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Catalytic and structural properties of alginate lyases from bacterial epiphytes of Sargassum (Phaeophyceae)

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Catalytic and structural properties of alginate lyases from bacterial epiphytes of Sargassum (Phaeophyceae)
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Romeo, Tony, 1956-
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English
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vii, 151 leaves : ill. ; 28 cm.

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Subjects / Keywords:
Alginates ( jstor )
Bacteria ( jstor )
Depolymerization ( jstor )
Dimers ( jstor )
Enzymes ( jstor )
Gels ( jstor )
Polymers ( jstor )
Sargassum ( jstor )
Sodium ( jstor )
Uronic acids ( jstor )
Dissertations, Academic -- Microbiology and Cell Science -- UF
Energy crops ( lcsh )
Lyases ( lcsh )
Microbiology and Cell Science thesis Ph. D
Phaeophyceae ( lcsh )
Sargassum ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1986.
Bibliography:
Bibliography: leaves 140-150.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Tony Romeo.

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ATALYTIC AND STRUCTURAL PROPERTIES OF ALGINATE
LYASES FROM BACTERIAL EPIPHYTES OF
SARGASSUM (PHAEOPHYCEAE)
By
TONY ROMEO
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
1986


ACKNOWLEDGEMENTS
I offer my sincere gratitude to Dr. James Preston who
has provided immeasurable encouragement, support, and
guidance to me throughout my graduate studies. The members
of my graduate committee have offered a great deal of
helpful advice for which I am grateful: Dr. L. Ingram, Dr.
W. Gurley, Dr. H. Aldrich, and Dr. P. McGuire. I also wish
to thank Dr. J. Gander for valuable advice and comment on
parts of this work. The students in Dr. Preston's lab have
been good friends and helpful colleagues to me. I wish them
success and happiness.
The skilled efforts of Donna Huseman in preparing the
figures for this work and Adele Koehler in typing the
manuscript are greatly appreciated.
Jeanette Reinhardt provided electron microscopy of
Sargassum protoplasts. B. Parten and Dr. B. Dunn carried
out amino acid analyses and N-terminal sequence analysis of
alginate lyase. The NMR analyses of substrates were
obtained by Sandra Bonetti and Cynthia Jackson. The amino
acid sequence similarity search was conducted by Dr. Michael
Little in the Dept, of Biochemistry of the- University of
Arizona. J.C. Bromley, D.R. Preston, and J. Beiswanger
provided assistance in culturing and harvesting bacteria for
enzyme isolations. All of these
valuable to my work.
contributions have been


for her
I would
patience and
like to thank my beloved wife, Lori,
moral support.
in


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
ABSTRACT vi
CHAPTER
I OBJECTIVES AND BACKGROUND 1
Objectives and Rationale 1
Background 3
IIALGINATE LYASES FROM BACTERIAL EPIPHYTES OF
SARGASSUM: SUBSTRATE SPECIFICITIES, MECH
ANISMS OF ALGINATE DEPOLYMERIZATION, ATTEMPTS
TO FORM PROTOPLASTS FROM SARGASSUM BY LYASE-
MEDIATED DEGRADATION OF CELL WALLS 18
Introduction 18
Isolation, Properties, and Growth of Alginate
Lyase Producing Bacteria 19
Intracellular and Extracellular Alginate
Lyases: Substrate Specificities and
Cleavage Patterns 24
Digestion of Alginate Present in Sargassum
Tissues by Alginate Lyases 30
Preparation of Protoplasts by Mechanical
Disruption of Tissue 37
Discussion 39
IIIHPLC ANALYSIS OF THE DEPOLYMERIZATION OF
(1-4)-8-D-MANNURONAN BY EXTRACELLULAR AND
INTRACELLULAR ALGINATE LYASES FROM A MARINE
BACTERIUM 46
Introduction 46
Experimental 48
Results and Discussion 56
IV


Page
IVPURIFICATION AND STRUCTURAL PROPERTIES OF AN
EXTRACELLULAR (1-4)- 3-D-MANNURONAN SPECIFIC
ALGINATE LYASE FROM A MARINE BACTERIUM 78
Introduction 78
Materials and Methods 79
Results 86
Discussion 104
VDEPOLYMERIZATION OF ALGINATE BY AN EXTRA
CELLULAR ALGINATE LYASE FROM A MARINE BAC
TERIUM: SUBSTRATE SPECIFICITY, ACCUMULATION
OF REACTION PRODUCTS, AND EFFECTS OF PRODUCTS
ON THE REACTION RATE Ill
Introduction Ill
Materials and Methods 113
Results 116
Discussion 128
VI CONCLUSIONS 138
REFERENCES 140
BIOGRAPHICAL SKETCH 151
V


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
CATALYTIC AND STRUCTURAL PROPERTIES OF ALGINATE
LYASES FROM BACTERIAL EPIPHYTES OF
SARGASSUM (PHAEOPHYCEAE)
by
Tony Romeo
August 1986
Chairman: James F. Preston, III
Major Department: Microbiology and Cell Science
Enzymes which catalyze depolymerization of alginate,
the major cell wall polymer of brown algae (Phaeophyceae)
were isolated and characterized from bacteria which were
part of the epiphytic flora of healthy tissues of Sargassum
species. Both Gram negative obligate aerobes and
facultative anaerobes produced activities with a range of
substrate specificities. Extracellular preparations of the
bacteria were highly endolytic while cell extracts were more
exolytic in their overall mechanisms. The activities were
capable of extensive degradation of the alginate present in
tissues of Sargassum.
A method for analysis of the depolymerization of (1-4)-
8-D-mannuronan (poly(ManA)) by high performance liquid
chromatography (HPLC) demonstrated that the single
extracellular alginate lyase from a fermentative isolate
generated unsaturated oligouronides which included dimeric
vi


through pentameric products. The trimer was the major
product and could not be further depolymerized; the tetramer
was converted to trimer at a low rate; the pentamer was
readily converted to trimer and dimer forms. The
intracellular preparation differed in that it generated a
considerable amount of monomer.
The extracellular alginate lyase was purified to
chromatographic and electrophoretic (SDS-PAGE) homogeneity
by gel filtration on Sephadex G-75, anion exchange HPLC on a
Mono Q HR 5/5 column, and gel filtration HPLC on an Ultrapac
TSK-G4000SW column. The enzyme was composed of a single
polypeptide of 29 kDa. The amino terminal sequence was
determined through the first 6 amino acids, and 19 of the
first 30 amino acids were assigned. Several closely
migrating forms were separated by isoelectric focusing,
which have pi values ranging from 4.2 to 5.0, suggesting
posttranslational modification. The secondary structure was
approximately 74% ct-helix by CD spectroscopy.
The extent of depolymerization of well characterized
alginates and block regions of alginate by the purified
enzyme was strongly correlated with the frequency of
mannuronic acid triad in the polymers and not to the
mannuronic acid diad frequency or mannuronic acid content,
suggesting a minimal recognition site of three sequential
residues. Unsaturated trimer and tetramer end products of
the reaction did not show appreciable inhibition of
activity. A model for the specificity of the active site of
the enzyme is presented.
vi 1


CHAPTER I
OBJECTIVES AND BACKGROUND
Objectives and Rationale
The general objective of these studies was to identify
and characterize enzymatic systems capable of depolymerizing
the major cell wall polymer of the brown algae (Phaeo-
phyceae), alginate. A major impetus for the work has been a
need for methods which would allow members of this group of
organisms, in particular, species of the genus Sargassum, to
be readily cultured. A long range goal for this project has
been the development of systems of introduction of new
genetic material into the Sargassum species to alter their
growth characteristics and/or biochemical properties for
improvement of their biomass potential. Identification of
enzymes which specifically degrade the carbohydrate polymers
present in cell walls of the brown algae should allow
methods for producing protoplasts of the algae to be
developed. As has been the case with work on the higher
plants, this capability should simplify procedures for
culturing these organisms and allow the development methods
for the eventual genetic manipulation of Sargassum species.
The isolation and thorough characterization of enzymes
capable of catalyzing depolymerization of alginate will also
-1-


-2-
provide specific tools for analysis of brown algal cell wall
structure and biosynthesis and for modification and analysis
of alginate structure. Fermentative bacterial isolates
which degrade alginate may prove useful in improving the
rate and/or extent of conversion of brown algal tissue to
methane during anaerobic digestion.
The specific objectives of this project were to
1) Isolate bacteria from Sargassum tissue, which are capable
of growth upon alginate as a sole carbon and energy source,
and evaluate the secreted and the intracellular enzymatic
activities which catalyze depolymerization of alginate.
This included measurements of relative levels of enzyme
activities produced by each isolate, determination of
substrate specificities and general mechanisms, i.e., endo-
or exolytic nature of the activities which depolymerize
alginate, and comparisons of the abilities of various
preparations to degrade Sargassum tissues. 2) Develop
methods which permit the depolymerization of alginate to be
quantitatively and conveniently analyzed, including methods
for identification and quantification of specific products,
determination of the rates of product accumulation and/or
depletion, and determination of the limit products of a
particular depolymerization reaction. 3) Isolate and
purify at least one of the bacterial alginate lyases to
homogeneity and characterize its subunit composition,
primary and secondary structure, and its mechanism of
substrate depolymerization.


-3-
Background
Biomass Potential of Phaeophyceae
The marine brown algae inhabit oceanic coastal waters
throughout the world. Although several species have been
commercially exploited for their anionic carbohydrates, in
particular the alginates, the bulk of the world's supply
remains untapped. The giant kelp Macrocystis pyrifera has
been evaluated for its bioconversion to methane and shown to
provide methane yields that are competitive with other
biomass and waste sources (Chynoweth et al., 1981). Studies
are in progress to determine the feasibility of farming
Macrocystic pyrifera (Neushul, 1977) and Laminaria
saccharina (Brinkhuis et al., 1984) for the production of
feedstocks for methane generation. Species of the genus
Sargassum represent an alternative source which includes
benthic species common to the colder waters and pelagic as
well as benthic species found along the coast of Florida and
other subtropical and warmer temperate waters. Trawling
collections of the pelagic species, S^. natans and £3.
fluitans, have placed their estimated biomass in the
Sargasso sea alone at 4 to 40 million metric tons (Parr,
1939). The high carbohydrate content of these algae and
their present lack of commercial exploitation make them
attractive as a potential source of biomass for conversion
to methane.


-4-
Carbohydrate Polymers of Phaeophyceae
The brown algae produce large amounts of carbohydrates,
e.g., almost 50% of the dry weight of the giant kelp,
Macrocyst is pyrifera (Chynoweth et al., 1981). The
functions of the major carbohydrates are, in general,
related to the roles in maintenance of structural integrity
or in supplying short or long term energy reserves for the
plants. The present discussion will focus upon the
structural carbohydrates which are localized in the cell
walls of the organisms and are depicted in Fig. 1-1.
The major wall component is alginate, a linear 1-4
polymer of ¡3-D-mannuronic acid and a-L-guluronic acid which
generally comprises 10 to 25% of the dry weight of brown
algae (Table 1-1). Alginate is a compound of considerable
commercial importance; over 8,000 tons are utilized annually
in the United States (Wells, 1977). The commercial value of
alginate has been partially responsible for generating
interest in alginate; a relatively large body of information
has been obtained regarding its fine structure and solution
properties.
Studies of Haug and coworkers have established that the
two constituent uronic acids of alginate are arranged into
homo- and heteropolymeric block regions of DP (degree of
polymerization) around 20 for the homopolymers, which are
interspersed in native alginate (Haug et al 1966; Haug et
al. 1967). These polymers were obtained in relatively pure
form by mild acid hydrolysis of alginate, followed by


-5-
STRUCTURAL CARBOHYDRATES
OF PHAEOPHYTA
H
Figure 1-1. Chemical structures of the three polymers
which comprise the bulk of marine brown algal
cell walls (Phaeophyceae). Unit saccharides,
glycan bonds, and modifications of the
saccharides are indicated. Configurational
aspects of structures are not implied.


-6-
Table 1-1. Alginate levels in Phaeophyta species.
Species
Alginic acid % dry wt.
S. fluitans
1'
00

00
D>
S. natans
19.9a
S. filipndula
13.3-23.5b
S. polyceratium
20.3a
S. vulgare
17.9a
L. cloustoni
14-22c
M. pyrifera
14. ld
a ,b ,c ,dVa]_ues are from Aponte de Otaola et al (1983), Davis
(1950), Black (1950), and Chynoweth et al (1981).


-7-
fractionations based upon differential acid solubility of
the individual polymers. Isolated mannuronan regions,
poly(ManA), and guluronan regions, poly(GulA), have been
subjected to x-ray crystallographic studies which indicated
that the configuration the poly(ManA) resembles a flattened
ribbon, whereas that of the poly(GulA) forms a buckled chain
(Atkins et al., 1971). Nuclear magnetic resonance analyses
of alginate (Penman and Sanderson, 1972) suggest the Cl
conformation for the 3-linked mannuronic acid residues,
indicating diequiator ial linkages to adjacent residues, and
the 1C conformation for the a-linked guluronic residues,
which would form diaxial linkages, further supporting the
polymer conformations which were based upon x-ray crystal
lography .
The conformations of the block regions of alginate and
their relative abundance in the native polymer have
important effects on the solution properties and biological
functions of alginate. Only the poly(GulA) regions of
alginate bind to calcium and certain other divalent ions
with high affinity, thus rendering the purified poly(GulA)
insoluble or causing the native polymer to form a gel
(Smidsrod and Haug, 1965; Haug and Smidsrod, 1965a; Haug and
Smidsrod, 1965b; Kohn et al 1968). Circular dichroism
studies of the interaction of calcium with poly(GulA)
sequences suggest that calcium mediates cooperative
interactions of regions containing at least 20 sequential
guluronic acid residues and thereby allows stable interchain


-8-
dimerization to occur. The investigators of this process
have developed a model to describe the calcium guluronate
complexes which has been dubbed the "egg box model" (Grant
et al. 1973; Morris et al., 1978 ; Rees et al., 1982). A
gel or three dimensional lattice is formed by interaction of
native alginate with calcium ions, wherein insoluble calcium
guluronate complexes are flanked by the soluble poly(ManA)
regions and heteropolymeric poly(ManA, GulA) regions. Since
the brown algae in almost all instances are marine
organisms, alginate is present in the cell walls as a gel
containing a mixture of metal ions, although due to the high
selectivity of alginate for calcium, and the relatively high
concentration of calcium ions in seawater; this is probably
the most prevalent ion (Percival and McDowell, 1967).
The relative amount of poly(GulA) present in alginate
will affect its gelling properties, and this can vary
depending upon the source of the alginate (Penman and
Sanderson, 1972; Haug et al., 1974). Some species of brown
algae have been shown to form tough, firm, holdfast tissues
using alginate with high levels of guluronic acid and in the
same plant produce flexible apical tissues of alginate
containing high levels of mannuronic acid (Haug et al.,
1974; Andresen et al., 1977).
The algae are apparently capable of increasing the
poly(GulA) content of the alginate in a given part of the
plant as it ages (Haug et al 1974). This might be
accomplished by either degrading the alginate originally


-9-
present and replacing it with new polymers of different
structure or by altering the uronic acid composition of the
polymer which is already present. Laminaria d ig i tata has
been shown to contain an enzyme which depolymerizes alginate
(Madgwick et al 1973a) allowing for the former
possibility; the presence of an epimerase capable of
converting mannuronic acid residues of alginate to guluronic
acid has been detected in Pelvetia canaliculata (Madgwick et
al., 1973b), allowing for the latter mechanism. A
combination of these two means of altering alginate
composition may also occur. Interestingly, a mannuronan-
epimerase from Azotobacter vinelandii has been purified, and
it has been shown to be capable of altering the distribution
of the guluronic acid residues which it generates, in
response to calcium ion concentration (Skajak-Braek and
Larsen, 1985). Perhaps the activities of algal epimerases
are also responsive to conditions which may require the
formation of alginates with varying structural and
functional properties.
A polymer which is quite similar to alginate of the
brown algae is produced by certain bacteria, including
Azotobacter vinelandii (Larsen and Haug, 1971) and certain
isolates of Pseudomonas aeruginosa (Evans and Linker, 1973) .
These bacterial alginates differ from the algal polymer in
containing acetylations of certain hydroxyl groups. The
isolates of P. aeruginosa are of interest in that alginate
producing organisms are frequently isolated from patients
with cystic fibrosis, where the organisms and the alginate


-10-
which they produce are believed to increase the morbidity of
the disease (Hoiby et al. 1977). Studies of the immune
response of cystic fibrosis patients to alginate (Bryan et
al 1983; Speert et al., 1984) and the genetics of alginate
biosynthesis by P. aeruginosa (Roehl et al., 1983; Banerjee
et al. 1985; Goldberg and Ohman, 1984) are areas of recent
interest which should increase our understanding of the
chemical properties and biosynthesis of alginate.
Fucoidin or fucoidan is a sulfated fucose polymer,
which is found in levels from less than 1% of the dry tissue
weight for species such as Macrocyst is pyrifera (Chynoweth
et al 1981) to 24% for species such as Fucus spirilis and
Pelvetia canaliculata which are extensively exposed to air
during growth (Percival and McDowell, 1967). It consists of
a,l -> 2 linked L-fucose with sulfate esterified primarily
at position 4 (Fig. 1-1). The molecule is probably branched
at positions 3 and 4 and as isolated, may contain other
saccharides, including galactose, xylose, and uronic acids,
and metal ions as well (Percival and McDowell, 1967).
Fucoidin is believed to reside in extracellular mucilaginous
material and non-fibriliar portions of the wall, and as a
result of its hygroscopic properties, may protect the brown
algae against dehydration (Percival and McDowell, 1967;
McCully, 1966; Evans et al., 1973).
Cellulose is a 3,1 -> 4 unbranched glucan which is an
important structural component of higher plant cell walls
and is consistently found in the cell wall of brown algae,


-11-
in levels which range from about 1 to 10% of the dry weight
of the plant (Percival and McDowell, 1967). Studies on the
histology of the walls of Fucus have identified crystalline
components which consist at least in part of cellulose
(McCully, 1970). Studies on the zygotes of Fucus indicate
that the shape is maintained to a large extent by cellulose.
Procedures which extract the other components, including
alginate, leave an intact sack-like structure which retains
the original form of the zygote (Quatrano and Stevens, 1976).
Enzymatic Depolymerization of Alginate
Alginate has been shown to be chemically depolymerized
by acid hydrolysis, base-catalyzed 6-elimination, and by a
free radical mechanism (Haug et al., 1963; Smidsrod et al.,
1963; Smidsrod et al., 1965). Although enzyme activities
capable of depolymer izing alginate were reported as early as
1934 by Waksman et al., the first definitive study of
alginate depolymerization which identified the 6-elimination
reaction as the mechanism of bond cleavage was presented by
Preiss and Ashwell in 1962. This established that the
enzyme(s) responsible was a lyase, as opposed to a
hydrolase. Since that time virtually all alginolytic
enzymes examined, with one exception (Stevens and Levin,
1976a; Stevens and Levin, 1976b), have proven to be lyases.
This type of reaction also has been observed for enzymes
which depolymerize other uronic acid containing polymers,
including pectin and pectic acid (Collmer et al.,
1982;


-12-
Fogarty and Kelly, 1983), heparin (Yang et al., 1984),
chondroitin (Linn et al., 1983), the acidic heteropoly
saccharides of Rhizobium trifo 1 i (Hollingsworth et al.,
1984), and many others.
The elimination reaction catalyzed by these lyases
relies upon the electron withdrawing carboxyl group of the
substrate (C-6), an extractable a-proton (at C-5), and an 0-
linked uronide (at C-4) which is the leaving group (Kiss,
1974). In the case of endolytic cleavage (internal to the
polymer), the new reducing terminus of the products is
identical to that which would be produced hydrolytically,
and the new nonreducing terminus is a 4,5 unsaturated
residue (see Fig. 1-2).
Assays for bond scission by alginate lyases measure
either generation of reducing or nonreducing termini.
Unsaturated termini absorb UV with a maximum at
approximately 232 nm (Preiss and Ashwell, 1962a), although
the unsaturated monomer, 4-deoxy-L-erythro-5-hexoseu1 ose
uronic acid does not do so appreciably. A specific and
sensitive assay for both unsaturated monomer and unsaturated
nonreducing terminal residues is based upon the soectrophoto-
metric determination at 548 nm of the chromogen generated
upon the reaction of 2-th iobarbi tur ic acid (TBA) with
periodate treated products (Preiss and Ashwell, 1962a).
Alginate lyases have been shown to be produced by and
in some cases isolated and characterized from marine
invertebrates (Nakada and Sweeny, 1967; Favorov and


-13-
Gul
Gul Man
Alginate lyase
t
Gul Gul Man Unsat.
Gul
Figure 1-2. The alginate lyase reaction as catalyzed by
an endo-poly(ManA) lyase. A new reducing end,
indistinguishable from that formed by
hydrolysis, and a 4,5 unsaturated nonreducing
end which confers UV absorbance properties
and reactivity of the lyase products in the
TBA assay are the result. The configurations
of the poly(GulA) and poly(ManA) regions of
alginate are indicated.


-14-
Vaskovsky, 1971; Elyakova and Favorov, 1974; Favorov et al.,
1979; Muramatsu et al., 1977; Muraraatsu, 1984; Jacober et
al., 1980), fungi (Wainwright and Sherbrock-Cox, 1981),
marine bacteria (Kashiwabara et al., 1969; Fujibayashi et
al., 1970; Min et al., 1977; Davidson et al 1976; Quatrano
and Caldwell, 1978; Doubet and Quatrano, 1982; Doubet and
Quatrano, 1984; Pitt and Raisbeck, 1978; Southerland and
Keene, 1981; Preston et al., 1985a), terrestrial bacteria
(Boyd and Turvey, 1977; Boyd and Turvey, 1978; Hansen et
al., 1984), and brown algae (Madgwick et al., 1973).
The work of Nakada and Sweeny (1967) initially
demonstrated that an alginate lyase from abalone showed a
preference for alginate containing a high mannuronic acid
content, and a second enzyme from the same source was more
active with alginates that were high in guluronic acid. All
alginate lyases which have been subsequently examined are
selective in their substrate specificities; i.e., no enzyme
has been isolated which shows significant activity upon both
poly(ManA) and poly(GulA). However, there are reports of
two lyases which will cleave both the GulA-GulA bonds of
poly(GulA) and the GulA-ManA bonds of poly(ManA, GulA) (Boyd
and Turvey, 1978; Min et al., 1977).
Alginate lyases may attack their substrates by either
endolytic or exolytic mechanisms, keeping in mind that some
polyuronide degrading enzymes act by making apparent random
initial endolytic cleavages followed by non-random cleavages
in the later stages of substrate depolymerization (Thibault,


-15-
1983). The mechanism of substrate attack by alginate lyases
was first examined by viscometric analyses (Nakada and
Sweeny, 1967) and thence has also been examined by analysis
of reaction products, using methods which have included
conventional gel filtration and ion exchange column
chromatography (Boyd and Turvey, 1977; Favorov et al.,
1979), paper electrophoresis (Davidson et al., 1977), and
gel electrophoresis (Doubet and Quatrano, 1984).
The rigorous examination of the catalytic activities
and structural properties of an enzyme require its
purification and characterization at least to an extent
which establishes that a single activity has been obtained.
Preferably, the enzyme will have been purified to a
single homogeneous protein. Alginate lyase enzymes have,
however, in most cases been analyzed in impure states.
Davidson et al. (1976) reported that they had purified a
poly(GulA) lyase from a marine bacterium, although the data
which were presented did not establish their claims.
Enzymes from marine molluscs have been purified, all of
which were poly(ManA) lyases (Elyakova and Favorov, 1974;
Muramatsu et al., 1977). Recently, a bacterial poly(GulA)
lyase was purified to electrophoretic homogeneity (Doubet
and Quatrano, 1984). Studies of Muramatsu and coworkers on
two isoenzymes from the snail Turbo cornutus represent the
only thorough studies of the structural properties of
alginate lyases up to this time (Muramatsu and Egawa, 1982;
Muramatsu et al 1984). These two enzymes were each
composed of a single subunit of 32,000 kDa by SDS


-16-
polyacrylamide gel electrophoresis. Native masses of 25 kDa
were established by Sephadex G-100 chromatography. The
isoelectric points were 7.5 and 7.7 for SP1 and SP2 enzymes,
respectively. The enzymes were glycoproteins and were
composed primarily of 3-sheet secondary structure.
Enzymatic Digestion of Brown Algal Cell Walls
The presence of cellulose in the cell wall of the brown
algal wall suggests a requirement for cellulases, in
addition to alginate lyases specific for each of the block
regions of alginate, to effect complete digestion of wall
material. Quatrano (1982) reported that the cellulose which
is deposited during early development of Fucus is subject to
depolymerization by cellulases. The cellulases have been
extensively studied (Enari, 1983) and are readily available
from a variety of commercial sources.
Although the fucoidin of the cell wall may not
contribute to the shape or structural base of brown algal
tissue, it may pose a barrier to cellulases and alginases
and thereby prevent or impede the removal of the cell wall.
There are reports of bacteria having been isolated which
produce enzymes capable of depolymerizing fucoidin (Quatrano
and Caldwell, 1978; Morinaga et al., 1981; Yaphe and Morgan,
1959). However, no such enzymes have been isolated and
examined in detail.
There is a recent report of a method for enzymatically
removing the cell walls of Sargassum species to generate


-17-
protoplasts by the use of extracts of the hepatopancreas of
abalone (Preston et al 1985b). The method is, however,
not yet reproducible, and the origin(s) of the difficulties
are at this time unknown. A reasonable approach to
developing reliable methods for obtaining protoplasts from
Sargassum species might be to fractionate and reconstitute
extracts of abalone. Individual enzymatic factors necessary
for generation of protoplasts might then be identified and
isolated more reproducibly from bacterial and/or fungal
sources.


CHAPTER II
ALGINATE LYASES FROM BACTERIAL EPIPHYTES OF SARGASSUM:
SUBSTRATE SPECIFICITIES, MECHANISMS OF ALGINATE
DEPOLYMERIZATION, ATTEMPTS TO FORM PROTOPLASTS FROM
SARGASSUM BY LYASE-MEDIATED DEGRADATION OF CELL WALLS
Introduction
Certain members of the brown algae, or Phaeophyceae,
have been exploited for their anionic carbohydrates,
specifically alginate, for many years (Percival and
McDowell, 1967; Steiner and McNeely, 1954). However, the
following studies were prompted by the more recent interest
in their potential as a source of biomass for conversion to
methane (Preston et al 1985b). Species of Sargassum are
particularly attractive in this regard, as they are not
commercially exploited at present; i.e., there are no
competitive uses for them, and they contain significant
quantities of the carbohydrates alginate (Aponte de Otaola
et al 1983) and mannitol (Preston and Jiminez, 1986). The
ability to reproducibly generate protoplasts from Sargassum
species would aid efforts to develop methods for tissue
culture and for genetic improvement of these algae (Preston
et al., 1985b).
The goal of the work described in this chapter has been
to explore potential enzyme systems for degrading the cell
-18-


-20-
Gainesville, Florida, where identifications were confirmed,
voucher specimens saved, and epiphytic bacteria isolated.
Sargassum tissue (1 g quantities) was subjected to mild
sonication in sterile sea water (Instant Ocean from Aquarium
Systems, Mentor, OH) to dislodge epiphytic bacteria.
Dilutions of the sea water were plated onto solid alginate
medium (2% agar, 1% sodium alginate in PESI, Provasoli's
enriched seawater, supplemented with 0.27 g/L iodine)
(Provasoli, 1968; Polne-Fuller et al., 1984). Colonies
which exhibited substantial clearing of the calcium alginate
haze of the medium after several days of growth were
selected for further studies. Purity of the cultures has
been established by subculturing on solid alginate medium,
growth in liquid alginate medium (0.1% sodium alginate, PESI
containing 1.0 mM calcium and 5.5 mM magnesium), and growth
on a rich solid medium (2% agar, 1% glucose, 0.8% nutrient
broth, 1% yeast extract in PESI).
Some of the morphological and physiological properties
of the bacteria have been described (Preston et al., 1985a)
and along with other properties are shown in Table 2-1. All
isolates are Gram negative, polarly flagellated rods. Of
seven organisms isolated, four are oxidative and three
fermentative. All oxidative organisms are oxidase positive
and all fermentative isolates oxidase negative. All
fermentative isolates, but none of the oxidative bacteria,
produce acid in liquid glucose medium (Table 2-1). None of
the isolates showed evidence of gas production on glucose or


Table 2-1. Morphological and biochemical properties of alginase secreting bacteria
associated with Sargassum species.
Isolate
Morphology3
Oxidase0
Reaction
Glucose0
+o2 -o2
Alginate
+2 -2
PH6
S012382 FM
short
rod
+
+
+
5.84
SFFB080483
A
0.9-1.1
X
1.6-2.2
-
+
+
+
+
4.69
SNFB080483
B
0.5-0.6
X
2.1-2.6
+
+
-
+
-
6.61
SNFB080483
C
0.7-0.9
X
1.6-2.0
+
+
-
+
-
6.91
SNFB080483
D
0.4-0.6
X
1.4-2.1
-
+
+
+
+
5.27
SFFB080483
F
0.5
X
1.2-2.0
+
+
-
+
-
6.73
SFFB08048 3
G
0.5
X
1.1-1.3
+
+
+
+
+
4.98
aMorpho1ogies and dimensions in um determined by measurements from scanning electron
micrographs with the exception of isolate FM, which was analyzed with the optical
microscope. All isolates were Gram negative.
^Oxidase reactions were carried out according to methods described by Preston et al.
(1985) .
cGlucose medium formulation consisted of 1% glucose, 0.8% nutrient broth, 1% yeast extract
in PESI. A positive reaction indicates growth relative to controls which had no added
g1ucose.
'^Alginate medium formulation consisted of 0.1% sodium alginate in PESI.
epH determinations were carried out directly on cultures grown on glucose medium incubated
under aerobic conditions for 4 days; uninoculated glucose medium had a pH of 6.53.
21-


-22-
alginate containing media. Morphological, physiological,
and DNA base composition data allowed the assignment of
aerobes to the genus Alteromonas (Preston et al., 1985a).
The fermentative isolates, although morphologically and
physiologically similar to bacteria of the genus
Photobacterium, are excluded from this genus by their DNA
base composition and have not been assigned to any existing
genus.
Figure 2-1 shows the growth of two representative
fermentative isolates, A (SFFB080483 A) and G (SFFB080483
B) in liquid alginate medium at 22C with rapid gyrotory
shaking. Duplicate flasks of media were periodically
sampled, the turbidity was measured, and cells were removed
by centrifugation prior to measurements of alginate
utilization and generation of alginate degradation products.
Isolates A and G removed alginate from the medium during
growth, as did all of the isolates selected for these
studies (data not shown) as measured by a loss in total
uronic acid equivalents from the medium (Blumenkrantz and
Asboe-Hansen, 1973). The depolymerization of alginate to
form oligomeric uronides which possessed unsaturated
nonreducing terminal residues, as measured by the method of
Preiss and Ashwell (1962a), occurred during the growth of
the isolates. This indicated that alginate was being
degraded by enzymes which were transeliminases or lyases.


009
-23-
Figure 2-1. Growth of bacterial isolates in liquid alginate
medium. Isolates A (a) and G (b) were cul
tured as described in the text. Growth was
monitored by measuring turbidity (A^OO), total
uronic acid equivalents (A^O) and unsaturated
nonreducing terminal residues (A~^).


