Carbohydrate Polymers 38 (1999) 319–328

Polysaccharases for microbial exopolysaccharides

Ian W. Sutherland 1
Institute of Cell and Molecular Biology, Edinburgh University, Mayfield Road, Edinburgh EH9 3JH, UK
Received 6 April 1998; accepted 5 August 1998

Abstract
Microbial exopolysaccharides (EPS) are the substrates for a wide range of enzymes most of which are highly specific. The enzymes are
either endoglycanases or polysaccharide lyases and their specificity is determined by carbohydrate structure with uronic acids often playing a
major role. The presence of various acyl substituents frequently has little effect on the action of many of the polysaccharases but markedly
inhibits some of the polysaccharide lyases including alginate and gellan lyases. The commonest sources of such enzymes can be either
microorganisms or bacteriophages. These specific polysaccharide-degrading enzymes can yield oligosaccharide fragments, which are
amenable to NMR and other analytical techniques. They have thus proved to be extremely useful in providing information about microbial
polysaccharide structures and were routinely used in many such studies. Complex systems containing various mixtures of enzymes may also
be effective in the absence of single enzymes but may be difficult to obtain with reproducible activities. Such preparations may also cause
extensive degradation of the polysaccharide structure and thus prove less useful in providing information. Commercially available enzyme
preparations have seldom proved capable of degrading microbial heteropolysaccharides, although some are active against bacterial alginates
and homopolysaccharides including bacterial cellulose and curdlan. 䉷 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Exopolysaccharide; Polysaccharases,Bacteriophage

1. Polysaccharases releasing glucose, laminaribiose, laminaritriose and other

oligosaccharides in the b -1,3-linked series. The other endo-
Microbial exopolysaccharides may be degraded either by glucanase yields the pentasaccharide laminaripentaose.
polysaccharide hydrolases (polysaccharases) or by polysac- Scleroglucan, a fungal b -d-glucan carrying 1,6-b -d-gluco-
charide lyases. The latter cleave the linkage between a syl side-chains, is susceptible to the commercially available
neutral monosaccharide and the C4 of a uronic acid with (1!3†-b -d-glucanase preparation marketed as ‘zymolyase’
simultaneous introduction of a double bond at the C4 and (Catley and Fraser, 1988). Similarly, degradation of bacter-
C5 of the uronic acid. Both types of enzyme are commonly ial cellulose (and cellulose from other sources) requires the
found to degrade microbial EPS as well as eukaryotic poly- concerted action of various endo- and exo-b -d-glucanases
mers (Sutherland (1995). Although the enzymes degrading (Gilbert and Hazlewood, 1993), yielding a complex mixture
microbial exopolysaccharides may be endo- or exo-acting of glucose and cellodextrins. In contrast to these systems,
leading to rapid or slow breakdown of the polymer chain the hydrolysis of many microbial heteropolysaccharides is
respectively, almost all those studied in detail have proved commonly achieved by a single, highly specific, endo-
to be endoglycanases or endo-acting polysaccharide lyases. acting polysaccharase which yields a series of oligo-
Only for microbial extracellular homopolysaccharides is a saccharides representing either the repeating unit structure
wider range of exo- and endo-acting enzymes usually avail- or multiples of it.
able. As was pointed out by Mishra and Robbins (1995), b -
d-glucanases with a variety of specificities can be used to
elucidate the structures of this group of polysaccharides. 2. Sources of polysaccharases
Thus, even the linear 1,3-b -d-glucan curdlan is degraded
by four different enzymes. Two exo-glucanases release d- Polysaccharases may derive from three major sources –
glucose and laminaribiose respectively. Two endo-gluca- endogenously from the polysaccharide-synthesising micro-
nases differ in their mode of action. One attacks randomly, organisms; exogenously from a wide range of other
eukaryotic or prokaryotic microorganisms; and thirdly,
1
Tel.: 0131 650 5331; Fax 0131 650 5392; E-mail: i.w.sutherland@ from bacteriophage particles or phage-induced bacterial
ed.ac.uk lysates. Most of the enzymes acting on microbial EPS
0144-8617/99/$ - see front matter 䉷 1999 Elsevier Science Ltd. All rights reserved.
PII: S0144-861 7(98)00114-3
320 I.W. Sutherland / Carbohydrate Polymers 38 (1999) 319–328

