setiferus) and brown shrimp (Penaeus aztecus) were obtained from

a local seafood store. Grass prawn or Taiwanese black tiger shrimp

(Penaeus monodon) frozen in dry ice was provided by Dr. J. S. Yang, Food

Industry Research and Development Institute, Hsinchu, Taiwan, Republic of

China. Lobster cuticle, shrimp cephalothorax (head), and potato and apple

peels were frozen in liquid nitrogen and ground into a fine powder using

a Waring blender. The ground powder was stored at -200C until needed.


Extraction and Purification of Lobster PPO

Frozen lobster tails were thawed at room temperature. After the

cuticle was separated from the flesh, it was frozen in liquid nitrogen and

ground into a fine powder in a Waring blender. The cuticle powder was

stored at -200C and used as required.

PPO was extracted according to the procedure of Simpson et al.

(1988a). One part lobster cuticle powder was added to three parts (w/v)

0.05 M sodium phosphate buffer (pH 7.2) containing 1 M NaCl and 0.2% Brij

35 (Fisher Scientific Co., Orlando, FL). The extract was stirred for 3 hr

at 40C and the suspension was centrifuged at 8,000g (40C) for 30 min. The

supernatant was then dialyzed at 40C overnight against 3 changes (4L) of

0.05 M sodium phosphate buffer (pH 6.5).


I








19

Each enzyme was purified further using a nondenaturing preparative

polyacrylamide gel electrophoresis (PAGE) system. Equipment utilized

included a gel tube chamber (Bio-Rad Model 175, Richmond, CA) and a Bio-

Rad power supply (Model EPS 500/400). A one-mL aliquot of crude enzyme
extract was applied to each of eight gel tubes (1.4 cm I.D. x 12 cm

length) containing 5% acrylamide/ 0.13% bisacrylamide gel prepared

according to the method of Sigma Bulletin No. MKR-137 (Sigma Chemical Co.,
1984), and ran at a constant current of 10 mA/tube in a buffer (pH 8.3)

containing 5 mM Tris-HC1 and 38 mM glycine. PPO was visualized using a

specific enzyme-substrate staining method (Constantinides and Bedford,
1967); 10 mM DL-P-3,4-dihydroxyphenylalanine (DL-DOPA) in 0.05 M sodium

phosphate buffer (pH 6.5) was used as a substrate. One tube was used to

determine the migration of the enzyme relative to the dye front (Rf). The

remaining gels were then sectioned at the determined Rf and the enzyme was

eluted from the gel by homogenization in 0.05 M sodium phosphate buffer

(pH 6.5) utilizing a Dounce manual tissue grinder (Wheaton, Millville,

NJ). The homogenates were filtered through Whatman No. 4 filter papers,

pooled, and concentrated using an Amicon stirred cell (Model 8050, Amicon

Co., Danvers, MA) fitted with a 10 K filter (Pharmacia LBK Biotechnology

Inc., Piscataway, NJ).

Extraction and Purification of Potato PPO

The method of Patil and Zucker (1965) with some modifications was

used. After ammonium sulfate precipitation and dialysis, crude PPO
preparation was subjected to chromatography with a DEAE-cellulose (0.95

meq/g, Sigma) column (40 cm length x 26 mm i.d., K 26/40 Pharmacia Fine









20
Chemicals) which had been equilibrated with 1.0 mM potassium phosphate

buffer (pH 7.0). Unbound phenolic compounds and proteins were washed off

using 250 mL 1 mM phosphate buffer (pH 7.0) at 24 mL/hr for 3 hr. Elution

of PPO was performed using a linear gradient (0 1.0 M) of NaC1 in 1.0 mM

potassium phosphate buffer at 24 mL/hr for 18 hr. Four-mL fractions were

collected, and protein was estimated by spectrophotometry at 280 nm.
Fractions showing PPO activity were pooled and concentrated using an

Amicon stirred cell fitted with an Amicon YM 10 filter.

The partially purified enzyme preparation was loaded onto a Sephadex
G-100 (Pharmacia) gel filtration column (Pharmacia K 26/40)

pre-equilibrated with 1.0 mM potassium phosphate buffer (pH 7.0). The

column was eluted at 40C with 400 mL 1.0 mM potassium phosphate buffer (pH
7.0) at 24 mL/hr for 15 hr. Four-mL fractions were collected and protein

estimated as above; fractions showing PPO activity were pooled and

concentrated using an Amicon stirred cell. Concentrated samples were

dialyzed at 40C overnight against 3 changes of 2L elution buffer.

Extraction and Purification of Apple PPO

The method of Stelzig et al. (1972) with some modification was
followed. Crude apple PPO after partial purification and dialysis against

H20 was loaded onto an HT hydroxylapatite (Bio-Rad) column (K 26/40). The
enzyme was desorbed from the gel using 250 mL 0.005 0.3 M (linear

gradient) sodium phosphate buffer (pH 7.6) containing 5% ammonium sulfate
at 24 mL/hr for 18 hr. Four-mL fractions were collected and protein
estimated by absorbance at 280 nm; fractions showing PPO activity were

pooled and dialyzed overnight (4C) against 2 changes of 4L H20. The









21
dialysate was further concentrated using an Amicon stirred cell fitted
with a YM 10 filter.

Extraction and Purification of White Shrimp and Grass Prawn PPO

The methods of Chen et al. (1991a) and Rolle et al. (1991) with
slight modification were followed to purify white shrimp and grass prawn

PPO, respectively. Shrimp cephalothorax powder was suspended in 3 volumes

(w/v) 0.05 M sodium phosphate buffer (pH 7.2) containing 1 M NaCl
(extraction buffer) and 0.2% (v/v) Brij 35, and stirred at 40C for 3 hr.
Following centrifugation at 23,000g (40C) for 30 min, the supernatant was
fractionated with ammonium sulfate between 0 40% saturation; protein

precipitate was collected by centrifugation at 23,500g at 40C for 30 min.

For white shrimp, the precipitate was dissolved in 0.05 M phosphate
buffer (pH 7.2) and dialyzed at 40C overnight against 3 changes of 4L of
0.05 M phosphate buffer (pH 7.2). The dialyzed PPO was loaded onto a

DEAE-cellulose (0.95 meq/g) column (K 26/40) pre-equilibrated with 0.05 M
phosphate buffer (pH 7.2). Sixty-mL of 0.05 M sodium phosphate buffer (pH

7.2) was used to desorb unbound phenolic compounds and proteins at 0.2

mL/min for 1.5 hr. Elution of PPO was performed using a 300 mL 0.05 M
sodium phosphate buffer (pH 7.2) containing a linear gradient (0 1.0 M)
of NaC1. Three-mL fractions were collected and the protein estimated by

absorbance at 280 nm. Fractions possessing PPO activity were pooled and
concentrated using an Amicon stirred cell fitted with a YM 10 filter.