-24-
For enzyme isolation, bacteria were grown in Fernbach
flasks containing 1 1 of liquid alginate medium at room
temperature (22C) with rapid gyrotory shaking and were
harvested at late exponential phase.
Intracellular and Extracellular Alginate Lyases:
Substrate Specificities and Cleavage Patterns
Isolation of Bacterial Enzymes
Bacterial cells were removed from culture medium by
centrifugation, frozen in liquid nitrogen or at -70C, and
stored at -70C. For analysis of extracellular enzymes the
spent medium was concentrated by tangential flow filtration
using a Millipore Pellicon cassette system with a
polysulfone (PTGC) membrane which allowed retention of
molecules larger than 10 kDa, and dialyzed against
distilled, deionized water.
For intracellular preparations cells were thawed,
suspended in 4 volumes of ice cold 0.1 M sodium phosphate
buffered at pH 7.5, and disrupted with a French pressure
cell at 16,000 lb in-^. Unbroken cells and cell debris were
removed by centrifugation at 10,000 x g for 15 min, and
acidic polymers were rendered insoluble in the supernatant
solution by adding 5% streptomycin sulfate dropwise with
stirring to a beaker at 0C to give a final concentration of
2%. After stirring the mixture for 10 minutes at 0C, the
resulting precipitate was removed by centrifugation and the
supernatant solution containing alginate lyase was treated


-25-
with solid ammonium sulfate (to 65% saturation). The
protein precipitate was pelleted by centrifugation at 10,000
x g, 10 min, 4C, redissolved in pH 7.5 phosphate buffer,
and dialyzed against distilled deionized water or sodium
phosphate buffered at the desired pH.
Preparation of Substrates
Sodium alginate was purchased from Fisher Scientific
Company as a purified grade originally isolated from
Macrocystis. Prior to use in viscometric determinations
alginate was centrifuged at 100,000 x g for 5 h (Smidsrod
and Haug, 1968). Poly(ManA) and poly(GulA) were obtained
from HC1 hydrolyzed alginate, following the methods
developed by Haug et al. (1967). Preparations of poly(GulA)
and poly(ManA) were further fractionated on Sephadex G-50
with 0.5 M NaCl as eluant, and selected fractions analyzed
by reducing sugar and total carbohydrate assays (Nelson,
1944 ; Dubois et al 1956; Haug and Larsen, 1962) and 3H and
13C NMR (Grasdalen et al., 1979; Grasdalen et al., 1981), to
assess uniformity of size and purity of substrates. The
poly(ManA) fraction contained approximately 89% B-D-
mannuronic acid residues and contained polymers with degree
of polymerization (DP) values of 16-20; the poly(GulA)
contained 89% a-L-guluronic acid residues and had an average
DP of 22.


-26-
Substrate Specificities of Intracellular and
Extracellular Preparations
Alginate lyase was quantified by the TBA assay (Preiss
and Ashwell, 1962a; Weissbach and Hurwitz, 1959). Substrate
mixtures contained either 0.1% sodium alginate, poly G, poly
M, or no carbohydrate (controls for endogenous substrate),
in 0.05 M KCl, buffered with 0.03 M sodium phosphate from pH
5 to 8, or 0.05 M sodium acetate at pH 4.
With three exceptions, activities of the enzyme
preparations with each substrate were maximal at pH 8.
Extracellular preparations from facultative organisms A and
D were most active on poly(GulA) at pH 7 and extracellular
activity of isolate G was highest with poly(ManA) at pH 7.
Figure 2-2 compares intracellular and extracellular
activities from isolate A on poly(GulA), poly(ManA), and
alginate under several pH conditions.
Comparison of levels of activities of intracellular and
extracellular preparations on poly(GulA), poly(ManA), and
alginate at pH 8.0 are shown in Table 2-2. Intracellular
preparations were, in all cases, most active on poly(ManA)
and generally slightly higher on alginate than poly(GulA).
Extracellular preparations generally were highly active on
alginate. Fermentative isolates A and D showed little or no
activity toward poly(GulA) extracellularly, and fermentative
isolate G showed little activity on poly(ManA). Levels of
extracellular activities of the oxidative isolates, FM, B,
and C, were comparable on poly(GulA) and poly(ManA) .


% Maximal activity
-27-
4 6 8
pH
Figure 2-2. Activities of intracellular and extracellular
preparations from isolate A, under various pH
conditions, toward poly(GulA), poly(ManA),
and alginate. Values have been normalized to
the condition which allowed the maximal number
of bonds to be cleaved, as determined using
the TBA assay.


Table 2-2. Substrate specificities and modes of cleavage of alginate by bacterial lyases
at pH 8.0.
Intracellular9
Extracellular*3
4>spe
spe
Isolate0 Poly(GulA) Poly(ManA) Alginate
A548
Poly(GulA) Poly(ManA) Alginate A*48
FM
0.190
0.527
0.235
1.2
0.300
0.569
0.647
20.0
A
0.689
1.140
0.764
8.6
0.000
0.370
0.225
12.0
B
0.026
0.091
0.032

0.276
0.287
0.367

C
0.015
0.042
0.018

0.423
0.373
0.648

D
0.825
0.917
0.880

0.087
1.058
0.981

G
1.287
2.445
1.200
7.3
2.230
0.347
3.135
10.5
Isolate^
FM
0.80
2.24
1.00
0.46
0.87
1.00
A
0.90
1.46
1.00
0.00
1.64
1.00
B
0.81
2.84
1.00
0.75
0.78
1.00
C
0.83
2.33
1.00
0.65
0.58
1.00
D
0.94
1.04
1.00
0.09
1.08
1.00
G
1.07
2.04
1.00
0.71
0.11
1.00
28-


Table 2-2. continued
aIntrace 1lu 1 ar activities were obtained after disrupting bacteria in a French pressure
cell followed by partial purification to remove anionic polymers. This fraction may
include activities bound to the cell surface as well as those which are truly
intracellular.
Extrace 11u1ar activities were measured in the medium after concentration and dialysis but
without further purification.
cValues in the upper panel are u moles of unsaturated product formed per min per g (wet
weight) of cells. All activities presented were calculated after subtracting activities
observed in the absence of added substrate.
^Values in the lower panel are calculated as ratios of lyase activity dependent upon added
substrate divided by the activity dependent upon native alginate.
eSlopes of straight lines obtained by plotting specific fluidity, 4> sp, against the results
of TBA assays, A^ ^, measured during depolymerization of alginate, indicate relative level
of endolytic vs. exolytic cleavage.
29-


-30-
Patterns of Substrate Cleavage
Measurement of the relative level of endo- and
exoeliminase activities in intracellular versus
extracellular preparations was carried out at pH 8.0. The
decrease in the viscosity of alginate during
depolymerization was measured by capillary viscometry (McKie
and Brandts, 1982), and the rate of glycan bond cleavage was
determined by the TBA assay. Plots of the reciprocal of
specific viscosity, i.e., specific fluidity, sp, versus
periodic acid generated TBA reactive products produced
straight lines with slopes proportional to the relative
level of endolytic activity. In the organisms examined, the
slopes were greatest, and therefore the endolytic activities
highest, in the extracellular fractions. The oxidative
isolate FM, in particular, shows striking partitioning of
exo- and endolytic activities. The comparisons of these
slopes are given in Table 2-2.
Digestion of Alginate Present in Sargassum Tissues
by Alginate Lyases
Digestion of S. filipndula Tissues by Intracellular
and Extracellular Alginate Lyase Activities from
Isolate A
Active apical tissue with no visible epiphytic growth
was excised and subjected to mild sonication, weighed,
finely chopped with a scalpel, and incubated with enzyme
preparations from facultative isolate A under conditions
described in Table 2-3. The total number of unsaturated


-31-
Table 2-3.
Degradation of S.
intracellular and
preparations from
filipendula tissue by
extracellular alginate
isolate A.a
lyase
T ime
h
K
Direct Assay
n.r. ends u moles
+Enz. Mixture0
n.r. ends u moles
Intra.
Extra.
Intra.
Extra.
0
0.005
0.004


2
0.131
0.049
0.140
0.163
6
0.317
0.167
0.371
0.519
10
0.419
0.226
0.432
0.703
24
0.651
0.334


aTwo 25 rag apical portions of sonicated S. fi 1ipendula
tissue, containing an estimated 2.5 u moles of uronic acid
residues each, were finely chopped and incubated with enzyme
preparations in PESI lacking added Ca++ and Mg++, and
containing 1.2 mM EDTA. The activities of both enzyme
solutions at the start of the experiment were 0.0198 u
mole/min per ml with alginate as a substrate.
Samples of sea water media were removed at indicated times
and assayed for products using the TBA assay, which measures
nonreducing (n.r.) unsaturated terminal residues.
c
Samples of the seawa
removed at the indie
intra- and extracell
containing activitie
source, incubated fu
of the lyase reactio
ter solutions (exclud
ated times and added
ular alginate lyase p
s of 0.099 u mole/min
rther for 12 h and as
ns as above.
ing tissue) were
to a mixture of
reparations
per ml from each
sayed for products


-32-
nonreducing termini produced by the intracellular extract
was at each time point greater than that produced by the
extracellular extract. However, when samples of each
reaction mixture (minus tissue) were removed, mixed with a
combination of intracellular and extracellular enzymes, and
allowed to incubate further, the material released from the
tissue by the extracellular preparation was observed to be
accessible to further depolymerization, whereas the material
released by the intracellular preparation was not. The most
plausible explanation for this is that the extracellular
preparation, which was shown in Table 2-2 to be highly
endolytic, depolymerized the alginate of the tissue to yield
oligomers which were substrates for the enzymes in the
second incubation. The intracellular enzymes were also
capable of releasing and depolymerizing alginate from the
tissue, but released less total mass of alginate. The
material released by the intracellular preparation was in a
more highly depolymerized state than that released by the
extracellular preparation and could not be further degraded
by the enzyme mixture in the second incubation. Protoplasts
were not quantitatively released from the tissues in any of
the above digestions, and under light microscopy the tissue
appeared relatively intact.


-33-
Degradation of Tissue-Bound Alginate by Extracellular
Enzymes from Isolates A and G
In order to examine the capacities of extracellular
preparations which release unsaturated oligomeric products
from alginate in tissues of S. filipendula and S. fluitans,
the experiment described in Table 2-4 was carried out. The
preparation from isolate G, which was active primarily on
poly(GulA), generated approximately 4-fold as many
unsaturated termini from S. filipendula as did the enzyme
from isolate A, which is active with poly(ManA) but not
poly(GulA). When samples from individual wells were added
to the complementary enzyme preparations and incubated
further, the products generated by extensive degradation of
tissue with isolate G enzyme preparation (24 h) could not be
further depolymerized in the second incubation. The
products from 24 h degradation by isolate A enzyme
preparation were depolymerized to yield 2-fold more
unsaturated termini. The total wall mass released by the
preparation from isolate G was therefore 2-fold greater than
that released by the preparation from isolate A.
Both enzyme preparations released approximately the
same amount of wall mass from the S. f1uitans tissue,
although the preparation from isolate G released 2-fold more
unsaturated termini. In addition, the tissue from
fluitans, unlike that from S. filipendula, was depolymerized
more effectively after treatment with EDTA.


Table 2-4. Degradation of S. f i 1ipendula and S. fluitans tissues by extracellular
alginate lyases from bacterial isolates A and G.
u moles n.r.
+/- second
termini,
enzyme3
4 h
8 h
24 h
Organism3
Enzyme^
EDTA
+
+
+
S.
fi 1ipendula
A
-
0.036
0.107
0.066
0.155
0.132
0.237
A
+
0.032
0.087
0.061
0.135
0.129
0.233
G
-
0.144
0.220
0.229
0.324
0.529
0.517
G
+
0.133
0.172
0.248
0.250
0.489
0.473
none3
-
0.000
0.008
0.000
0.012
0.000
0.019
none
+
0.000
0.010
0.002
0.018
0.009
0.006
s.
fluitans
A

0.111
0.218
0.157
0.429
0.013
0.006
A
+
0.146
0.424
0.207
0.587
0.019
0.008
G
-
0.220
0.307
0.282
0.405
0.018
0.009
G
+
0.303
0.422
0.490
0.542
0.112
0.007
none
-
0.000
0.009
0.002
0.015
0.002
0.011
none
+
0.002
0.019
0.002
0.021
0.016
0.015
34-


Table 2-4. continued
aApical foliar tissues from S. fi 1ipendula and S. fluitans were sonicated and 12.5 mg
portions were finely chopped, placed in wells of a microtiter plate, and incubated 15 min
in PESI lacking added Ca^" and Mg + + in the presence or absence of 1.0 mM EDTA.
The PESI solutions were removed and replaced with enzyme solutions prepared from isolate
A or G containing 198 nmoles/min ml of activity, as assayed with alginate as the
substrate. The enzyme solutions were buffered at pH 7.8 with 0.1 M sodium hydrogen
phosphate and contained 0.5 M NaCl.
cThese tissue samples were incubated in buffer with no added enzyme.
At 4, 8, and 24 h the contents of wells were sampled, excluding pieces of tissue, and the
content of unsaturated nonreducing terminal residues generated by the action of alginate
lyase on the tissues was determined by the TBA assay. Additional samples from tissues
incubated with alginate lyase activity from isolate A or with buffer solution were added
to equal amounts of alginate lyase activity from isolate G, and samples from wells
containing tissues incubated with enzyme from isolate G were added to equal volumes of
enzyme from isolate A. These mixtures were incubated for 14 h before measuring
unsaturated terminal residues.
3 5-


-36-
It is interesting that the level of products from S.
fluitans decreases between 8 and 24 h. In a similar
experiment, this was observed to occur only when tissues of
this species are degraded, since three other Sargassum
species, S. fi1ipendula, S. furcatum, and S. hystrix did not
show similar decreases (data not shown). The tissues of
fluitans, from which the bacterial isolates were obtained,
may harbor more bacteria capable of utilizing the products
which accumulate. Alternatively, other species of algae may
produce inhibitory compounds which prevent bacterial growth
and subsequent utilization of depolymerized alginate.
Effect of Cellulase on Cell Wall Digestion
by Alginate Lyase
Cellulose which is present in the cell walls of brown
algae may present a barrier to release of protoplasts from
algal tissues and/or impede the digestion of alginate by
lyases. The effect of cellulase on the release of alginate
fragments by alginate lyase from S. fluitans was examined as
follows. Apical tissue was sonicated briefly to remove
epiphytes, and 100 mg samples were sectioned into small
pieces (< 0.5 mm in width). These were incubated for 10 h
in individual wells of a microtiter plate in artificial
seawater which lacked Ca++ and Mg++ salts and contained 50
itiM EDTA. Seawater solutions were removed and fresh
solutions containing alginate lyase or alginate lyase plus
cellulase were added to the wells. The cellulase was


-37-
prepared from Trichoderma viride (purchased
from Sigma Chemical Co., desalted by chromatography on
Biogel P-6) and the alginate lyase was from an acetone
powder preparation of abalone entrails (from Sigma Chemical
Co., desalted by P-6 chromatography). Each preparation was
dissolved at a concentration of 20 mg/ml. At this
concentration 1 ml of the alginate lyase generated 0.55
umoles of product/min from sodium alginate and the cellulase
solution, as prepared, was sufficiently active to
quantitatively convert calli of cultured Caucus carota to
protoplasts in less than 30 min.
Figure 2-3 shows that the addition of cellulase did not
improve the conversion of cell wall bound alginate to
soluble unsaturated uronides by the alginate lyase
preparation, although this does not imply that cellulase is
not removing cellulose from the walls. This combination of
cellulase and alginate lyase enzymes was not effective in
quantitatively converting algal tissue to protoplasts.
Preparation of Protoplasts by Mechanical
Disruption of Tissue
During efforts to enzymatically remove the cell walls
of Sargassum sp., protoplasts were observed consistently to
be generated in low numbers (1000-2000 per 0.10 g of
tissue), although the bulk of the tissue remained intact.
These protoplasts were produced by the mechanical disruption


N.R. TERMINI (/tunles)
-38-
TIME (h)
Figure 2-3. Digestion of S. fluitans tissue with alginate
lyase in the presence and absence of cellulase.
Apical tissue was prepared from fluitans,
incubated in a microtiter plate with alginate
lyase from Haliotus (abalone) in the presence
(closed triangles) or absence of cellulase
(open circles) from Trichoderma viride,
and the contents of the wells sampled and
assayed for nonreducing termini by the TBA
method according to the text. Controls with
no added enzymes are indicated by open
squares.


-39-
of tissue, in the absence of any added enzymatic activity,
by slicing tissue into small pieces which were then
incubated in PESI solution which lacked Ca++ and Mg++.
Figure 2-4 shows what are apparently intact as well as
damaged protoplasts. The intact protoplasts also showed
neutral red staining of vacuoles (Stadelmann and Kinzel,
1972), trypan blue dye exclusion, and intense red
fluorescence of chloroplasts under UV light (data not
shown), which are indicative of viability (Berliner, 1981).
The protoplasts rapidly lysed upon exposure to distilled
water and had smooth outer surfaces as viewed by scanning
electron microscopy (Fig. 2-5), suggesting a membrane
surface which is free of cell wall. At least some of the
protoplasts of a given preparation remained viable for
several hours, although their capacity for regeneration or
continued growth was not tested.
Discussion
This chapter and our previous work (Preston et al.,
1985a; Romeo et al., 1986) document observations that
fermentative as well as oxidative marine bacteria are
capable of producing alginate lyases of varied substrate
specificities. Marine bacterial poly(GulA) lyases were
previously described by several investigators (Fujibayashi
et al 1970; Davidson et al 1976; Sutherland and Keen,
1981) and oxidative bacteria from Fucus, which produced both
poly(GulA) and poly(ManA) lyases were isolated by Doubet and


-40-
Figure 2-4. Light microscopy of protoplasts from S.
fluitans. Healthy apical tissues of S.
fluitans were sonicated briefly to remove
epiphytes, weighed (0.1 g), and sliced into
small pieces with a scalpel, and incubated
0.5 h in PESI lacking Ca^+ and Mg++. The
suspension was drawn into a Pasteur pipette,
avoiding large pieces of tissue, transferred
to a clean well of a microtiter plate, and
observed at 400X, with an Olympus inverted
microscope.


-41-
Figure 2-5. Scanning electron microscopy of protoplasts
from S. fluitans. Panel a depicts a single
protoplast emerging from algal tissue; panel
b shows two protoplasts resting upon a piece
of Sargassum tissue (1600X).


-42-
Quatrano (1982, 1984). Intracellular extracts from our
isolates were high in poly(ManA) lyase, making them similar
to the isolates from Fucus (Doubet and Quatrano, 1982).
Extracellular preparations, except from isolates A and D,
were most active on native alginate. Levels of poly(GulA)
and poly(ManA) lyases were comparable in the extracellular
preparations from all oxidative isolates that were examined.
On the other hand, fermentative isolate G produced much
more poly(GulA) lyase than poly(ManA) lyase, and isolates A
and D produced poly(ManA) lyase in large excess over
poly(GulA) lyase. These last two organisms are in contrast
with the isolates from Fucus which tended to produce higher
levels of poly(GulA) lyase in the extracellular fractions.
An organism such as isolate A, which produces little if any
extracellular poly(GulA) lyase but makes intracellular
poly(GulA) lyase may depend upon other bacteria in the
environment to secrete endo poly(GulA) lyases for the
further depolymerization of alginate.
The observation that endolytic activities are higher in
the extracellular fractions as compared to the intracellular
fraction presumably reflects a requirement for degradation
of large native alginate molecules to allow entry into the
bacterial cells. By retaining exoeliminases either in the
cytoplasm or bound to the cell surface, these organisms avoid
producing large amounts of metabolizable monomers in the
external environment which through diffusion and/or
utilization by other organisms would be lost to the bacteria
producing the enzymes.


-43-
A model for bacterial utilization of alginate based on
this study and the work of others is shown in Fig. 2-6.
Native alginate is endolytically depolymerized to fragments
possessing unsaturated nonreducing ends. These fragments
are internalized and exolytically degraded, possibly
undergoing degradation during the entry process. The
metabolic pathway for degradation of the monomer product by
a pseudomonad has been described (Preiss and Ashwell,
1962b).
Under conditions of anaerobic digestion, the
metabolites of the monomer should be readily converted to
methane by a consortium of bacteria. Shiralipour et al .
(1984) have observed that improved yields of methane can be
obtained from digestion of Sargassum tissues using
microflora associated with Sargassum. The rate and/or
extent of methane production might be further improved by
supplementary inocula of organisms such as the facultative
isolates of this study, which produce alginate lyases with a
spectrum of substrate specificities and modes of cleavage,
or by addition of alginate lyase enzymes to the fermentor.
The exposure of active Sargassum tissues to bacterial
alginate lyase preparations is effective in removing
considerable amounts of alginate from the cell walls
(virtually all of the alginate may be removed from the
tissues by endolytic extracellular alginate lyase
preparations). Although alginate lyase activities alone or
in combination with a cellulase preparation did not effect


-44-
EXTRACELLULAR
Endo Poly (GulA) Lyase
AX-G v
A X-G-G )
A X-G-M (
A X-M-G >
A X-M-M (
A X-M )
Unsaturated
Fragments
of Alginate
INTRACELLULAR
Transport
AX-G
A X-G-G
A X-G-M
A X-M-G
A X-M-M
A X-M
CHO
HOCH
HOCH
l
Exolyases HCH
_ Further
Metabolism
C =
COOH
4-Deoxy-5-Keto
Uronic Acid
Figure 2-6. Proposed model for metabolism of alginate by-
bacteria which colonize tissues of Phaeophyceae.
Alginate polymers are released from a gel-like
state in the wall by secreted endolytic bac
terial alginate lyases. The initial products
are depolymerized to an extent which allows the
bacteria to assimilate them. Exolytic enzymes
continue the depolymerization of oligomers
which enter the cell, with the eventual produc
tion of the monomer, which may be used as a
source of carbon and energy.


-45-
quant i tat ive release of protoplasts from tissue, the
combined effects of these activities and activities toward
other cell wall polymers, e.g., fucoidin and/or proteins,
may be successful. The mechanical production of viable
protoplasts, although with low yields, is encouraging, and
supports the feasibility of developing an enzyme-mediated
method. Recently, crude extracts of the hepatopancreas of
Holiotus sp. have allowed preparation of protoplasts from
Sargassum hystrix (Preston et al., 1985b), although with
some inconsistency of yields which is not yet understood.
Fractionation of this type of crude extract and
identification of specific enzymatic components necessary
for protoplast formation may provide information which will
allow well defined bacterial and/or fungal enzymes to be
applied with more reproducible results.


-47-
Kashiwabara et al., 1969; Linker and Evans, 1984; Davidson
et al., 1977) and paper (Davidson et al., 1976; Davidson et
al., 1977), and gel electrophoresis (Hansen et al., 1984;
Doubet and Quatrano, 1984).
The isolation and characterization of alginate lyase
producing oxidative and fermentative marine bacteria
associated with actively growing tissues of marine brown
algae, genus Sargassum, have been described previously
(Preston et al ., 1985a; Romeo et al., 1986). One of the
facultative anaerobes, isolate A (SFFB080483 A, see Table 2-
1), was shown to produce extracellular lyase activity which
was specific for poly(ManA) versus poly(GulA) and endolytic
in its action on alginate. The intracellular activities of
this bacterium depolymerized both poly(ManA) and poly(GulA)
and in comparison with the extracellular preparation showed
a greater ratio of bond cleavage to increase in fluidity
with native alginate, suggestive of exolytic as well as
endolytic mechanisms. Here the extracellular activity is
shown to belong to a single enzyme. A method based upon
HPLC separation of small oligomers is described, and it has
allowed a kinetic evaluation of the depolymerization of
poly(ManA) by both of these preparations. The limit
products generated by the poly(ManA) specific extracellular
enzyme have also been established.


-48-
Experimental
Materials
Chemicals were analytical grade except as indicated.
Acetonitrile (Fisher Scientific) and tetrabutylammonium
hydroxide (Fisher Scientific and Sigma Chemical Co.) were
HPLC grade. Commercially available electrophoresis grade
reagents were used for electrophoretic analyses. All
aqueous solutions were prepared with water which was
deionized and glass distilled.
Preparation of Substrates
Sodium alginate was purchased from Fisher Scientific
Company as a purified grade originally isolated from
Macrocystis. Poly(GulA) and poly(ManA) were obtained from
HC1 hydrolyzed alginate following the methods developed by
Haug et al. (1967). These preparations were further
fractionated on Sephadex G-50 with 0.5 M NaCl as eluant, and
selected fractions were analyzed for reducing termini
(Nelson, 1944) and total carbohydrate (Dubois et al., 1956;
Haug and Larsen, 1962) to obtain substrates of uniform size.
Both *H and ^C NMR (Grasdalen et al 1979; Grasdalen et
al., 1981) analyses were carried out to assess the purity of
substrates. Poly(ManA) preparations contained 11%
guluronate, and poly(GulA) contained 11% mannuronate.


-49-
Enzyme Assays
Alginate lyase activity was quantified by spectrophoto-
metric determination at 548 nm of the chromophore formed
upon reaction of thiobarbi turic acid (TBA) with periodate
treated products (Preiss and Ashwell, 1962; Weissbach and
Hurwitz, 1959). This method allows the specific measure of
unsaturated nonreducing termini of oligomeric products and
the unsaturated monomer, 4-deoxy-L-erythro-5-hexoseulose
uronic acid. Substrate mixtures contained either 0.1%
alginate, poly(GulA), or poly(ManA), and 0.05 M KC1,
buffered with 0.03 M sodium phosphate at the desired pH.
Enzyme was mixed with 9 volumes of substrate to start the
reactions, and reactions were terminated after 10 min by
addition of periodic acid solution. One enzyme unit is
defined as that amount of activity which will catalyze the
formation of 1 nmole of nonreducing termini and/or monomer
at pH 7.5 in one min at 22C. Protein was routinely
estimated by absorbance at 280 nm. For more quantitative
measurements the assay of Bradford (1976) was utilized with
bovine serum albumin as a standard protein.
Enzyme Isolation
The bacterium used in this study was obtained from
healthy, apical tissue of Sargassum fluitans and initially
identified as an organism which secretes alginate degrading
activity, based on the appearance of extensive clearing zones


-50-
surrounding colonies grown on solid alginate medium. The
organism used for the work described here has been desig
nated isolate A (complete designation SFFB080483 A) and has
been described (Preston et al., 1985a; Romeo et al., 1986).
Biochemical and morphological properties of this bacterium
suggest its assignment to the genus Photobacterium, although
its DNA has a GC fraction of 0.454, somewhat greater than
that of other species currently included in this genus
(0.398-0.429). The organism has been maintained by monthly
transfer on solid alginate medium (Preston et al., 1985a).
For enzyme isolations the organism was grown in 0.1%
liquid alginate medium (Preston et al., 1985a) with rapid
gyrotory shaking at room temperature. In all subsequent
purification steps the extracellular and intracellular
preparations were kept at approximately 4C. Bacterial
cells viere harvested at late exponential phase by
centrifugation at 10,000 x g for 10 min, washed twice with
water by resuspension and centrifugation, frozen in liquid
nitrogen, and stored at -70C. The spent medium was
concentrated and dialyzed against distilled, deionized water
by tangential flow filtration using a Millipore Pellicon
cassette system with a polysulfone membrane (PTGC) which
allowed retention of proteins larger than 10 kDa.
For extracellular enzyme preparations the alginate
lyase activity was precipitated along with remaining
alginate products by dropwise addition of 10% polytheyleni-
mine (PEI) to concentrated medium while stirring on ice.


-51-
The PEI was obtained as a 50% aqueous solution from Sigma
Chemical Co. Prior to use, this solution was diluted with
water, titrated to pH 7.5 with 12 N HC1, and centrifuged at
10,000 x g for 10 min to remove insoluble particles. The
relative volume of PEI necessary for maximal precipitation
of enzyme was found to be critical and was determined for
each batch of enzyme. Typically 1 ml of 10% PEI would yield
maximal precipitation of enzyme from 125 ml of concentrated
medium derived from 10 ul of spent medium. The precipitate
was collected by centrifugation at 10,000 x g for 15 min and
resuspended in distilled, deionized water using a Potter-
Elvehjem homogenizer driven by a variable speed motor. The
resulting suspension was centrifuged and the pellet
homogenized in 0.25 M NaCl, 0.1 M sodium phosphate at pH
7.5, to elute the enzyme. The suspension was centrifuged
for 1 h at. 150,000 x g, and the supernatant solution was
subjected to gel permeation chromatography on Sephacryl S-
200 (2.5 x 133 cm) with 0.1 M sodium phosphate at pH 7.0.
To obtain protein concentration sufficient for preparative
digestions and for quantification with the Bradford assay,
fractions were concentrated by ultrafiltration using an
Amicon cell with a YM 10 filter (10 kDa cutoff).
For intracellular enzyme preparations, cells were
thawed, suspended in 4 volumes of ice cold 0.1 M sodium
phosphate, pH 7.5, and disrupted with a French pressure cell
at 16,000 PSI. Unbroken cells and debris were removed by
centrifugation at 10,000 x g for 15 min, and the supernatant


-52-
solution brought to 2% streptomycin sulfate with the
addition of a 5% streptomycin sulfate stock solution. After
mixing for 10 min, the precipitate containing anionic
polymers was removed by centrifugation at 10,000 x g for 15
min and protein precipitated from the supernatant solution
with the slow addition of solid ammonium sulfate (to 65%
saturation). After centrifugation at 10,000 x g for 15 min,
the protein pellet, containing alginate lyase, was dissolved
in 0.1 M sodium phosphate buffer, pH 7.0, and chromato-
graphically fractionated on the Sephacryl S-200 column
(2.5 x 133 cm) with the same buffer.
Electrophoresis
The method for native gel electrophoresis was
described by Shuster (1971) and used the discontinuous
buffer system of Davis (1964). The running gel had a final
concentration of 7.5% acrylamide and 0.2% bisacrylamide, was
buffered at pH 8.9 with 0.38 M Tris HC1, and was polymerized
with 0.07% ammonium persulfate and 0.058% N,N,N',N'-
tetramethylethylene-diamine (TEMED). The stacking gel was
composed of 2.5% acrylamide, 0.5% bisacrylamide, 0.062 M pH
6.8 Tris HC1, and was polymerized with 0.058% TEMED and
0.01 mM riboflavin phosphate, using a fluorescent light to
activate the polymerization process. The running buffer was
composed of 0.3% Tris base, 1.44% glycine at pH 8.9.
Vertical slab gels were 0.15 cm in thickness and were
subjected to electrophoresis at 30 mA per gel until the


-53-
bromophenol tracking dye had reached the end of the gel.
The gels were cut into 0.5 cm slices which were incubated at
room temperature with 200 ul of sodium alginate pH 7.5
substrate mixture. Following the incubations 100 uL of the
solutions were withdrawn and assayed for products of the
lyase reaction.
Analysis of Alginate Lyase Generated Products
Analytical chromatographic separation of products was
accomplished by ion-paired reversed phase HPLC using a
system which is a modification of that developed by Voragen
et al (1982) to fractionate pectate products. The column
was a C18 uBondapak 8MB 10 u column housed on a Z-Module
radial compression system (Waters). A Rainin 0.5 u
stainless steel filter and a Waters RCSS Guard-Pak C18
prefilter cartridge were positioned between the column and
the injector. The column was run at room temperature in an
isocratic mode with a solvent system of 10% acetonitrile,
10 mM tetrabutylammonium hydroxide, 0.1 M sodium phosphate
buffered at pH 6.5. Unsaturated oligomers were detected by
monitoring UV absorbance of the effluent from the column
at 230 nm with a Gilson Holocnrome variable wavelength
detector equipped with a 1.0 cm flow cuvette. A Waters Tri-
Mod system was used for programming a 6000A pump,
integration of peak areas, and for automated injection of


-54-
samples onto the column. A Waters U6K injector was used for
manual injection of samples.
Preparative fractionation of products generated from
poly(ManA) by the extracellular enzyme was carried out by
gel filtration using a Biogel P-2 column (2.5 x 133 cm)
eluted with 0.1 M ammonium bicarbonate, collecting 5.8 ml
per tube. For a successful isolation of the products, 110
mg of poly(ManA) was dissolved in a 1.6 ml solution of the
extracellular enzyme (900 units/ml) buffered at pH 7.0 with
0.1 M sodium phosphate, and incubated for 12 h at room
temperature. Under these conditions the digestion was not
complete at the time the reaction mixture was applied to the
P-2 column, allowing some of the larger oligomeric
mannuronans to be obtained. Absorbance at 230 nm was
determined for each tube.
Contents of tubes comprising each peak from the P-2
column were pooled, lyophilized, and stored at -20C over
anhydrous calcium sulfate. The lyophilized products were
dissolved in 0.1 M sodium phosphate buffer, pH 7.0, and were
analyzed for unsaturated nonreducing termini by measuring
TBA reactive material generated by periodate oxidation
(Preiss and Ashwell, 1962a; Weissbach and Hurwitz, 1959) with
3-deoxy-D-manno-octulosonic acid (KDO, Sigma Chemical Co.)
as a standard. For preparation of the KDO standard, the
compound was desiccated overnight in vacuo. The chromophore
generated by the reaction of TBA with the 8-formylpyruvate


-55-
formed by periodic acid oxidation of the KDO was quantified
spectrophotometrically according to Preiss and Ashwell
(1962) and according to Koseki et al. (1978) which indicated
that the sample was 90% and 85% pure, respectively. Total
uronic acid content was measured by the method of
Blumenkrantz and Asboe-Hansen (1973) using D-mannurono-
lactone (Sigma Chemical Co.) as a standard. Based upon the
expected extinction at 520 nm for the chromophore from
D-mannuronolactone, the desiccated standard was 84% pure.
Absorbance at 232 nm was measured in a 1.00 cm cuvette after
diluting samples 200-fold with 0.01 N HCl.
Samples of lyophilized fractions from the P-2 column
effluent were sent to Triangle Laboratories, Inc., Research
Triangle Park, North Carolina, for fast atom bombardment
(FAB) mass spectrometry under the direction of Ronald Hass.
Analyses were performed on a VG 7070H mass spectrometer with
a VG11-250 data system. The acceleration voltage was 3 kV
for the trimer analysis and 2 kV for the other samples. An
Iontech saddle field ion source was used with xenon as the
bombarding species. The gun was operated at 7 keV with a
discharge current of ca. 1.5 mA. The samples were analyzed
after dissolving in water and applying 1-2 ul of the
solution to thioglycerol on the probe. The mass spectrometer
was scanned at 5 s per dec of mass from 1200-100, at a mass
resolution of 1000.