Table 1
Glycan depolymerases associated with exopolysaccharide synthesis

Bacterial species Enzyme Mol. Mass Reference

Acetobacter xylinum Endoglucanase (CM-Cellulase) 35.6 kDa Standal et al. (1994)

Agrobacterium tumefaciens Endoglucanase (CM-Cellulase) ⫺ Matthysse et al. (1995)
Azotobacter chroococcum Alginate lyase 43 kDa Pecina and Paneque (1994)
(polymannuronate lyase)
Azotobacter vinelandii Alginate lyase ⫺ Kennedy et al., (1992)
(polymannuronate lyase)
Cellulomonas flavigena 1,3-b -d-Glucanase Voepel and Buller (1990)
Escherichia coli K5 N-acetyl-heparosan lyase 70–89 kDa Legoux et al. (1996)
Pseudomonas aeruginosa Alginate lyase 39 kDa Schiller et al. (1993)
(polymannuronate lyase)
Pseudomonas fluorescens Alginate lyase ⫺ Hughes (1997)
(polymannuronate lyase)
Pseudomonas fluorescens Galactoglucanase ⫺ Osman et al., (1993)
(marginalis)
Pseudomonas mendocina Alginate lyase ⫺ Sengha et al.(1989)
(polymannuronate lyase)
Pseudomonas putida Alginate lyase ⫺ Conti et al. (1994)
(polymannuronate lyase)
Rhizobium meliloti Succinoglycan depolymerase ⫺ Glucksman et al. (1993)
Rhizobium meliloti Polyglucuronic acid lyase Michaud et al. (1997)
Sphingomonas spp. Gellan lyase ⫺ Sutherland and Kennedy (1996).
⫺ Yamazaki et al. (1996)
Streptococcus equi Hyaluronidase ⫺

have to be obtained de novo, few are available commer- the same physiological conditions, which favour EPS synth-
cially and few of the commercially available enzymes act esis. If the enzymes are present, they may only be released
on microbial polysaccharides. An exception was demon- slowly as cells lyse. This may nevertheless cause a rapid
strated for xanthan. In its disordered form, the cellulose reduction in both the polymer mass and the solution viscos-
backbone of the polysaccharide revealed limited suscept- ity. The mass of alginates from Pseudomonas fluorescens
ibility to some commercial cellulase preparations (Rinaudo and Pseudomonas putida fell approximately 50% per 24h at
and Milas, 1980; Sutherland, 1984). 30⬚C in shaken cultures through the action of endogenous
poly-d-mannuronate-specific lyases after cell growth had
ceased (Conti et al., 1994). This resulted in greatly reduced
3. The endogenous production of polysaccharases solution viscosity. In alginate-synthesising strains of Pseu-
domonas aeruginosa, the algL gene similarly controls an
It is very rare for a microbial species to use its exopoly- alginate lyase capable of limited action on the highly acety-
saccharide as a source of carbon and energy. The curdlan- lated alginate from this bacterium (Schiller et al., 1993).
synthesising bacterium Cellulomonas flavigena is one
exception as it produces an extracellular enzyme capable
of degrading the EPS to utilisable products (Voepel and 4. The exogenous production of polysaccharases
Buller, 1990). A more common finding follows recent mole-
cular studies on exopolysaccharide synthesis, which have The source of exopolysaccharide-degrading enzyme
revealed that glycanases or polysaccharide lyases are gene mixtures may either be single microbial species or more
products which are often associated with the biosynthesis of commonly mixed cultures. In nature, microorganisms coex-
the exopolysaccharide itself. Such enzymes have now been ist in close proximity and are frequently found as consortia
found in a wide range of exopolysaccharide-synthesising capable of degrading complex substrates, which cannot be
bacterial species but do not allow the microorganism to utilised by the individual species. Polysaccharases may
utilise its own EPS as a carbon source (Table 1). Thorough therefore be produced to enable microorganisms to degrade
genetic analysis of more species will probably extend the polysaccharide substrates and utilise their component
list further. In many examples which were studied, the genes monomers as carbon and energy sources. The enzymes
for the polysaccharases formed part of the operons or gene themselves usually represent a complex mixture of activities
cassettes regulating synthesis, polymerisation and excretion even when the substrate is a homopolysaccharide. As well
of the exopolysaccharide (e.g. Glucksman et al., 1993; as enzymes capable of degrading the polysaccharide
Matthysse et al., 1995). It is not yet clear whether these substrate to oligosaccharides, other enzymes capable of
enzymes are always expressed or whether they respond to acting on these smaller fragments reduce them to smaller
 I.W. Sutherland / Carbohydrate Polymers 38 (1999) 319–328 321