22
PPO was loaded onto a Sephadex G-100 gel column (K 26/40) pre-

equilibrated with 0.05 M sodium phosphate buffer (pH 7.2) and then eluted

with 300 mL 0.05 M sodium phosphate buffer (pH 7.2) at 0.15 mL/min.

Three-mL fractions were collected and protein was estimated by absorbance

at 280 nm; fractions showing PPO activity were pooled and concentrated

using an Amicon stirred cell fitted with YM 10 filter. Concentrated PPO

was then dialyzed at 40C overnight against 3 changes of 2L H20.

For grass prawn, the precipitate was resuspended in extraction

buffer containing 40% ammonium sulfate. After homogenization using a

Dounce manual tissue grinder, the sample was centrifuged at 23,500g (40C)

for 20 min. The precipitate was homogenized in extraction buffer and

centrifuged as previously described. The resulting precipitate was

homogenized in extraction buffer, then subjected to high performance

hydrophobic interaction chromatography at 40C using a preparative Phenyl

Sepharose CL-4B (Sigma) column (K 16/40) attached to a Dionex gradient

pump (Dionex Corp., Sunnyvale, CA). The column was pre-equilibrated with

extraction buffer.

PPO was eluted with a stepwise gradient of elution buffer [100%

extraction buffer (9 mL), 50% extraction buffer in water (24 mL), and 10%

extraction buffer in water (24 mL)], 50% ethylene glycol (12 mL), and then

distilled water (150 mL) at a flow of 0.2 mL/min. Four-mL fractions were

collected and fractions exhibiting PPO activity were pooled and

concentrated via ultrafiltration utilizing an Amicon stirred cell fitted

with an XM 50 filter (Amicon).


I











Protein Ouantitation and Enzyme Purity Determination


The protein contents of the various PPO preparations were

quantitated using the Bio-Rad Protein Assay kit with bovine serum albumin

(Sigma) as standard. Enzyme purity was examined using a mini gel system

(Mini-Protean II Dual Slab Cell) (Bio-Rad, 1985b). Plant and crustacean

PPO's (20 pg protein/well) were loaded and electrophoresis was carried out

at constant voltage (200 V) in a buffer (pH 8.3) containing 25 mM Tris-HC1

and 0.19 M glycine for 35 min. The purity of enzyme preparations was

determined by comparing gels stained with 10 mM DL-DOPA in 0.05 M sodium

phosphate buffer (pH 6.5) and then with a Commassie blue R-250 (Eastman

Kodak Co., Rochester, NY) solution.


PPO Activity Determination


Lobster PPO activity was determined spectrophotometrically by

monitoring at 475 nm the rate of dopachrome formation from DL-DOPA

(Savagaon and Sreenivasan, 1978). The assay was run at 250C for 10 min by

mixing 40 pL of enzyme extract with 560 pL of 10 mM DL-DOPA in 0.05 M

sodium phosphate buffer (pH 6.5). Two molecules of DOPA produce one

molecule of dopaquinone which has a molar absorption coefficient (a,,) of

3,600 M'cmI' (Fling et al., 1963). PPO activity was defined as pmoles

dopachrome formed per min at 250C.

PPO activities of shrimp, potato, and apple were measured by adding

60 pL enzyme preparations to 840 pL 10 mM DL-DOPA in 0.05 M sodium

phosphate buffer (pH 6.5) and monitored at 250C for 5 min. Maximal initial

velocity was determined as AA4 rn/min and one unit of PPO activity was

defined as an increase in absorbance of 0.001/min at 25C. Unless


- I









24
otherwise specified, experiments were carried out three times in

duplicate. For this study, the enzyme activities of potato, apple, spiny

lobster, Australian lobster, white shrimp, and grass prawn PPOs were

determined to be 10,900, 97,400, 7,000, 3,000, 5,400, and 900 units/mg

protein, respectively.

DH Optima and Stability of Lobster PPO

The modified method of Gormori (1955) was followed for preparation

of various buffer solutions including 0.1 M sodium citrate-0.1 M HC1, pH

2.0, 3.0, and 4.0; 0.05 M sodium phosphate, pH 5.0, 6.0, 6.5, 7.0, 7.5,

and 8.0; 0.1 M glycine-0.1 M NaOH, pH 9.0, and 10.0; and 0.1 M sodium

phosphate-0.1 M NaOH, pH 11.0 and 12.0. The assay was performed at 250C

by adding 40 AL enzyme solution to a mixture containing 280 pL of buffer

solution and an equal volume of 10 mM DL-DOPA in distilled water. The

mean velocity for dopachrome formation was determined at 475 nm using a

DU-7 spectrophotometer (Beckman Instruments, Inc., Irvine, CA).

After enzyme mixtures containing 40 AL enzyme preparation and 120 pL

of each of the previously described buffer systems were incubated at 250C

for 30 min, a 40 AL aliquot was removed and added to 560 pL of 10 mM DL-

DOPA solution in distilled water. Dopachrome formation was monitored

spectrophotometrically.

Activation Enerqy and Thermostabilitv of Lobster PPO


Reaction mixtures containing 40 pL enzyme extract and 560 AL 10 mM
DL-DOPA solution were incubated at various temperatures ranging from 200

to 600C. Activation energy (Ea) was determined according to the Arrhenius









25

equation by measuring the initial rate of reaction at different

temperatures and plotting the logarithmic value of Vm versus 1/T (Segel,

1976).

To determine the thermostability, a 40 pL aliquot of lobster PPO was

sealed in a quartz cell and incubated in a DU-7 spectrophotometer for 30

min at different temperatures ranging from 20-600C. Following

equilibration to room temperature, the enzyme extract was mixed with 560

AL of 10 mM DL-DOPA and then monitored spectrophotometrically as described
above.

Molecular Weight Determination of Lobster PPO

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

was used for molecular weight determination of the enzyme isoforms. Slab

gels (16 cm x 20 cm) at 1.50 mm thickness, consisting of stacking gel (4%

acrylamide/ 0.1% bisacrylamide) and separating gel (7.5% acrylamide/ 0.2%

bisacrylamide), were prepared according to the ProteanTM II Slab Cell

Instruction Manual (Bio-Rad Labs., 1985a). Electrophoresis was carried

out in a Protean II Slab Cell system equipped with a Bio-Rad Model

3000/300 power supply. A constant current of 13 mA/gel and 18 mA/gel was

applied to the stacking and separating gel, respectively in a buffer (pH
8.3) containing 5 mM Tris-HC1 and 38 mM glycine. Enzyme samples were

diluted with 4 volumes of SDS reducing buffer and then heated at 950C for

4 min. Fifty-&g aliquots were applied to each sample well. An SDS-6H

Molecular Weight Marker Kit (Sigma) containing carbonic anhydrase

(29,000), egg albumin (45,000), bovine albumin (66,000), phosphorylase B

(97,000), f-galactosidase (116,000), and myosin (205,000) was used. The









26

molecular weights of the proteins were determined following the methods of

Weber and Osborn (1969) and Weber et al. (1972).