-56-
Results and Discussion
Chromatographic and Electrophoretic Behavior of the
Extracellular and Intracellular Activities
The extracellular preparation from a fermentative
marine bacterium, designated isolate A, was shown previously
to be highly active on poly(ManA) and native alginate, but
inactive or possessing only trace activity with poly(GulA)
(Preston et al., 1985a; Romeo et al. 1986). The
preparation was endolytic with alginate as substrate, as
shown by comparing the rate of bond cleavage with the
increase in the reciprocal of specific viscosity, i.e.,
specific fluidity. For purification and characterization of
extracellular poly(ManA) lyases, the concentrated
preparation was first treated with 10% PEI to remove the
partially degraded alginate which remained after dialysis of
the medium. This procedure is effective in reducing the
viscosity, allowing a greater quantity of enzyme to be
applied to the gel filtration column. When subjected to gel
filtration on Sephacryl S-200, a single peak of lyase
activity eluted at 0.67 column bed volumes (Fig. 3-la). The
column allowed complete removal of remaining products
derived from alginate, which appeared as TBA reactive
material eluting at 0.94 column bed volumes and separation
of some of the contaminating proteins from the enzyme. The
pooled enzyme fractions represented recovery of 82% of the
alginate degrading activity loaded onto the column. At this
stage of purification, the specific activity of the pooled


-57-
-3
4
E
c
o
co
eg
u
c
(0
A
h.
O
co
A
<
Figure 3-1. Chromatographic fractionation of extracellular
and intracellular alginate lyase activities.
Samples of the extracellular and intracellular
fractions were obtained as described in the
text and subjected to chromatography at 4 C on
Sephacryl S-200. The column was eluted with
0.1 M sodium phosphate at pH 7.0, and 7.8 ml
fractions collected and assayed for protein by
absorbance at 280 nm, and for alginate lyase
activity by the TBA assay, absorbance at 548 nm.
The column was calibrated with molecular weight
standards, bovine serum albumin (BSA, 67 kDa),
6-lactoglobulin ($-L, 37 kDa), blue dextran
(to establish the void volume, Vo) and NaCl
(total column volume, Vt). The extracellular
preparation (a) was derived from medium which
yielded 3.7 g wet weight of cells and the
intracellular (b) from 13.8 g wet weight of
cells.


-58-
enzyme fraction was typically 3500 units per mg of protein
with alginate as the substrate and the ratio of activities
on poly(GulA) versus poly(ManA) was 0.14.
Samples from individual tubes containing the
extracellular alginate lyase fraction which eluted from the
Sephacryl S-200 column were subjected to native polyacryla
mide gel electrophoresis followed by detection of activity.
The individual fractions comprising the alginate lyase peak
eluting from the S-200 column showed the same single
activity component which migrated as a homogeneous band
(Fig. 3-2a), indicating that the extracellular fraction
contained a single enzymatic activity.
Intracellular activities eluted from the same Sephacryl
200 column as two small and one large peak (Fig. 3-lb). The
major peak eluted in a volume corresponding to an estimated
molecular mass of 40 KDa and contained approximately 15% of
the alginate degrading activity originally loaded onto the
column. The enzymes present in the tubes from centers of
the three activity peaks were active on both poly(ManA) and
poly(GulA) and therefore were probably mixtures of two or
more alginate lyases. The ratios of activities on
poly(ManA) versus poly(GulA) were 6.8, 1.8, and 1.2 for the
contents of tubes 31, 37, and 44, respectively.
The peak tube from the major S-200 intracellular lyase
fraction was electrophoretically resolved into at least
three activities (Fig. 3-2b), indicating that the major
fraction eluting from this column included more than one


-59-
Figure 3-2. Electrophoretic analysis of (a) extracellular
and (b) intracellular alginate lyase activities.
The extracellular preparation was fractionated
by gel filtration on Sephacryl S-200. A sample
was removed from the peak tube and subjected to
native polyacrylamide gel electrophoresis and
subsequent detection of activity according to
methods described in the text. A sample from
the peak tube from the Sephacryl S-200 frac
tionated intracellular extract was subjected
to electrophoresis on the same native poly
acrylamide slab gel used for analysis of the
extracellular lyase preparation.


-60-
alginate degrading enzyme. The activity with the highest
electrophoretic mobility could be identical to the single
major extracellular enzyme based upon electrophoretic
migration; however, this has not been confirmed by other
analyses.
HPLC Analysis of Poly(ManA) Depolymerization
When the extracellular enzyme (14 units) from the S-200
column was incubated at room temperature with 5 mg of
poly(ManA) in 0.5 ml of 0.1 M sodium phosphate buffered at
pH 7.0, unsaturated oligomers were produced which could be
fractionated by HPLC. Profiles generated after 15 min and
4 h of depolymerization are shown in Figs. 3-3a and 3-3b,
respectively. At least six peaks were detected by
absorbance at 230 nm, and five of these were sufficiently
distinct to be integrated by the data analyzing system.
Individual oligomers, detected as A230 peaks (Fig. 3-3b),
were designated numerically from 1 to 5 in the order of
their elution with retention times (in minutes) of 5.87,
7.54, 10.00, 13.87, and 19.83, respectively. As will
be demonstrated, these represent the unsaturated dimer
through hexamer, respectively. When the depolymerization
was monitored over a 30 h period (Fig. 3-4) several features
of interest were noted. Component 1, the dimer, and to a
lesser extent component 2, the trimer, exhibited initial
lags in their rates of accumulation. Components 4 and 5,
the pentamer and hexamer,
increased until the digestion had


Absorbance (230nm)
-61-
0.04 n
0.02-
0.00-
0.08-
0.04-
0.00-
i 1 I 1 1 1 1
0 10 20 30
Retention Time (min)
Figure 3-3. Liquid chromatographic analysis of products
generated by digestion of poly (ManA) by the
extracellular lyase. The unsaturated oligomers
produced from poly(ManA) by the extracellular
enzyme were resolved by HPLC, as described in
the text. Sample volumes of 10 ul were
delivered with automatic injection (Waters,
WISP) and eluted isocratically at 1.0 ml/min.
Profiles of the products which had accumulated
after 15 min (a) and 4 h (b) are shown.


Relative peak area
-62-
Figure 3-4. Kinetic analysis of poly(ManA) depolymeriza
tion by extracellular alginate lyase. The
depolymerization reaction described in Fig.
3-3 was sampled periodically over 30 h, and
samples subjected to HPLC as described in the
text. The peak areas of the major products
are plotted against the times at which the
reaction was sampled. The individual products
were given number designations according to
their order of elution from the column, start
ing with the fastest moving compound, 1,
representing a dimer, through 5, which repre
sents a hexamer. The dimer peak integrates
as 0.11 area units per nmole.


-63-
continued for 6 and 4 h, respectively, and thereafter
decreased. The dimer, trimer, and tetramer never showed
declines, although the rates of accumulation of trimer and
tetramer decreased at approximately 6 h, and the rate of
dimer accumulation began to decrease gradually at 6 to 10 h.
The delays in appearance of the two smaller products, dimer
and trimer, indicated that these compounds were generated to
a significant extent from products which accrued from
initial depolymerization reactions. The decrease in the
concentration of the larger compounds, pentamer and hexamer,
after initial accumulation suggested that these must be
subject to depolymerization by the enzyme, and that their
relative rates of formation and degradation determine the
levels at any given time. The unsaturated monomer is not
readily detected by absorbance at 230 nm due to
tautomerization to the a-keto acid form (Preiss and Ashwell,
1962). Detection at 205 nm revealed a minor component which
eluted prior to the dimer, and analysis by the TBA method of
fractions collected from a reversed phase separation of a
preparative poly(ManA) digest also showed a minor component
of reactive material eluting prior to the dimer (data not
shown). This, presumed to be the monomer, represented less
than 4% of the products including dimers to pentamers
produced by the depolymerization of poly(ManA) catalyzed by
this enzyme.
The depolymerization of poly(ManA) by the intracellular
preparation was analyzed by HPLC using the methods described
for the extracellular preparation. Figure
3-5 shows the


-64-
products which have accumulated at 3 h and 21.5 h. During
an initial phase of the reaction, up to 8-10 h, four
products predominate. The mobilities of three of these are
identical to those of major products generated by the
extracellular enzyme, as established by direct comparisons
at the time of analysis (data not shown). Direct
comparisons were necessary due to changes in column
performance which occurred with usage. Products included
the trimer, with a retention time of 6.9 min, the tetramer,
with a retention time of 8.8 min, and the pentamer, with a
retention time of 11.5 min. The peak which was generated
only by the intracellular preparation presumably represents
the monomer compound, 4-deoxy-L-erythro-5-hexoseulose uronic
acid, and has a retention time of 4.7 min. The identity of
the monomer is indicated by its ratio of absorbance at 230
nm/205 nm, 0.16, as compared with those of the dimer through
tetramer, 0.9-1.3, and by its elution position, which is
prior to the dimer. Due to the poor absorption of the
monomer at 230 nm, the peak area for the monomer
underestimates its concentration relative to other products
several-fold. A fifth compound accumulates to a
considerable extent in the reaction after a lag of 10 h and
is seen in Fig. 3-5 in the lower profile. This product has
the retention time of the dimer compound, 5.95 min. Figure
3-6 shows that the dimer and trimer, peaks 2 and 3, continue
to accumulate at later times in the digest, long after the
concentrations of the monomer, peak 1, and tetramer, peak 4,
have become constant.


ABSORBANCE (230nm)
-65-
0.02-1
0.00-

0.08-
(O
iD
(0
0.04-
0.00 J
D
T
0 10 20
RETENTION TIME (min)
i
30
Figure 3-5. Liquid chromatographic analysis of products
generated by digestion of poly(ManA) by the
intracellular alginate lyase activities. The
unsaturated oligomeric products generated from
poly(ManA), 10 mg/ml, by the third peak of
activity from the S-200 column, tubes 42-47,
13 units/ml, were resolved by HPLC as described
in the Materials and Methods. Profiles of the
products which had accumulated at 3 h, upper
profile, and 21.5 h, lower profile, are
shown.


RELATIVE PEAK AREA
-66-
TIME (h)
Figure 3-6. Kinetic analysis of poly(ManA) depolymeriza
tion by intracellular alginate lyase activities.
The depolymerization reaction described in
Fig. 3-5 was monitored over 33.5 h, by
periodically subjecting 10 ul samples to
HPLC analysis. Products are numbered accord
ing to their elution positions. Peak 1 is the
presumptive monomer, and peaks 2-4 are the
unsaturated dimer through tetramer.


-67-
The poly(ManA) lyase activity from the intracellular
preparation differs from the extracellular activity in
generating a considerable quantity of apparent monomer. The
physiological necessity to produce monomer on the inside of
a bacterial cell or at the cell surface is obvious, although
the mechanisms of the enzymes responsible for monomer
production in the reaction are not yet established. A
single exolytic enzyme might generate monomer as it degrades
poly(ManA) from either the reducing or nonreducing termini.
Alternatively, the monomer may be generated from some
intermediate degradation products by one or more enzymes
with glycosidase-1ike activities.
The unique appearance of the dimer product after a lag
of approximately 10 h is quite unexpected. The fact that
the preparation may contain more than one alginate degrading
activity does not allow a definitive explanation for this.
However, a number of possibilities might be envisaged. If
the monomer is being produced primarily from a dimer product
by a glycosidase-like activity, the loss of this activity
would be expected to allow dimer to accumulate. A second
possibility is that the dimer begins to accumulate as some
intermediate depolymerization product reaches a
concentration sufficient to allow its recognition by the
enzyme which is capable of generating dimer.


-68-
Purification and Characterization of the
Reaction Products
In order to obtain sufficient quantities of the
oligomeric products for characterization, 110 mg of
poly(ManA) was digested with extracellular enzyme for 12 h
and the products were resolved by chromatography on Biogel
P-2 eluted with 0.1 M ammonium bicarbonate. Four components
measured by absorbance at 230 nm were resolved from one
another. These were eluted at column volumes of 0.46, 0.50,
0.56, and 0.64 and designated as fractions 1, 2, 3, and 4,
respectively (Fig. 3-7). A small fraction, approximately 9%
of the total absorbance at 230 nm, eluted between the void
volume of the column and the leading edge of the peak
designated as fraction 1. Chromatography in ammonium
formate led to a similar profile; however, the lyophi1ization
of these fractions resulted in discoloration of some of the
fractions and subsequent HPLC analysis demonstrated
significant degradation. Products obtained after elution
with ammonium bicarbonate were, after lyophi1ization, fluffy
and white, although they were quite hygroscopic.
Samples of the peak tubes from the P-2 ammonium
bicarbonate column were analyzed by HPLC (data not shown),
which identified the contents of tubes 52, 57, 63, and 72 as
the oligomers comprising peaks 4, 3, 2, and 1, respectively,
of the HPLC profiles (Fig. 3-3). Absorbance spectra in the
UV range for samples diluted in 0.01 N HC1 showed maxima


Absorbance (230nm)
-69-
Figure 3-7. Preparative fractionation of the unsaturated
mannuronides. A 110 mg sample of poly(ManA)
was digested by 1,400 units of extracellular
enzyme in a 1.6 ml volume for 12 h at room
temperature and the products fractionated by
gel filtration with a P-2 column, as described
in the text. Volumes of 5.8 ml were collected
in tubes and assayed for absorbance at 230 nm.
Native alginate and galacturonic acid were
subjected to chromatography on the P-2 column
and their elution positions are indicated.
The contents of tubes comprising 4 fractions
were pooled and lyophilized: fraction 1,
tubes 52, 53; fraction 2, tubes 55-57; frac
tion 3, tubes 62-64; fraction 4, tubes 71-73.


-70-
at approximately 232 am (profiles not shown), typical of
products generated by alginate lyases.
The pooled, lyophilized fractions from the P-2 column
were further analyzed to establish the molecular sizes of
the unsaturated products. Comparisons of the uronic acid
content with content of nonreducing residues (Table 3-1)
allowed an estimation of the degree of polymerization (DP)
of each product. The DP values estimated using the ratio of
total uronic acid to nonreducing termini show a trend
consistent with assigning the unsaturated oligomers in
fractions 1 through 4 as the pentamer, tetramer, trimer, and
dimer compounds, respectively.
Based upon the concentration of the nonreducing
unsaturated terminal residues estimated with the TBA assay
of the periodate treated products and the abosrbance values
at 232 nm of products in 0.01 N HC1, an estimation of the
molar absorptivities for the dimer to pentamer series,
presumed to contain a single unsaturated residue in each
molecule, ranged from 5,160 (Fr 1) to 5,420 (Fr 3) M-^cm--'-.
The concentrations of each component, based upon gravimetric
preparations of lyophilized samples and calculated molecular
weights of each as an ammonium salt, were calculated on the
assumption that fractions 1, 2, 3, and 4 represented the
pentamer, tetramer, trimer, and dimer, respectively. These
values, as divisors for the A232 values listed in Table 3-1,
led to calculated molar absorptivities which ranged from


-71-
Table 3-1. Analysis of unsaturated oligomers from
lyophilized P-2 fractions.3
Fraction
no .
Abs k
2 32 nm
Unsat.c
termini
mM
Uronicd
acids
mM
DP
uronic
acids
unsat.
termini
1
39.2
7.59
40.5
5.3
2
42.0
7.94
37.5
4.7
3
64.0
11.8
36.3
3.1
4
84.6
16.2
45.6
2.8
aLyophilized products from the Biogel P-2 fractionated
preparative digest were obtained according to methods
described in the text and dissolved in 0.1 M sodium
phosphate buffer, pH 7.0, to a final concentration by
weight of 10 mg/ml.
Solutions were analyzed for absorbance at 232 nm in a 1.00
cm quartz cuvette, after diluting 200-fold with 0.01 N HCl.
cUnsaturated nonreducing
by the TBA assay of per
a standard. Values wer
apparent 90% purity of
terminal residues we
iodate treated produc
e adjusted to correct
the standard.
re determined
ts, with KDO
for the
as
dUronic acid
lactone as a
the apparent
the expected
Asboe-Hansen
residues were determined using D-mannurono-
standard. Values were adjusted to correct
84% purity of our standard compound, based
yield of cnromophore given by Blumenkrantz
(1973) .
for
on
and


-72-
3350 (Fr 2 as the tetramer) to 3890 (Fr 1 as the pentamer).
The individual fractions, in particular the putative dimer,
were sufficiently unstable to heating to preclude high
temperature desiccation, and these lower molar absorptivi-
ties, in comparison to those determined from the concentra
tions determined with the TBA assay, may reflect the
presence of water not removed by the lyophilization process.
Samples of unsaturated dimeric and tetrameric products
obtained from digestion of bacterial alginate with a
poly(ManA) lyase from a Pseudomonas aeruginosa isolate
(Linker and Evans, 1984) were graciously provided by Dr.
Alfred Linker and shown to possess the same HPLC mobilities
as our dimer and tetramer, respectively; molar absorptivi-
ties at 232 nm in 0.01 N HC1 of 6,400 and 5,500 M-^cm-^ were
obtained for this dimer and tetramer, respectively (data not
shown).
Further evidence of the DP of the major products was
obtained by FAB mass spectrometry (Table 3-2) of the
lyophilized fractions. Spectra from each of the products
contained major ions which correspond to within 1 mass unit
of the calculated M+NH^ and the M+NH4 + H2O ions. The triiner
product showed an additional ion which represents the M+NH^
+ thioglycerol, and the spectrum of the dimer showed two
major ions (373, 391) which could not be explained, based on
the expected structure of the dimer. The dimer sample was
the only one which was not white in color, and we assume


-73-
Table 3-2. Analysis of unsaturated oligomers by FA3 mass
spectrometry.a
Product
Observed ions
m+nh4
m+nh4+h
m+nh4 +
20 thioglycerol
Unidentified
Pentamer
898
916
Tetramer
722
740
Trimer
546
564
654
Dimer
370
388
373, 391
aSamples of lyophilized fractions from the P-2 column were
analyzed by FAB mass spectrometry according to methods
described in the text.


-74-
that these ions resulted from some decomposition of the
dimer, which occurred in transit or in handling prior to FAB
spectrometry.
Activity of the Extracellular Enzyme on Unsaturated
Oligomeric Products
Although the HPLC kinetic analysis of poly(ManA)
digestion yields valuable information about the reaction,
the complex nature of the process, wherein several products
compete for binding to the enzyme and some products are
degraded as they accumulate, does not allow detailed
consideration of the activity of the enzyme on individual
molecular species. To test the capability of the
extracellular enzyme to further degrade products which
accumulate during depolymerization of poly(ManA), the
lyophilized trimer, tetramer, and pentamer purified by P-2
column chromatography were individually incubated with
extracellular enzyme and the reactions sampled at 5 min and
5 h and subjected to HPLC. The conditions and the resulting
profiles are shown in Fig. 3-8. Profiles generated from
poly(ManA) digestion (al and a2) are included for
comparison, as the retention times for the products had
changed over several months of column use since the profiles
shown in Fig. 3-3 were obtained. It is clear that the
trimer is not subject to depolymerization by the enzyme, as
profiles bl and b2 are identical. The tetramer compound (cl
and c2) is not a good substrate for the enzyme, as predicted


Absorbance (230nm) Absorbance (230nm)
-75-
Figure 3-8. Activity of the extracellular enzyme on puri
fied trimer, tetramer, and pentamer products.
The lyophilized products obtained after P-2
column chromatography were dissolved in 0.1 M
sodium phosphate at pH 7.0 such that the final
concentration in the reaction mixtures was
5 mg per ml, and enzyme was added to a final
concentration of 100 units per ml to start the
reactions. The mixtures were incubated at room
temperature and 5 ul samples withdrawn at
5 min and 5 h, and subjected to reversed phase
HPLC. The chromatographic profiles generated
from poly(ManA), trimer, tetramer, and pentamer
are designated a, b, c, and d, respectively,
and numbers 1 and 2 indicate sampling times
of 5 min and 5 h.


-76-
from the kinetic analysis (Fig. 3-4); however, a small
amount of trimer was generated from the tetramer over the 5
h period. An equal amount of monomer should also have been
produced, although it would not be readily detected by
absorbance at 230 nm as noted above. The pentamer was
readily degraded by the enzyme, which converted almost 50%
of the initial quantity to equal amounts of dimer and
trimer, but produced little or no tetramer in 5 h of
incubation.
Few other bacterial poly(ManA) lyases have been
characterized to an extent which would allow a detailed
comparison with the extracellular enzyme analyzed in this
study. Doubet and Quatrano demonstrated that a cell-bound
enzyme from a marine bacterium could degrade poly(ManA) by
an apparent exolytic mechanism (Doubet and Quatrano, 1984).
Davidson et al. (1977) described an endolytic poly(ManA)
lyase which was induced by phage infection of Azotobacter
vinelandii. This enzyme seems to be quite similar to the
one which we have studied, in that it generates a series of
unsaturated products ranging from dimers through pentamers,
although neither the relative levels of the products nor the
limit products were determined. Kashiwabara et al. (1969)
measured poly(ManA) (SM) degrading activities in crude
extracts of two marine pseudomonads. The activities were
weak in relation to the endogenous poly(GulA) lyase
activities, and although the reaction products were not well
characterized, an unsaturated trimer was shown to be the


-77-
major product. Linker and Evans (1984) examined an
intracellular poly(ManA) lyase from a Pseudomonas aeruginosa
isolate which generated unsaturated oligomers ranging from
dimeric through pentameric compounds. This enzyme was
apparently incapable of producing monomer and cleaved the
unsaturated tetramer to form dimeric products. Although the
major reaction products generated by the enzyme which we
have studied are similar to those produced by the P.
aeruginosa enzyme, the catalytic mechanisms of the two
enzymes clearly differ, as shown by the HPLC analysis of the
conversion of unsaturated tetramer to trimer.
The approach utilizing reversed phase ion-pairing HPLC
to evaluate the mechanisms of the lytic depolymerization of
alginate is being applied to study alginate lyases with
different substrate specificies, such as the extracellular
preparation from isolate G, and enzymes from other marine
bacteria (work in progress, J.F. Preston and T. Romeo).
This method should also prove useful for the study of lyases
acting on other glycuronans. Detection of products based on
refractive index or absorbance of UV at shorter wave
lengths (e.g., 205 nm) should extend the applicability of
the method to hydrolytic systems as well.


CHAPTER IV
PURIFICATION AND STRUCTURAL PROPERTIES OF AN
EXTRACELLULAR (1-4)-g-D-MANNURONAN SPECIFIC
ALGINATE LYASE FROM A MARINE BACTERIUM
Introduction
Previous studies utilizing alginate lyases have
examined the structure of alginate (Min et al., 1977; Boyd
and Turvey, 1978), the composition of alginate containing
cell walls of brown algae (Quatrano and Peterman, 1980), and
the feasibility of generating protoplasts of brown algal
species (Preston et al 1985b; Romeo et al., 1986). The
possibility that the alginate produced by Pseudomonas
aeruginosa strains colonizing the lungs of cystic fibrosis
patients is involved in the morbidity of that disease has
recently led to the identification of alginate lyases in
isolates of clinical origin (Linker and Evans, 1984; Dunne
and Buckmire, 1985).
With few exceptions alginate lyase enzymes have been
examined as impure mixtures of proteins, or even as
preparations containing more than one activity, disallowing
firm conclusions to be drawn about their substrate
specificities, mechanisms, and their structural properties.
The result is that the only investigations on the structures
of these enzymes, with the exception of molecular weight
-78-


-79-
determinations, have been carried out on two isozymes from
the mid-gut gland of the wreath shell, Turbo cornutus
(Muramatsu and Egawa, 1982; Muramatsu et al., 1984).
We previously reported the isolation of an
extracellular alginate lyase capable of depolymerizing
poly(1-4)-8-D-mannuronan, poly(ManA), derived from alginate,
and an analysis of the products of this enzymatic reaction
(Chapter III). A method for purification of the
extracellular enzyme to homogeneity using HPLC is now
described. Structural properties of the enzyme which have
been examined include the molecular mass, pi, amino acid
composition, content of helical secondary structure, and the
N-terminal amino acid sequence. Some of the properties are
compared with those of other alginate depolymerizing
enzymes.
Materials and Methods
Reagents
Chemicals were analytical grade except as indicated.
Commercially available electrophoresis grade reagents were
used for SDS polyacrylamide gel electrophoresis. Reagents
and chemicals for amino acid analysis and N-terminal
sequencing were commercially available ultrapure grades.
Water for all aqueous solutions was deoinized and glass
distilled.


-80-
Sodium alginate was purchased as a purified grade
(Fisher Scientific Co.) originally obtained from
Macrocystis. The content of mannuronic acid was determined
to be 67% by NMR, using methods established by Penman and
Sanderson (1972) and Grasdalen et al. (1979). The
poly(ManA) was prepared from HC1 hydrolysed alginate
according to Haug et al. (1967) and fractionated by size
through Sephadex G-50 with 0.5 M NaCl as eluent. The
fraction used for these studies was shown to contain 89%
mannuronic acid by -'-H NMR. Comparison of total carbohydrate
(Dubois et al., 1956) to reducing termini (Nelson, 1944)
indicated that the range for the degree of polymerization
was 16-22.
Enzyme Assays
The poly(ManA) lyase activity was quantified by
spectrophotometric determination of the chromophore
generated upon reaction of thiobarbi turic acid, TBA, with
periodate treated products (Preiss and Asnwell 1962a;
Weissbach and Hurwitz, 1959). The following conditions have
been used for the enzyme reactions, unless otherwise noted:
pH 7.5, 0.03 M sodium hydrogen phosphate, 0.05 M KCl, 0.10%
sodium alginate, incubated for 10 min at room temperature,
22 C. One unit of activity will generate one nmole of
unsaturated termini and/or unsaturated monomer in 1 minute.
The quantity of protein present in fractions at various
stages of purification was determined by the Coomassie blue


-81-
binding assay of Bradford (1976), using bovine serum albumin
as the standard. When a more accurate estimate of the
protein concentration of the purified poly(ManA) lyase was
needed, spectrophotornetric analysis at 205 and 280 nm was
used (Scopes, 1974). This method, unlike that of Bradford
(see Tal et al., 1985; Compton and Jones, 1985), has
relatively little variation of response to proteins of
differing chemical constitution.
Purification of Poly(ManA) Lyase
The poly(ManA) lyase was purified from a bacterium
originally isolated from healthy tissues of Sargassum
fluitans; the bacterium grew on alginate as sole
carbon source and secreted significant alginate lyase
activity. The properties of this fermentative marine
bacterium, designated as isolate A, or SFFB080483 A, have
been described (Preston et al., 1985a; Romeo et al., 1986).
The purification steps were carried out at 4 C, except for
chromatography in the HPLC systems, which was at room
temperature. For enzyme isolations the organism was grown
to late exponential phase in 0.1% liquid alginate medium
(Preston et al. 1985a), with rapid gyrotory shaking at 22 C.
Bacterial cells were separated from the medium by
centrifugation (10,000 x g, 10 min), and the medium was
concentrated and dialyzed against water by tangential flow
filtration using a Millipore Pellicon Cassette System with a


-82-
polysulfone membrane (PTCG) which allowed retention of
proteins larger than 10 kDa.
The enzyme was precipitated along with remaining
products of alginate degradation by dropwise addition of 10%
polyethylenimine (PEI, purchased from Sigma Chemical Co.,
St. Louis, MO, titrated to pH 7.5 with concentrated HC1,
diluted with water, and centrifuged at 10,000 x g to remove
insoluble particles) to the concentrated medium. The
relative volume of PEI needed for maximal precipitation of
alginate lyase activity was determined by titrating soluble
alginate lyase activity. The PEI precipitate was collected
by centrifugation (10 min at 10,000 x g) and washed by
resuspension in water with a Potter-Elvehjem homogenizer
driven by a variable speed motor. The suspension was
centrifuged and enzyme activity eluted from the precipitate
by homogenization in 0.25 M NaCl, 0.1 M sodium hydrogen
phosphate at pH 7.5. Insoluble material was removed by
centrifugation at 150,000 x g for 2.5 h, and the supernatant
solution subjected to gel filtration chromatography.
A preparation which was derived from 43 1 of growth
medium (yielding 71.3 g of wet cells) was fractionated on a
column of Sephadex G-75 superfine grade (5 x 79 cm) at 4 C
with 0.1 M sodium phosphate buffered at pH 7.0. Fractions
were collected (7.8 ml) and assayed for enzyme activity and
protein (absorbance at 280 nm). The recovered activity was
concentrated by pressure filtration in an Amicon stirred
cell (Amicon Corp.,
Lexington, MA) with a YM 10 membrane.


-83-
The concentrated activity was applied in 3 separate
runs to a Mono Q HR 5/5 anion exchange column (5 x 50 mm,
Pharmacia, Inc., Piscataway, NJ) and eluted at room
temperature with a gradient of NaCl (0-1.0 M) buffered at pH
7.0 with 0.01 M sodium hydrogen phosphate at a flow rate of
0.5 ml/min. The chromatography system included an LK3
Ultrachrome GTi HPLC system (2152 Controller, 2150 Pump,
2154-002 Injector, LKB-Produkter AB, Bromma, Sweden). A
Gilson Holochrome variable wavelength detector fitted with a
1.00 cm cell (Gilson Medical Electronics, Inc., Middleton,
WI) was used to measure absorbance at 280 nm, which was
recorded with a Linear 800 Versagraph (Linear Instruments
Corp., Irvine, CA). Fractions of 0.5 ml were collected and
assayed for alginate lyase activity.
Enzymatically active fractions from the 3 Mono Q column
runs were concentrated and desalted using the Amicon cell
and again applied to the Mono Q column and eluted as above.
Activity was recovered and concentrated and subjected to gel
filtration HPLC using an UltroPac TSK-G4000 SW column (7.5 x
600 mm, LKB) run at room temperature with a buffer of 0.1 M
sodium hydrogen phosphate, pH 7.0, containing 0.1 M NaCl, at
a flowrate of 0.2 ml/min. The column was fitted to the HPLC
system described above.
Electrophoresis
The SDS polyacrylamide gel electrophoresis was
performed (Laemmli, 1970) using single dimension 1.5 mm


-84-
thick slab gels, 9.73% acrylamide, 0.27% bisacrylamide, pH
8.8 for the running gel and 3.85% acrylamide, 0.11%
bisacrylamide, pH 6.8 for the stacking gel. The conditions
for the analyses are described in detail in the Hoefer
Scientific Instruments Catalog (Hoefer Scientific
Instruments, San Francisco, CA).
Isoelectric focusing gels were purchased as 1.0 mm
thick prepared gels, Ampholine PAGplates pH 3.5-9.5 (LKB)
and were run using an LKB Multiphore system according to
instructions provided with the gels. The isoelectric point
of the enzyme was determined by comparison with standard
proteins. Enzyme activity in the gel was determined by
sectioning the gel into 0.5 cm slices which were incubated
overnight with 200 ul of alginate under standard conditions.
Samples (100 ul) of the solutions were withdrawn and assayed
for unsaturated products. Proteins present in both SDS and
isoelectric focusing gels were fixed with acetic
acid/ethanol/water (1:5:4) and were visualized by staining
at 65 C for 30 min with Coomassie brilliant blue R-250, 0.46
g/400 ml of destain solution (acetic acid/ethanol/water,
1:2.5:6.5) and destaining of the gels with several changes
of destain solution.
Circular Dichroism Spectroscopy
Analyses were performed using a Jasco J500C
Spectropolarimeter. The scan speed was 20 nm/min, at a
sensitivity of 1 mdeg/cm using a spectral bandwidth of 1 nm


-85-
and a time constant of 2 sec. The samples were contained in
a 0.1 cm pathlength cuvette. Data processing was
accomplished with an Oki IF 800 Model 30 computer to provide
scan averaging and molar elipticity values.
Amino Acid Composition
Purified alginate lyase was dialyzed against water and
concentrated using a Centricon-10 unit (Amicon), lyophilized,
and hydrolyzed in 6 N HC1 under N2 in sealed tubes for 24 h
at 100 C. The amino acids were resolved and quantified
using a Beckman 6300 Amino Acid Analyzer with a Nelson
Analytical Data Acquisition System. The amino acid analyses
were carried out by B. Parten and B. Dunn in the Department
of Biochemistry at the University of Florida.
The content of cystine plus cysteine was obtained after
hydrolysis in the presence of dimethylsulfoxide, which
converts these amino acids to cysteic acid (Spencer and
Wold, 1969). Tryptophan content was estimated after
hydrolysis in the presence of 4% t'nioglycolic acid
(Matsubara and Sasaki, 1969).
Serine and threonine are knov/n to be degraded slightly
under the conditions of hydrolysis, and their levels were
estimated by extrapolation to 0 h of hydrolysis from their
levels at 24 and 48 h of hydrolysis (Hirs et al.,
1954) .