Fig. 1. Enzymes acting on the succinoglycan group of microbial exopolysaccharides

oligosaccharides or to monosaccharides which can then 33 kDa secreted by a consortium of heat-stable, salt-tolerant
enter the uptake and utilisation systems of the enzyme- bacteria. It only acted on side-chains carrying pyruvate ketal
producing bacteria. Thus, a mixture of polysaccharases groups on the terminal mannose residues. The problem with
and glycosidases may be present. This appears to be true such bacterial mixtures is to ensure that they are stable and
for homopolysaccharides such as bacterial cellulose, that the necessary micro-organisms are present in the
curdlan, scleroglucan and dextrans including mutan or for mixture in sufficient numbers to provide the required
heteropolymers such as xanthan. enzyme. Although a pure bacterial culture secreting one or
The enzyme systems needed for degradation of cellulose more xanthan-degrading enzymes has also been found by
provide a good example of the complexity. Typically such Cadmus et al. (1982), more commonly, as was demonstrated
mixtures comprise several examples of each of three types in several laboratories, a complex mixture of polysacchar-
of enzyme -b -1,4-endoglucanases cleaving internal b -1,4- ide-degrading micro-organisms was obtained. Mixed micro-
glucosidic bonds; cellobiohydrolases releasing cellobiose bial cultures were found to synthesise a complex enzyme
from the non-reducing terminus of the cellulose molecule; mixture which achieved extensive degradation (e.g.Hou et
and b -d-glucosidase degrading the cellobiose so formed al., 1986). Another example of a pure bacterial culture was
(Gilbert and Hazlewood, 1993). the succinoglycan-degrading bacterial species Cytophaga
It has proved relatively rare to find a pure bacterial culture arvensicola (Abe et al., 1980; Oyaizu et al., 1982; Harada,
capable of degrading an exopolysaccharide following 1994). This bacterium produced two different enzymes,
normal enrichment procedures. However, early studies on which acted jointly to yield the octasaccharide repeat units
degradation of the EPS of Streptococcus pneumoniae type 8 of succinoglycans and the two component tetrasaccharides
yielded a bacterium Bacillus palustris which excreted a respectively (Fig. 1). The enzyme degrading the octasac-
lyase degrading the polymer (Becker and Pappenheimer, charide repeat units of succinoglycans may well be of
1966). Xanthan lyases were obtained from several mixed wider specificity than first thought as it also appears to
bacterial cultures (Sutherland, 1987; Ahlgren, 1991). The degrade some galactoglucans in addition to succinoglycan
preparation described by Ahlgren represented a protein of (I.W.Sutherland, unpublished results). The enzyme action

Table 2
Examples of polysaccharide depolymerases from heterologous microorganisms