Enzyme Kinetics Study

Kinetic parameters (K, and Vma) of the two lobster PPO's were

determined using the Lineweaver-Burk equation (Lineweaver and Burk, 1934).

DL-DOPA and catechol solutions at concentrations varying from 1.67 to 9.92

mM in 0.05 M phosphate buffer (pH 6.5) were used as substrates. PPO

activity on catechol was defined as Amoles of benzoquinone formed per min

at 250C. One molecule of catechol produced one molecule of benzoquinone,

which has a molar absorption coefficient (am,) of 1,350 M"1 cm"1 (Whitaker,

1972).

In addition, Michaelis constants (K,) for mushroom, potato, apple,

white shrimp, and grass prawn PPOs were determined. Sixty-pL PPO was

added to 10 mM DL-DOPA in 0.05 M sodium phosphate buffer (pH 6.5). The

total volume in the cuvette was 1.0 mL and the final concentration of DL-

DOPA varied from 1.4 to 8.9 mM. The reaction was monitored at 475 nm and

250C for 10 min.

Results and Discussion


Effect of pH on Lobster PPO Activity

PPO enzymes isolated from Western Australian lobster and Florida

spiny lobster exhibited similar patterns of sensitivity to pH changes.

Florida spiny lobster PPO had a pH optimum of 6.5, which was a half unit

less than that for Western Australian lobster PPO (Figure 3). These

values for the two lobster PPO's were similar to that of gulf (brown)














s.0 s-

Q) q0) -







0 0
4> --J
C L






.- VI V)

I-g




C~J
-41



=U











S.- =
X- X
..o CO









0 00






o 4)m









4 .4 *L
+> 0








S- -
L- )0-
o W





















4. CO







0. uI -




U. 0 =
1->
C C S
0 i 3
0) *I-1
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u-O~













L.

0 0
o c





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co CDt cv
> 0 000

m$ S O









29
shrimp (Madero and Finne, 1982). PPO from white shrimp (Penaeus

setiferus) had a pH optimum at 7.5, while that from pink shrimp (Penaeus

duorarum) was 8.0 (Simpson et al., 1987, 1988a). Ohshima and Nagayama

(1980) showed that catecholase isolated from antarctic krill (Euphausia

superba) had a pH optimum at 6.5. PPO isolated from grass prawn was shown

to have a pH optimum at 6.0 (Rolle et al., 1991). For plant PPO, the pH

optimum of 7.0 was observed for mushroom (Dawson and Mager, 1962), apple

(Stelzig et al., 1972), and banana (Galeazzi and Sgarbieri, 1978),

respectively, while pear PPO was reported to have a pH optimum at 4.0

(Rivas and Whitaker, 1973).

With respect to the pH-related relative activity, the Western

Australian lobster PPO had a broader pH range (from 5 to 9) than that

observed for the Florida spiny lobster PPO. The behavior of Florida

lobster PPO to various pH environments observed in this study was similar

to that of IP01 (inert phenoloxidase) and TAP01 (trypsin activated

phenoloxidase) forms from a study by Ferrer et al. (1989a). For white and

pink shrimp PPO, the optimal pH environments ranged from 6-7.5 and 6.5-

9.0, respectively (Simpson et al., 1988a).

Data in Table 1 indicated that crustacean (Florida spiny lobster,

Western Australian lobster, pink shrimp, white shrimp, brown shrimp, and

grass prawn) PPOs had a narrow range of optimum pH between 6 to 8.

However, a broad range of pH optimum between 4 and 7 was observed for

mushroom and other plant (potato, apple, peach, pear, and banana) PPOs.

Aylward and Haisman (1969) proposed that the optimum pH of PPO activity,

which usually ranged between pH 4 and 7, varied with enzyme sources and

substrates used.



































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Effect of DH on Lobster PPO Stability

Enzyme stability over a broad pH range (2 12) revealed that PPO

obtained from both lobster species exhibited optimum stability at pH 7

(Figure 4). When PPO was pre-incubated between pH of 5 and 9, both

Florida spiny lobster and Western Australian lobster PPOs still retained

at least 60% of their relative activity when compared at pH 6.5.

Conformational changes at the active site due to dramatic pH changes may

have caused the significant decline in enzyme activity between pH 2 and 5,

and between 9 and 12. Similar changes were reported to occur with TAP02

(trypsin activated phenoloxidase) form of the Florida spiny lobster PPO

(Ferrer et al., 1989a). PPO from pink shrimp (Simpson et al., 1988a),

white shrimp (Simpson et al., 1987), and grass prawn (Rolle et al., 1991)

all exhibited optimal activity within the neutral to alkaline pH range (pH

6-8), and showed maximal stability at pH 8.

Effect of Temperature on Lobster PPO Activity and Stability

Both lobster PPOs showed temperature-related changes in enzyme

activity (Figure 5). Results obtained for this study showed that Florida

spiny lobster and Western Australian lobster PPO had the temperature

optimum at 350 and 300C, respectively, (Figure 5). These values were lower

than those observed for the PPO of pink shrimp (40C, Simpson et al.,

1988a), white shrimp (450C, Simpson et al., 1987), and grass prawn (450C,

Rolle et al., 1991). Compared to crustacean PPO, the activity of peach

PPO was found to increase from 30 to 370C and then declined up to 450C
(Vamos-Vigyazo, 1981). In apple, the enzyme reached its maximum activity

at 300C when chlorogenic acid was used as substrate (Vamos-Vigyazo, 1981);














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36

; while 37C was reported for banana PPO using dopamine as substrate

(Palmer, 1963). Regarding potato and grape PPO activity, the optimum

temperature of 25C was reported for the former (Lavollay et al., 1963),

while 10-15C for the latter (Cash et al., 1976). The data available

indicate that the temperature optimum of the enzyme also depends

essentially on the same factors as the pH optimum (Vamos-Vigyazo, 1981).

It is noted that most crustacean PPOs had higher temperature optima than

those observed for plant PPOs. With the exception of banana PPO, which

showed an unusually high temperature optimum at 370C (Palmer, 1963).