-86-
N-Terminal Amino Acid Sequence
Sequence analyses were carried out by B. Parten and B.
Dunn. An Applied Biosystem Model 470A Gas Phase Protein
Sequencer was used for automated Edman degradation. The
program (02RPTH) ran 30 cycles with 2 nmole protein. The
repetitive yield was 94% and the initial yield was 70-80%
with a myoglobin standard. The PTH-amino acids were
identified and quantified using reversed phase HPLC with a
Waters Model Trimod system including a 721 Programadle
System Controller, 730 Data Module, and a WISP 710B
automatic injector, using a Waters Model 440 Absorbance
Detector to monitor absorbance at 254 nm. A Novapak C-18
column (3.9 x 150 mm) was eluted with a gradient of
methanol, 10-90% in 0.025% acetic acid, to resolve the PTH-
amino acids.
Results
Purification of Poly(ManA) Lyase
Table 4-1 summarizes the purification procedures. The
first step in the purification of the enzyme from the crude
extract, precipitation with PEI (see Fig. 4-1 for titration
of soluble activity) followed by'elution of enzyme activity
with 0.1 M sodium hydrogen phosphate containing 0.25 M NaCl,
separates a large amount of acidic carbohydrate from the
enzyme activity, decreases the viscosity of the preparation
and allows a greater amount of activity to be fractionated
on the Sephadex column.


Table 4-1. Purification of poly(ManA) lyase.
Fraction
Total3
activity
units
Totalb
protein
ug
Specific
activity
units/ug
Yield
%
Fold
pur i ficationc
1.
Crude
17,000


100
2.
PEI eluate
9,000
9,300
0.97
53
1.0
3.
Sephadex G-75
7,300
2,700
2.7
43
hJ

00
4.
Mono Q-l
6,700
260
26
39
27
5.
Mono Q-2
5,100
110
46
30
47
6.
TSK
4,100
86
48
24
49
aBased on the TBA assay for unsaturated nonreducing termini generated, as described in the
Materials and Methods. The initial crude preparation was derived from 431 of growth
med i urn.
^Assayed according to Bradford (1976) with bovine serum albumin as the standard protein.
cCalculated relative to the PEI eluate. The high level of acidic carbohydrate in the
crude extract prevented assay of its protein content.
87-


ABSORBANCE (548nm)
-88-
Figure 4-1. Titration of soluble alginate lyase activity
with addition of 10% polyethyleneimine. A
crude extracellular preparation was concen
trated 300-fold and dialyzed against water
using the Pellicon system, as described in the
Materials and Methods. Samples (100 ul) of
the preparation were vortexed at 0 C with
added volumes of PEI, the precipitates were
removed by centrifugation (13,000 x g, 5 min),
and the supernatant solutions (10 ul samples)
were assayed for enzyme activity (absorbance
at 548 nm).


-89-
Chromatography on Sephadex G-75 separated alginate
lyase activity as a single peak at approximately 0.5 column
volumes (see Fig. 4-2) from some of the larger contaminating
proteins which eluted in the void volume of the column.
Remaining alginate degradation products eluted near the
total column volume (data not shown) and were detected by
absorbance at 232 nm and oxidation with periodate to TBA
reactive compounds. The enzyme activity was completely
dependent upon the addition of exogenous substrate after
this step.
Anion exchange HPLC using a Mono Q column afforded a
purification of 27-fold. Less than 1 h was required for
recovery of the enzyme. The conditions shown in Fig. 4-3
allowed optimum separation of enzyme activity, which eluted
at approximately 0.4 M NaCl, from proteins with similar
charge properties. The procedure was repeated one time with
slight (1.8-fold) improvement in specific activity (Fig.
4-3b) .
A final step in the purification was gel filtration
HPLC using a Biosil TSK-G4000 SW column (Fig. 4-4, panel a).
Although this procedure yielded only 1.04-fold purification
(Table 4-1) of the enzyme for the preparation considered
here, in purification of other batches of enzyme the fold
purification was as high as 1.2-fold, and the step has
been used routinely in the purification sequence. Samples
containing up to 250 ug of protein from the Mono Q column
have been successfully purified with the TSK column, and the


280
-90-
TUBE NUMBER
Figure 4-2. Fractionation of alginate lyase activity by
Sephadex G-75 chromatography. A preparation
of activity which was obtained from the PEI
procedure was subjected to gel filtration
chromatography using Sephadex G-75, as
described in the Materials and Methods.
Enzyme activity ^543) and a relative measure
of protein (A28O) were determined for frac
tions which were collected, and the fractions
containing alginate lyase activity were pooled
and concentrated by pressure ultrafiltration
with an Amicon stirred cell.


A 2 80
-91-
0.2
0.1
0
0.2
0.1
0
0 16 32 48 64
RETENTION TIME (min)
Figure 4-3. Anion exchange HPLC of poly(ManA) lyase. A
preparation containing 730 units of activity
and 270 ug protein from the Sephadex G-75 column
was fractionated with a Mono Q column using a
gradient of NaCl (up to 1.0 M, top portion of
gradient not shown) to elute the activity (panel
a). The single peak which possessed alginate
lyase activity is indicated by an arrow.
Activity peaks from three column runs were
pooled, concentrated and desalted, and this
combined preparation was subjected again to
anion exchange chromatography with the Mono Q
column (panel b).


-92-
RETENTION TIME (min)
Figure 4-4. Gel filtration HPLC and SDS-polyacrylamide gel
electrophoresis of poly(ManA) lyase. A sample of
lyase activity (10 ug of protein) from the second
pass through the Mono Q column was analyzed by gel
filtration HPLC on a TSK4000 column as described
in the Materials and Methods section (panel a).
The elution of standard proteins shown for com
parison are as follows: 1, 6-amylase, 200 kDa;
2, bovine serum albumin, 66 kDa; 3 egg albumin,
45 kDa and 6-lactoglobulin, 37 kDa; 4, carbonic
anhydrase, 29 kDa; 5, trypsinogen, 24 kDa; 6,
lysozyme, 14.3 kDa.
Activity from the second pass through the
Mono Q column was denatured in the presence of
SDS and 2-mercaptoethanol and analyzed in the
presence and absence of standard proteins (panel
b). Lane 1 contains standard proteins only,
lane 2 contains 10 ug poly(ManA) lyase, and
lane 3 contains lyase plus standards.


Full Text

ATALYTIC AND STRUCTURAL PROPERTIES OF ALGINATE
LYASES FROM BACTERIAL EPIPHYTES OF
SARGASSUM (PHAEOPHYCEAE)
By
TONY ROMEO
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
1986

ACKNOWLEDGEMENTS
I offer my sincere gratitude to Dr. James Preston who
has provided immeasurable encouragement, support, and
guidance to me throughout my graduate studies. The members
of my graduate committee have offered a great deal of
helpful advice for which I am grateful: Dr. L. Ingram, Dr.
W. Gurley, Dr. H. Aldrich, and Dr. P. McGuire. I also wish
to thank Dr. J. Gander for valuable advice and comment on
parts of this work. The students in Dr. Preston's lab have
been good friends and helpful colleagues to me. I wish them
success and happiness.
The skilled efforts of Donna Huseman in preparing the
figures for this work and Adele Koehler in typing the
manuscript are greatly appreciated.
Jeanette Reinhardt provided electron microscopy of
Sargassum protoplasts. B. Parten and Dr. B. Dunn carried
out amino acid analyses and N-terminal sequence analysis of
alginate lyase. The NMR analyses of substrates were
obtained by Sandra Bonetti and Cynthia Jackson. The amino
acid sequence similarity search was conducted by Dr. Michael
Little in the Dept, of Biochemistry of the- University of
Arizona. J.C. Bromley, D.R. Preston, and J. Beiswanger
provided assistance in culturing and harvesting bacteria for
enzyme isolations. All of these
valuable to my work.
contributions have been

for her
I would
patience and
like to thank my beloved wife, Lori,
moral support.
in

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
ABSTRACT vi
CHAPTER
I OBJECTIVES AND BACKGROUND 1
Objectives and Rationale 1
Background 3
IIALGINATE LYASES FROM BACTERIAL EPIPHYTES OF
SARGASSUM: SUBSTRATE SPECIFICITIES, MECH¬
ANISMS OF ALGINATE DEPOLYMERIZATION, ATTEMPTS
TO FORM PROTOPLASTS FROM SARGASSUM BY LYASE-
MEDIATED DEGRADATION OF CELL WALLS 18
Introduction 18
Isolation, Properties, and Growth of Alginate
Lyase Producing Bacteria 19
Intracellular and Extracellular Alginate
Lyases: Substrate Specificities and
Cleavage Patterns 24
Digestion of Alginate Present in Sargassum
Tissues by Alginate Lyases 30
Preparation of Protoplasts by Mechanical
Disruption of Tissue 37
Discussion 39
IIIHPLC ANALYSIS OF THE DEPOLYMERIZATION OF
(1-4)-8-D-MANNURONAN BY EXTRACELLULAR AND
INTRACELLULAR ALGINATE LYASES FROM A MARINE
BACTERIUM 46
Introduction 46
Experimental 48
Results and Discussion 56
IV

Page
IVPURIFICATION AND STRUCTURAL PROPERTIES OF AN
EXTRACELLULAR (1-4)- 3-D-MANNURONAN SPECIFIC
ALGINATE LYASE FROM A MARINE BACTERIUM 78
Introduction 78
Materials and Methods 79
Results 86
Discussion 104
VDEPOLYMERIZATION OF ALGINATE BY AN EXTRA¬
CELLULAR ALGINATE LYASE FROM A MARINE BAC¬
TERIUM: SUBSTRATE SPECIFICITY, ACCUMULATION
OF REACTION PRODUCTS, AND EFFECTS OF PRODUCTS
ON THE REACTION RATE Ill
Introduction Ill
Materials and Methods 113
Results 116
Discussion 128
VI CONCLUSIONS 138
REFERENCES 140
BIOGRAPHICAL SKETCH 151
V

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
CATALYTIC AND STRUCTURAL PROPERTIES OF ALGINATE
LYASES FROM BACTERIAL EPIPHYTES OF
SARGASSUM (PHAEOPHYCEAE)
by
Tony Romeo
August 1986
Chairman: James F. Preston, III
Major Department: Microbiology and Cell Science
Enzymes which catalyze depolymerization of alginate,
the major cell wall polymer of brown algae (Phaeophyceae)
were isolated and characterized from bacteria which were
part of the epiphytic flora of healthy tissues of Sargassum
species. Both Gram negative obligate aerobes and
facultative anaerobes produced activities with a range of
substrate specificities. Extracellular preparations of the
bacteria were highly endolytic while cell extracts were more
exolytic in their overall mechanisms. The activities were
capable of extensive degradation of the alginate present in
tissues of Sargassum.
A method for analysis of the depolymerization of (1-4)-
6-D-mannuronan (poly(ManA)) by high performance liquid
chromatography (HPLC) demonstrated that the single
extracellular alginate lyase from a fermentative isolate
generated unsaturated oligouronides which included dimeric
vi

through pentameric products. The trimer was the major
product and could not be further depolymerized; the tetramer
was converted to trimer at a low rate; the pentamer was
readily converted to trimer and dimer forms. The
intracellular preparation differed in that it generated a
considerable amount of monomer.
The extracellular alginate lyase was purified to
chromatographic and electrophoretic (SDS-PAGE) homogeneity
by gel filtration on Sephadex G-75, anion exchange HPLC on a
Mono Q HR 5/5 column, and gel filtration HPLC on an Ultrapac
TSK-G4000SW column. The enzyme was composed of a single
polypeptide of 29 kDa. The amino terminal sequence was
determined through the first 6 amino acids, and 19 of the
first 30 amino acids were assigned. Several closely
migrating forms were separated by isoelectric focusing,
which have pi values ranging from 4.2 to 5.0, suggesting
posttranslational modification. The secondary structure was
approximately 74% ct-helix by CD spectroscopy.
The extent of depolymerization of well characterized
alginates and block regions of alginate by the purified
enzyme was strongly correlated with the frequency of
mannuronic acid triad in the polymers and not to the
mannuronic acid diad frequency or mannuronic acid content,
suggesting a minimal recognition site of three sequential
residues. Unsaturated trimer and tetramer end products of
the reaction did not show appreciable inhibition of
activity. A model for the specificity of the active site of
the enzyme is presented.
vi 1

CHAPTER I
OBJECTIVES AND BACKGROUND
Objectives and Rationale
The general objective of these studies was to identify
and characterize enzymatic systems capable of depolymerizing
the major cell wall polymer of the brown algae (Phaeo-
phyceae), alginate. A major impetus for the work has been a
need for methods which would allow members of this group of
organisms, in particular, species of the genus Sargassum, to
be readily cultured. A long range goal for this project has
been the development of systems of introduction of new
genetic material into the Sargassum species to alter their
growth characteristics and/or biochemical properties for
improvement of their biomass potential. Identification of
enzymes which specifically degrade the carbohydrate polymers
present in cell walls of the brown algae should allow
methods for producing protoplasts of the algae to be
developed. As has been the case with work on the higher
plants, this capability should simplify procedures for
culturing these organisms and allow the development methods
for the eventual genetic manipulation of Sargassum species.
The isolation and thorough characterization of enzymes
capable of catalyzing depolymerization of alginate will also
-1-

-2-
provide specific tools for analysis of brown algal cell wall
structure and biosynthesis and for modification and analysis
of alginate structure. Fermentative bacterial isolates
which degrade alginate may prove useful in improving the
rate and/or extent of conversion of brown algal tissue to
methane during anaerobic digestion.
The specific objectives of this project were to
1) Isolate bacteria from Sargassum tissue, which are capable
of growth upon alginate as a sole carbon and energy source,
and evaluate the secreted and the intracellular enzymatic
activities which catalyze depolymerization of alginate.
This included measurements of relative levels of enzyme
activities produced by each isolate, determination of
substrate specificities and general mechanisms, i.e., endo-
or exolytic nature of the activities which depolymerize
alginate, and comparisons of the abilities of various
preparations to degrade Sargassum tissues. 2) Develop
methods which permit the depolymerization of alginate to be
quantitatively and conveniently analyzed, including methods
for identification and quantification of specific products,
determination of the rates of product accumulation and/or
depletion, and determination of the limit products of a
particular depolymerization reaction. 3) Isolate and
purify at least one of the bacterial alginate lyases to
homogeneity and characterize its subunit composition,
primary and secondary structure, and its mechanism of
substrate depolymerization.

-3-
Background
Biomass Potential of Phaeophyceae
The marine brown algae inhabit oceanic coastal waters
throughout the world. Although several species have been
commercially exploited for their anionic carbohydrates, in
particular the alginates, the bulk of the world's supply
remains untapped. The giant kelp Macrocystis pyrifera has
been evaluated for its bioconversion to methane and shown to
provide methane yields that are competitive with other
biomass and waste sources (Chynoweth et al., 1981). Studies
are in progress to determine the feasibility of farming
Macrocystic pyrifera (Neushul, 1977) and Laminaria
saccharina (Brinkhuis et al., 1984) for the production of
feedstocks for methane generation. Species of the genus
Sargassum represent an alternative source which includes
benthic species common to the colder waters and pelagic as
well as benthic species found along the coast of Florida and
other subtropical and warmer temperate waters. Trawling
collections of the pelagic species, S. natans and £3.
fluitans, have placed their estimated biomass in the
Sargasso sea alone at 4 to 40 million metric tons (Parr,
1939). The high carbohydrate content of these algae and
their present lack of commercial exploitation make them
attractive as a potential source of biomass for conversion
to methane.

-4-
Carbohydrate Polymers of Phaeophyceae
The brown algae produce large amounts of carbohydrates,
e.g., almost 50% of the dry weight of the giant kelp,
Macrocyst is pyrifera (Chynoweth et al., 1981). The
functions of the major carbohydrates are, in general,
related to the roles in maintenance of structural integrity
or in supplying short or long term energy reserves for the
plants. The present discussion will focus upon the
structural carbohydrates which are localized in the cell
walls of the organisms and are depicted in Fig. 1-1.
The major wall component is alginate, a linear 1-4
polymer of B-D-mannuronic acid and a-L-guluronic acid which
generally comprises 10 to 25% of the dry weight of brown
algae (Table 1-1). Alginate is a compound of considerable
commercial importance; over 8,000 tons are utilized annually
in the United States (Wells, 1977). The commercial value of
alginate has been partially responsible for generating
interest in alginate; a relatively large body of information
has been obtained regarding its fine structure and solution
properties.
Studies of Haug and coworkers have established that the
two constituent uronic acids of alginate are arranged into
homo- and heteropolymeric block regions of DP (degree of
polymerization) around 20 for the homopolymers, which are
interspersed in native alginate (Haug et al . , 1966; Haug et
al. , 1967). These polymers were obtained in relatively pure
form by mild acid hydrolysis of alginate, followed by

-5-
STRUCTURAL CARBOHYDRATES
OF PHAEOPHYTA
H
Figure 1-1. Chemical structures of the three polymers
which comprise the bulk of marine brown algal
cell walls (Phaeophyceae). Unit saccharides,
glycan bonds, and modifications of the
saccharides are indicated. Configurational
aspects of structures are not implied.

-6-
Table 1-1. Alginate levels in Phaeophyta species.
Species
Alginic acid % dry wt.
S. fluitans
1—'
00
•
00
D>
S. natans
19.9a
S. filipéndula
13.3-23.5b
S. polyceratium
20.3a
S. vulgare
17.9a
L. cloustoni
14-22c
M. pyrifera
14. ld
a ,b ,c ,dVa]_ues are from Aponte de Otaola et al . (1983), Davis
(1950), Black (1950), and Chynoweth et al . (1981).

-7-
fractionations based upon differential acid solubility of
the individual polymers. Isolated mannuronan regions,
poly(ManA), and guluronan regions, poly(GulA), have been
subjected to x-ray crystallographic studies which indicated
that the configuration the poly(ManA) resembles a flattened
ribbon, whereas that of the poly(GulA) forms a buckled chain
(Atkins et al., 1971). Nuclear magnetic resonance analyses
of alginate (Penman and Sanderson, 1972) suggest the Cl
conformation for the 3-linked mannuronic acid residues,
indicating diequiator ial linkages to adjacent residues, and
the 1C conformation for the a-linked guluronic residues,
which would form diaxial linkages, further supporting the
polymer conformations which were based upon x-ray crystal¬
lography .
The conformations of the block regions of alginate and
their relative abundance in the native polymer have
important effects on the solution properties and biological
functions of alginate. Only the poly(GulA) regions of
alginate bind to calcium and certain other divalent ions
with high affinity, thus rendering the purified poly(GulA)
insoluble or causing the native polymer to form a gel
(Smidsrod and Haug, 1965; Haug and Smidsrod, 1965a; Haug and
Smidsrod, 1965b; Kohn et al . , 1968). Circular dichroism
studies of the interaction of calcium with poly(GulA)
sequences suggest that calcium mediates cooperative
interactions of regions containing at least 20 sequential
guluronic acid residues and thereby allows stable interchain

-8-
dimerization to occur. The investigators of this process
have developed a model to describe the calcium guluronate
complexes which has been dubbed the "egg box model" (Grant
et al. , 1973; Morris et al., 1978 ; Rees et al., 1982). A
gel or three dimensional lattice is formed by interaction of
native alginate with calcium ions, wherein insoluble calcium
guluronate complexes are flanked by the soluble poly(ManA)
regions and heteropolymeric poly(ManA, GulA) regions. Since
the brown algae in almost all instances are marine
organisms, alginate is present in the cell walls as a gel
containing a mixture of metal ions, although due to the high
selectivity of alginate for calcium, and the relatively high
concentration of calcium ions in seawater; this is probably
the most prevalent ion (Percival and McDowell, 1967).
The relative amount of poly(GulA) present in alginate
will affect its gelling properties, and this can vary
depending upon the source of the alginate (Penman and
Sanderson, 1972; Haug et al., 1974). Some species of brown
algae have been shown to form tough, firm, holdfast tissues
using alginate with high levels of guluronic acid and in the
same plant produce flexible apical tissues of alginate
containing high levels of mannuronic acid (Haug et al.,
1974; Andresen et al., 1977).
The algae are apparently capable of increasing the
poly(GulA) content of the alginate in a given part of the
plant as it ages (Haug et al . , 1974). This might be
accomplished by either degrading the alginate originally

-9-
present and replacing it with new polymers of different
structure or by altering the uronic acid composition of the
polymer which is already present. Laminaria d ig i tata has
been shown to contain an enzyme which depolymerizes alginate
(Madgwick et al . , 1973a) allowing for the former
possibility; the presence of an epimerase capable of
converting mannuronic acid residues of alginate to guluronic
acid has been detected in Pelvetia canaliculata (Madgwick et
al., 1973b), allowing for the latter mechanism. A
combination of these two means of altering alginate
composition may also occur. Interestingly, a mannuronan-
epimerase from Azotobacter vinelandii has been purified, and
it has been shown to be capable of altering the distribution
of the guluronic acid residues which it generates, in
response to calcium ion concentration (Skajak-Braek and
Larsen, 1985). Perhaps the activities of algal epimerases
are also responsive to conditions which may require the
formation of alginates with varying structural and
functional properties.
A polymer which is quite similar to alginate of the
brown algae is produced by certain bacteria, including
Azotobacter vinelandii (Larsen and Haug, 1971) and certain
isolates of Pseudomonas aeruginosa (Evans and Linker, 1973) .
These bacterial alginates differ from the algal polymer in
containing acetylations of certain hydroxyl groups. The
isolates of P. aeruginosa are of interest in that alginate
producing organisms are frequently isolated from patients
with cystic fibrosis, where the organisms and the alginate

-10-
which they produce are believed to increase the morbidity of
the disease (Hoiby et al. , 1977). Studies of the immune
response of cystic fibrosis patients to alginate (Bryan et
al . , 1983; Speert et al., 1984) and the genetics of alginate
biosynthesis by P. aeruginosa (Roehl et al., 1983; Banerjee
et al. , 1985; Goldberg and Ohman, 1984) are areas of recent
interest which should increase our understanding of the
chemical properties and biosynthesis of alginate.
Fucoidin or fucoidan is a sulfated fucose polymer,
which is found in levels from less than 1% of the dry tissue
weight for species such as Macrocyst is pyrifera (Chynoweth
et al . , 1981) to 24% for species such as Fucus spirilis and
Pelvetia canaliculata which are extensively exposed to air
during growth (Percival and McDowell, 1967). It consists of
a,l -> 2 linked L-fucose with sulfate esterified primarily
at position 4 (Fig. 1-1). The molecule is probably branched
at positions 3 and 4 and as isolated, may contain other
saccharides, including galactose, xylose, and uronic acids,
and metal ions as well (Percival and McDowell, 1967).
Fucoidin is believed to reside in extracellular mucilaginous
material and non-fibriliar portions of the wall, and as a
result of its hygroscopic properties, may protect the brown
algae against dehydration (Percival and McDowell, 1967;
McCully, 1966; Evans et al., 1973).
Cellulose is a 3,1 -> 4 unbranched glucan which is an
important structural component of higher plant cell walls
and is consistently found in the cell wall of brown algae,

-11-
in levels which range from about 1 to 10% of the dry weight
of the plant (Percival and McDowell, 1967). Studies on the
histology of the walls of Fucus have identified crystalline
components which consist at least in part of cellulose
(McCully, 1970). Studies on the zygotes of Fucus indicate
that the shape is maintained to a large extent by cellulose.
Procedures which extract the other components, including
alginate, leave an intact sack-like structure which retains
the original form of the zygote (Quatrano and Stevens, 1976).
Enzymatic Depolymerization of Alginate
Alginate has been shown to be chemically depolymerized
by acid hydrolysis, base-catalyzed 3-elimination, and by a
free radical mechanism (Haug et al., 1963; Smidsrod et al.,
1963; Smidsrod et al., 1965). Although enzyme activities
capable of depolymer izing alginate were reported as early as
1934 by Waksman et al., the first definitive study of
alginate depolymerization which identified the 3-elimination
reaction as the mechanism of bond cleavage was presented by
Preiss and Ashwell in 1962. This established that the
enzyme(s) responsible was a lyase, as opposed to a
hydrolase. Since that time virtually all alginolytic
enzymes examined, with one exception (Stevens and Levin,
1976a; Stevens and Levin, 1976b), have proven to be lyases.
This type of reaction also has been observed for enzymes
which depolymerize other uronic acid containing polymers,
including pectin and pectic acid (Collmer et al.,
1982;

-12-
Fogarty and Kelly, 1983), heparin (Yang et al., 1984),
chondroitin (Linn et al., 1983), the acidic heteropoly¬
saccharides of Rhizobium trifo 1 i (Hollingsworth et al.,
1984), and many others.
The elimination reaction catalyzed by these lyases
relies upon the electron withdrawing carboxyl group of the
substrate (C-6), an extractable a-proton (at C-5), and an 0-
linked uronide (at C-4) which is the leaving group (Kiss,
1974). In the case of endolytic cleavage (internal to the
polymer), the new reducing terminus of the products is
identical to that which would be produced hydrolytically,
and the new nonreducing terminus is a 4,5 unsaturated
residue (see Fig. 1-2).
Assays for bond scission by alginate lyases measure
either generation of reducing or nonreducing termini.
Unsaturated termini absorb UV with a maximum at
approximately 232 nm (Preiss and Ashwell, 1962a), although
the unsaturated monomer, 4-deoxy-L-erythro-5-hexoseulose
uronic acid does not do so appreciably. A specific and
sensitive assay for both unsaturated monomer and unsaturated
nonreducing terminal residues is based upon the spectrophoto-
metric determination at 548 nm of the chromogen generated
upon the reaction of 2-th iobarbi tur ic acid (TBA) with
periodate treated products (Preiss and Ashwell, 1962a).
Alginate lyases have been shown to be produced by and
in some cases isolated and characterized from marine
invertebrates (Nakada and Sweeny, 1967; Favorov and

-13-
Gul
Gul Man
Alginate lyase
t
Gul Gul Man Unsat.
Gul
Figure 1-2. The alginate lyase reaction as catalyzed by
an endo-poly(ManA) lyase. A new reducing end,
indistinguishable from that formed by
hydrolysis, and a 4,5 unsaturated nonreducing
end which confers UV absorbance properties
and reactivity of the lyase products in the
TBA assay are the result. The configurations
of the poly(GulA) and poly(ManA) regions of
alginate are indicated.

-14-
Vaskovsky, 1971; Elyakova and Favorov, 1974; Favorov et al.,
1979; Muramatsu et al., 1977; Muraraatsu, 1984; Jacober et
al., 1980), fungi (Wainwright and Sherbrock-Cox, 1981),
marine bacteria (Kashiwabara et al., 1969; Fujibayashi et
al., 1970; Min et al., 1977; Davidson et al . , 1976; Quatrano
and Caldwell, 1978; Doubet and Quatrano, 1982; Doubet and
Quatrano, 1984; Pitt and Raisbeck, 1978; Southerland and
Keene, 1981; Preston et al., 1985a), terrestrial bacteria
(Boyd and Turvey, 1977; Boyd and Turvey, 1978; Hansen et
al., 1984), and brown algae (Madgwick et al., 1973).
The work of Nakada and Sweeny (1967) initially
demonstrated that an alginate lyase from abalone showed a
preference for alginate containing a high mannuronic acid
content, and a second enzyme from the same source was more
active with alginates that were high in guluronic acid. All
alginate lyases which have been subsequently examined are
selective in their substrate specificities; i.e., no enzyme
has been isolated which shows significant activity upon both
poly(ManA) and poly(GulA). However, there are reports of
two lyases which will cleave both the GulA-GulA bonds of
poly(GulA) and the GulA-ManA bonds of poly(ManA, GulA) (Boyd
and Turvey, 1978; Min et al., 1977).
Alginate lyases may attack their substrates by either
endolytic or exolytic mechanisms, keeping in mind that some
polyuronide degrading enzymes act by making apparent random
initial endolytic cleavages followed by non-random cleavages
in the later stages of substrate depolymerization (Thibault,

-15-
1983). The mechanism of substrate attack by alginate lyases
was first examined by viscometric analyses (Nakada and
Sweeny, 1967) and thence has also been examined by analysis
of reaction products, using methods which have included
conventional gel filtration and ion exchange column
chromatography (Boyd and Turvey, 1977; Favorov et al.,
1979), paper electrophoresis (Davidson et al., 1977), and
gel electrophoresis (Doubet and Quatrano, 1984).
The rigorous examination of the catalytic activities
and structural properties of an enzyme require its
purification and characterization at least to an extent
which establishes that a single activity has been obtained.
Preferably, the enzyme will have been purified to a
single homogeneous protein. Alginate lyase enzymes have,
however, in most cases been analyzed in impure states.
Davidson et al. (1976) reported that they had purified a
poly(GulA) lyase from a marine bacterium, although the data
which were presented did not establish their claims.
Enzymes from marine molluscs have been purified, all of
which were poly(ManA) lyases (Elyakova and Favorov, 1974;
Muramatsu et al., 1977). Recently, a bacterial poly(GulA)
lyase was purified to electrophoretic homogeneity (Doubet
and Quatrano, 1984). Studies of Muramatsu and coworkers on
two isoenzymes from the snail Turbo cornutus represent the
only thorough studies of the structural properties of
alginate lyases up to this time (Muramatsu and Egawa, 1982;
Muramatsu et al . , 1984 ). These two enzymes were each
composed of a single subunit of 32,000 kDa by SDS

-16-
polyacrylamide gel electrophoresis. Native masses of 25 kDa
were established by Sephadex G-100 chromatography. The
isoelectric points were 7.5 and 7.7 for SP1 and SP2 enzymes,
respectively. The enzymes were glycoproteins and were
composed primarily of 3-sheet secondary structure.
Enzymatic Digestion of Brown Algal Cell Walls
The presence of cellulose in the cell wall of the brown
algal wall suggests a requirement for cellulases, in
addition to alginate lyases specific for each of the block
regions of alginate, to effect complete digestion of wall
material. Quatrano (1982) reported that the cellulose which
is deposited during early development of Fucus is subject to
depolymerization by cellulases. The cellulases have been
extensively studied (Enari, 1983) and are readily available
from a variety of commercial sources.
Although the fucoidin of the cell wall may not
contribute to the shape or structural base of brown algal
tissue, it may pose a barrier to cellulases and alginases
and thereby prevent or impede the removal of the cell wall.
There are reports of bacteria having been isolated which
produce enzymes capable of depolymerizing fucoidin (Quatrano
and Caldwell, 1978; Morinaga et al., 1981; Yaphe and Morgan,
1959). However, no such enzymes have been isolated and
examined in detail.
There is a recent report of a method for enzymatically
removing the cell walls of Sargassum species to generate

-17-
protoplasts by the use of extracts of the hepatopancreas of
abalone (Preston et al . , 1985b). The method is, however,
not yet reproducible, and the origin(s) of the difficulties
are at this time unknown. A reasonable approach to
developing reliable methods for obtaining protoplasts from
Sargassum species might be to fractionate and reconstitute
extracts of abalone. Individual enzymatic factors necessary
for generation of protoplasts might then be identified and
isolated more reproducibly from bacterial and/or fungal
sources .