Polysaccharide Enzyme action Linkage cleaved Enzyme source Reference

Endoglycanases
Bradyrhizobium sp. Endorhamnosidase 1,4-b -l-Rha Bacillus sp. Cadmus et al. (1988)
Dextran Endoglucosidase 1,3-b -d-Glc Bacillus sp. Bertram et al., (1993).
Dextran Endoglucosidase 1,2-a -d-Glc Flavobacterium sp. Mitsuishi et al., (1980)
Dextran Endoglucosidase 1,2-a -d-Glc Streptococcus mutans Pulkownik and Walker (1977)
Streptococcus pneumoniae Endoglucosidase 1,3-b -d-Glc-1,4-b -d-GlcA Bacillus palustris Torriani and Pappenheimer
type 3 (1962)
Lyases
Streptococcus pneumoniae 1,4-a -d-Gal-b -d-GlcA- Bacillus palustris Becker and Pappenheimer
type 8 (1966)
Sphingomonas sp. (Gellan) 1,4-b-d-Glc-b-d-GlcA- Pseudomonad Kennedy and Sutherland (1994)
Sphingomonas sp. (Gellan) 1,4-b-d-Glc-b-d-GlcA- Bacillus sp. Hashimoto et al. (1997)
322 I.W. Sutherland / Carbohydrate Polymers 38 (1999) 319–328

Table 3
Examples of phage-associated polysaccharide depolymerases

Bacterial host/Polysaccharide Enzyme action Linkage Cleaved Reference

Endoglycanases
Klebsiella type K3 Endogalactosidase Dutton et al. (1986)
Klebsiella type K8 Endogalactosidase Sutherland (1976)
Klebsiella type K13 Endogalactosidase Niemann et al. (1978)
Klebsiella type K18 Endogalactosidase 1,4-b -d-Gal Dutton et al. (1980)
Klebsiella type K22 Endogalactosidase 1,4-b -d-Glc Stirm (1994)
Klebsiella type K25 Endogalactosidase Niemann et al. (1977)
Klebsiella type K26 Endogalactosidase DiFabio et al. (1986)
Escherichia coli type K34 Endogalactosidase 1,2-b -d-GlcA Dutton and Kuma-Mintah (1987)
Escherichia col type K36 Endogalactosidase 1,3-b -d-GalA Parolis et al. (1988)
Klebsiella type K36 Endogalactosidase 1,3-b -l-Rha Dutton et al. (1981)
Klebsiella type K43 Endogalactosidase 1,3-a -d-Man Aereboe et al. (1993)
Klebsiella type K51 Endogalactosidase 1,3-a -d-Gal Chakraborty (1985)
Klebsiella sp. Endogalactosidase Yurewicz et al. (1971)
Klebsiella type K74 Endogalactosidase 1,2-b -d-Man Dutton et al. (1981)
Erwinia amylovora Endogalactosidase 1,3-b -d-Gal Nimtz et al. (1996)
Escherichia coli K36 Endogalactosidase 1,3-b -d-GlcA Parolis et al. (1988)
Escherichia coli K103 Endogalactosidase 1,4-a -d-Gal Grue et al. (1994)
Klebsiella serotype K11 Endoglucosidase 1,3-b -d-GlcA Stirm et al. (1972); Bessler et al. (1973)
Klebsiella serotype K124 Endoglucosidase 1,2-b -d-GlcA Annison et al. (1988)