Both lobster PPOs exhibited similar thermostability characteristics,

although the Australian lobster PPO showed decreased activity when pre-

incubated at temperatures greater than 300C (Figure 6). However, Florida

lobster PPO showed greater stability at a preincubation temperature of

350C, which was within the range of EAPO (endogenously activated

phenoloxidase) and TAPO1 forms but slightly different from the IP01 form

of the Florida spiny lobster PPO (Ferrer et al., 1989a). The

thermostability characteristics of both lobster PPO's behave similarly to

that of grass prawn (Rolle et al., 1991) and pink shrimp PPO (20-300C,

Simpson et al., 1988a) but varied from that of white shrimp PPO (25-500C,

Simpson et al., 1987). Florida spiny lobsters grew in warm water while

Western Australian lobsters are found in cold water. These differences in

their habitats may account for the difference in the optimal

thermostability between these two enzymes. PPO enzymes from shrimp are

usually stable at temperatures ranging between 300 and 500C (Madero and

Finne, 1982; Simpson et al., 1987, 1988a). Most PPO enzymes are heat














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39

labile; a short exposure of the enzyme to temperatures at 70-900C is

sufficient to cause partial or total irreversible denaturation. Crude PPO

isolated from deep sea crab was shown to be inactivated at 70C (Marshall

et al., 1984). Similarly, apple PPO (Walker, 1964) and grape PPO (Vamos-

Vigyazo, 1981) were found to be markedly inactivated at temperatures above

700C. For banana PPO, an exposure to 800C for 15 min was required to

inactivate enzymes completely (Galeazzi and Sgarbieri, 1978). Out of 22

cultivars of different stone fruits, peach PPO was found the least

thermostable and the greater heat stable enzyme from plum and cherry was

accompanied by higher activity when compared to peach PPO (Dang and

Yankov, 1970).

Vamos-Vigyazo (1981) pointed out that the thermotolerance of PPO is

dependent on the source of enzyme. In addition, different molecular forms

of PPO from the same source may behave differently in thermostabilities.

Activation Energy (Ea) of Lobster PPO

The activation energies, Ea, for Florida and Australian lobster PPO

were 6.9 and 7.5 Kcal/mole, respectively. These Ea values were similar to

TAP01 (7.8 Kcal/mole) from Florida spiny lobster PPO (Ferrer et al.,

1989a), and comparable to shrimp PPO (Ea = 5.2 Kcal/mole) (Bailey et al.,

1954), but somewhat different from those of the PPO prepared from white

shrimp (Ea = 13.9 Kcal/mole; Simpson et al., 1987), pink shrimp (Ea = 11.5

Kcal/mole; Simpson et al., 1988a), and grass prawn (E, = 13.3 Kcal/mole;
Rolle et al., 1991). The Ea of banana PPO was found to be 4.4 Kcal/mole

when catechol was used as substrate (Palmer, 1963).











Molecular Weight and Isoform Determination of Lobster PPO

Figure 7 shows the SDS-PAGE (reduction condition) patterns for both

lobster PPO enzymes; the Florida spiny lobster PPO had three isoforms and

the Western Australian lobster PPO had two. The molecular masses of the

Florida lobster PPO subunits were determined to be 82, 88, and 97 kD,

while those of the Australian lobster were 87 and 92 kD. The molecular

masses of these subunits were higher than those of white shrimp (30 kD),

pink shrimp (40 kD), grass prawn (63 and 80 kD) and antarctic krill (75

and 83 kD) (Simpson et al., 1987, 1988a; Ohshima and Nagayama, 1980; Rolle

et al., 1991), but lower than that of brown shrimp (210 kD) (Madero and

Finne, 1982). Multiple molecular forms with different molecular masses

were observed for PPO from various plant sources (Vamos-Vigyazo, 1981).

Mushroom PPO was reported to have 4 isoforms (isozymes), each had

molecular masses of 34.5 kD (Bouchilloux et al., 1963). Apple PPO was

shown to have 3 isoforms with molecular masses of 24, 67, and 134 kD,

respectively (Demenyuk et al., 1974). PPO from banana was reported to

possess two isoforms with molecular masses of 12 and 60 kD, respectively

(Palmer, 1963). Regarding potato, multiple isoforms of PPO bearing varied

molecular masses have been reported by many workers (Anisimov et al.,1978;

Constantinides and Bedford, 1967; Patil and Evans, 1963; Thomas et al.,

1978). Although Florida spiny lobster and Western Australian lobster were

grown in different habitats, a recent study using immunological techniques

with rabbit antisera against the Florida spiny lobster PPO revealed that

these two lobsters shared cross-reactivity (Rolle et al., 1991).





























Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-
PAGE, 7.5% Gel) Profile of Polyphenol Oxidase from Florida
Spiny Lobster (FSL) and Western Australian Lobster (WAL); PPO
Samples Were Diluted with 4 Volumes of SDS Reducing Buffer
Containing #-Mercaptoethanol and Heated at 950C for 4 Min.
Fifty-pg PPO Was Loaded onto Each Sample Well.


Figure 7.

















200 kDa


116 kDa
97.4 kDa
i '
66 kDa

43 kDa


Protein WAL
Standard PPO


FSL
PPO


.- fMH e ^








43

PPO from most sources, as mentioned previously, have been reported

to be present in different molecular forms. The number of these forms

depends on the enzyme source and on methods used to prepare them. It has

been revealed that part of molecular forms are due to association-
dissociation phenomena which is attributed to (1) association of various
degrees of polymerization of similar units; (2) various combinations of

different subunits; (3) conformational changes of a single protein; or
combinations of these three possibilities (Vamos-Vigyazo, 1981). Much

attention has been paid to differences in the properties of multiple forms

of the enzyme and to the possible physiological significance of such
differences. These include differences in affinity and specificity to

phenolic substrates and to oxygen (Harel et al., 1964; Kahn, 1976; Taneja

and Sachar, 1974), sensitivity to inhibitors (Constantinides and Bedford,

1967; Harel et al., 1964), pH optima (Takeo and Uritani, 1965; Wong et

al., 1971), and inactivation by heat (Ben-Shalom et al., 1977; Fling et

al., 1963; Sussman, 1961). In addition, differences in isozyme patterns
were also reported in connection with subcellular organelles (Harel et

al., 1965), the stage of tissue development (Takeo and Baker, 1973; Taneja

et al., 1974), as result of attack by pathogens (Hyodo and Uritani, 1964),

or of treatment with plant hormones (Taneja and Sachar, 1977a, b).