CHAPTER II
ALGINATE LYASES FROM BACTERIAL EPIPHYTES OF SARGASSUM:
SUBSTRATE SPECIFICITIES, MECHANISMS OF ALGINATE
DEPOLYMERIZATION, ATTEMPTS TO FORM PROTOPLASTS FROM
SARGASSUM BY LYASE-MEDIATED DEGRADATION OF CELL WALLS
Introduction
Certain members of the brown algae, or Phaeophyceae,
have been exploited for their anionic carbohydrates,
specifically alginate, for many years (Percival and
McDowell, 1967; Steiner and McNeely, 1954). However, the
following studies were prompted by the more recent interest
in their potential as a source of biomass for conversion to
methane (Preston et al . , 1985b). Species of Sargassum are
particularly attractive in this regard, as they are not
commercially exploited at present; i.e., there are no
competitive uses for them, and they contain significant
quantities of the carbohydrates alginate (Aponte de Otaola
et al . , 1983) and mannitol (Preston and Jiminez, 1986). The
ability to reproducibly generate protoplasts from Sargassum
species would aid efforts to develop methods for tissue
culture and for genetic improvement of these algae (Preston
et al., 1985b).
The goal of the work described in this chapter has been
to explore potential enzyme systems for degrading the cell
-18-

-19-
walls of Sargassum. In particular, alginate lyases, which
depolymerize the major wall polymer, alginate, have been
assessed for their activities on Sargassum cell walls and
for their capacities to generate protoplasts of Sargassum
species.
The natural bacterial flora of Sargassum have provided
the source of the alginate degrading enzymes for this study.
The substrate specificities and overall mechanisms of the
intracellular, or cell-bound enzymes, and of the secreted,
or extracellular enzymes, of the bacteria have been
examined. The effects of these activities on algal tissue
have been quantified by a sensitive and specific assay for
the unsaturated nonreducing terminal residues which are
generated by the eliminative cleavage of alginate by
alginate lyases (Preiss and Ashwell, 1962a). This has
allowed comparisons to be made of the efficacies of
individual alginate lyase preparations, which differ in
substrate specificities and mechanisms, to remove alginate
from Sargassum cell walls.
Isolation, Properties, and Growth of Alginate
Lyase Producing Bacteria
Viable tissues of Sargassum natans and Sargassum
f1uitans provided the source for isolation of alginate lyase
producing bacteria, except in the case of isolate FM, which
was obtained from decaying Sargassum. The algae were
transported from the Atlantic Coast of Florida to

-20-
Gainesville, Florida, where identifications were confirmed,
voucher specimens saved, and epiphytic bacteria isolated.
Sargassum tissue (1 g quantities) was subjected to mild
sonication in sterile sea water (Instant Ocean from Aquarium
Systems, Mentor, OH) to dislodge epiphytic bacteria.
Dilutions of the sea water were plated onto solid alginate
medium (2% agar, 1% sodium alginate in PESI, Provasoli's
enriched seawater, supplemented with 0.27 g/L iodine)
(Provasoli, 1968; Polne-Fuller et al., 1984). Colonies
which exhibited substantial clearing of the calcium alginate
haze of the medium after several days of growth were
selected for further studies. Purity of the cultures has
been established by subculturing on solid alginate medium,
growth in liquid alginate medium (0.1% sodium alginate, PESI
containing 1.0 mM calcium and 5.5 mM magnesium), and growth
on a rich solid medium (2% agar, 1% glucose, 0.8% nutrient
broth, 1% yeast extract in PESI).
Some of the morphological and physiological properties
of the bacteria have been described (Preston et al., 1985a)
and along with other properties are shown in Table 2-1. All
isolates are Gram negative, polarly flagellated rods. Of
seven organisms isolated, four are oxidative and three
fermentative. All oxidative organisms are oxidase positive
and all fermentative isolates oxidase negative. All
fermentative isolates, but none of the oxidative bacteria,
produce acid in liquid glucose medium (Table 2-1). None of
the isolates showed evidence of gas production on glucose or

Table 2-1. Morphological and biochemical properties of alginase secreting bacteria
associated with Sargassum species.
Isolate
Morphology3
Oxidase0
Reaction
Glucose0
+o2 -o2
Alginate°
+°2 -°2
PH6
S012382 FM
short
rod
+
+
_
+
_
5.84
SFFB080483
A
0.9-1.1
X
1.6-2.2
-
+
+
+
+
4.69
SNFB080483
B
0.5-0.6
X
2.1-2.6
+
+
-
+
-
6.61
SNFB080483
C
0.7-0.9
X
1.6-2.0
+
+
-
+
-
6.91
SNFB080483
D
0.4-0.6
X
1.4-2.1
-
+
+
+
+
5.27
SFFB080483
F
0.5
X
1.2-2.0
+
+
-
+
-
6.73
SFFB08048 3
G
0.5
X
1.1-1.3
+
+
+
+
+
4.98
aMorpho1ogies and dimensions in urn determined by measurements from scanning electron
micrographs with the exception of isolate FM, which was analyzed with the optical
microscope. All isolates were Gram negative.
^Oxidase reactions were carried out according to methods described by Preston et al.
(1985) .
cGlucose medium formulation consisted of 1% glucose, 0.8% nutrient broth, 1% yeast extract
in PESI. A positive reaction indicates growth relative to controls which had no added
g1ucose.
'^Alginate medium formulation consisted of 0.1% sodium alginate in PESI.
epH determinations were carried out directly on cultures grown on glucose medium incubated
under aerobic conditions for 4 days; uninoculated glucose medium had a pH of 6.53.
21-

-22-
alginate containing media. Morphological, physiological,
and DNA base composition data allowed the assignment of
aerobes to the genus Alteromonas (Preston et al., 1985a).
The fermentative isolates, although morphologically and
physiologically similar to bacteria of the genus
Photobacterium, are excluded from this genus by their DNA
base composition and have not been assigned to any existing
genus.
Figure 2-1 shows the growth of two representative
fermentative isolates, A (SFFB080483 A) and G (SFFB080483
B) , in liquid alginate medium at 22°C with rapid gyrotory
shaking. Duplicate flasks of media were periodically
sampled, the turbidity was measured, and cells were removed
by centrifugation prior to measurements of alginate
utilization and generation of alginate degradation products.
Isolates A and G removed alginate from the medium during
growth, as did all of the isolates selected for these
studies (data not shown) as measured by a loss in total
uronic acid equivalents from the medium (Blumenkrantz and
Asboe-Hansen, 1973). The depolymerization of alginate to
form oligomeric uronides which possessed unsaturated
nonreducing terminal residues, as measured by the method of
Preiss and Ashwell (1962a), occurred during the growth of
the isolates. This indicated that alginate was being
degraded by enzymes which were transeliminases or lyases.

009
-23-
Figure 2-1. Growth of bacterial isolates in liquid alginate
medium. Isolates A (a) and G (b) were cul¬
tured as described in the text. Growth was
monitored by measuring turbidity (A^OO), total
uronic acid equivalents (A^O) and unsaturated
nonreducing terminal residues (A~^®).

-24-
For enzyme isolation, bacteria were grown in Fernbach
flasks containing 1 1 of liquid alginate medium at room
temperature (22°C) with rapid gyrotory shaking and were
harvested at late exponential phase.
Intracellular and Extracellular Alginate Lyases:
Substrate Specificities and Cleavage Patterns
Isolation of Bacterial Enzymes
Bacterial cells were removed from culture medium by
centrifugation, frozen in liquid nitrogen or at -70°C, and
stored at -70°C. For analysis of extracellular enzymes the
spent medium was concentrated by tangential flow filtration
using a Millipore Pellicon cassette system with a
polysulfone (PTGC) membrane which allowed retention of
molecules larger than 10 kDa, and dialyzed against
distilled, deionized water.
For intracellular preparations cells were thawed,
suspended in 4 volumes of ice cold 0.1 M sodium phosphate
buffered at pH 7.5, and disrupted with a French pressure
cell at 16,000 lb in-^. Unbroken cells and cell debris were
removed by centrifugation at 10,000 x g for 15 min, and
acidic polymers were rendered insoluble in the supernatant
solution by adding 5% streptomycin sulfate dropwise with
stirring to a beaker at 0°C to give a final concentration of
2%. After stirring the mixture for 10 minutes at 0°C, the
resulting precipitate was removed by centrifugation and the
supernatant solution containing alginate lyase was treated

-25-
with solid ammonium sulfate (to 65% saturation). The
protein precipitate was pelleted by centrifugation at 10,000
x g, 10 min, 4°C, redissolved in pH 7.5 phosphate buffer,
and dialyzed against distilled deionized water or sodium
phosphate buffered at the desired pH.
Preparation of Substrates
Sodium alginate was purchased from Fisher Scientific
Company as a purified grade originally isolated from
Macrocystis. Prior to use in viscometric determinations
alginate was centrifuged at 100,000 x g for 5 h (Smidsrod
and Haug, 1968). Poly(ManA) and poly(GulA) were obtained
from HC1 hydrolyzed alginate, following the methods
developed by Haug et al. (1967). Preparations of poly(GulA)
and poly(ManA) were further fractionated on Sephadex G-50
with 0.5 M NaCl as eluant, and selected fractions analyzed
by reducing sugar and total carbohydrate assays (Nelson,
1944 ; Dubois et al . , 1956; Haug and Larsen, 1962) and 3H and
13C NMR (Grasdalen et al., 1979; Grasdalen et al., 1981), to
assess uniformity of size and purity of substrates. The
poly(ManA) fraction contained approximately 89% B-D-
mannuronic acid residues and contained polymers with degree
of polymerization (DP) values of 16-20; the poly(GulA)
contained 89% a-L-guluronic acid residues and had an average
DP of 22.

-26-
Substrate Specificities of Intracellular and
Extracellular Preparations
Alginate lyase was quantified by the TBA assay (Preiss
and Ashwell, 1962a; Weissbach and Hurwitz, 1959). Substrate
mixtures contained either 0.1% sodium alginate, poly G, poly
M, or no carbohydrate (controls for endogenous substrate),
in 0.05 M KCl, buffered with 0.03 M sodium phosphate from pH
5 to 8, or 0.05 M sodium acetate at pH 4.
With three exceptions, activities of the enzyme
preparations with each substrate were maximal at pH 8.
Extracellular preparations from facultative organisms A and
D were most active on poly(GulA) at pH 7 and extracellular
activity of isolate G was highest with poly(ManA) at pH 7.
Figure 2-2 compares intracellular and extracellular
activities from isolate A on poly(GulA), poly(ManA), and
alginate under several pH conditions.
Comparison of levels of activities of intracellular and
extracellular preparations on poly(GulA), poly(ManA), and
alginate at pH 8.0 are shown in Table 2-2. Intracellular
preparations were, in all cases, most active on poly(ManA)
and generally slightly higher on alginate than poly(GulA).
Extracellular preparations generally were highly active on
alginate. Fermentative isolates A and D showed little or no
activity toward poly(GulA) extracellularly, and fermentative
isolate G showed little activity on poly(ManA). Levels of
extracellular activities of the oxidative isolates, FM, B,
and C, were comparable on poly(GulA) and poly(ManA) .

% Maximal activity
-27-
4 6 8
pH
Figure 2-2. Activities of intracellular and extracellular
preparations from isolate A, under various pH
conditions, toward poly(GulA), poly(ManA),
and alginate. Values have been normalized to
the condition which allowed the maximal number
of bonds to be cleaved, as determined using
the TBA assay.

Table 2-2. Substrate specificities and modes of cleavage of alginate by bacterial lyases
at pH 8.0.
Intracellular9
Extracellular*3
4>spe
spe
Isolate0 Poly(GulA) Poly(ManA) Alginate
A548
Poly(GulA) Poly(ManA) Alginate A~*48
FM
0.190
0.527
0.235
1.2
0.300
0.569
0.647
20.0
A
0.689
1.140
0.764
8.6
0.000
0.370
0.225
12.0
B
0.026
0.091
0.032
—
0.276
0.287
0.367
—
C
0.015
0.042
0.018
—
0.423
0.373
0.648
—
D
0.825
0.917
0.880
—
0.087
1.058
0.981
—
G
1.287
2.445
1.200
7.3
2.230
0.347
3.135
10.5
Isolate^
FM
0.80
2.24
1.00
0.46
0.87
1.00
A
0.90
1.46
1.00
0.00
1.64
1.00
B
0.81
2.84
1.00
0.75
0.78
1.00
C
0.83
2.33
1.00
0.65
0.58
1.00
D
0.94
1.04
1.00
0.09
1.08
1.00
G
1.07
2.04
1.00
0.71
0.11
1.00
28-

Table 2-2. continued
aIntrace 1lu 1 ar activities were obtained after disrupting bacteria in a French pressure
cell followed by partial purification to remove anionic polymers. This fraction may
include activities bound to the cell surface as well as those which are truly
intracellular.
°Extrace 11u1ar activities were measured in the medium after concentration and dialysis but
without further purification.
cValues in the upper panel are u moles of unsaturated product formed per min per g (wet
weight) of cells. All activities presented were calculated after subtracting activities
observed in the absence of added substrate.
^Values in the lower panel are calculated as ratios of lyase activity dependent upon added
substrate divided by the activity dependent upon native alginate.
eSlopes of straight lines obtained by plotting specific fluidity, sp, against the results
of TBA assays, A^ ®, measured during depolymerization of alginate, indicate relative level
of endolytic vs. exolytic cleavage.
29-

-30-
Patterns of Substrate Cleavage
Measurement of the relative level of endo- and
exoeliminase activities in intracellular versus
extracellular preparations was carried out at pH 8.0. The
decrease in the viscosity of alginate during
depolymerization was measured by capillary viscometry (McKie
and Brandts, 1982), and the rate of glycan bond cleavage was
determined by the TBA assay. Plots of the reciprocal of
specific viscosity, i.e., specific fluidity, sp, versus
periodic acid generated TBA reactive products produced
straight lines with slopes proportional to the relative
level of endolytic activity. In the organisms examined, the
slopes were greatest, and therefore the endolytic activities
highest, in the extracellular fractions. The oxidative
isolate FM, in particular, shows striking partitioning of
exo- and endolytic activities. The comparisons of these
slopes are given in Table 2-2.
Digestion of Alginate Present in Sargassum Tissues
by Alginate Lyases
Digestion of S. filipéndula Tissues by Intracellular
and Extracellular Alginate Lyase Activities from
Isolate A
Active apical tissue with no visible epiphytic growth
was excised and subjected to mild sonication, weighed,
finely chopped with a scalpel, and incubated with enzyme
preparations from facultative isolate A under conditions
described in Table 2-3. The total number of unsaturated

-31-
Table 2-3.
Degradation of S.
intracellular and
preparations from
filipendula tissue by
extracellular alginate
isolate A.a
lyase
T ime
h
K
Direct Assay
n.r. ends u moles
+Enz. Mixture0
n.r. ends u moles
Intra.
Extra.
Intra.
Extra.
0
0.005
0.004
—
—
2
0.131
0.049
0.140
0.163
6
0.317
0.167
0.371
0.519
10
0.419
0.226
0.432
0.703
24
0.651
0.334
—
—
aTwo 25 rag apical portions of sonicated S. fi 1ipendula
tissue, containing an estimated 2.5 u moles of uronic acid
residues each, were finely chopped and incubated with enzyme
preparations in PESI lacking added Ca++ and Mg++, and
containing 1.2 mM EDTA. The activities of both enzyme
solutions at the start of the experiment were 0.0198 u
mole/min per ml with alginate as a substrate.
^Samples of sea water media were removed at indicated times
and assayed for products using the TBA assay, which measures
nonreducing (n.r.) unsaturated terminal residues.
c
Samples of the seawa
removed at the indie
intra- and extracell
containing activitie
source, incubated fu
of the lyase reactio
ter solutions (exclud
ated times and added
ular alginate lyase p
s of 0.099 u mole/min
rther for 12 h and as
ns as above.
ing tissue) were
to a mixture of
reparations
per ml from each
sayed for products

-32-
nonreducing termini produced by the intracellular extract
was at each time point greater than that produced by the
extracellular extract. However, when samples of each
reaction mixture (minus tissue) were removed, mixed with a
combination of intracellular and extracellular enzymes, and
allowed to incubate further, the material released from the
tissue by the extracellular preparation was observed to be
accessible to further depolymerization, whereas the material
released by the intracellular preparation was not. The most
plausible explanation for this is that the extracellular
preparation, which was shown in Table 2-2 to be highly
endolytic, depolymerized the alginate of the tissue to yield
oligomers which were substrates for the enzymes in the
second incubation. The intracellular enzymes were also
capable of releasing and depolymerizing alginate from the
tissue, but released less total mass of alginate. The
material released by the intracellular preparation was in a
more highly depolymerized state than that released by the
extracellular preparation and could not be further degraded
by the enzyme mixture in the second incubation. Protoplasts
were not quantitatively released from the tissues in any of
the above digestions, and under light microscopy the tissue
appeared relatively intact.

-33-
Degradation of Tissue-Bound Alginate by Extracellular
Enzymes from Isolates A and G
In order to examine the capacities of extracellular
preparations which release unsaturated oligomeric products
from alginate in tissues of S. filipendula and S. fluitans,
the experiment described in Table 2-4 was carried out. The
preparation from isolate G, which was active primarily on
poly(GulA), generated approximately 4-fold as many
unsaturated termini from S. filipendula as did the enzyme
from isolate A, which is active with poly(ManA) but not
poly(GulA). When samples from individual wells were added
to the complementary enzyme preparations and incubated
further, the products generated by extensive degradation of
tissue with isolate G enzyme preparation (24 h) could not be
further depolymerized in the second incubation. The
products from 24 h degradation by isolate A enzyme
preparation were depolymerized to yield 2-fold more
unsaturated termini. The total wall mass released by the
preparation from isolate G was therefore 2-fold greater than
that released by the preparation from isolate A.
Both enzyme preparations released approximately the
same amount of wall mass from the S. f1uitans tissue,
although the preparation from isolate G released 2-fold more
unsaturated termini. In addition, the tissue from
fluitans, unlike that from S. filipendula, was depolymerized
more effectively after treatment with EDTA.

Table 2-4. Degradation of S. f i 1ipendula and S. fluitans tissues by extracellular
alginate lyases from bacterial isolates A and G.
u moles n.r.
+/- second
termini,
enzyme3
4 h
8 h
24 h
Organism3
Enzyme'3
EDTA
+
+
+
S.
fi 1ipendula
A
-
0.036
0.107
0.066
0.155
0.132
0.237
A
+
0.032
0.087
0.061
0.135
0.129
0.233
G
-
0.144
0.220
0.229
0.324
0.529
0.517
G
+
0.133
0.172
0.248
0.250
0.489
0.473
none3
-
0.000
0.008
0.000
0.012
0.000
0.019
none
+
0.000
0.010
0.002
0.018
0.009
0.006
s.
fluitans
A
—
0.111
0.218
0.157
0.429
0.013
0.006
A
+
0.146
0.424
0.207
0.587
0.019
0.008
G
-
0.220
0.307
0.282
0.405
0.018
0.009
G
+
0.303
0.422
0.490
0.542
0.112
0.007
none
-
0.000
0.009
0.002
0.015
0.002
0.011
none
+
0.002
0.019
0.002
0.021
0.016
0.015
34-

Table 2-4. continued
aApical foliar tissues from S. fi 1ipendula and S. fluitans were sonicated and 12.5 mg
portions were finely chopped, placed in wells of a microtiter plate, and incubated 15 min
in PESI lacking added Ca^" and Mg + + in the presence or absence of 1.0 mM EDTA.
°The PESI solutions were removed and replaced with enzyme solutions prepared from isolate
A or G containing 198 nmoles/min ml of activity, as assayed with alginate as the
substrate. The enzyme solutions were buffered at pH 7.8 with 0.1 M sodium hydrogen
phosphate and contained 0.5 M NaCl.
cThese tissue samples were incubated in buffer with no added enzyme.
At 4, 8, and 24 h the contents of wells were sampled, excluding pieces of tissue, and the
content of unsaturated nonreducing terminal residues generated by the action of alginate
lyase on the tissues was determined by the TBA assay. Additional samples from tissues
incubated with alginate lyase activity from isolate A or with buffer solution were added
to equal amounts of alginate lyase activity from isolate G, and samples from wells
containing tissues incubated with enzyme from isolate G were added to equal volumes of
enzyme from isolate A. These mixtures were incubated for 14 h before measuring
unsaturated terminal residues.
3 5-

-36-
It is interesting that the level of products from S.
fluitans decreases between 8 and 24 h. In a similar
experiment, this was observed to occur only when tissues of
this species are degraded, since three other Sargassum
species, S. fi1ipendula, S. furcatum, and S. hystrix did not
show similar decreases (data not shown). The tissues of
fluitans, from which the bacterial isolates were obtained,
may harbor more bacteria capable of utilizing the products
which accumulate. Alternatively, other species of algae may
produce inhibitory compounds which prevent bacterial growth
and subsequent utilization of depolymerized alginate.
Effect of Cellulase on Cell Wall Digestion
by Alginate Lyase
Cellulose which is present in the cell walls of brown
algae may present a barrier to release of protoplasts from
algal tissues and/or impede the digestion of alginate by
lyases. The effect of cellulase on the release of alginate
fragments by alginate lyase from S. fluitans was examined as
follows. Apical tissue was sonicated briefly to remove
epiphytes, and 100 mg samples were sectioned into small
pieces (< 0.5 mm in width). These were incubated for 10 h
in individual wells of a microtiter plate in artificial
seawater which lacked Ca++ and Mg++ salts and contained 50
mM EDTA. Seawater solutions were removed and fresh
solutions containing alginate lyase or alginate lyase plus
cellulase were added to the wells. The cellulase was

-37-
prepared from Trichoderma viride (purchased
from Sigma Chemical Co., desalted by chromatography on
Biogel P-6) and the alginate lyase was from an acetone
powder preparation of abalone entrails (from Sigma Chemical
Co., desalted by P-6 chromatography). Each preparation was
dissolved at a concentration of 20 mg/ml. At this
concentration 1 ml of the alginate lyase generated 0.55
umoles of product/min from sodium alginate and the cellulase
solution, as prepared, was sufficiently active to
quantitatively convert calli of cultured Caucus carota to
protoplasts in less than 30 min.
Figure 2-3 shows that the addition of cellulase did not
improve the conversion of cell wall bound alginate to
soluble unsaturated uronides by the alginate lyase
preparation, although this does not imply that cellulase is
not removing cellulose from the walls. This combination of
cellulase and alginate lyase enzymes was not effective in
quantitatively converting algal tissue to protoplasts.
Preparation of Protoplasts by Mechanical
Disruption of Tissue
During efforts to enzymatically remove the cell walls
of Sargassum sp., protoplasts were observed consistently to
be generated in low numbers (1000-2000 per 0.10 g of
tissue), although the bulk of the tissue remained intact.
These protoplasts were produced by the mechanical disruption

N.R. TERMINI (/tunóles)
-38-
TIME (h)
Figure 2-3. Digestion of S. fluitans tissue with alginate
lyase in the presence and absence of cellulase.
Apical tissue was prepared from fluitans,
incubated in a microtiter plate with alginate
lyase from Haliotus (abalone) in the presence
(closed triangles) or absence of cellulase
(open circles) from Trichoderma viride,
and the contents of the wells sampled and
assayed for nonreducing termini by the TBA
method according to the text. Controls with
no added enzymes are indicated by open
squares.

-39-
of tissue, in the absence of any added enzymatic activity,
by slicing tissue into small pieces which were then
incubated in PESI solution which lacked Ca++ and Mg++.
Figure 2-4 shows what are apparently intact as well as
damaged protoplasts. The intact protoplasts also showed
neutral red staining of vacuoles (Stadelmann and Kinzel,
1972), trypan blue dye exclusion, and intense red
fluorescence of chloroplasts under UV light (data not
shown), which are indicative of viability (Berliner, 1981).
The protoplasts rapidly lysed upon exposure to distilled
water and had smooth outer surfaces as viewed by scanning
electron microscopy (Fig. 2-5), suggesting a membrane
surface which is free of cell wall. At least some of the
protoplasts of a given preparation remained viable for
several hours, although their capacity for regeneration or
continued growth was not tested.
Discussion
This chapter and our previous work (Preston et al.,
1985a; Romeo et al., 1986) document observations that
fermentative as well as oxidative marine bacteria are
capable of producing alginate lyases of varied substrate
specificities. Marine bacterial poly(GulA) lyases were
previously described by several investigators (Fujibayashi
et al . , 1970; Davidson et al . , 1976; Sutherland and Keen,
1981) and oxidative bacteria from Fucus, which produced both
poly(GulA) and poly(ManA) lyases were isolated by Doubet and

-40-
Figure 2-4. Light microscopy of protoplasts from S.
fluitans. Healthy apical tissues of S.
fluitans were sonicated briefly to remove
epiphytes, weighed (0.1 g), and sliced into
small pieces with a scalpel, and incubated
0.5 h in PESI lacking Ca^+ and Mg++. The
suspension was drawn into a Pasteur pipette,
avoiding large pieces of tissue, transferred
to a clean well of a microtiter plate, and
observed at 400X, with an Olympus inverted
microscope.

-41-
Figure 2-5. Scanning electron microscopy of protoplasts
from S. fluitans. Panel a depicts a single
protoplast emerging from algal tissue; panel
b shows two protoplasts resting upon a piece
of Sargassum tissue (1600X).

-42-
Quatrano (1982, 1984). Intracellular extracts from our
isolates were high in poly(ManA) lyase, making them similar
to the isolates from Fucus (Doubet and Quatrano, 1982).
Extracellular preparations, except from isolates A and D,
were most active on native alginate. Levels of poly(GulA)
and poly(ManA) lyases were comparable in the extracellular
preparations from all oxidative isolates that were examined.
On the other hand, fermentative isolate G produced much
more poly(GulA) lyase than poly(ManA) lyase, and isolates A
and D produced poly(ManA) lyase in large excess over
poly(GulA) lyase. These last two organisms are in contrast
with the isolates from Fucus which tended to produce higher
levels of poly(GulA) lyase in the extracellular fractions.
An organism such as isolate A, which produces little if any
extracellular poly(GulA) lyase but makes intracellular
poly(GulA) lyase may depend upon other bacteria in the
environment to secrete endo poly(GulA) lyases for the
further depolymerization of alginate.
The observation that endolytic activities are higher in
the extracellular fractions as compared to the intracellular
fraction presumably reflects a requirement for degradation
of large native alginate molecules to allow entry into the
bacterial cells. By retaining exoeliminases either in the
cytoplasm or bound to the cell surface, these organisms avoid
producing large amounts of metabolizable monomers in the
external environment which through diffusion and/or
utilization by other organisms would be lost to the bacteria
producing the enzymes.

-43-
A model for bacterial utilization of alginate based on
this study and the work of others is shown in Fig. 2-6.
Native alginate is endolytically depolymerized to fragments
possessing unsaturated nonreducing ends. These fragments
are internalized and exolytically degraded, possibly
undergoing degradation during the entry process. The
metabolic pathway for degradation of the monomer product by
a pseudomonad has been described (Preiss and Ashwell,
1962b).
Under conditions of anaerobic digestion, the
metabolites of the monomer should be readily converted to
methane by a consortium of bacteria. Shiralipour et al .
(1984) have observed that improved yields of methane can be
obtained from digestion of Sargassum tissues using
microflora associated with Sargassum. The rate and/or
extent of methane production might be further improved by
supplementary inocula of organisms such as the facultative
isolates of this study, which produce alginate lyases with a
spectrum of substrate specificities and modes of cleavage,
or by addition of alginate lyase enzymes to the fermentor.
The exposure of active Sargassum tissues to bacterial
alginate lyase preparations is effective in removing
considerable amounts of alginate from the cell walls
(virtually all of the alginate may be removed from the
tissues by endolytic extracellular alginate lyase
preparations). Although alginate lyase activities alone or
in combination with a cellulase preparation did not effect

-44-
EXTRACELLULAR
Endo Poly (GulA) Lyase
AX-G v
A X-G-G )
A X-G-M (
A X-M-G >
A X-M-M (
A X-M )
Unsaturated
Fragments
of Alginate
INTRACELLULAR
Transport
AX-G
A X-G-G
A X-G-M
A X-M-G
A X-M-M
A X-M
CHO
HOCH
HOCH
l
Exolyases HCH
_ Further
Metabolism
C = °
COOH
4-Deoxy-5-Keto
Uronic Acid
Figure 2-6. Proposed model for metabolism of alginate by-
bacteria which colonize tissues of Phaeophyceae.
Alginate polymers are released from a gel-like
state in the wall by secreted endolytic bac¬
terial alginate lyases. The initial products
are depolymerized to an extent which allows the
bacteria to assimilate them. Exolytic enzymes
continue the depolymerization of oligomers
which enter the cell, with the eventual produc¬
tion of the monomer, which may be used as a
source of carbon and energy.

-45-
quant i tat ive release of protoplasts from tissue, the
combined effects of these activities and activities toward
other cell wall polymers, e.g., fucoidin and/or proteins,
may be successful. The mechanical production of viable
protoplasts, although with low yields, is encouraging, and
supports the feasibility of developing an enzyme-mediated
method. Recently, crude extracts of the hepatopancreas of
Holiotus sp. have allowed preparation of protoplasts from
Sargassum hystrix (Preston et al., 1985b), although with
some inconsistency of yields which is not yet understood.
Fractionation of this type of crude extract and
identification of specific enzymatic components necessary
for protoplast formation may provide information which will
allow well defined bacterial and/or fungal enzymes to be
applied with more reproducible results.

CHAPTER III
HPLC ANALYSIS OF THE DEPOLYMERIZATION OF (1-4)-3-D-
MANNURONAN BY EXTRACELLULAR AND INTRACELLULAR
ALGINATE LYASES FROM A MARINE BACTERIUM
Introduction
Alginate lyases have been sought as specific probes to
study the structure of soluble alginates (Davidson et al . ,
1976; Boyd and Turvey, 1978; Fujibayashi et al . , 1970) and
polymers contributing to the structure of the cell walls of
the brown algae (Doubet and Quatrano, 1984). These enzymes
have recently been evaluated for the production of
protoplasts from Sargassum species (Preston et al., 1985b;
Romeo et al., 1986). Information about the substrate
specificities and the modes of substrate cleavage by
alginate lyases would be valuable in defining their
catalytic properties and their potential uses. Previous
studies on the mechanisms of alginate depolymerization have
included viscometric analyses (Kashiwabara et al., 1969;
Nakada and Sweeny, 1967; Elyakova and Favorov, 1974) and the
characterization of enzyme generated products utilizing a
number of methods including gel filtration (Boyd and Turvey,
1977; Favorov et al., 1979), ion exchange column
chromatography (Preiss and As’nwell, 1962a; Linker and Evans,
1984), paper chromatography (Preiss and Ashwell, 1962a;
-46-

-47-
Kashiwabara et al., 1969; Linker and Evans, 1984; Davidson
et al., 1977) and paper (Davidson et al . , 1976; Davidson et
al., 1977), and gel electrophoresis (Hansen et al., 1984;
Doubet and Quatrano, 1984).
The isolation and characterization of alginate lyase
producing oxidative and fermentative marine bacteria
associated with actively growing tissues of marine brown
algae, genus Sargassum, have been described previously
(Preston et al., 1985a; Romeo et al., 1986). One of the
facultative anaerobes, isolate A (SFFB080483 A, see Table 2-
1), was shown to produce extracellular lyase activity which
was specific for poly(ManA) versus poly(GulA) and endolytic
in its action on alginate. The intracellular activities of
this bacterium depolymerized both poly(ManA) and poly(GulA)
and in comparison with the extracellular preparation showed
a greater ratio of bond cleavage to increase in fluidity
with native alginate, suggestive of exolytic as well as
endolytic mechanisms. Here the extracellular activity is
shown to belong to a single enzyme. A method based upon
HPLC separation of small oligomers is described, and it has
allowed a kinetic evaluation of the depolymerization of
poly(ManA) by both of these preparations. The limit
products generated by the poly(ManA) specific extracellular
enzyme have also been established.