Klebsiella serotype K25 Endoglucosidase Niemann et al. (1977)
Klebsiella serotype K39 Endoglucosidase 1,3-b -d-Glc Anderson et al., (1987)
Klebsiella serotype K44 Endoglucosidase 1,4-a -d-GlcA Dutton and Karunaratne (1985)
Klebsiella serotype K60 Endoglucosidase DiFabio et al. (1984)
Klebsiella serotype K63 Endoglucosidase Dutton and Merrifield (1982)
Escherichia coli serotype 29 Endoglucosidase 1,3-b -d-GlcA Fehmel et al. (1975)
Escherichia coli serotype 39 Endoglucosidase 1,6-b -d-Glc Parolis et al. (1989)
Klebsiella pneumoniae SK1 Endoglucanase 1,3-b -d-Glc Cescutti and Paoletti (1994)
(Endoglucosida se)
Klebsiella serotype K2 Endoglucosidase 1,4-b -d-Glc Geyer et al., (1983)
Klebsiella serotype K6 Endoglucanase Elsässer-Beile and Stirm (1981)
Escherichia coli Type 8 Endomannosidase 1,3-a -d-Man Prehm and Jann (1976)
Klebsiella serotypeK30 Endomannosidase Ravenscroft et al. (1988)
Klebsiella serotypeK69 Endomannosidase 1,4-b -d-Glc Hackland et al. (1988)
Escherichia coli Type 44 Endo-N-acetyl-b -d- b -d-GlcA Dutton et al. (1988)
galactosaminidase 1,
Acetobacter methanolicus Endorhamnosidase Grimmeke et al., (1994a)
Klebsiella serotype K17 Endorhamnosidase 1,4-b -d-Glc Dutton et al. (1981)
Klebsiella serotype K19 Endorhamnosidase 1,2-a -d-Glc Beurret and Joseleau (1986)
Pseudomonas syringae pv. Endorhamnosidase Smith et al. (1994)
morsprunorum
Escherichia coli Endofucosidase 1,3-b -d-Glc Sutherland (1971)
Neuraminidases/Sialidases
Neuraminidase Hallenbeck et al. (1987)
Escherichia coli Endo-N-acetylneuraminidase 2,8-a -NeuNAc Kwiatkowski et al. (1983)
Escherichia coli K1 Phage E endosialidase Long et al. (1995)
Escherichia coli KDO-KDO glycanase Nimmich (1997)
Escherichia coli KDO-KDO glycanase b -d-KDOf ! Ribp Altmann et al. (1986) andAltmann et al.,
(1987)
a -d-KDOf ! Ribp
Lyases
Azotobacter vinelandii Alginate lyase Davidson et al. (1977)
Streptococcus Hyaluronidase Niemann et al. (1976)
Klebsiella serotype K5 Polysaccharide lyase b -d-Manp-(1 ! 4)- van Dam et al. (1985)
b -d-GlcpA
Klebsiella serotype K14 Polysaccharide lyase b -d-Manp-(1 ! 4)- Parolis et al. (1988)
b -d-GlcpA
Escherichia coli K5 Polysaccharide lyase a -d-GlcpNAc-(1 ! Hänfling et al. (1996)
4)-b -d-GlcpA
Rhizobium spp. Polysaccharide lyase McNeil et al. (1986)
 I.W. Sutherland / Carbohydrate Polymers 38 (1999) 319–328 323