Enzyme Kinetics of Lobster PPO

Double-reciprocal plots for the oxidation of DL-DOPA and catechol by

both lobster PPOs are shown in Figures 8 and 9, respectively. Both

lobster PPOs were capable of catalyzing DL-DOPA and catechol. Australian

lobster PPO displayed a relatively greater Michaelis constant (Km = 3.57














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48
mM) and a lower maximum velocity (Vmx = 0.008 mmol/liter/min) with DL-DOPA

than with catechol (Km = 3.09 mM and V, = 0.041 mmol/liter/min) (Table

2). Similar results were also noted for the Florida lobster PPO (Table

2). These two PPOs had a higher affinity for catechol than for DL-DOPA.

Using DL-DOPA as a substrate, Simpson et al. (1987, 1988a) showed that

pink shrimp PPO had a lower Michaelis constant (1.6 mM) than white shrimp

(2.8 mM). Rolle et al. (1991) characterized grass prawn PPO and they

determined the Km of enzyme was 4.45 mM when DL-DOPA was used as substrate.

Summers (1967) showed that blood PPO from fiddler crab had a Km value of

0.50 mM.

Australian lobster PPO exhibited a higher affinity for DL-DOPA and

catechol than Florida lobster PPO. However, the latter showed a greater

rate for oxidizing DL-DOPA and catechol than the former. It has been

reported by Lavollay et al. (1963) that no relationship could be found

between Km and V., values obtained for a substrate with a given PPO

preparation. Instead, the term of Vx/Km has been recommended by some

authors (Lavollay et al., 1963; Pollock, 1965) to express the efficiency

of a given substrate for a given enzyme. Table 2 indicates that Florida

lobster PPO not only showed a higher specific activity but also had a

higher turnover number than the Australian lobster PPO. The Florida

lobster PPO also showed a higher physiological efficiency for both

substrates than the Australian lobster PPO (Table 2), which could account

for why Florida spiny lobsters were more susceptible to melanosis than

Western Australian lobsters. The kinetic and molecular properties of PPO

from other sources are also summarized in Table 1. With DL-DOPA as

substrate, different Michaelis constants (Km) were observed for mushroom,


I
















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50

potato, apple, grass prawn, pink shrimp, and white shrimp PPOs. Varied Km

values were also reported for peach (K, = 4.2 mM, Wong et al., 1971), pear

(K, = 20.9 mM, Rivas and Whitaker, 1973), and banana (K, = 0.63 mM, Palmer,
1963) PPOs when different phenolic compounds were used as substrates

(Table 2). Data obtained from this study showed that mushroom, potato,

and apple PPOs had comparatively lower K, than those of crustacean PPOs,

indicating that the former PPOs had a higher affinity for DL-DOPA than the

latter ones. It has been reported that the affinity of PPO towards a

given substrate may vary widely, even if isozymes of the same origin are

used. The differences might be due, at least partly, to steric factors

connected to differences in protein structure (Vamos-Vigyazo, 1981).

Substrate specificity varies considerably for PPO from various

sources (Aurand and Wood, 1977; deMan, 1985). Simpson et al. (1988a)

identified higher levels of free tyrosine and phenylalanine as natural

substrates of melanosis in the carapace of pink shrimp. Further studies

by Ali (1991) showed PPO isolated from Florida spiny lobster was capable

of hydroxylating free tyrosine to DOPA. Tyramine, produced by bacterial

activity on free L-tyrosine (Veciana-Nogues et al., 1989; Santos-Buelga et

al., 1986) was also identified as the substrate of crab PPO (Summers,

1967). For mushroom (Agaricus bisporum) PPO, DOPA and catechol were,

respectively, reported by Bouchilloux et al. (1962) and Nakamura et al.

(1966) as specific substrates. For potato and apple PPOs, chlorogenic

acid (Patil and Zucker, 1965) was considered a specific substrate for the

former, while 4-methylcatechol for the latter (Stelzig, 1972). Compared

to pear PPO with pyrocatechol (Rivas and Whitaker, 1973) and banana PPO

with dopamine (Palmer, 1963), D-catechin was demonstrated as the specific









51

substrate of peach (Luh and Phithakpol, 1972). Results from this study

show that DOPA was the unanimous substrate of crustacean PPOs. For

mushroom and other plant PPOs, the specific substrate varied with the

sources of enzyme. Mason (1955) reported that tyrosine and DOPA were

specific substrates of animal tissue PPO, while Mayer and Harel (1979)

listed a wide variety of mono- and o-diphenols as the substrates of fungal

and plant PPOs. Besides the previously discussed characteristics

(substrate specificity, K,, pH and temperature effects, enzyme isoforms,

and molecular weights), other biochemical properties concerning the

catalytical function (mechanism), response to the activator/inhibitor, and

isoelectric profile (pI) of PPO also varied with the enzyme sources.

Conclusion

PPO isolated from Florida spiny lobster and Western Australian

lobster showed very similar patterns in response to effects of pH and

temperature on enzyme activity and in SDS-PAGE profile. Using DL-DOPA and

catechol as substrates, the Western Australian lobster PPO was shown to

have higher affinity but lower physiological efficiency than the Florida

spiny lobster PPO. These two PPOs showed distinctly different properties

for catalyzing the oxidation of phenolic substrates; this may explain

differences in susceptibility of spiny lobster to melanosis compared to

Western Australian lobster. Results indicate that PPO from various plant

and crustacean sources vary in substrate specificity, kinetic properties,

molecular weights, isoforms, activity and stability to pH and temperature

effects, activation energy, and isoelectric profiles.














STRUCTURAL COMPARISON OF CRUSTACEAN, PLANT, AND MUSHROOM
POLYPHENOL OXIDASES

Introduction


Polyphenol oxidase (PPO) (E.C. 1.14.18.1.), also known as

tyrosinase, polyphenolase, phenolase, catechol oxidase, cresolase, and

catecholase, is widely distributed in nature (Schwimmer, 1981). The

unfavorable enzymatic browning caused by PPO on the surface of many plants

and seafood products has been of a great concern to food processors and

scientists. Although the formation of melanin (blackening spot) does not

affect the nutrient content of food products, it does however connote

spoilage by consumers (Eskin et al., 1971). Economic loss resulting from

this action has caused great concern among food processors. Enzymatic

browning of fruits, vegetables, and crustaceans due to PPO activity has

been extensively studied (Chen et al., 1991a; Ferrer et al., 1989a;

Flurkey and Jen, 1978; Macrae and Duggleby, 1968; Sciancalepore and

Longone, 1984; Simpson et al., 1989a; Walker, 1964).