-48-
Experimental
Materials
Chemicals were analytical grade except as indicated.
Acetonitrile (Fisher Scientific) and tetrabutylammonium
hydroxide (Fisher Scientific and Sigma Chemical Co.) were
HPLC grade. Commercially available electrophoresis grade
reagents were used for electrophoretic analyses. All
aqueous solutions were prepared with water which was
deionized and glass distilled.
Preparation of Substrates
Sodium alginate was purchased from Fisher Scientific
Company as a purified grade originally isolated from
Macrocystis. Poly(GulA) and poly(ManA) were obtained from
HC1 hydrolyzed alginate following the methods developed by
Haug et al. (1967). These preparations were further
fractionated on Sephadex G-50 with 0.5 M NaCl as eluant, and
selected fractions were analyzed for reducing termini
(Nelson, 1944) and total carbohydrate (Dubois et al., 1956;
Haug and Larsen, 1962) to obtain substrates of uniform size.
Both ■*‘H and ^C NMR (Grasdalen et al . , 1979; Grasdalen et
al., 1981) analyses were carried out to assess the purity of
substrates. Poly(ManA) preparations contained 11%
guluronate, and poly(GulA) contained 11% mannuronate.

-49-
Enzyme Assays
Alginate lyase activity was quantified by spectrophoto-
metric determination at 548 nm of the chromophore formed
upon reaction of thiobarbi turic acid (TBA) with periodate
treated products (Preiss and Ashwell, 1962; Weissbach and
Hurwitz, 1959). This method allows the specific measure of
unsaturated nonreducing termini of oligomeric products and
the unsaturated monomer, 4-deoxy-L-erythro-5-hexoseulose
uronic acid. Substrate mixtures contained either 0.1%
alginate, poly(GulA), or poly(ManA), and 0.05 M KC1,
buffered with 0.03 M sodium phosphate at the desired pH.
Enzyme was mixed with 9 volumes of substrate to start the
reactions, and reactions were terminated after 10 min by
addition of periodic acid solution. One enzyme unit is
defined as that amount of activity which will catalyze the
formation of 1 nmole of nonreducing termini and/or monomer
at pH 7.5 in one min at 22°C. Protein was routinely
estimated by absorbance at 280 nm. For more quantitative
measurements the assay of Bradford (1976) was utilized with
bovine serum albumin as a standard protein.
Enzyme Isolation
The bacterium used in this study was obtained from
healthy, apical tissue of Sargassum fluitans and initially
identified as an organism which secretes alginate degrading
activity, based on the appearance of extensive clearing zones

-50-
surrounding colonies grown on solid alginate medium. The
organism used for the work described here has been desig¬
nated isolate A (complete designation SFFB080483 A) and has
been described (Preston et al., 1985a; Romeo et al., 1986).
Biochemical and morphological properties of this bacterium
suggest its assignment to the genus Photobacterium, although
its DNA has a GC fraction of 0.454, somewhat greater than
that of other species currently included in this genus
(0.398-0.429). The organism has been maintained by monthly
transfer on solid alginate medium (Preston et al., 1985a).
For enzyme isolations the organism was grown in 0.1%
liquid alginate medium (Preston et al., 1985a) with rapid
gyrotory shaking at room temperature. In all subsequent
purification steps the extracellular and intracellular
preparations were kept at approximately 4°C. Bacterial
cells viere harvested at late exponential phase by
centrifugation at 10,000 x g for 10 min, washed twice with
water by resuspension and centrifugation, frozen in liquid
nitrogen, and stored at -70°C. The spent medium was
concentrated and dialyzed against distilled, deionized water
by tangential flow filtration using a Millipore Pellicon
cassette system with a polysulfone membrane (PTGC) which
allowed retention of proteins larger than 10 kDa.
For extracellular enzyme preparations the alginate
lyase activity was precipitated along with remaining
alginate products by dropwise addition of 10% polytheyleni-
mine (PEI) to concentrated medium while stirring on ice.

-51-
The PEI was obtained as a 50% aqueous solution from Sigma
Chemical Co. Prior to use, this solution was diluted with
water, titrated to pH 7.5 with 12 N HC1, and centrifuged at
10,000 x g for 10 min to remove insoluble particles. The
relative volume of PEI necessary for maximal precipitation
of enzyme was found to be critical and was determined for
each batch of enzyme. Typically 1 ml of 10% PEI would yield
maximal precipitation of enzyme from 125 ml of concentrated
medium derived from 10 ul of spent medium. The precipitate
was collected by centrifugation at 10,000 x g for 15 min and
resuspended in distilled, deionized water using a Potter-
Elvehjem homogenizer driven by a variable speed motor. The
resulting suspension was centrifuged and the pellet
homogenized in 0.25 M NaCl, 0.1 M sodium phosphate at pH
7.5, to elute the enzyme. The suspension was centrifuged
for 1 h at- 150,000 x g, and the supernatant solution was
subjected to gel permeation chromatography on Sep’nacryl S-
200 (2.5 x 133 cm) with 0.1 M sodium phosphate at pH 7.0.
To obtain protein concentration sufficient for preparative
digestions and for quantification with the Bradford assay,
fractions were concentrated by ultrafiltration using an
Amicon cell with a YM 10 filter (10 kDa cutoff) .
For intracellular enzyme preparations, cells were
thawed, suspended in 4 volumes of ice cold 0.1 M sodium
phosphate, pH 7.5, and disrupted with a French pressure cell
at 16,000 PSI. Unbroken cells and debris were removed by
centrifugation at 10,000 x g for 15 min, and the supernatant

-52-
solution brought to 2% streptomycin sulfate with the
addition of a 5% streptomycin sulfate stock solution. After
mixing for 10 min, the precipitate containing anionic
polymers was removed by centrifugation at 10,000 x g for 15
min and protein precipitated from the supernatant solution
with the slow addition of solid ammonium sulfate (to 65%
saturation). After centrifugation at 10,000 x g for 15 min,
the protein pellet, containing alginate lyase, was dissolved
in 0.1 M sodium phosphate buffer, pH 7.0, and chromato-
graphically fractionated on the Sephacryl S-200 column
(2.5 x 133 cm) with the same buffer.
Electrophoresis
The method for native gel electrophoresis was
described by Shuster (1971) and used the discontinuous
buffer system of Davis (1964). The running gel had a final
concentration of 7.5% acrylamide and 0.2% bisacrylamide, was
buffered at pH 8.9 with 0.38 M Tris HC1, and was polymerized
with 0.07% ammonium persulfate and 0.058% N,N,N',N'-
tetramethylethylene-diamine (TEMED). The stacking gel was
composed of 2.5% acrylamide, 0.5% bisacrylamide, 0.062 M pH
6.8 Tris HC1, and was polymerized with 0.058% TEMED and
0.01 mM riboflavin phosphate, using a fluorescent light to
activate the polymerization process. The running buffer was
composed of 0.3% Tris base, 1.44% glycine at pH 8.9.
Vertical slab gels were 0.15 cm in thickness and were
subjected to electrophoresis at 30 mA per gel until the

-53-
bromophenol tracking dye had reached the end of the gel.
The gels were cut into 0.5 cm slices which were incubated at
room temperature with 200 ul of sodium alginate pH 7.5
substrate mixture. Following the incubations 100 uL of the
solutions were withdrawn and assayed for products of the
lyase reaction.
Analysis of Alginate Lyase Generated Products
Analytical chromatographic separation of products was
accomplished by ion-paired reversed phase HPLC using a
system which is a modification of that developed by Voragen
et al . (1982) to fractionate pectate products. The column
was a C18 uBondapak 8MB 10 u column housed on a Z-Module
radial compression system (Waters). A Rainin 0.5 u
stainless steel filter and a Waters RCSS Guard-Pak C18
prefilter cartridge were positioned between the column and
the injector. The column was run at room temperature in an
isocratic mode with a solvent system of 10% acetonitrile,
10 mM tetrabutylammonium hydroxide, 0.1 M sodium phosphate
buffered at pH 6.5. Unsaturated oligomers were detected by
monitoring UV absorbance of the effluent from the column
at 230 nm with a Gilson Holoc’nrome variable wavelength
detector equipped with a 1.0 cm flow cuvette. A Waters Tri-
Mod system was used for programming a 6000A pump,
integration of peak areas, and for automated injection of

-54-
samples onto the column. A Waters U6K injector was used for
manual injection of samples.
Preparative fractionation of products generated from
poly(ManA) by the extracellular enzyme was carried out by
gel filtration using a Biogel P-2 column (2.5 x 133 cm)
eluted with 0.1 M ammonium bicarbonate, collecting 5.8 ml
per tube. For a successful isolation of the products, 110
mg of poly(ManA) was dissolved in a 1.6 ml solution of the
extracellular enzyme (900 units/ml) buffered at pH 7.0 with
0.1 M sodium phosphate, and incubated for 12 h at room
temperature. Under these conditions the digestion was not
complete at the time the reaction mixture was applied to the
P-2 column, allowing some of the larger oligomeric
mannuronans to be obtained. Absorbance at 230 nm was
determined for each tube.
Contents of tubes comprising each peak from the P-2
column were pooled, lyophilized, and stored at -20°C over
anhydrous calcium sulfate. The lyophilized products were
dissolved in 0.1 M sodium phosphate buffer, pH 7.0, and were
analyzed for unsaturated nonreducing termini by measuring
TBA reactive material generated by periodate oxidation
(Preiss and Ashwell, 1962a; Weissbach and Hurwitz, 1959) with
3-deoxy-D-manno-octulosonic acid (KDO, Sigma Chemical Co.)
as a standard. For preparation of the KDO standard, the
compound was desiccated overnight in vacuo. The chromophore
generated by the reaction of TBA with the 8-formylpyruvate

-55-
formed by periodic acid oxidation of the KDO was quantified
spectrophotometrically according to Preiss and Ashwell
(1962) and according to Koseki et al. (1978) which indicated
that the sample was 90% and 85% pure, respectively. Total
uronic acid content was measured by the method of
Blumenkrantz and Asboe-Hansen (1973) using D-mannurono-
lactone (Sigma Chemical Co.) as a standard. Based upon the
expected extinction at 520 nm for the chromophore from
D-mannuronolactone, the desiccated standard was 84% pure.
Absorbance at 232 nm was measured in a 1.00 cm cuvette after
diluting samples 200-fold with 0.01 N HCl.
Samples of lyophilized fractions from the P-2 column
effluent were sent to Triangle Laboratories, Inc., Research
Triangle Park, North Carolina, for fast atom bombardment
(FAB) mass spectrometry under the direction of Ronald Hass.
Analyses were performed on a VG 7070H mass spectrometer with
a VG11-250 data system. The acceleration voltage was 3 kV
for the trimer analysis and 2 kV for the other samples. An
Iontech saddle field ion source was used with xenon as the
bombarding species. The gun was operated at 7 keV with a
discharge current of ca. 1.5 mA. The samples were analyzed
after dissolving in water and applying 1-2 ul of the
solution to thioglycerol on the probe. The mass spectrometer
was scanned at 5 s per dec of mass from 1200-100, at a mass
resolution of 1000.

-56-
Results and Discussion
Chromatographic and Electrophoretic Behavior of the
Extracellular and Intracellular Activities
The extracellular preparation from a fermentative
marine bacterium, designated isolate A, was shown previously
to be highly active on poly(ManA) and native alginate, but
inactive or possessing only trace activity with poly(GulA)
(Preston et al., 1985a; Romeo et al. , 1986). The
preparation was endolytic with alginate as substrate, as
shown by comparing the rate of bond cleavage with the
increase in the reciprocal of specific viscosity, i.e.,
specific fluidity. For purification and characterization of
extracellular poly(ManA) lyases, the concentrated
preparation was first treated with 10% PEI to remove the
partially degraded alginate which remained after dialysis of
the medium. This procedure is effective in reducing the
viscosity, allowing a greater quantity of enzyme to be
applied to the gel filtration column. When subjected to gel
filtration on Sephacryl S-200, a single peak of lyase
activity eluted at 0.67 column bed volumes (Fig. 3-la). The
column allowed complete removal of remaining products
derived from alginate, which appeared as TBA reactive
material eluting at 0.94 column bed volumes and separation
of some of the contaminating proteins from the enzyme. The
pooled enzyme fractions represented recovery of 82% of the
alginate degrading activity loaded onto the column. At this
stage of purification, the specific activity of the pooled

-57-
<
E
c
o
co
CNJ
o
c
(0
A
w
O
co
A
<
Figure 3-1. Chromatographic fractionation of extracellular
and intracellular alginate lyase activities.
Samples of the extracellular and intracellular
fractions were obtained as described in the
text and subjected to chromatography at 4 C on
Sephacryl S-200. The column was eluted with
0.1 M sodium phosphate at pH 7.0, and 7.8 ml
fractions collected and assayed for protein by
absorbance at 280 nm, and for alginate lyase
activity by the TBA assay, absorbance at 548 nm.
The column was calibrated with molecular weight
standards, bovine serum albumin (BSA, 67 kDa),
6-lactoglobulin (8-L, 37 kDa), blue dextran
(to establish the void volume, Vo) and NaCl
(total column volume, Vt). The extracellular
preparation (a) was derived from medium which
yielded 3.7 g wet weight of cells and the
intracellular (b) from 13.8 g wet weight of
cells.

-58-
enzyme fraction was typically 3500 units per mg of protein
with alginate as the substrate and the ratio of activities
on poly(GulA) versus poly(ManA) was 0.14.
Samples from individual tubes containing the
extracellular alginate lyase fraction which eluted from the
Sephacryl S-200 column were subjected to native polyacryla¬
mide gel electrophoresis followed by detection of activity.
The individual fractions comprising the alginate lyase peak
eluting from the S-200 column showed the same single
activity component which migrated as a homogeneous band
(Fig. 3-2a) , indicating that the extracellular fraction
contained a single enzymatic activity.
Intracellular activities eluted from the same Sephacryl
200 column as two small and one large peak (Fig. 3-lb). The
major peak eluted in a volume corresponding to an estimated
molecular mass of 40 KDa and contained approximately 15% of
the alginate degrading activity originally loaded onto the
column. The enzymes present in the tubes from centers of
the three activity peaks were active on both poly(ManA) and
poly(GulA) and therefore were probably mixtures of two or
more alginate lyases. The ratios of activities on
poly(ManA) versus poly(GulA) were 6.8, 1.8, and 1.2 for the
contents of tubes 31, 37, and 44, respectively.
The peak tube from the major S-200 intracellular lyase
fraction was electrophoretically resolved into at least
three activities (Fig. 3-2b), indicating that the major
fraction eluting from this column included more than one

-59-
Figure 3-2. Electrophoretic analysis of (a) extracellular
and (b) intracellular alginate lyase activities.
The extracellular preparation was fractionated
by gel filtration on Sephacryl S-200. A sample
was removed from the peak tube and subjected to
native polyacrylamide gel electrophoresis and
subsequent detection of activity according to
methods described in the text. A sample from
the peak tube from the Sephacryl S-200 frac¬
tionated intracellular extract was subjected
to electrophoresis on the same native poly¬
acrylamide slab gel used for analysis of the
extracellular lyase preparation.

-60-
alginate degrading enzyme. The activity with the highest
electrophoretic mobility could be identical to the single
major extracellular enzyme based upon electrophoretic
migration; however, this has not been confirmed by other
analyses.
HPLC Analysis of Poly(ManA) Depolymerization
When the extracellular enzyme (14 units) from the S-200
column was incubated at room temperature with 5 mg of
poly(ManA) in 0.5 ml of 0.1 M sodium phosphate buffered at
pH 7.0, unsaturated oligomers were produced which could be
fractionated by HPLC. Profiles generated after 15 min and
4 h of depolymerization are shown in Figs. 3-3a and 3-3b,
respectively. At least six peaks were detected by
absorbance at 230 nm, and five of these were sufficiently
distinct to be integrated by the data analyzing system.
Individual oligomers, detected as A230 peaks (Fig. 3-3b),
were designated numerically from 1 to 5 in the order of
their elution with retention times (in minutes) of 5.87,
7.54, 10.00, 13.87, and 19.83, respectively. As will
be demonstrated, these represent the unsaturated dimer
through hexamer, respectively. When the depolymerization
was monitored over a 30 h period (Fig. 3-4) several features
of interest were noted. Component 1, the dimer, and to a
lesser extent component 2, the trimer, exhibited initial
lags in their rates of accumulation. Components 4 and 5,
the pentamer and hexamer,
increased until the digestion had

Absorbance (230nm)
-61-
0.04 n
0.02-
0.00-
0.08-
0.04-
0.00-
i 1 I 1 1 1 1
0 10 20 30
Retention Time (min)
Figure 3-3. Liquid chromatographic analysis of products
generated by digestion of poly(ManA) by the
extracellular lyase. The unsaturated oligomers
produced from poly(ManA) by the extracellular
enzyme were resolved by HPLC, as described in
the text. Sample volumes of 10 ul were
delivered with automatic injection (Waters,
WISP) and eluted isocratically at 1.0 ml/min.
Profiles of the products which had accumulated
after 15 min (a) and 4 h (b) are shown.

Relative peak area
-62-
Figure 3-4. Kinetic analysis of poly(ManA) depolymeriza¬
tion by extracellular alginate lyase. The
depolymerization reaction described in Fig.
3-3 was sampled periodically over 30 h, and
samples subjected to HPLC as described in the
text. The peak areas of the major products
are plotted against the times at which the
reaction was sampled. The individual products
were given number designations according to
their order of elution from the column, start¬
ing with the fastest moving compound, 1,
representing a dimer, through 5, which repre¬
sents a hexamer. The dimer peak integrates
as 0.11 area units per nmole.

-63-
continued for 6 and 4 h, respectively, and thereafter
decreased. The dimer, trimer, and tetramer never showed
declines, although the rates of accumulation of trimer and
tetramer decreased at approximately 6 h, and the rate of
dimer accumulation began to decrease gradually at 6 to 10 h.
The delays in appearance of the two smaller products, dimer
and trimer, indicated that these compounds were generated to
a significant extent from products which accrued from
initial depolymerization reactions. The decrease in the
concentration of the larger compounds, pentamer and hexamer,
after initial accumulation suggested that these must be
subject to depolymerization by the enzyme, and that their
relative rates of formation and degradation determine the
levels at any given time. The unsaturated monomer is not
readily detected by absorbance at 230 nm due to
tautomerization to the a-keto acid form (Preiss and Ashwell,
1962). Detection at 205 nm revealed a minor component which
eluted prior to the dimer, and analysis by the TBA method of
fractions collected from a reversed phase separation of a
preparative poly(ManA) digest also showed a minor component
of reactive material eluting prior to the dimer (data not
shown). This, presumed to be the monomer, represented less
than 4% of the products including dimers to pentamers
produced by the depolymerization of poly(ManA) catalyzed by
this enzyme.
The depolymerization of poly(ManA) by the intracellular
preparation was analyzed by HPLC using the methods described
for the extracellular preparation. Figure
3-5 shows the

-64-
products which have accumulated at 3 h and 21.5 h. During
an initial phase of the reaction, up to 8-10 h, four
products predominate. The mobilities of three of these are
identical to those of major products generated by the
extracellular enzyme, as established by direct comparisons
at the time of analysis (data not shown). Direct
comparisons were necessary due to changes in column
performance which occurred with usage. Products included
the trimer, with a retention time of 6.9 min, the tetramer,
with a retention time of 8.8 min, and the pentamer, with a
retention time of 11.5 min. The peak which was generated
only by the intracellular preparation presumably represents
the monomer compound, 4-deoxy-L-erythro-5-hexoseulose uronic
acid, and has a retention time of 4.7 min. The identity of
the monomer is indicated by its ratio of absorbance at 230
nm/205 nm, 0.16, as compared with those of the dimer through
tetramer, 0.9-1.3, and by its elution position, which is
prior to the dimer. Due to the poor absorption of the
monomer at 230 nm, the peak area for the monomer
underestimates its concentration relative to other products
several-fold. A fifth compound accumulates to a
considerable extent in the reaction after a lag of 10 h and
is seen in Fig. 3-5 in the lower profile. This product has
the retention time of the dimer compound, 5.95 min. Figure
3-6 shows that the dimer and trimer, peaks 2 and 3, continue
to accumulate at later times in the digest, long after the
concentrations of the monomer, peak 1, and tetramer, peak 4,
have become constant.

ABSORBANCE (230nm)
-65-
0.02-1
0.00-

0.08-
(O
iD
(0
0.04-
0.00 J
“D
T
0 10 20
RETENTION TIME (min)
“l
30
Figure 3-5. Liquid chromatographic analysis of products
generated by digestion of poly(ManA) by the
intracellular alginate lyase activities. The
unsaturated oligomeric products generated from
poly(ManA), 10 mg/ml, by the third peak of
activity from the S-200 column, tubes 42-47,
13 units/ml, were resolved by HPLC as described
in the Materials and Methods. Profiles of the
products which had accumulated at 3 h, upper
profile, and 21.5 h, lower profile, are
shown.

RELATIVE PEAK AREA
-66-
TIME (h)
Figure 3-6. Kinetic analysis of poly(ManA) depolymeriza¬
tion by intracellular alginate lyase activities.
The depolymerization reaction described in
Fig. 3-5 was monitored over 33.5 h, by
periodically subjecting 10 ul samples to
HPLC analysis. Products are numbered accord¬
ing to their elution positions. Peak 1 is the
presumptive monomer, and peaks 2-4 are the
unsaturated dimer through tetramer.

-67-
The poly(ManA) lyase activity from the intracellular
preparation differs from the extracellular activity in
generating a considerable quantity of apparent monomer. The
physiological necessity to produce monomer on the inside of
a bacterial cell or at the cell surface is obvious, although
the mechanisms of the enzymes responsible for monomer
production in the reaction are not yet established. A
single exolytic enzyme might generate monomer as it degrades
poly(ManA) from either the reducing or nonreducing termini.
Alternatively, the monomer may be generated from some
intermediate degradation products by one or more enzymes
with glycosidase-1ike activities.
The unique appearance of the dimer product after a lag
of approximately 10 h is quite unexpected. The fact that
the preparation may contain more than one alginate degrading
activity does not allow a definitive explanation for this.
However, a number of possibilities might be envisaged. If
the monomer is being produced primarily from a dimer product
by a glycosidase-like activity, the loss of this activity
would be expected to allow dimer to accumulate. A second
possibility is that the dimer begins to accumulate as some
intermediate depolymerization product reaches a
concentration sufficient to allow its recognition by the
enzyme which is capable of generating dimer.

-68-
Purification and Characterization of the
Reaction Products
In order to obtain sufficient quantities of the
oligomeric products for characterization, 110 mg of
poly(ManA) was digested with extracellular enzyme for 12 h
and the products were resolved by chromatography on Biogel
P-2 eluted with 0.1 M ammonium bicarbonate. Four components
measured by absorbance at 230 nm were resolved from one
another. These were eluted at column volumes of 0.46, 0.50,
0.56, and 0.64 and designated as fractions 1, 2, 3, and 4,
respectively (Fig. 3-7). A small fraction, approximately 9%
of the total absorbance at 230 nm, eluted between the void
volume of the column and the leading edge of the peak
designated as fraction 1. Chromatography in ammonium
formate led to a similar profile; however, the lyophi1ization
of these fractions resulted in discoloration of some of the
fractions and subsequent HPLC analysis demonstrated
significant degradation. Products obtained after elution
with ammonium bicarbonate were, after lyophi1ization, fluffy
and white, although they were quite hygroscopic.
Samples of the peak tubes from the P-2 ammonium
bicarbonate column were analyzed by HPLC (data not shown),
which identified the contents of tubes 52, 57, 63, and 72 as
the oligomers comprising peaks 4, 3, 2, and 1, respectively,
of the HPLC profiles (Fig. 3-3). Absorbance spectra in the
UV range for samples diluted in 0.01 N HC1 showed maxima

Absorbance (230nm)
-69-
Figure 3-7. Preparative fractionation of the unsaturated
mannuronides. A 110 mg sample of poly(ManA)
was digested by 1,400 units of extracellular
enzyme in a 1.6 ml volume for 12 h at room
temperature and the products fractionated by
gel filtration with a P-2 column, as described
in the text. Volumes of 5.8 ml were collected
in tubes and assayed for absorbance at 230 nm.
Native alginate and galacturonic acid were
subjected to chromatography on the P-2 column
and their elution positions are indicated.
The contents of tubes comprising 4 fractions
were pooled and lyophilized: fraction 1,
tubes 52, 53; fraction 2, tubes 55-57; frac¬
tion 3, tubes 62-64; fraction 4, tubes 71-73.

-70-
at approximately 232 am (profiles not shown), typical of
products generated by alginate lyases.
The pooled, lyophilized fractions from the P-2 column
were further analyzed to establish the molecular sizes of
the unsaturated products. Comparisons of the uronic acid
content with content of nonreducing residues (Table 3-1)
allowed an estimation of the degree of polymerization (DP)
of each product. The DP values estimated using the ratio of
total uronic acid to nonreducing termini show a trend
consistent with assigning the unsaturated oligomers in
fractions 1 through 4 as the pentamer, tetramer, trimer, and
dimer compounds, respectively.
Based upon the concentration of the nonreducing
unsaturated terminal residues estimated with the TBA assay
of the periodate treated products and the abosrbance values
at 232 nm of products in 0.01 N HC1, an estimation of the
molar absorptivities for the dimer to pentamer series,
presumed to contain a single unsaturated residue in each
molecule, ranged from 5,160 (Fr 1) to 5,420 (Fr 3) M-^cm--'-.
The concentrations of each component, based upon gravimetric
preparations of lyophilized samples and calculated molecular
weights of each as an ammonium salt, were calculated on the
assumption that fractions 1, 2, 3, and 4 represented the
pentamer, tetramer, trimer, and dimer, respectively. These
values, as divisors for the A232 values listed in Table 3-1,
led to calculated molar absorptivities which ranged from

-71-
Table 3-1. Analysis of unsaturated oligomers from
lyophilized P-2 fractions.3
Fraction
no .
Abs . k
2 32 nm
Unsat.c
termini
mM
Uronicd
acids
mM
DP
uronic
acids
unsat.
termini
1
39.2
7.59
40.5
5.3
2
42.0
7.94
37.5
4.7
3
64.0
11.8
36.3
3.1
4
84.6
16.2
45.6
2.8
aLyophilized products from the Biogel P-2 fractionated
preparative digest were obtained according to methods
described in the text and dissolved in 0.1 M sodium
phosphate buffer, pH 7.0, to a final concentration by
weight of 10 mg/ml.
“Solutions were analyzed for absorbance at 232 nm in a 1.00
cm quartz cuvette, after diluting 200-fold with 0.01 N HCl.
cUnsaturated nonreducing
by the TBA assay of per
a standard. Values wer
apparent 90% purity of
terminal residues we
iodate treated produc
e adjusted to correct
the standard.
re determined
ts, with KDO
for the
as
dUronic acid
lactone as a
the apparent
the expected
Asboe-Hansen
residues were determined using D-mannurono-
standard. Values were adjusted to correct
84% purity of our standard compound, based
yield of c’nromophore given by Blumenkrantz
(1973) .
for
on
and

-72-
3350 (Fr 2 as the tetramer) to 3890 (Fr 1 as the pentamer).
The individual fractions, in particular the putative dimer,
were sufficiently unstable to heating to preclude high
temperature desiccation, and these lower molar absorptivi-
ties, in comparison to those determined from the concentra¬
tions determined with the TBA assay, may reflect the
presence of water not removed by the lyophilization process.
Samples of unsaturated dimeric and tetrameric products
obtained from digestion of bacterial alginate with a
poly(ManA) lyase from a Pseudomonas aeruginosa isolate
(Linker and Evans, 1984) were graciously provided by Dr.
Alfred Linker and shown to possess the same HPLC mobilities
as our dimer and tetramer, respectively; molar absorptivi-
ties at 232 nm in 0.01 N HC1 of 6,400 and 5,500 M-^cm-^ were
obtained for this dimer and tetramer, respectively (data not
shown).
Further evidence of the DP of the major products was
obtained by FAB mass spectrometry (Table 3-2) of the
lyophilized fractions. Spectra from each of the products
contained major ions which correspond to within 1 mass unit
of the calculated M+NH^ and the M+NH4 + H2O ions. The triiner
product showed an additional ion which represents the M+NH^
+ thioglycerol, and the spectrum of the dimer showed two
major ions (373, 391) which could not be explained, based on
the expected structure of the dimer. The dimer sample was
the only one which was not white in color, and we assume

-73-
Table 3-2. Analysis of unsaturated oligomers by FA3 mass
spectrometry.a
Product
Observed ions
m+nh4
m+nh4+h
m+nh4 +
20 thioglycerol
Unidentified
Pentamer
898
916
Tetramer
722
740
Trimer
546
564
654
Dimer
370
388
373, 391
aSamples of lyophilized fractions from the P-2 column were
analyzed by FAB mass spectrometry according to methods
described in the text.

-74-
that these ions resulted from some decomposition of the
dimer, which occurred in transit or in handling prior to FAB
spectrometry.
Activity of the Extracellular Enzyme on Unsaturated
Oligomeric Products
Although the HPLC kinetic analysis of poly(ManA)
digestion yields valuable information about the reaction,
the complex nature of the process, wherein several products
compete for binding to the enzyme and some products are
degraded as they accumulate, does not allow detailed
consideration of the activity of the enzyme on individual
molecular species. To test the capability of the
extracellular enzyme to further degrade products which
accumulate during depolymerization of poly(ManA), the
lyophilized trimer, tetramer, and pentamer purified by P-2
column chromatography were individually incubated with
extracellular enzyme and the reactions sampled at 5 min and
5 h and subjected to HPLC. The conditions and the resulting
profiles are shown in Fig. 3-8. Profiles generated from
poly(ManA) digestion (al and a2) are included for
comparison, as the retention times for the products had
changed over several months of column use since the profiles
shown in Fig. 3-3 were obtained. It is clear that the
trimer is not subject to depolymerization by the enzyme, as
profiles bl and b2 are identical. The tetramer compound (cl
and c2) is not a good substrate for the enzyme, as predicted

Absorbance (230nm) Absorbance (230nm)
-75-
0.08
0.04
0.08
0.04
Figure 3-8. Activity of the extracellular enzyme on puri¬
fied trimer, tetramer, and pentamer products.
The lyophilized products obtained after P-2
column chromatography were dissolved in 0.1 M
sodium phosphate at pH 7.0 such that the final
concentration in the reaction mixtures was
5 mg per ml, and enzyme was added to a final
concentration of 100 units per ml to start the
reactions. The mixtures were incubated at room
temperature and 5 ul samples withdrawn at
5 min and 5 h, and subjected to reversed phase
HPLC. The chromatographic profiles generated
from poly(ManA), trimer, tetramer, and pentamer
are designated a, b, c, and d, respectively,
and numbers 1 and 2 indicate sampling times
of 5 min and 5 h.

-76-
from the kinetic analysis (Fig. 3-4); however, a small
amount of trimer was generated from the tetramer over the 5
h period. An equal amount of monomer should also have been
produced, although it would not be readily detected by
absorbance at 230 nm as noted above. The pentamer was
readily degraded by the enzyme, which converted almost 50%
of the initial quantity to equal amounts of dimer and
trimer, but produced little or no tetramer in 5 h of
incubation.
Few other bacterial poly(ManA) lyases have been
characterized to an extent which would allow a detailed
comparison with the extracellular enzyme analyzed in this
study. Doubet and Quatrano demonstrated that a cell-bound
enzyme from a marine bacterium could degrade poly(ManA) by
an apparent exolytic mechanism (Doubet and Quatrano, 1984).
Davidson et al. (1977) described an endolytic poly(ManA)
lyase which was induced by phage infection of Azotobacter
vinelandii. This enzyme seems to be quite similar to the
one which we have studied, in that it generates a series of
unsaturated products ranging from dimers through pentamers,
although neither the relative levels of the products nor the
limit products were determined. Kashiwabara et al. (1969)
measured poly(ManA) (SM) degrading activities in crude
extracts of two marine pseudomonads. The activities were
weak in relation to the endogenous poly(GulA) lyase
activities, and although the reaction products were not well
characterized, an unsaturated trimer was shown to be the

-77-
major product. Linker and Evans (1984) examined an
intracellular poly(ManA) lyase from a Pseudomonas aeruginosa
isolate which generated unsaturated oligomers ranging from
dimeric through pentameric compounds. This enzyme was
apparently incapable of producing monomer and cleaved the
unsaturated tetramer to form dimeric products. Although the
major reaction products generated by the enzyme which we
have studied are similar to those produced by the P.
aeruginosa enzyme, the catalytic mechanisms of the two
enzymes clearly differ, as shown by the HPLC analysis of the
conversion of unsaturated tetramer to trimer.
The approach utilizing reversed phase ion-pairing HPLC
to evaluate the mechanisms of the lytic depolymerization of
alginate is being applied to study alginate lyases with
different substrate specificies, such as the extracellular
preparation from isolate G, and enzymes from other marine
bacteria (work in progress, J.F. Preston and T. Romeo).
This method should also prove useful for the study of lyases
acting on other glycuronans. Detection of products based on
refractive index or absorbance of UV at shorter wave
lengths (e.g., 205 nm) should extend the applicability of
the method to hydrolytic systems as well.