appeared to be largely unaffected by the different acyl (e.g. Rieger-Hug and Stirm, 1981). Although many EPS-
groups found in succinoglycans from a wide range of producing bacteria have proved to be hosts for polysacchar-
bacterial sources. In contrast, the endogenous succinoglycan ase-inducing bacteriophage (see e.g. Stirm, 1994), others
depolymerases (gene products ExoK and ExsH) from Rhizo- including Xanthomonas campestris have proved recalcitrant
bium meliloti, proved to be affected by the presence of both and viruses of this type were difficult to isolate. Stirm (1994)
acetyl and succinyl groups on their succinoglycan substrate has provided excellent protocols for the isolation of poly-
(York and Walker, 1998). Acetylation inhibited the action saccharase-inducing phages and the subsequent preparation
of both polysaccharases while the presence of succinyl of the enzymes and of their products.
groups stimulated action. Similarly, phages destroying their lipopolysaccharide
Generally speaking, mixed cultures yield a range of both (LPS) receptors are well known and in one example, the
polysaccharases and glycosidases. Many of the enzymes tail spike protein was fully characterised and functions in
produced in mixed culture only act on the initial degradation both adhesion to the host cell surface and receptor destruc-
products, causing further breakdown of the oligosaccharides tion (Baxa et al., 1996; Steinbacher et al., 1997). The
to monosaccharides, disaccharides or trisaccharides, frag- enzyme in this phage is an endorhamnosidase that cleaves
ments utilisable by the microbial cells. the 1,3-a -O-glycosidic bond between L-rhamnose and D-
As well as the endogenous polysaccharide lyase found galactose, yielding octasaccharide fragments (2 repeat
in gellan-synthesising Sphingomonas spp. (Sutherland and units) from the LPS side-chains as the major product. As
Kennedy, 1996), other sources of similar enzymes were no enzymes acting on EPS have yet been characterised in
found. A group of Gram negative rods produced a gellan such detail with regard to their protein structure, it is not
lyase acting on deacylated gellan (Kennedy and Suther- clear whether this enzyme protein represents the typical
land, 1994) as did a red-pigmented Gram positive Bacil- structure and configuration for phage-associated polysac-
lus sp. (Hashimoto et al., 1997; Hashimoto et al., 1998). charide depolymerases.
In the Bacillus sp., intracellular glycosidases further Phage enzymes were used both in crude bacterial lysates
degraded the tetrasaccharide released by the lyase. and after extensive purification. Protocols for their use can
Neither of the lyase preparations acted on any of the be found in Dutton et al. (1981) or in Stirm (1994). These
closely related polysaccharides produced by other Sphin- phage-induced enzymes acting on exopolysaccharide
gomonas sp. (or on the chemically deacylated deriva- substrates have revealed a very wide range of specificities
tives). They were also strongly inhibited by the acyl as indicated by the examples inTable 3. Although several
groups present on the native gellan polysaccharide. endoglucosidases, endogalactosidases, or endorhamnosi-
Some of these enzymes are listed in Table 2. dases are listed, each is distinct in its specificity. It is
clear from the table, however, that the majority of the
enzymes, which were reported, are either endoglucosidases
5. Bacteriophage or endogalactosidases. Cleavage of other types of glycosidic
linkage was less commonly described. Acyl groups seldom
Many of the bacteria which are surrounded by poly- affect the action. An example was reported in which the
saccharide slime or capsules are also hosts for virulent same phage enzyme (from Klebsiella type 5) degrades
bacteriophage. As demonstrated byStirm and Freund- both the EPS of the host strain which is acetylated on posi-
Mölbert, 1971) and Eichholtz et al. (1975), the phages them- tion 2 of a glucose residue and that of E. coli K55 in which
selves vary greatly in their structures. To gain access to their the same polysaccharide structure contains an O-acetylated
primary receptors on the bacterial walls, most of the phage mannose (Anderson and Parolis, 1989). Enzyme activity is
possess associated polysaccharide-degrading enzymes. As generally greatly influenced by the residues adjacent to the
with polysaccharide-degrading enzymes in general, these bond cleaved. Side-chains and charged groups appear to
enzymes may act either hydrolytically, cleaving specific play an important role in determining the specificity. It is
linkages in the polysaccharides, or they may be poly- perhaps of interest that many of the phage-induced enzymes
saccharide lyases acting by eliminative cleavage at a mono- appear to preferentially attack either (1 ! 3)-a - or (1 ! 3)-
saccharide-uronic acid linkage and introducing an b -bonds. Further, the residue targeted is very often adjacent
unsaturated bond at the C4 and C5 of the non-reducing to an anionic residue such as glucuronic acid, which may be
uronic acid terminal. The polysaccharide depolymerase part of the main chain or attached as a side-chain. In some
activity may be associated with small spikes attached to EPS however, including that of E. coli K103, uronic acids
the viral base-plate. Several studies have separated the are absent but charge is conferred by the presence of a
spikes and demonstrated enzyme activity. In addition to pyruvate ketal (Grue et al., 1994). The enzymes are usually
the phage-associated enzyme, further activity is usually highly specific. It is rare for one such enzyme to act on more
found in the soluble proteins in the cell lysates following than one polysaccharide substrate unless the structures are
viral maturation. Bacteriophage have thus provided a very very similar, although a few examples are known. Chemical
extensive range of highly specific polysaccharases which modification of the substrate such as carboxyl reduction of
were widely used in structural studies on bacterial EPS uronic acids to the corresponding neutral hexose results in
324 I.W. Sutherland / Carbohydrate Polymers 38 (1999) 319–328