Differences in the secondary structure between endogenously

activated (EAPO) and trypsin activated (TAPO) forms of Florida spiny

lobster PPO was recently demonstrated by Rolle et al. (1991) using

circular dichroism (CD) spectropolarimetry. A study conducted by Chen et

al. (1991b) showed that PPOs from plant and crustacean sources not only

varied with respect to catalytic activity in the oxidation of DL-0-3,4-

dihydroxyphenylalanine (DL-DOPA) but also had different sensitivities to
52









53
the inhibitor, kojic acid (5-hydroxy-2-hydroxymethyl--y-pyrone). Since the

conformational structures of these fungal, plant and crustacean PPOs has

not been well documented, this study was undertaken to elucidate whether

conformational differences exists among these PPOs using immunological

techniques and circular dichroism spectropolarimetry.


Materials and Methods

Fresh Florida spiny lobster (Panulirus argus) tails obtained from

the Whitney Marine Laboratory at Marineland, FL, were transported in ice

to the laboratory and stored at -200C. Mushroom (Agaricus bispora)

tyrosinase with an activity of 2,200 units/mg solid was purchased from

Sigma Chemical Co. Russet potato was purchased from a local supermarket.

White shrimp (Penaeus setiferus) and brown shrimp (Penaeus aztecus) were

obtained from a local seafood store. Lobster cuticle, shrimp

cephalothorax (head), and potato peel were each frozen in liquid nitrogen

and ground into a fine powder using a Waring blender. The individual

ground powder was stored at -200C until needed.

Extraction of Mushroom, Potato, Lobster, and Shrimp PPO


PPO was extracted according to the procedure of Simpson et al.

(1988a). One part ground powder was added to three parts (w/v) 0.05 M

sodium phosphate buffer (pH 7.2) containing 1 M NaCl and 0.2% Brij 35.

The extract was stirred for 5 min at 40C and the suspension was centrifuged

at 8,000g (40C) for 30 min. The supernatant was then dialyzed at 40C

overnight against 3 changes of 4L 0.05 M sodium phosphate buffer (pH 6.5).


I










Purification of Mushroom, Potato, Lobster, and Shrimp PPO

Crude PPO preparation was purified further using a nondenaturing

preparative polyacrylamide gel electrophoresis (PAGE) system. Equipment

utilized included a gel tube chamber (Model 175, Bio-Rad Labs.) and a

power supply (Model EPS 500/400, Pharmacia LKB Biotechnology Inc.). A

one-mL aliquot of crude enzyme extract (potato, lobster, or shrimp) was

applied to each of eight gel tubes (1.4 cm I.D. x 12 cm length) containing

5% acrylamide/ 0.13% bisacrylamide gel and subjected to a constant current

of 10 mA/tube in a buffer (pH 8.3) containing 5 mM Tris-HC1 and 38 mM

glycine (Sigma Chemical CO., 1984). PPO was visualized using a specific

enzyme-substrate staining method (Constantinides and Bedford, 1967); 10 mM

DL-DOPA in 0.05 M sodium phosphate buffer (pH 6.5) was used as substrate.

After the migration of the enzyme relative to the dye front (Rf) was

determined using one of the eight gels, the remaining gels were sectioned

at the determined R,. PPO was eluted from the gel by homogenization in

0.05 M sodium phosphate buffer (pH 6.5) utilizing a Dounce manual tissue

grinder. The homogenates were filtered through Whatman No. 4 filter

paper, pooled, and concentrated using an Amicon stirred cell (Model 8050).

Mushroom PPO (0.25 mg/mL) in 0.05 M sodium phosphate buffer (pH 6.5) was

further purified according to the procedures previously described.

Protein Quantitation and Enzyme Purity Determination

Protein content of the various PPO preparations was quantitated

using the Bio-Rad Protein Assay kit with bovine serum albumin as standard.

Enzyme purity was examined using a mini gel system (Mini-Protean II Dual

Slab Cell) (Bio-Rad, 1985b). Mushroom, potato, and crustacean PPOs (20 Ag









55

protein/well) were loaded and electrophoresis was carried out at constant

voltage (200 V) in a buffer (pH 8.3) containing 25 mM Tris-HCl and 0.19 M

glycine for 35 min. Purity of the preparations was determined by
comparing gels stained with 10 mM DL-DOPA in 0.05 M sodium phosphate
buffer (pH 6.5) and then with a Coomassie blue R-250 solution.

Enzyme Activity Assay

PPO activities were measured by adding 60 AL PPO to 840 iL 10 mM DL-

DOPA in 0.05 M sodium phosphate buffer (pH 6.5) and monitoring at 250C for

5 min in a Beckman DU7 spectrophotometer at 475 nm. Maximal initial

velocity was determined as AA475 n/min and one unit of PPO activity was

defined as an increase in absorbance of 0.001/min at 250C. Unless

otherwise specified, experiments were carried out twice in triplicates.

For this study, the enzyme activities of mushroom, potato, lobster, white

shrimp, and brown shrimp PPO were determined to be 96,000, 120,000, 4,500,
1,000, and 1,200 units/mg protein, respectively.

Anti-lobster PPO Antibody Production and Purification

One-mL purified lobster PPO containing 100 ag protein was used as an

antigen to inject into a hen biweekly. Eggs laid by the immunized hen

were collected and anti-lobster PPO antibody was isolated and purified
from the egg yolk using the method of Polson et al. (1985). One part egg
yolk was added to 4 parts (v/v) 0.1 M sodium phosphate buffer (pH 7.6).

The mixture was made up 3.5% (w/v) with polyethyleneglycol (PEG) and

stirred for 5 min. Following centrifugation at 5,000g (10C) for 20 min,

the supernatant collected was made up with 8.5% (w/v) PEG. The suspension









56

was allowed to stand for 10 min followed by centrifugation at 5,000g (100C)

for 25 min. The pellet was dissolved in 2.5 volumes (v/v) phosphate

buffer (pH 7.6) and the suspension was made up with PEG to 12% (v/v).

Again, the suspension was allowed to stand for 10 min and then centrifuged

at 5,000g (100C) for 25 min. The pellet was resuspended in 1/4 volume
phosphate buffer and cooled to 00C before adding an equivalent volume of
50% ethanol (-200C). Following centrifugation at 10,000g (40C) for 25 min,

the precipitate was dissolved in 1/4 volume phosphate buffer and the

suspension was dialyzed overnight (40C) against 4L 0.1 M sodium phosphate

buffer (pH 7.6). After dialysis, the antibody preparation was made up

with NaN3 to 0.1% and stored in the refrigerator until needed.