CHAPTER IV
PURIFICATION AND STRUCTURAL PROPERTIES OF AN
EXTRACELLULAR (1-4)-g-D-MANNURONAN SPECIFIC
ALGINATE LYASE FROM A MARINE BACTERIUM
Introduction
Previous studies utilizing alginate lyases have
examined the structure of alginate (Min et al., 1977; Boyd
and Turvey, 1978), the composition of alginate containing
cell walls of brown algae (Quatrano and Peterman, 1980), and
the feasibility of generating protoplasts of brown algal
species (Preston et al . , 1985b; Romeo et al., 1986). The
possibility that the alginate produced by Pseudomonas
aeruginosa strains colonizing the lungs of cystic fibrosis
patients is involved in the morbidity of that disease has
recently led to the identification of alginate lyases in
isolates of clinical origin (Linker and Evans, 1984; Dunne
and Buckmire, 1985).
With few exceptions alginate lyase enzymes have been
examined as impure mixtures of proteins, or even as
preparations containing more than one activity, disallowing
firm conclusions to be drawn about their substrate
specificities, mechanisms, and their structural properties.
The result is that the only investigations on the structures
of these enzymes, with the exception of molecular weight
-78-

-79-
determinations, have been carried out on two isozymes from
the mid-gut gland of the wreath shell, Turbo cornutus
(Muramatsu and Egawa, 1982; Muramatsu et al., 1984).
We previously reported the isolation of an
extracellular alginate lyase capable of depolymerizing
poly(1-4)-8-D-mannuronan, poly(ManA), derived from alginate,
and an analysis of the products of this enzymatic reaction
(Chapter III). A method for purification of the
extracellular enzyme to homogeneity using HPLC is now
described. Structural properties of the enzyme which have
been examined include the molecular mass, pi, amino acid
composition, content of helical secondary structure, and the
N-terminal amino acid sequence. Some of the properties are
compared with those of other alginate depolymerizing
enzymes.
Materials and Methods
Reagents
Chemicals were analytical grade except as indicated.
Commercially available electrophoresis grade reagents were
used for SDS polyacrylamide gel electrophoresis. Reagents
and chemicals for amino acid analysis and N-terminal
sequencing were commercially available ultrapure grades.
Water for all aqueous solutions was deoinized and glass
distilled.

-80-
Sodium alginate was purchased as a purified grade
(Fisher Scientific Co.) originally obtained from
Macrocystis. The content of mannuronic acid was determined
to be 67% by NMR, using methods established by Penman and
Sanderson (1972) and Grasdalen et al. (1979). The
poly(ManA) was prepared from HC1 hydrolysed alginate
according to Haug et al. (1967) and fractionated by size
through Sephadex G-50 with 0.5 M NaCl as eluent. The
fraction used for these studies was shown to contain 89%
mannuronic acid by NMR. Comparison of total carbohydrate
(Dubois et al., 1956) to reducing termini (Nelson, 1944)
indicated that the range for the degree of polymerization
was 16-22.
Enzyme Assays
The poly(ManA) lyase activity was quantified by
spectrophotometric determination of the chromophore
generated upon reaction of thiobarbituric acid, TBA, with
periodate treated products (Preiss and As’nwell , 1962a;
Weissbach and Hurwitz, 1959). The following conditions have
been used for the enzyme reactions, unless otherwise noted:
pH 7.5, 0.03 M sodium hydrogen phosphate, 0.05 M KCl, 0.10%
sodium alginate, incubated for 10 min at room temperature,
22 C. One unit of activity will generate one nmole of
unsaturated termini and/or unsaturated monomer in 1 minute.
The quantity of protein present in fractions at various
stages of purification was determined by the Coomassie blue

-81-
binding assay of Bradford (1976), using bovine serum albumin
as the standard. When a more accurate estimate of the
protein concentration of the purified poly(ManA) lyase was
needed, spectrophotornetric analysis at 205 and 280 nm was
used (Scopes, 1974). This method, unlike that of Bradford
(see Tal et al., 1985; Compton and Jones, 1985), has
relatively little variation of response to proteins of
differing chemical constitution.
Purification of Poly(ManA) Lyase
The poly(ManA) lyase was purified from a bacterium
originally isolated from healthy tissues of Sargassum
fluitans; the bacterium grew on alginate as sole
carbon source and secreted significant alginate lyase
activity. The properties of this fermentative marine
bacterium, designated as isolate A, or SFFB080483 A, have
been described (Preston et al., 1985a; Romeo et al., 1986).
The purification steps were carried out at 4 C, except for
chromatography in the HPLC systems, which was at room
temperature. For enzyme isolations the organism was grown
to late exponential phase in 0.1% liquid alginate medium
(Preston et al. , 1985a), with rapid gyrotory shaking at 22 C.
Bacterial cells were separated from the medium by
centrifugation (10,000 x g, 10 min), and the medium was
concentrated and dialyzed against water by tangential flow
filtration using a Millipore Pellicon Cassette System with a

-82-
polysulfone membrane (PTCG) which allowed retention of
proteins larger than 10 kDa.
The enzyme was precipitated along with remaining
products of alginate degradation by dropwise addition of 10%
polyethylenimine (PEI, purchased from Sigma Chemical Co.,
St. Louis, MO, titrated to pH 7.5 with concentrated HC1,
diluted with water, and centrifuged at 10,000 x g to remove
insoluble particles) to the concentrated medium. The
relative volume of PEI needed for maximal precipitation of
alginate lyase activity was determined by titrating soluble
alginate lyase activity. The PEI precipitate was collected
by centrifugation (10 min at 10,000 x g) and washed by
resuspension in water with a Potter-Elvehjem homogenizer
driven by a variable speed motor. The suspension was
centrifuged and enzyme activity eluted from the precipitate
by homogenization in 0.25 M NaCl, 0.1 M sodium hydrogen
phosphate at pH 7.5. Insoluble material was removed by
centrifugation at 150,000 x g for 2.5 h, and the supernatant
solution subjected to gel filtration chromatography.
A preparation which was derived from 43 1 of growth
medium (yielding 71.3 g of wet cells) was fractionated on a
column of Sephadex G-75 superfine grade (5 x 79 cm) at 4 C
with 0.1 M sodium phosphate buffered at pH 7.0. Fractions
were collected (7.8 ml) and assayed for enzyme activity and
protein (absorbance at 280 nm). The recovered activity was
concentrated by pressure filtration in an Amicon stirred
cell (Amicon Corp.,
Lexington, MA) with a YM 10 membrane.

-83-
The concentrated activity was applied in 3 separate
runs to a Mono Q HR 5/5 anion exchange column (5 x 50 mm,
Pharmacia, Inc., Piscataway, NJ) and eluted at room
temperature with a gradient of NaCl (0-1.0 M) buffered at pH
7.0 with 0.01 M sodium hydrogen phosphate at a flow rate of
0.5 ml/min. The chromatography system included an LK3
Ultrachrome GTi HPLC system (2152 Controller, 2150 Pump,
2154-002 Injector, LKB-Produkter AB, Bromma, Sweden). A
Gilson Holochrome variable wavelength detector fitted with a
1.00 cm cell (Gilson Medical Electronics, Inc., Middleton,
WI) was used to measure absorbance at 280 nm, which was
recorded with a Linear 800 Versagraph (Linear Instruments
Corp., Irvine, CA). Fractions of 0.5 ml were collected and
assayed for alginate lyase activity.
Enzymatically active fractions from the 3 Mono Q column
runs were concentrated and desalted using the Amicon cell
and again applied to the Mono Q column and eluted as above.
Activity was recovered and concentrated and subjected to gel
filtration HPLC using an UltroPac TSK-G4000 SW column (7.5 x
600 mm, LKB) run at room temperature with a buffer of 0.1 M
sodium hydrogen phosphate, pH 7.0, containing 0.1 M NaCl, at
a flowrate of 0.2 ml/min. The column was fitted to the HPLC
system described above.
Electrophoresis
The SDS polyacrylamide gel electrophoresis was
performed (Laemmli, 1970) using single dimension 1.5 mm

-84-
thick slab gels, 9.73% acrylamide, 0.27% bisacrylamide, pH
8.8 for the running gel and 3.85% acrylamide, 0.11%
bisacrylamide, pH 6.8 for the stacking gel. The conditions
for the analyses are described in detail in the Hoefer
Scientific Instruments Catalog (Hoefer Scientific
Instruments, San Francisco, CA).
Isoelectric focusing gels were purchased as 1.0 mm
thick prepared gels, Ampholine PAGplates pH 3.5-9.5 (LKB)
and were run using an LKB Multiphore system according to
instructions provided with the gels. The isoelectric point
of the enzyme was determined by comparison with standard
proteins. Enzyme activity in the gel was determined by
sectioning the gel into 0.5 cm slices which were incubated
overnight with 200 ul of alginate under standard conditions.
Samples (100 ul) of the solutions were withdrawn and assayed
for unsaturated products. Proteins present in both SDS and
isoelectric focusing gels were fixed with acetic
acid/ethanol/water (1:5:4) and were visualized by staining
at 65 C for 30 min with Coomassie brilliant blue R-250, 0.46
g/400 ml of destain solution (acetic acid/ethanol/water,
1:2.5:6.5) and destaining of the gels with several changes
of destain solution.
Circular Dichroism Spectroscopy
Analyses were performed using a Jasco J500C
Spectropolarimeter. The scan speed was 20 nm/min, at a
sensitivity of 1 mdeg/cm using a spectral bandwidth of 1 nm

-85-
and a time constant of 2 sec. The samples were contained in
a 0.1 cm pathlength cuvette. Data processing was
accomplished with an Oki IF 800 Model 30 computer to provide
scan averaging and molar elipticity values.
Amino Acid Composition
Purified alginate lyase was dialyzed against water and
concentrated using a Centricon-10 unit (Amicon), lyophilized,
and hydrolyzed in 6 N HC1 under N2 in sealed tubes for 24 h
at 100 C. The amino acids were resolved and quantified
using a Beckman 6300 Amino Acid Analyzer with a Nelson
Analytical Data Acquisition System. The amino acid analyses
were carried out by B. Parten and B. Dunn in the Department
of Biochemistry at the University of Florida.
The content of cystine plus cysteine was obtained after
hydrolysis in the presence of dimethylsulfoxide, which
converts these amino acids to cysteic acid (Spencer and
Wold, 1969). Tryptophan content was estimated after
hydrolysis in the presence of 4% t'nioglycolic acid
(Matsubara and Sasaki, 1969).
Serine and threonine are known to be degraded slightly
under the conditions of hydrolysis, and their levels were
estimated by extrapolation to 0 h of hydrolysis from their
levels at 24 and 48 h of hydrolysis (Hirs et al.,
1954) .

-86-
N-Terminal Amino Acid Sequence
Sequence analyses were carried out by B. Parten and B.
Dunn. An Applied Biosystem Model 470A Gas Phase Protein
Sequencer was used for automated Edman degradation. The
program (02RPTH) ran 30 cycles with 2 nmole protein. The
repetitive yield was 94% and the initial yield was 70-80%
with a myoglobin standard. The PTH-amino acids were
identified and quantified using reversed phase HPLC with a
Waters Model Trimod system including a 721 Programadle
System Controller, 730 Data Module, and a WISP 710B
automatic injector, using a Waters Model 440 Absorbance
Detector to monitor absorbance at 254 nm. A Novapak C-18
column (3.9 x 150 mm) was eluted with a gradient of
methanol, 10-90% in 0.025% acetic acid, to resolve the PTH-
amino acids.
Results
Purification of Poly(ManA) Lyase
Table 4-1 summarizes the purification procedures. The
first step in the purification of the enzyme from the crude
extract, precipitation with PEI (see Fig. 4-1 for titration
of soluble activity) followed by'elution of enzyme activity
with 0.1 M sodium hydrogen phosphate containing 0.25 M NaCl,
separates a large amount of acidic carbohydrate from the
enzyme activity, decreases the viscosity of the preparation
and allows a greater amount of activity to be fractionated
on the Sephadex column.

Table 4-1. Purification of poly(ManA) lyase.
Fraction
Total3
activity
units
Totalb
protein
ug
Specific
activity
units/ug
Yield
%
Fold
pur i ficationc
1.
Crude
17,000
—
—
100
2.
PEI eluate
9,000
9,300
0.97
53
1.0
3.
Sephadex G-75
7,300
2,700
2.7
43
00
•
CM
4.
Mono Q-l
6,700
260
26
39
27
5.
Mono Q-2
5,100
110
46
30
47
6.
TSK
4,100
86
48
24
49
aBased on the TBA assay for unsaturated nonreducing termini generated, as described in the
Materials and Methods. The initial crude preparation was derived from 431 of growth
med i urn.
^Assayed according to Bradford (1976) with bovine serum albumin as the standard protein.
cCalculated relative to the PEI eluate. The high level of acidic carbohydrate in the
crude extract prevented assay of its protein content.
87-

ABSORBANCE (548nm)
-88-
Figure 4-1. Titration of soluble alginate lyase activity
with addition of 10% polyethyleneimine. A
crude extracellular preparation was concen¬
trated 300-fold and dialyzed against water
using the Pellicon system, as described in the
Materials and Methods. Samples (100 ul) of
the preparation were vortexed at 0 C with
added volumes of PEI, the precipitates were
removed by centrifugation (13,000 x g, 5 min),
and the supernatant solutions (10 ul samples)
were assayed for enzyme activity (absorbance
at 548 nm).

-89-
Chromatography on Sephadex G-75 separated alginate
lyase activity as a single peak at approximately 0.5 column
volumes (see Fig. 4-2) from some of the larger contaminating
proteins which eluted in the void volume of the column.
Remaining alginate degradation products eluted near the
total column volume (data not shown) and were detected by
absorbance at 232 nm and oxidation with periodate to TBA
reactive compounds. The enzyme activity was completely
dependent upon the addition of exogenous substrate after
this step.
Anion exchange HPLC using a Mono Q column afforded a
purification of 27-fold. Less than 1 h was required for
recovery of the enzyme. The conditions shown in Fig. 4-3
allowed optimum separation of enzyme activity, which eluted
at approximately 0.4 M NaCl, from proteins with similar
charge properties. The procedure was repeated one time with
slight (1.8-fold) improvement in specific activity (Fig.
4-3b).
A final step in the purification was gel filtration
HPLC using a Biosil TSK-G4000 SW column (Fig. 4-4, panel a).
Although this procedure yielded only 1.04-fold purification
(Table 4-1) of the enzyme for the preparation considered
here, in purification of other batches of enzyme the fold
purification was as high as 1.2-fold, and the step has
been used routinely in the purification sequence. Samples
containing up to 250 ug of protein from the Mono Q column
have been successfully purified with the TSK colunn, and the

280
-90-
TUBE NUMBER
Figure 4-2. Fractionation of alginate lyase activity by
Sephadex G-75 chromatography. A preparation
of activity which was obtained from the PEI
procedure was subjected to gel filtration
chromatography using Sephadex G-75, as
described in the Materials and Methods.
Enzyme activity ^543) and a relative measure
of protein (A28O) were determined for frac¬
tions which were collected, and the fractions
containing alginate lyase activity were pooled
and concentrated by pressure ultrafiltration
with an Amicon stirred cell.

A 2 80
-91-
0.2
0.1
0
0.2
0.1
0
0 16 32 48 64
RETENTION TIME (min)
Figure 4-3. Anion exchange HPLC of poly(ManA) lyase. A
preparation containing 730 units of activity
and 270 ug protein from the Sephadex G-75 column
was fractionated with a Mono Q column using a
gradient of NaCl (up to 1.0 M, top portion of
gradient not shown) to elute the activity (panel
a). The single peak which possessed alginate
lyase activity is indicated by an arrow.
Activity peaks from three column runs were
pooled, concentrated and desalted, and this
combined preparation was subjected again to
anion exchange chromatography with the Mono Q
column (panel b).

-92-
RETENTION TIME (min)
Figure 4-4. Gel filtration HPLC and SDS-polyacrylamide gel
electrophoresis of poly(ManA) lyase. A sample of
lyase activity (10 ug of protein) from the second
pass through the Mono Q column was analyzed by gel
filtration HPLC on a TSK4000 column as described
in the Materials and Methods section (panel a).
The elution of standard proteins shown for com¬
parison are as follows: 1, 6-amylase, 200 kDa;
2, bovine serum albumin, 66 kDa; 3 egg albumin,
45 kDa and 6-lactoglobulin, 37 kDa; 4, carbonic
anhydrase, 29 kDa; 5, trypsinogen, 24 kDa; 6,
lysozyme, 14.3 kDa.
Activity from the second pass through the
Mono Q column was denatured in the presence of
SDS and 2-mercaptoethanol and analyzed in the
presence and absence of standard proteins (panel
b). Lane 1 contains standard proteins only,
lane 2 contains 10 ug poly(ManA) lyase, and
lane 3 contains lyase plus standards.

-93-
method has allowed the detection of minor contaminants which
were not apparent in the SDS polyacrylamide gel analyses
(data not shown). The poly(ManA) lyase was judged to be
pure when enzyme activity eluted as a single homogenous peak
of UV absorbing material (280 nm) on gel filtration HPLC, a
single band was observed on SDS gel electrophoresis (see
Fig. 4-4 panel b), and a specific activity of approximately
48 units/ug (as calculated in Table 4-1) was obtained.
Molecular Mass of the Active Enzyme and Its
Single Subunit
The molecular mass of the native enzyme was estimated
by gel filtration HPLC (Fig. 4-4, panel a; Fig. 4-5, panel
a) to be 29 kDa. Polyacrylamide gel electrophoresis in the
presence of SDS showed a single band at approximately 29 kDa
(Fig. 4-4, panel b; Fig. 4-5, panel b) indicating that the
active enzyme is composed of a single polypeptide.
UV Spectrum
The absorbance spectrum of the purified enzyme shows a
maximum at 280 nm, a minimum at 250 nm, a ratio of
absorbance at 280 to 250 of 2.6 (Fig. 4-6), and no measurable
absorbance in the visible range (data not shown) . The
absorbance at 280 nm of a 1 mg/ml solution of enzyme at pH 7
was 1.6 when the protein concentration is calculated by the
method of Scopes (1974). The dye binding assay of Bradford
(1976), using bovine serum albumin as a standard protein,

-94-
RETENTION TIME (min) MOBILITY (cm)
Figure 4-5. Estimation of the molecular mass of the native
poly(ManA) lyase and its denatured subunit.
The molecular mass of the native enzyme was
estimated by comparison of its retention time
on the TSK column (marked with an arrow,
panel a) relative to the retention times of
standard proteins and was essentially iden¬
tical to that of carbonic anhydrase (29 kDa).
The mobility of the denatured enzyme
(10 ug) on SDS polyacrylamide gel electro¬
phoresis is compared in panel b with that of
5 standard proteins.

-95-
250 300 350
A(nm)
Figure 4-6. Ultraviolet spectroscopy of alginate lyase.
The protein in a solution of 0.10 M NaCl
buffered at pH 7.0, with 0.1 M sodium hydrogen
phosphate, was analyzed for its UV absorbance
from 235 to 325 nm.

-96-
underest imated the protein concentration of the purified
enzyme relative to the method of Scopes (1974) by
approximately 50%.
Isoelectric pH of Poly(ManA) Lyase
Isoelectric focusing showed that the purified enzyme
migrated as several closely spaced bands of protein with pi
values from approximately 4.2 to 5.0 (Fig. 4-7a). Analysis
of several batches of purified enzyme showed similar
focusing patterns, and the pattern was visible, though
somewhat obscured by other bands, in preparations of enzyme
which were purified only through Sephadex G-75
chromatography (profiles not shown). Enzyme activity was
detected over a similar pH range (Fig. 4-7b).
Optimum pH
Sodium phosphate and Tris HC1 buffers which were
utilized yielded a reasonably continuous pH profile which
identified pH 7.8 as optimal with alginate as the substrate
(Fig. 4-8). Less than 10% of the maximum activity was
observed below pH 5.8 or above pH 9.1.
Salt Requirement
When poly(ManA) was used as the substrate for the
lyase, enzyme activity was found to require significant
levels of salts for maximal activity (Fig. 4-9). Optimal

I
Figure 4-7. Isoelectric focusing of poly(ManA) lyase. Panel a, horizontal IEF poly¬
acrylamide gel electrophoresis was used to analyze the poly(ManA) lyase
as described in the Materials and Methods: lane 1, standard proteins;
lane 2, poly(ManA) lyase purified through HPLC Mono Q and TSK steps
(4 ug); 3, purified poly(ManA) lyase plus standard proteins. Panel b,
a lane containing poly(ManA) lyase was sliced and enzyme activity was
detected as described in the Materials and Methods. The relative
positions of standard proteins are also shown.
97-

-98-
J I I 1
6 8 10
pH
Figure 4-8. Effect of pH on poly(ManA) lyase activity.
The enzyme was assayed in reaction mixtures
containing final concentrations of 0.1% sodium
alginate, 0.25 M NaCl, and 0.05 M sodium
hydrogen phosphate (solid line) or 0.05 M Tris
HC1 (broken line).

-99-
0.01 0.20 0.60 1.00
CONCENTRATION (M)
Figure 4-9. Effect of salt concentration on enzyme
activity. The enzyme was assayed in reaction
mixtures containing 0.1% poly(ManA), 5 mM
Tris HC1 at pH 7.8, and the indicated salt
concentrations. Curves are numbered to indi¬
cate salts which were present: 1, CaCl2;
2, MgCl2; 3, NaCl; 4, KC1.

-100-
activities were obtained at approximately 0.05 M with
divalent cations and between 0.3 and 0.4 M with monovalent
cations. The ratios of maximal activities obtained for
chloride salts were 1:0.76:0.60:0.46 for Ca:Mg:Na:K.
CD Spectrum
The CD spectrum of the enzyme at pH 7.0 in 0.01 M
sodium hydrogen phosphate is shown in Fig. 4-10. In the
presence of NaCl (0.05 to 1.0 M) the spectrum was not
observably altered. The helical content of the protein was
calculated to be 74% based upon the molar elipticity at 208
nm (Greenfield and Fasman, 1969).
Amino Acid Composition
The amino acid composition was determined after the
hydrolysis of the protein in 6 N HC1 for 24 h, with the
exceptions noted for tryptophan, serine and threonine, and
cysteine (Table 4-2). The content of potentially acidic
residues is high, asx (aspartic acid plus asparagine), 27
residues or 14.9%, glx (glutamic acid plus glutamine), 31
residues or 12.5%. Serine is also abundant with 20
residues. The contents of cys (cysteine and/or 1/2
cystine), methionine, and histidine are low; four residues
of each are present.

-101-
A(nm)
Figure 4-10. Circular dichroism spectroscopy of poly(ManA)
lyase. The CD spectrum of the purified enzyme
at a concentration of 104 ug/ml in 0.01 M
sodium hydrogen phosphate buffer was obtained
(scanned 10 times and averaged). The molar
elipticity from 190 to 250 nm is shown. No
apparent changes in the CD spectrum of the
protein were observed when the ionic strength
was increased by addition of NaCl up to 1.0 M
(data not shown).

-102-
Table 4-2.
Amino acid composition of the
poly(ManA) lyase.
No. residues3
Amino acid
Molecule
Integer
Asx
37.18
37
Thrb
12.99
19
Serb
19.99
20
Glx
31.14
31
Pro
6.73
7
Gly
11.51
12
Ala
16.16
16
Val
16.12
16
Met
3.50
4
lie
13.94
14
Leu
14.87
15
Tyr
16.57
17
Phe
7.25
7
His
3.97
4
Lys
11.32
11
Arg
9.36
9
Cysc
4.43
4
Trpd
12.38
12
aBased upon
an estimated molecular mass of
29,000 kDa.
bThe contents of serine and threonine were corrected for
slight decomposition by extrapolation to 0 h of hydrolysis
using values obtained at 24 and 48 h (Hirs et al., 1954) .

-103-
Table 4-2. continued
cTotal cysteine plus cystine was estimated by conversion to
cysteic acid by hydrolysis in the presence of dimethyl-
sulfoxide (Spencer and Wold, 1969). The value was
corrected for an approximate yield of 67.5%.
^Tryptophan was estimated after hydrolysis in the presence
of 4% thioglycolic acid (Matsubara and Sasaki, 1969). The
value was corrected for 83% recovery.

-104-
Amino-Terminal Sequence
Thirty cycles of Edman degradation allowed 19 of the 30
amino terminal residues of the protein to be unambiguously
assigned (Fig. 4-11). The first 6 cycles established a
continuous amino terminal sequence containing no ambiguous
residues. The HPLC profiles of PTH-derivatives at cycles 9
and 17 showed no assignable residues, possibly indicating
the presence of cysteine, threonine, or arginine, which are
not readily detected. The sample from cycle 19 was lost due
to malfunction of the HPLC. The chromatography system did
not resolve PTH-methionine and PTH-valine, or PTH-isoleucine
and PTH-phenylalanine. However, the peak shapes of the
latter two derivatives are sufficiently different to allow a
preference to be indicated in some cases.
Discussion
This work represents the first time that an enzyme
which specifically depolymerizes poly(ManA) has been
purified to homogeneity from a bacterial source. Previously
such enzymes have been purified from invertebrates
(Muramatsu et al., 1977; Elyakova and Favorov, 1974) and
have been examined in partially purified states from marine
(Doubet and Quatrano, 1984) and terrestrial bacteria
(Sutherland and Keen, 1981; Hansen et al., 1984; Dunne and
Buckmire, 1985) and from Azotobacter vinelandii after

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Asp-Ser-Ala-Pro-Tyr-Asp-Ile(Phe?)-Ala-X-Tyr-Gln-Ser-Met(Val?)-Leu-Asp-
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
lie(Phe?)-X-Lys-X-Gly(Gln?)-Ala-Pro-Asn-Ser-Met,Val-X-Tyr-Phe(lie?)-Ala-Met,Val
N-terminal amino acid sequence of poly(ManA) lyase. Ambiguous residues are
indicated as X. When the amino acid at a site has been narrowed to two
possibilities, the least likely one is indicated by enclosure in
parentheses, if neither is favored the two are punctuated by a comma.
Figure 4-11.
105-

-106-
bacteriophage infection (Davidson et al., 1977). The method
involves concentration of the spent growth medium of the
bacterium by tangential flow ultrafiltration and precipita¬
tion of the enzyme activity and acidic polymers with
polyethylenimine followed by elution of the enzyme with
buffered NaCl. A procedure involving precipitation with
polyethylenimine has been utilized routinely for removing
acidic polymers in the purification of RNA polymerase
(Jendrisak and Burgess, 1975). Further fractionation of the
preparation through a single gel filtration step with
Sephadex G-75, and two kinds of HPLC, anion exchange
chromatography using a Mono HR 5/5 column, and gel
filtration on an Ultropac G4000 SW column, yields enzyme
which is purified to homogeneity. The percentage yields for
the chromatography steps were quite good (from 77 to 91% for
individual steps). The use of HPLC allows the procedure to
be accomplished relatively rapidly (less than 1 h to elute
the enzyme from the Mono Q column and less than 2 h for the
TSK column). The total amount of enzyme protein obtained
from the medium was 4.1 ug/1 (assayed by the method of
Scopes, 1974). This protein yield is comparable to that
obtained for an intracellular poly(GulA) degrading enzyme
from another marine bacterium which yielded 8 ug/1 of
harvested cells (Davidson et al., 1976). The overall
purification of 49-fold indicates that the enzyme
constitutes 2% of the protein secreted. The fold
purification of the other bacterial extracellular alginate

-107-
lyase which has been purified, a poly(GulA) lyase, was 5.7-
fold (Doubet and Quatrano, 1984).
The pH optimum (7.8) and salt optimum (300-400 mM with
NaCl and 10-100 mM with CaC^) of the enzyme make it well
suited to its marine environment, where it is presumed to
depolymerize alginate present in the cell walls of
Sargassum algae. The products which are generated may
provide a pool of oligomeric uronides which is available to
bacterial epiphytes for further depolymerization by
intracellular or cell-bound lyases (Romeo et al., 1986), for
conversion to the monomer, 4-deoxy-L-erythro-5-hexoseulose
uronic acid (Preiss and Ashwell, 1962a).
The molecular mass of the native enzyme and that after
reduction and SDS treatment indicate that the enzyme is
composed of a single polypeptide. The gel pattern obtained
for the native enzyme after isoelectric focusing showed that
, the enzyme is heterogeneous in its charge properties. All
of the observed forms focus in a relatively narrow region of
the anodic side of the gel, pH 4.2-5.0. The acidic nature
of the enzyme is also indicated by its interaction with the
Mono Q column. This property may be accounted for by an
amino acid composition which is high in asx and glx, 14.9
and 12.5%, respectively, on a mole basis, and low in basic
residues, 4.5 and 3.8% for Lys and Arg, respectively. The
amino acid composition of the enzyme differs considerably
from that of two poly(ManA) lyase isoenzymes from a marine
mollusc, Turbo cornutus (Muramatsu and Egawa, 1982);

-108-
asx, glx, ala, val, ile, and tyr are higher in the bacterial
enzyme by 30 to 150% on a residue/molecule basis; gly, phe,
and his are 70 to 200% higher in the mollusc enzymes; levels
of other residues are similar in the two.
The low isoelectric pH range of this enzyme makes it
quite different in net charge from the poly(ManA) lyases of
T. cornutus (Muramatsu et al., 1984), which have pi values
of 7.5 and 7.7, but similar to that of an endolytic
poly(GulA) lyase present in growth media of a marine
bacterium, which was described as having a high net negative
charge at pH 8.5 (Doubet and Quatrano, 1984). These workers
suggested that their enzyme may be a glycoprotein. The
acidic charge of the extracellular bacterial enzymes is not
unexpected for proteins which must function in a saline
environment (Lanyi, 1974).
Although the specific chemical nature of the charge
heterogeneity of the poly(ManA) lyase is not established,
the isoelectric focusing pattern of the purified enzyme
suggests that the protein is subject to posttranslational
modification by one or more of the enzymatic or spontaneous
reactions which are known to alter the net charge of
proteins (see Wold, 1981; Wold and Moldave, 1984). The N-
terminal sequence analysis indicates that this region of the
protein does not account for the observed heterogeneity and
suggests that the enzyme is a single gene product. The
method does not rule out the possibility that glutamine or
asparagine of this region are partially deamidated, or that

-109-
gaps in the sequence may contain modified amino acids. The
single sharp band which is consistently observed for the
purified enzyme on SDS gel electrophoresis tends to rule out
proteolysis. It is relevant that enzyme activity is
detected as a peak which spans several 0.5 cm slices of the
isoelectric focusing gel, suggesting that some or all of the
observed protein bands are enzymatically active and are not
due to gross structural changes which destroy activity.
The CD spectrum of poly(ManA) lyase indicates that the
predominant structure (74%) is helix. This is quite
different from the results obtained for the two poly(ManA)
lyase isozymes from T. cornutus (Muramatsu et al., 1984),
which were predominantly beta structure. The functional
significance of these observations is not immediately
obvious, although it is clear that poly(ManA) lyases from
bacteria and invertebrate sources differ significantly in
their primary and secondary structures.
Comparisons of the structural features of the
poly(ManA) lyase with other bacterial alginate lyases is not
highly informative at this time due to a lack of information
on these enzymes. A marine bacterium was previously shown
to produce a poly(GulA) lyase with a molecular weight of
approximately 50,000 (Davidson, 1976). Two alginate lyases
from marine bacteria were shown to depolymerize poly(ManA)
and poly(GulA) and to have molecular weights of 100,000 and
35,000, respectively (Doubet and Quatrano, 1984). This last
report indicated that studies on the structure of the

-110-
poly(GulA) lyase are being pursued. Perhaps eventually the
features which allow alginate lyases to function, e.g., bind
to substrate and catalyze 3-elimination, will be understood
at the molecular level.
The identification of the N-terminal sequence of the
poly(ManA) lyase allowed a search to be conducted for
similarities with other known protein sequences. The
program FASTP (Lipman and Pearson, 1985) was run by Dr.
Michael Little, Dept, of Biochemistry, University of
Arizona, and provided a comparison with sequences in a
500,000 residue database. Partial homologies were observed
with several proteins, including leghemoglobins,
hemagglutinins, deoxyribonuclease I, and N,0-
diacetylmuramidase. The latter relationship is intriguing
since the partial homology is with the N-terminus.
Convincing evidence of either functional or
evolutionary relationships of the poly(ManA) lyase with
these or other proteins is not possible with the limited
sequence data currently available. Further information on
the amino acid sequence of the poly(ManA) lyase will
probably be obtained by cloning and sequencing the gene
which codes for the protein. The data provided by the N-
terminal sequence analysis may simplify such efforts by
providing a basis for the design of DMA probes (Itakura et
al., 1984).