loss of polysaccharase activity, indicating that the poly- hyaluronic acid by strains of Streptococcus spp. but have
anionic nature of the polymers appears to be an important apparently been overcome.
feature of enzyme specificity. However, removal of acyl Much depends on the nature of the enzyme action. Endo-
groups by mild alkali treatment usually has little if any acting polysaccharases will cause a rapid reduction in the
effect. Recent work in our laboratory using a phage origin- degree of polymerisation (DP) of the polymer substrate. The
ally isolated on a Pseudomonad host, isolated from a fresh- glycan chains may however remain associated with one
water biofilm has shown that this phage is unusual. It can another or with other macromolecules to some extent after
also form plaques on Enterobacter cloacae NCTC 5920 and a single bond is cleaved. Only when extensive bond break-
some colanic acid-producing E. coli K12 strains (Lopez and age has occurred will the polysaccharide be totally
Sutherland, unpublished results). On each host, the plaques dispersed. The environment of the polysaccharide will
are surrounded by large haloes. Assays using viscometry also be of major importance. In biofilms, the proximity of
and measurement of the reducing sugar released, clearly polysaccharides and micro-organisms may allow much
demonstrated enzyme action on the polysaccharides from greater action than would be the case with individual poly-
each bacterial host strain. Reducing sugar release was great- mers and single enzymes (Hughes, 1997). One of the few
est from the polysaccharide of the original host. This result successful examples of commercial enzymes acting on
appears to be exceptional although one other example of a microbial EPS was observed by Johansen et al. (1997) on
phage enzyme attacking two different EPS is known. The biofilms from several bacterial species. The preparation
Klebsiella K5 phage enzyme cleaves the main chain of the contained a range of different activities. Similarly, polysac-
polysaccharide from this strain (van Dam et al., 1985). It charides in flocs such as those found in waste–water puri-
does also have slight activity against xanthan in which the fication may be affected by the enzyme activities associated
same linkages are present in the side-chain of the polymer with these complexes (Frølund et al., 1995).
(I.W.Sutherland, unpublished results). The physical properties of the polysaccharide may occa-
sionally be enhanced, as is the case with the levans and
dextrans associated with dental plaque. Streptococcus
6. Effect of enzymes on exopolysaccharides mutans secretes an extracellular endo-dextranase that may
play a significant role in modifying the physicochemical
The effect of the phage enzyme attached to the viral base properties of mutans (Lawman and Bleiweis, 1991). The
plate, is to carve a path through polysaccharide capsules as enzymes attack a-1,6 linkages. The net effect is thus to
demonstrated by Bayer et al. (1979) for E. coli K29. As most increase the proportion of a-1,3 linkages and enhance the
of the polysaccharases described in this article are endo- hydrophobicity of the polysaccharide, rendering it increas-
glycanases they rapidly destroy solution viscosity along ingly insoluble in water and more adherent to the dental
with release of oligosaccharides representing the repeat surface.
units of the polysaccharides. This allows the phage particles Exo-acting enzymes only cause a slow reduction in DP and
to reach their primary receptors on the bacterial walls, inject a slow gradual release of oligosaccharide products. An
their nucleic acid and complete the lytic cycle. unusual bacterial dextranase from an Achromobacter sp.
In a study of attached growth of alginate-producing Pseu- proved to be an exo-glucanase releasing isomaltose from its
domonas aeruginosa, Boyd and Chakrabarty (1994) substrate (Sawai et al., 1974). Unlike the well documented
observed that increased expression of alginate lyase caused degradation of cellulose in which there is combined attack
alginate degradation and increased cell detachment. As the of endo- and exo-1,4-b -D-glucanases (Gilkes et al., 1991),
enzyme probably has limited activity against its substrate, it most exopolysaccharides subjected to enzymic degradation
probably caused cell detachment through reduced mass and are subjected to the action of a single endo-acting enzyme.
viscosity. This would correspond to the observations of Although action may be slow and incomplete, the presence
Conti et al. (1994) in other alginate-synthesising Pseudomo- of exo-acting enzymes may lead to alteration of the exposed
nas spp. The uncontrolled action of the enzymes does surfaces over a prolonged period. Such enzymes could cleave
however greatly reduce the molecular mass of bacterial exposed monosaccharide termini from either exopolysacchar-
alginates and thus reduce their potential value as products ides or glycoproteins. Removal of the terminal sugars would
of biotechnology. Similar problems are encountered in the also lead to possible changes in physical properties. A
commercial production of bacterial hyaluronic acid from debranching enzyme from a Flavobacterium sp. was shown
Streptococcus spp. As has already been mentioned, alginate to be highly specific for (1 ! 2)-b -D-glucosidic linkages in
lyases present in Pseudomonas spp. caused a considerable dextrans (Mitsuishi et al., 1980).
drop in the mass and solution viscosity of these polymers.
Similar problems were encountered during attempts to use
Azotobacter spp. as a source of commercial alginate produc- 7. Other enzymes acting on microbial
tion (Deavin et al., 1977). The rapid loss of polymer integ- exopolysaccharides
rity lead to the abandonment of the project. The same type
of problems were encountered in commercial production of Many microbial exopolysaccharide are acylated, the
 I.W. Sutherland / Carbohydrate Polymers 38 (1999) 319–328 325