Molecular Weight Determination of Anti-lobster PPO Antibody

Protein content of the antibody preparation was also quantitated

using the Bio-Rad Protein Assay kit. The molecular weight of the antibody

preparation was determined using SDS-PAGE (reduction condition) according

to the method of Laemmli (1970). Mini slab gels (7 cm x 8 cm) at 1.0 mm
thickness, consisting of stacking gel (4% acrylamide/ 0.1% bisacrylamide)

and separating gel (7.5% acrylamide/ 0.2% bisacrylamide) were prepared

according to the Mini-Protein II Dual Slab Cell Instruction Manual (Bio-

Rad, 1985b). Antibody preparations were diluted with 4 volumes of SDS

sample buffer containing P-mercaptoethanol, and heated for 4 min at 950C.
After 30 pg aliquots were applied to each sample well and electrophoresis
was carried out for 35 min at a constant voltages of 200 V. An SDS-6H

high Molecular Weight Protein Standards kit (Sigma) containing carbonic

anhydrase (29,000), egg albumin (45,000), bovine albumin (66,000),








57

phosphorylase (97,400), #-galactosidase (116,000), and myosin (205,000)
was used. Molecular weights of the antibody proteins were determined

following the methods of Weber and Osborn (1969) and Weber et al. (1972).

Antibody Titer Determination by Enzyme-linked Immunosorbent Assay (ELISA)

One hundred-pL lobster PPO containing 2.5 100 ng protein in 0.1 M

NaHCO3, pH 8.6, (coating buffer) was applied to the sample well of a
microplate (Immulon 2, Dynatech). Following overnight incubation at 40C,

the well was aspirated and washed 4 times with PBS-Tween [0.01 M sodium

phosphate, pH 7.4, containing 0.15 M NaC1 and 0.2% (v/v) Tween 20] using

the Nunc-Immuno Wash (A/S NUNC, Denmark). After 100 pL primary antibody

(anti-lobster PPO antibody) at amounts of 0.01 10 Ag in PBS-Tween was

added to the well, incubation was allowed to proceed at ambient

temperature for 1 hr. The aspirations and washings were repeated as

previously described before 0.1 mL secondary antibody (antichicken IgG-

alkaline phosphatase conjugate, Sigma) was added. Following another one-

hour incubation, the microplate was aspirated and washed again with PBS-

Tween. Following the addition of 0.1 mL p-nitrophenyl phosphate disodium

(1.0 mg/mL) in assay buffer (0.05 M Na CO3 and 0.05 M NaHCO3 containing
0.0005 M MgCl) to the well, the plate was incubated at ambient temperature

till a yellow color developed. The absorbance of the plate at 405 nm was

monitored every hour using an ELISA reader (Model 2550, Bio-Rad). In this

study, coating buffer without antigen was used as the negative control.










Analysis of Antigenic Properties of PPO

The competitive ELISA adopted from Seymour et al. (1991) was

employed to study whether PPO from mushroom, potato, white shrimp, and

brown shrimp possessed similar antigenic determinants as the Florida spiny

lobster. After the microplate was coated with lobster PPO at 10 ng/well
for one hour at room temperature, 100 AL aliquot of antibody-competitive

PPO mixture was added. The antibody-PPO mixture was prepared by mixing

the competitive PPO (either mushroom, potato, or lobster, white shrimp,

and brown shrimp) (0.2 2.0 Ag/mL) with an equal volume of primary

antibody solution (10 or 20 Ag/mL of IgY) for 1 hr at ambient temperature.

The assay procedures were conducted as previously described.

Immunoblotting

Purified mushroom, potato, lobster, white shrimp, and brown shrimp

PPO and a crude lobster PPO preparation were subjected to SDS-PAGE under

reduction condition (Laemmli, 1970) and then electro-transferred to

nitrocellulose membrane (Bio-Rad). Slab gels (16 cm x 20 cm) at 1.0 mm

thickness, consisting of stacking gel (4% acrylamide/ 0.1% bisacrylamide)

and separating gel (7.5% acrylamide/ 0.2% bisacrylamide), were prepared

according to the ProteanM II Slab Cell Instruction Manual (Bio-Rad,

1985a). A constant current of 13 mA/gel was applied during stacking and

18 mA/gel during separation. Fifty-pg aliquots of test samples were

applied to each well and run with the protein standards (SDS-6H Molecular

Weight Marker Kit).

Following electrophoresis, the gel was equilibrated in 500 mL of

Towbin transfer buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, and 20%









59

methanol; pH 8.3) for 15 min. Electro-transfer was performed according to

the Trans-Blot Electrophoretic Transfer Cell Instruction Manual (Bio-Rad,

1989) at a constant voltage of 50 V for 1.5 hr using Towbin buffer as the

electrolytic buffer. Complete transfer of proteins was verified by

staining the gel and the nitrocellulose membrane with a Coomassie blue

solution.

The nitrocellulose membrane following electro-transferring was

rinsed with phosphate-buffered saline (PBS) 3 times, and then incubated

with Blotto/Tween blocking solution (5% w/v nonfat dry milk, 0.2% v/v

Tween 20, and 0.02% w/v NaN3 in PBS; Harlow and Lane, 1988) at ambient

temperature with agitation for 2 hr. After washing twice for 5 min each

in PBS, the membrane was incubated overnight in the primary antibody (IgY)

solution (10 pg/mL). Following washing with 4 changes of PBS for 5 min

each, the membrane was treated for 1 hr with the secondary antibody

(antichicken IgG-alkaline phosphatase conjugate, Sigma) at a dilution of

1/2000. The membrane was then washed again with PBS as previously

described, and incubated with 100 mL alkaline phosphatase buffer (100 mM

NaCl, 5 mM MgCl2, and 100 mM Tris; pH 9.5) containing 0.016%

bromochloroindolyl phosphate (BCIP) and 0.032% nitro blue tetrazolium

(NBT) (Harlow and Lane, 1988) for 45 min. The reaction was stopped by

rinsing the membrane with PBS containing 20 mM EDTA.

Spectropolarimetric Analysis of PPO

The circular dichromic spectra of PPO was scanned at the far UV (250

- 200 nm) range using a Jasco J-20 automatic recording spectropolarimeter

(Japan Spectroscopic Co., Tokyo, Japan). A 1.0-cm Suprasil (Helma Cells)









60

cuvette with a 1.0-cm light path was used. Four-mL of PPO (10 20 pg/mL)

in 0.5 mM sodium phosphate buffer (pH 6.5) was analyzed at ambient

temperature. Calculations of the secondary structures were carried out by

computer analysis of the spectra using the SSE program (Japan

Spectroscopic Co., 1985) with myoglobin, cytochrome c, ribonuclease A,

lysozyme, and papain as references.