CHAPTER V
DEPOLYMERIZATION OF ALGINATE BY AN EXTRACELLULAR ALGINATE
LYASE FROM A MARINE BACTERIUM: SUBSTRATE SPECIFICITY,
ACCUMULATION OF REACTION PRODUCTS, AND EFFECTS OF
PRODUCTS ON THE REACTION RATE
Introduction
Knowledge of the substrate specificities of enzymes
which depolymerize carbohydrate polymers is of importance
for their successful application to investigations of
polymer structure and function, and for their use in various
kinds of practical applications. Alginate, a 1-4 polymer of
a-L-guluronic acid and B-D-mannuronic acid, is the main
component of the cell walls of brown algae (Aponte de Otaola
et al., 1983) and is produced by certain bacteria, including
clinically important strains of Pseudomonas aeruginosa
(Linker and Evans, 1973; Hoiby et al., 1977). Alginate
depolymerizing enzymes have been used to analyze the
arrangement of uronic acids in the heteropolymeric regions,
poly(ManA, GulA), of alginate and to determine the size of
the homopolymeric mannuronic acid regions, poly(ManA), of
alginate (Min et al., 1977; Boyd and Turvey, 1978). These
enzymes have also been sought as probes of brown algal cell
wall structure and biosynthesis (Doubet and Quatrano, 1984)
and to provide protoplasts for culture and genetic
-111-

-112-
manipulation of brown algae (Preston et al., 1985a; Romeo et
al. , 1986) .
All alginate lyase enzymes which have been isolated and
examined show preferences for either the poly(l-4)-8-
mannuronan, poly(ManA), or the poly(1-4)-o-L-guluronan,
poly(GulA), blocks of the polymer. Alginate lyases from a
variety of molluscs generally show preferences for
poly(ManA), and some of these enzymes have been isolated and
characterized (Nakada and Sweeny, 1967; Favorov et al . ,
1979; Muramatsu, 1984). Bacteria have been isolated which
produce enzyme activities with demonstrated preferences for
poly(ManA) (Dunne and Buckmire, 1985; Sutherland and Keene,
1981; Hansen et al., 1984), poly(GulA) (Kashiwabara et al.,
1969; Davidson et al., 1976; Boyd and Turvey, 1977), or
activities recognizing both of these regions of alginate
(Doubet and Quatrano, 1982; Preston et al., 1985; Romeo et
al., 1986). Depolymerization of the other region of
alginate which consists primarily of alternating sequences
of mannuronic and guluronic acid, poly(ManA, GulA), can
apparently be effected by some enzymes which are specific
for poly(GulA) over poly(ManA) (Boyd and Turvey, 1978; Min
et al., 1977) .
The characterization of the partially purified
extracellular poly(ManA) lyase activity from a fermentative
marine bacterium associated with actively growing Sargassum
has been previously reported (Chapter III). The activity
(in 10 min assays) was greater when poly(ManA) rather than
poly(GulA) or alginate was used as a substrate. An HPLC was

-113-
used to kinetically monitor the products which accumulated
during the endolytic depolymer-ization of poly(ManA) by this
enzyme. The unequivocal identity of the specific bond(s)
cleaved by the enzyme was not established. The enzyme has
been purified to homogeneity, and some of its structural
features have been reported (see Chapter IV).
Information about the specific type of bond which is
cleaved by the purified enzyme and the minimum region of
polymer necessary for substrate recognition is now
presented. The major products of alginate depolymerization
are identified by HPLC and the effects of products on the
reaction rate are examined.
Materials and Methods
Chemicals and Reagents
Chemicals were analytical or HPLC grade except as
indicated. Deuterium oxide was 99.996% isotopically pure
(Sigma Chemical Co., St. Louis, MO). Water for all aqueous
solutions was deionized and glass distilled.
Substrates
Sod
ium alginates
were
obtained
from
Fisher Scie
ntif ic
Company
(Spring field,
NJ)
and Kelco
(San
Diego , CA) ,
as
purified
grades originally
obtained
from
Macrocystis
(S-211
Fisher; KGHV, Kelco) and Laminaria (Manugel "DMD," Kelco).
Propylene glycol modified alginates were kindly provided

-114-
by Kelco. Homopolymeric block regions of alginate,
poly(ManA), and poly(GulA), and blocks with a high
percentage of alternating sequence, poly(ManA, GulA), were
prepared from HC1 hydrolyzed alginate using methods
developed by Haug et al. (1967) and fractionated by gel
filtration chromatography on Sephadex G-50 with 0.5 M NaCl
as eluant. The degree of polymerization, DP, of each
fraction was determined by comparison of total carbohydrate
(Dubois et al., 1956; Haug et al., 1962) to reducing termini
(Nelson, 1944). The DP values of fractions chosen for these
studies were 16-20, 22 (average), and 16-40, for poly(ManA),
poly(GulA), and poly(ManA, GulA), respectively. The uronic
acid compositions and the frequencies of mannuronic acid
diads for the substrates were determined by the use of
â– *-H NMR with a Nicolet NT-300 instrument operating in the
Fourier transform mode (Penman and Sanderson, 1972;
Grasdalen et al., 1979). The analyses were kindly provided
by Sandra Bonetti and Dr. John E. Gander in the Department
of Microbiology and Cell Science at the University of
Florida.
Enzyme Purification and Assay
The methods and materials for purifying poly(ManA)
lyase were described in Chapter IV, including the
measurement of activity with the thiobarbituric acid assay,
which quantifies unsaturated nonreducing residues and/or

-115-
monomer which are generated by the eliminative depolymer¬
ization of substrate (Preiss and Ashwell, 1962a).
Analysis of the Products from the Depolymerization
of Alginate by Poly(ManA) Lyase
A reaction mixture containing 6 mg/ml of alginate (from
Macrocystis, purchased from Fisher Scientific Co.) and 200
units of poly(ManA) lyase in 0.1 M sodium phosphate buffer
at pH 7.0 was incubated at room temperature and sampled
periodically over 15 h. Aliquots of 10 ul were subjected to
ion-pairing reversed phase HPLC as previously described
(Romeo and Preston, 1986).
Determination of and Measurement of Inhibition
by Limit Products
The conjugated tt electron system of the unsaturated
oligomeric products absorbs UV with a maximum at 232 nm
(Preiss and Ashwell, 1962a). Inhibition and Km data were
obtained by continuously monitoring absorbance at 232 nm of
the depolymerization reactions in 1.00 cm quartz cuvettes,
using a Gilford Model 2400 spectrophotometer (Gilford
Instrument Laboratories, Inc., Oberlin, OH) with a Honeywell
chart recorder (Ft. Washington, PA). Apparent initial
velocities were maintained for sufficient periods of time
(at least 1 min) to allow their direct graphical
interpretation.

-116-
Results
The Extents of Depolymerization of Alginates, Block Regions
of Alginate, and Propylene Glycol Alginates
by Poly(ManA) Lyase
The time course of the depolymerization of several
potential substrates by the enzyme with the formation of
oligomeric uronides containing unsaturated nonreducing
terminal residues is shown in Fig. 5-1. At the substrate
concentrations chosen for the reactions, 0.20 mg/ml, the
initial rates were indicative of the final extent to which
each substrate was depolymerized. At 25 h the reactions had
almost ceased, yet the addition of a small quantity of
substrate to three of the reactions demonstrated that the
enzyme had remained active (Fig. 5-1). The substrates were
therefore assumed to be degraded to a practical limit.
Table 5-1 quantitatively compares the extent to which
each substrate was depolymerized at 25 h. Poly(ManA)
allowed the greatest number of unsaturated terminal residues
to be formed and hence the greatest amount of depolymeriza¬
tion; native alginates allowed from 26 to 58% of the number
of cleavage reactions observed for poly(ManA), and the
alternating polymer and poly(GulA) allowed still less, 17
and 4%, respectively. For poly(ManA) the fraction of uronic
acid residues converted to unsaturated terminal residues at
25 h, F^x, was 0.16, or a mole fraction of 1 out of 6.3
residues.

ABSORBANCE (548nm)
-117-
Figure 5-1. Extent of depolymerization of alginates and puri¬
fied polymeric blocjc regions of alginate by
poly(ManA) lyase. Reaction mixtures containing
final concentrations of 0.20 mg/ml of substrates,
0.05 M Tris HC1 at pH 7.8, and 0.3 M NaCl were
incubated with poly(ManA) lyase in 1.5 ml capped
polypropylene vials. Samples (100 ul) were with¬
drawn at the indicated times and the extent of
depolymerization determined by the TBA assay
(1.475 ml final assay volume), measuring absorbance
at 548 nm. Curves are numbered to identify sub¬
strates: 1, poly(ManA); 2, sodium alginate
(Fisher); 3, sodium alginate (Kelco, KGHV); 4,
sodium alginate (Kelco, Manugel DMD); 5, poly(ManA,
GulA); 6, poly(GulA).
At 25 h 20 ug of poly(ManA) was added to the
reactions (indicated by an arrow) which contained
poly(ManA), poly(ManA, GulA), and poly (GulA) ,
curves 1, 5, and 6, respectively, and the reac¬
tions were allowed to continue for 10 min before
sampling and assaying.

Table 5-1. Extent of degradation of purified block regions of alginate, sodium alginates,
and propylene glycol modified alginates by poly(ManA) lyase.
e
f
g
h
Extent of
depolymer-
fmm
fmmm
fax
a
b
c
d
i zation
Substrate
fm
fmm
fmmm
F AX
Fmm P°ly(ManA)
FMMM P°ly(ManA)
fAx Poly(ManA)
Poly(ManA)
0.89
0.81
0.61
0. 16
1.00
1.00
1.00
Poly(ManA,
Gul A)
0.61
0.36
0.21
0.027
0.44
0.20
0.17
Poly(GulA)
0.11
0.043
0.025
0.006
0.053
0.04
0.04
Sod i um
Alginate-1
0.67
0.58
0.093
0.71
0.58
-2
0.66
0.53
0.086
0.65
0.54
-3
0.38
0.31
0.042
0.38
0.26
PG-
Alginate-50
0.14
0.016
0.10
-85
0.27
0.010
0.06
118-

Table 5-1.
continued
a01igomeric block regions were prepared from partial acid hydrolysates as described in the
Materials and Methods. Sodium alginate-1 was alginate which was originally purified from
Macrocystis (Fisher Scientific Co.), sodium alginates-2 and -3 were purified from
Marocystis (KGHV) and Laminaria (Manugel "DMD") , respectively, and were generously
provided by Kelco. Propylene glycol alginates, PG-alginate-50 and -85 were Kelcoloid LVF,
and Kelcoloid S, respectively, and were modified at 50 and 85% of their carboxylate
moieties, as determined by Kelco.
^The mole fraction of mannuronic acid, as determined by NMR.
cThe frequency of occurrence of the mannuronic acid diad in the substrates, as determined
by 1H NMR.
^The frequency of occurrence of the mannuronic acid triad sequence in block polymers,
based upon NMR data of Grasdalen et al . (1981).
eThe fraction of the total uronic acid residues which were converted to unsaturated
termini in 25 h of exposure to enzyme, as determined by the TBA assay.
^The frequency of mannuronic acid diad of the substrates normalized with respect to that
found for poly(ManA).
^The frequency of mannuronic acid triad normalized with respect to that of poly(ManA).
^The extent of depolymerization normalized as the ratio of that quantity of unsaturated
termini generated from a given substrate at 25 h versus the amount of unsaturated termini
generated from poly(ManA) in 25 h, as measured by the TBA assay.
119-

-120-
To interpret the values describing the extent of
depolymerization of poly(ManA) and other substrates in terms
of the specific kind of site which is recognized by the
enzyme, parameters which express quantitative estimates of
the content of mannuronic acid, and of its arrangement in
the polymers, are given (Table 5-1). The fraction of each
substrate which is composed of mannuronic acid, FM, and the
frequency of occurrence of the mannuronic acid diad, FMM,
were determined by NMR. The triad frequency, FMMM,
was taken from previously published -^C NMR data for
purified block polymers derived from alginate (Grasdalen et
al. , 1981) .
An average DP of 3.4 may be estimated for the dimeric
through hexameric products which are present late in the
reaction with poly(ManA), based on the HPLC quantification
which was previously described (see Chapter III). The DP is
equal to the quantity of total residues divided by the
quantity of total residues divided by the quantity of
unsaturated termini. Since the fraction of the residues in
poly(ManA) which were converted to unsaturated termini at 25
h was 0.16, an estimation of the fraction of the total
uronic acids converted to low DP products is 3.4 x 0.16 or
0.54. The mannuronic acid triad frequency of the
poly(ManA), 0.61, agrees fairly well with this, but the diad
frequency is considerably higher, 0.81.
When the mannuronic acid diad and triad frequencies of
other block polymers are normalized relative to those of
poly(ManA), and the fractions of residues converted to

-121-
unsaturated residues are similarly normalized, a direct
quantitative relationship is observed for the extent of
depolymerization relative to mannuronic acid triad
frequency. Although mannuronic acid triad frequencies were
not available for the alginates, the diad frequencies, as
for the isolated block polymers, overestimated the amount of
potential substrate.
The esterification of the carboxyl moieties of alginate
with propylene glycol appears to completely block enzyme
activity; alginates which were 50% and 85% esterified were
depolymerized to 10% and 6%, respectively, of the extent
measured for poly(ManA) (Table 5-1).
Products of Alginate Depolymerization
A reaction mixture which contained 200 units/ml of
poly(ManA) lyase and 5.0 mg/ml of sodium alginate
(Macrocystis alginate from Fisher Scientific Co.) in 0.1 M
sodium phosphate buffered at pH 7 was sampled periodically,
and 10 ul aliquots were subjected to reversed phase HPLC.
The elution profile of the major products is shown in Fig.
5-2. The major products generated from alginate were
indistinguishable from those generated from poly(ManA) based
on the HPLC analysis. A linear relationship exists between
the size (DP) of the products generated from alginate and
poly(ManA) versus the log of their retention times on HPLC,
as shown in Fig. 5-3. The DP values of the dimer through
pentamer generated from poly(ManA) by the poly(ManA) lyase

Figure 5-2. The HPLC analysis of products generated by depolymerization of alginate by
poly(ManA) lyase. The unsaturated oligomeric products which had accumulated
after 15.1 h of depolymerization of alginate (5 mg/ml) by the poly(ManA) lyase
(200 units/ml) were fractionated by reversed phase ion-paring HPLC. The major
peaks are numbered in the order of their elution from the column; peaks 1
through 5 represent the unsaturated dimer through hexamer, respectively, based
on comparison of their retention times with those of the products from
poly(ManA) depolymerization (see Fig. 5-3).
122-

RETENTION TIME (min)
-123-
Figure 5-3. The relationship of the DP values of unsaturated
uronic acid oligomers with their retention times
after reversed phase HPLC. The retention times
of unsaturated dimeric through pentameric pro¬
ducts which were generated from the depolymeriza¬
tion of poly(ManA) by the poly(ManA) lyase were
corrected for the dead volume of the chroma¬
tography system and plotted on semilog paper
against their DP values (previously established,
Romeo and Preston, 1986, shown as circles)
along with the retention times and inferred DP
values of the analogous products from alginate
depolymerization (squares).

-124-
were previously established (Chapter III); the other DP
values were inferred.
When the levels of products present in the alginate
depolymerization reaction were measured at various times by
HPLC (Fig. 5-4), the pattern of product accumulation and
disappearance was similar to that observed for poly(ManA)
(Chapter III). The ratios of the concentrations of products
changed as the reaction progressed. The concentrations of
dimer and trimer, relative to other products, increased
beyond the first time of sampling, approximately 0.1 h, and
the absolute level of the pentamer decreased slightly from
that time. The concentrations of the dimer and trimer
continued to increase from approximately 7 h to 15.1 h (the
final time of sampling), while concentrations of other
products remained constant. The levels of products at
similar extents of reaction for alginate and poly(ManA) are
shown in Table 5-2, at which time the concentration of
trimer > tetramer > dimer > pentamer > hexamer. The two
reactions differed in that the relative levels of the larger
unsaturated oligomers (pentamer and hexamer) present late in
the reactions were somewhat higher when alginate was the
substrate.
Effect of Reaction Products on Enzyme Activity
The Km of the reaction with poly(ManA) was determined
to be 1.6 mM, with respect to uronic acid residues, as shown
in Fig. 5-5. The V,nax was approximately 130 umole •min--'-• ug .

RELATIVE PEAK AREA
TIME (h)
Figure 5-4. Kinetic evaluation of the unsaturated uronides generated from alginate by
the poly(ManA) lyase. The reaction described in the legend of Fig. 5-2
was monitored over 15.1 h by periodically sampling the reaction and sub¬
jecting the products to reversed phased HPLC. The curves are numbered
according to the individual products which they represent (see Fig. 5-2).
The unsaturated dimer product from poly(ManA) has a peak area of 0.11 units
per nmole.
125

Table 5-2. Comparison
poly(ManA)
of the
lyase
relative levels
from alginate and
of unsaturated
poly(ManA).
products generated
by
Products0
Substrate
Dimer
Trimer
Tetramer
Pentamer
Hexamer
Alginate3
0.35
1.00
0.49
0.24
0.16
Poly(ManA)^
0.28
1.00
0.54
0.16
0.05
aThe reaction mixture contained an initial concentration of 5 mg/ml sodium alginate
(Fisher), 200 units/ml poly(ManA) lyase, buffered with 0.1 M sodium phosphate at pH 7.0.
°This reaction mixture contained 10 mg/ml poly(ManA) and 50 units/ml poly(ManA) lyase.
cThe peak areas for products which had accumulated after 1.9 and 31 h from alginate and
poly(ManA), respectively, which represent similar extents of depolymerization, were
calculated as fractions of the trimer peak area for each reaction.
126-

UIIJU
-127-
-g (mM'1)
Fiqure 5-5. Lineweaver Burk plot of poly(ManA)
depolymerization by poly(ManA) lyase. The
reactions were carried out at pH 7.8, in 0.05
M Tris HC1 containing 0.30 M NaCl, at 23 C.
The K was determined by linear regression.

-128-
Two products which were previously shown to accumulate in,
and were isolated from the reaction with poly(ManA) (Romeo
and Preston, 1986), showed only slight inhibition of the
depolymerization of poly(ManA) (Table 5-3), even at relative
uronic acid residue concentrations of 5.7 and 7.6-fold of
the tetramer and trimer, respectively, over poly(ManA). The
addition of poly(GulA) also did not inhibit the poly(ManA)
depolymerization, but allowed a slight increase in the
initial rate of the reaction.
Discussion
Previously a crude preparation of extracellular
alginate lyase from a facultative marine bacterium which was
isolated from actively growing Sargassum fluitans
(designated isolate A, or SFFB080483 A) was shown to
preferentially depolymerize poly(ManA) relative to alginate
and poly(GulA) as measured in 10 minute assays (Preston et
al., 1985a). The single alginate lyase activity from this
source was purified (see Chapter IV) and has now been shown
to depolymerize uronic acid polymers to an extent which is
closely related to the frequency of mannuronic acid triad
present. The extent of depolymerization is not as closely
related to the content of mannuronic acid of the polymers,
nor to the frequency of mannuronic acid diad. This is
evidence that the ManA-ManA bond is preferentially cleaved
and, furthermore, that the enzyme acts only upon portions of
alginate which contain uninterrupted sequences of three or
more mannuronic acid residues.

Table 5-3. Effects of unsaturated mannuronic acid trimer and tetramer and poly(GulA) on
the initial rate of poly(ManA) depolymerization by poly(ManA) lyase.3
[Trimer]
mM
[Tetramer]
mM
[Poly(GulA)]
mM
[Poly(ManA)]D
mM
0.02 0.4
O
•
4^
O
0.4
5.0
0.21
91
95
91
120
1.7
96 '83
—
68
—
aValues are reported as percentages of the apparent initial velocities measured in the
absence of added trimer, tetramer, or poly(GulA), as determined by measuring increasing
absorbance of the solutions at 232 nm.
°The concentration of poly(ManA) and poly(GulA) are reported as uronic acid residue
concentrations, mM.
cData for this reaction were obtained by incubation of the enzyme with trimer for
before addition of poly(ManA); all other reactions were started upon addition of
enzyme.
20 min
the
129-

-130-
This suggests a site on the enzyme which contacts three
mannuronic acid residues to allow productive interaction
with the substrate. However, it should be emphasized that
the data simply indicate a correlation between the
mannuronic acid triad frequency of the substrates and the
extent of depolymerization by the poly(ManA) specific enzyme
and do not exclude the possibility that the enzyme has
contact sites which interact with a greater number of
residues of the substrate. Subsite affinity studies of the
kind which have been carried out for amylase enzymes
(Suganuma et al., 1978; MacGregor and MacGregor, 1985; Sano
et al . , 1985) would help to clarify this point.
The selectivity of the enzyme for poly(ManA) versus
poly(GulA) and alternating blocks is similar to that of
alginate lyase VI from the mollusc Littorina (Favorov et
al. , 1979), and the specificity is more stringent than that
of two poly(GulA) lyases examined previously (Min et al.,
1977; Boyd and Turvey, 1978), which were characterized as
also being able to depolymerize the GulA-ManA bonds of the
alternating polymer.
The results suggest that difficulties which have been
encountered in attempts to correlate the extent of enzymatic
degradation of alginates and block polymers from alginate
with monomer composition (Boyd and Turvey, 1977), may be
explained by considering the diad and triad frequencies of
substrates, which may better approximate the true substrate
sites of alginate lyases.

-131-
The enzyme is apparently unable to act upon alginate
which has been modified by formation of propylene glycol
esters. This suggests a role for the carboxyl moieties of
alginate in substrate recognition, as the esterification of
car’ooyxl moieties of 1-4 linked uronic acid polymers
actually increases their susceptibility to base-catalyzed
elimination (Kiss, 1974). The effect on activity appears to
be similar to that observed for a poly(ManA) lyase from a
marine mollusc, which had 40% as much activity on PG-
alginate as with native alginate (Muramatsu et al., 1977),
although these results cannot be directly compared, since
the level of esterification of the alginate was not
reported.
The activity of the poly(ManA) lyase enzyme in a crude
extracellular preparation was previously shown to degrade
alginate endolytically, based on viscometric analysis (Romeo
et al., 1986). The extensive degradation of the sodium
alginates in the present study also indicates that the
purified enzyme is endolytic; regions of nonreactive
substrate, i.e., guluronic acid residues, in the high
molecular weight alginates should greatly limit the extent
of depolymerization carried out by strictly exolytic
enzymes. The variety of products generated from alginate,
which were measured by HPLC, could only arise from somewhat
random attack on alginate, further establishing the
endolytic mechanism.

-132-
Alginate and poly(ManA) are depolymerized to low DP
products with identical retention times in the HPLC system
employed and include dimeric through hexameric forms. The
identities of the products from alginate are inferred by
comparison of their mobilities with those of products from
poly(ManA) depolymerization. The latter have been
characterized by a number of criteria, including fast atom
bombardment mass spectrometry (Chapter III). The linear
relationship observed for the log of the retention times of
the unsaturated uronides in ion-pairing reversed phase HPLC
with their DP values is a feature of the method which should
allow facile determination of the size (DP values) of
uronides which belong to homologous series containing two or
more known members.
In addition to the low DP unsaturated uronic acid
oligomers from alginate, the enzyme must also release larger
fragments of alginate which should contain poly(GulA) and
poly(ManA, GulA) block regions of alginate. However, these
are not detected by the HPLC system due to their strong
interaction with column. The larger products should make up
a fairly small fraction of the reaction products on a mole
basis, based on the sizes of the poly(GulA) and poly(ManA,
GulA) regions of the polymer, although on a weight basis
they are significant.
The kinetics of the accumulation of the major low DP
products generated from alginate is similar to that of the

-133-
products from poly(ManA) depolymerization (Chapter III),
i.e., the relative abundance of the analogous oligomers is
similar at comparable extents of substrate depolymerization,
except for the relatively greater concentration of the
pentamer and hexamer late in the reaction with alginate.
This may be due to the presence of guluronic acid in these
products, which would be expected to make them refractory to
further depolymerization by the enzyme and should be present
to a greater extent in products from alginate than from
poly(ManA). Support for this possibility comes from the
previous observation that the unsaturated pentamer which was
obtained from partially depolymerized poly(ManA) is a good
substrate for the enzyme (Chapter III). The limit products
should not contain unsaturated pentamer unless it contains
some guluronate.
It is plausible that the dimer and trimer are composed
(in addition to their nonreducing terminal unsaturated
residues) primarily or completely of mannuronic acid
residues. This is derived from two lines of evidence. The
observation that the extent of depolymerization is
correlated closely with frequency of mannuronic acid triad
but not diad of the substrates indicates that the enzyme
requires at least three sequential mannuronic acid residues
for catalytic activity. Secondly, although a single trimer
peak is observed from the digestion of alginate by the
extracellular alginate lyase from isolate A, the HPLC system
is capable of partially resolving three forms of unsaturated

-134-
trimer uronides which are released from alginate by a crude
extracellular preparation of alginate lyase from another
facultative marine bacterium, isolate G (Preston and Romeo,
unpublished) which was shown to be active on both poly(ManA)
and poly(GulA) blocks of alginate (Romeo et al., 1986).
Low DP unsaturated products from the partial
degradation of poly(ManA) by the poly(ManA) lyase were
previously examined for their ability to act as substrates
of the enzyme (Chapter III). The trimer was shown to be
resistant to depolymerization; the tetramer was degraded
slowly, with 9% conversion to trimer product in 5 h, under
conditions which would have allowed 50% of the pentamer to
be converted to trimer and dimer. Neither the trimer nor
the tetramer are effective inhibitors of poly(ManA)
depolymerization. Also, poly(GulA) caused a slight increase
in the initial velocity of the reaction, presumably due to
contaminating mannuronic acid sequences. This is in
contrast with the results obtained by Boyd and Turvey (1977)
for a guluronate lyase from Klebsiella aerogenes, which is
competitively inhibited by a normal, guluronic acid trimer;
i.e., not containing a terminal unsaturated residue.
In considering the mechanism of the enzyme, the
observations that the tetramer is slowly converted to a
trimer and a monomer, but not to dimer, and that the
frequency of the mannuronic acid triad is correlated with
the extent to which a particular substrate is degraded,

-135-
suggest a cleavage reaction which requires the interaction
of the enzyme with at least three sequential mannuronic acid
residues and a location for the reaction between the second
and third residue of this site of interaction, or bond C, as
shown:
abed a b d
-M-M-M- -> -M-M X-
Cleavage of bond b appears to be ruled out based upon the
failure of the tetraraer to yield dimer. The data do not
rule out the possibility that bond a is broken. However, as
the configuration of the unsaturated terminal residue of the
tetramer is sufficiently different from that of a mannuronic
acid residue or guluronic acid residues which might share
bond a in a polymer, this is not expected to occur. One of
the advantages of the lyase mechanism may be that the new
nonreducing termini which are generated limit further
interaction with the enzyme, thus yielding products which
have much reduced affinities for the endolytic enzyme which
generated them. Transglycosylation and condensation
reactions, which occur under high substrate concentrations
for hydrolytic enzymes such amylases (Suganuma et al., 1978)
would then be avoided. In the reaction of the poly(ManA)
lyase with unsaturated products (Fig. 3-8), we did not
observe higher oligouronides to be formed, e.g., no
tetramer, pentamer, or hexamer was formed when the enzyme
was incubated with the unsaturated trimer. This, in
addition to the minimal product inhibition by unsaturated

-136-
trimer and tetramer, lends credence to this notion. In
effect then the lytic mechanism allows the depolymerization
reactions to proceed at rates independent of the
concentration of products generated by the reaction.
The Km of the poly(ManA) lyase for its preferred
substrate is relatively high, 1.6 mM (with respect to total
uronic acid residues of poly(ManA)). The Km of a guluronate
lyase from another marine bacterium is also high, 5.51 mM
(for guluronic acid residues, Davidson et al., 1976),
relative to alginate lyases from a marine mollusc, 0.19 mM
(Elyakova and Favorov, 1974) and from Klebsiella, 0.11 mM
(Boyd and Turvey, 1977). The high Km of the poly(ManA)
lyase for substrate and the apparent lack of product
inhibition may help it to avoid substrate and product
inhibition during activity on algal cell walls.
The enzyme activity examined in this study is the major
extracellular alginate degrading activity from a bacterium
which probably derives much of its carbon and energy from
alginate in its native environment, the surface of Sargassum
algae. The organism also produces intracellular or cell-
bound activities capable of degrading both poly(ManA) and
poly(GulA), and which are more exolytic in their overall
mechanisms (Romeo et al., 1986). Unlike the extracellular
enzyme, these are able to produce a considerable amount of
apparent monomer product from poly(ManA) (Chapter IV). This
organism, which produces a single extracellular poly(ManA)
specific lyase may, therefore, obtain partially degraded

-137-
poly(GulA) and/or poly(ManA, GulA) regions from the action
of the extracellular enzymes of other bacteria which share
the same unique microenvironment, and which have been shown
to produce extracellular enzymes active on poly(GulA)
(Preston et al . , 1985a; Romeo et al., 1986).

CHAPTER VI
CONCLUSIONS
These studies have demonstrated that the aerotolerant
bacteria which are associated with Sargassum species are an
abundant natural source of alginate lyase enzymes. All of
the alginate degrading isolates which were examined
possessed activities with varied substrate specificities and
mechanisms. These enzymes may provide tools for studies on
the structure and biosynthesis of brown algal cell walls,
and for developing methods for reproducible production of
protoplasts from brown algal species.
The analysis of alginate depolymerization using HPLC
has been shown to rapidly yield valuable quantitative
information about the mechanism of the reaction. Comparison
of the depolymerization of the poly(ManA) regions of
alginate by intracellular and extracellular activities from
a single bacterial isolate identified distinct patterns of
product accumulation. The method should be readily
adaptable to the analysis of the depolymerization of other
uronic acid polymers.
A poly(ManA) specific alginate lyase has been purified
to homogeneity for the first time from a bacterial source,
and some of its structural properties have been reported. A
-138-

-139-
relatively rapid procedure involving anion exchange and gel
filtration HPLC has been developed, which provided
reasonably good yields of enzyme. The enzyme was composed
of a single polypeptide, 29 kDa, and contained a homogeneous
N-terminus by automated Edman degradation. The protein was
resolved into several isoforms with pi's ranging from
approximately 4.2-5.0, suggesting posttranslational
modification. The secondary structure was approximately 74%
a-helix. It is clear that the structure of this enzyme
differs from other alginate lyases which have been
characterized from molluscs. Comparisons with other
bacterial alginate lyases should prove interesting; however,
these will have to await future studies.
A model for the specificity of the active site of the
extracellular poly(ManA) specific enzyme has been presented,
which was based upon the extent of depolymerization of well
characterized alginates and block regions of alginate and
the activity of the enzyme on reaction products. The
reaction products did not appreciably inhibit enzyme
activity, nor were transglycosylation reactions observed.
These observations suggest that endolytic lyases which are
capable of degrading uronic acid polymers may allow the
depolymerization reactions to proceed without interference
from end products. This might provide a selective advantage
for organisms which produce lyases as opposed to hydrolases.
This may explain the observation that alginate degrading
organisms produce lyases.

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BIOGRAPHICAL SKETCH
Tony Romeo was bor
Arkansas, to Edward and
B.S. degree from the Un
graduate studies there,
for the Ph.D. degree in
Cell Science.
n December 20, 1956, in Batesville,
Norma Romeo. After receiving the
iversity of Florida, he continued
where he is currently a candidate
the Department of Microbiology and
-151-

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
íAlámes E\ Preston, III, Chairman
Professor of Microbiology and
Cell Science
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
William B. Gurley s
Assistant Professor/qj
Microbiology and bell Science
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
liv
Peter M. McGuire
Assistant Professor of
Biochemistry and Molecular
B iology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate/ in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Professor of Microbiology and
Cell Science
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
4 /--‘V- i. ^ ... .• ...
Lonnie O. Ingram /
Professor of Mict-bbiology and
Cell Science
This dissertation was submitted to the Graduate Faculty of
the College of Agriculture and to the Graduate School and wa
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.
August 1986
Dean, College of Agriculture
Dean, Graduate School

UNIVERSITY OF FLORIDA
3 1262 08554 1539






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