commonest substituents being ketal-linked pyruvate or ester- exopolysaccharides can be used to produce oligosacchar-
linked acetyl groups. Removal of the acyl groups, especially ides. Pullulanase can thus be used for the quantitative
acetate (Sutherland, 1997) may greatly affect the physical preparation of maltotriose from pullulan (Catley, 1994),
properties of the polysaccharides. Esterase activities capable b -D-glucanases may allow production of laminaridextrins
of removing these substituents have not yet been demon- from curdlan (or laminaran). To ensure that there is no
strated although deacetylation of pectin was recently reported further degradation of the product, the enzyme must be
in a strain of Erwinia chrysanthemi (Shevchik, 1997) and an free of other glucosidase activities. It has also been
esterase acting on acetylated xylans was obtained from Bacil- suggested that oligosaccharide products of alginate lyases
lus pumilis (Degrassi et al., 1998). Phage-induced enzymes might promote growth in plants. Root elongation was
acting on the ‘Vi’ (poly2-amino-2-deoxy-D-galacturonic demonstrated in barley (Tomoda et al., 1993). Alginate
acid) antigen of Enterobacterial strains, also revealed esterase lyase preparations active against either marine algal alginate
activity against acetylated poly-D-galacturonic acid (Kwiat- or deacetylated bacterial alginate are routinely used to
kowski et al., 1975). As the pectic derivative is an analogue of degrade algal alginates in the walls of marine algae to
the natural substrate, this finding is not unexpected. Phage- form protoplasts but thus is not strictly an application to
induced esterases removing acetyl groups from LPS are also microbial exopolysaccharides. However, it is clear that the
known (Iwashita and Kanegasaki, 1976), while Shabtai and most widespread application of these enzymes was in
Gutnick (1985) demonstrated esterase activity against emul- studies of EPS structures. Release of relatively large quan-
san, the surface-active lipopolysaccharide-like polymer tities of oligosaccharide repeat units without the degradation
synthesised by Acinetobacter calcoaceticus strain RAG-1. and loss of acyl substituents associated with acid hydrolysis,
The emulsan esterase appeared to show specificity for this has proved extremely useful in determination of microbial
unusual polymer but also cleaved nitrophenyl esters, a feature exopolysaccharide structures. This will no doubt continue to
that was also observed for the acetylated xylan esterase. Now be the case! With the increasing interest and sophistication
that several prokaryotic exopolysaccharides are known to available in the modelling of oligosaccharide and polysac-
carry sulphate groups, sulphatases similar in action to those charide conformation, the oligosaccharides produced by
acting on glycosaminoglycans (Shaklee et al., 1985) may also enzymes from phage or other sources may also prove useful
be found. model systems.
A small number of glycosidases do act on polymeric
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