Results and Discussion

SDS-PAGE Profile of PPO

Various enzyme subunits with different molecular masses were

observed for crude mushroom (7 isozymes), potato (5 isozymes), lobster (3

isozymes), white shrimp (2 isozymes), and brown shrimp (2 isozymes) PPO

preparations after the nondenaturing preparative polyacrylamide gel was

stained with DL-DOPA. After subjecting to SDS-PAGE (without treatment of

f-mercaptoethanol), mushroom and potato PPO chosen for this study were

shown to have a subunit of lower molecular mass than crustacean (lobster,

white shrimp, and brown shrimp) PPO, estimated as 66 and 148 kD,

respectively (Figure 10). These are in agreement with the previously

reported data of Anisimov et al. (1978) and Bouchilloux et al. (1963). As

to lobster, white shrimp, and brown shrimp PPO, the molecular masses were

determined as 200, 190, and 190 kD, respectively. Brown shrimp PPO of 190

kD was close to that of the Gulf brown shrimp PPO (210 kD) (Madero and

Finne, 1982). However, the molecular mass of white shrimp PPO determined

in this study varied with that reported previously by Simpson et al.

(1988). The use of different preparation and analytical methods in these

studies could have contributed to such discrepancy.
















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Antibody Production and Molecular Weight Determination


The production of lobster PPO specific antibody did not occur until

after the second boosting; peak activity occurred on day 18 (Figure 11).

Antibody activity decreased gradually with time following the third and

fourth boosting. The serum eventually lost antibody activity after day

35. The animals apparently had become tolerant to the antigen after the

third boosting.

Antibody produced on day 17 and 18 having the highest activities was

pooled for molecular weight determination. Three distinct bands were

observed on the SDS-PAGE gel (Figure 12). The molecular weight for the

lower band was estimated as 59,000 which was close to the value reported

for the heavy chain of IgY (Jensenius et al., 1981).

Antibody Titer Determination

Dose-related interactions of various PPO (mushroom, potato, lobster,

white shrimp, and brown shrimp) at 2.5 10 ng with lobster PPO-specific

antibody at 2.5 jig are shown in Figure 13. All test samples showed

affinity for the antibody. Therefore, these antigens were partially

cross-reactive and shared similar antigenic determinants. The appropriate

dose range of the interaction of the various PPOs with the antibody was

determined to be 2.5 10 ng/well. When PPO was added at more than 10

ng/well, the antigen-antibody reaction was saturated and the sensitivity

of the ELISA became dull.















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Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE, 7.5% Gel) of Anti-lobster PPO Antibody and Protein
Standard (SDS-6H); Anti-lobster PPO Antibody Was Diluted with
4 Volumes of SDS Sample Buffer Containing P-Mercaptoethanol
and Heated at 950C for 4 Min. Thirty-pg Antibody Preparation
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Immunological Characteristics of PPO

The absorbance at 405 nm of a lobster PPO-antibody reaction mixture

in the presence of various competitors (lobster, white shrimp, brown

shrimp, mushroom, and potato PPO) was determined by ELISA (Figure 14).

The color intensity was reduced by 18 79% of the control (Abs. = 0.967)

when the various competitor-antibody mixtures containing 12.5 ng

competitor and 5 pg antibody per sample well were added to the microplate.

The increase of the competitor up to 100 ng/well slightly reduced the

color intensity. For mushroom PPO, the competition with lobster PPO for

antibody however was not enhanced even when mushroom PPO was added at 100

ng/well. The result again indicated that these PPOs were partially cross-

reactive and therefore shared similar structural components. From the

extent of competition for lobster PPO-specific antibody, white shrimp and

brown shrimp PPO were shown to share more antigenic similarity to lobster

PPO than potato and mushroom PPO (in descending order).

Complete transfer of PPO bands along with protein standards from an

acrylamide gel onto a nitrocellulose membrane was achieved following

electro-transferring; the staining of the treated gels with Coomassie blue

revealed no protein bands. The purified lobster, white shrimp, brown

shrimp, mushroom, and potato PPO had only one protein band, while crude

lobster PPO had 3 isozymes (Figure 15). The nitrocellulose membrane,

following staining with lobster PPO-specific antibody, was found to

contain dark bands corresponding to potato, lobster, white shrimp, and

brown shrimp PPO and the crude lobster PPO preparation. Three dark bands

were observed for both the potato PPO and the crude lobster PPO

preparation, while only one dark band was found for purified lobster,














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Figure 15.


SDS-PAGE Profile of Protein Standard (SDS-6H), Purified
Mushroom (M), Potato (P), Florida Spiny Lobster (L), White
Shrimp (W), and Brown Shrimp (B) PPO, and a Crude Lobster PPO
Preparation (CL) under Reduction Condition on a Nitrocellulose
Membrane



























-205 kD






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75

white shrimp, and brown shrimp PPO (Figure 16). Incomplete protein

transfer or torsional changes in protein structure following electro-

transfer process was possibly responsible for the failure of mushroom PPO

that not to form a dark band with lobster PPO-antibody. Result from this

study did not support the competitive ELISA experiment obtained for

mushroom PPO. However, our previous findings that PPO from potato and

crustacean sources shared similar structural components was further

demonstrated.


Spectropolarimetric Analysis of PPO

Lobster, white shrimp, and brown shrimp PPO had similar circular

dichroic spectra (Figures 19, 20, and 21), which were different from those

of mushroom and potato (Figures 17 and 18). They all varied in their

secondary structures (a-helix, P-sheet, f-turn, and random coil) (Table

3). For example, white shrimp PPO had a higher percentage of

a-helix than brown shrimp PPO; they both showed the same broad negative

ellipticity between 207 and 220 nm. The percentage of a-helix of

mushroom, potato, and crustacean PPOs estimated using the SSE program were

close to the values that calculated according to the formula of Greenfield

and Fasman (1969). The crustacean PPO, in general, had a higher

percentage of a-helix and lower percentage of f-turn than mushroom and

potato PPO (Table 3). The percentages of the secondary structures of

these PPO estimated from the SSE program may not represent the absolute

values. However, the results from this study showed that PPO from various

sources possessed varied secondary structures, with the crustacean sources

producing PPO which showed very similar secondary structure.






























Figure 16.


Determination of the Specific Reactivity of Anti-lobster PPO
Antibody with a Crude Lobster PPO Preparation (CL), Purified
Mushroom (M), Potato (P), Florida Spiny Lobster (L), White
Shrimp (W), and Brown Shrimp (B) PPO as well as SDS-6H Protein
Standards as Analyzed by Immunoblotting Following SDS-PAGE


















































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