International Dairy Journal 11 (2001) 675–685

Homopolysaccharides from lactic acid bacteria

Pierre Monsan*, Sophie Bozonnet, C!ecile Albenne, Gilles Joucla,Ren!e-Marc Willemot, Magali Remaud-Sim!eon

Centre de Bioing !enierie Gilbert Durand, DGBA-INSA, UMR CNRS 5504, UMR INRA 792, INSA, Dept de Genie Biochimique and Alimentaire,

135 avenue de Rangueil, 310077 Toulouse Cedex 4, France

Abstract

In addition to heteropolysaccharides of complex structure, lactic bacteria produce a variety of homopolysaccharides containing

only either d-fructose or d-glucose. These fructans and glucans have a common feature in being synthesized by extracellulartransglycosylases (glycansucrases) using sucrose as glycosyl donor. The energy of the osidic bond of sucrose enables the efficienttransfer of a d-fructosyl or d-glucosyl residue via the formation of a covalent glycosyl-enzyme intermediate. In addition to thesynthesis of high molecular weight homopolysaccharides, glycansucrases generally catalyse the synthesis of low molecular weight

oligosaccharides or glycoconjugates when efficient acceptors, like maltose, are added to the reaction medium. While the enzymaticsynthesis of fructans (levan and inulin) is poorly documented at the molecular level, the field of Streptococcus and Leuconostocglucansucrases (glucosyltransferases and dextransucrases) has been well studied, both at the mechanistic and gene structure levels.

The nutritional applications of the corresponding polysaccharides and oligosaccharides account for this increasing interest. r 2001Elsevier Science Ltd. All rights reserved.

Keywords: Fructan; Levan; Inulin; Glucan; Dextran; Mutan; Alternan; Fructansucrase; Glucansucrase; Glucosyltransferase

1. Introduction

Lactic acid bacteria produce a wide variety ofexopolysaccharides (van Geel-Schutten, 2000), whichare mainly involved in cell adhesion and protection.Until recently, industrial interest has resulted from theirphysical–chemical properties, but these polysaccharideshave now raised new interest due to their potential fornutritional and health applications.In addition to heteropolysaccharides composed of

glucose, galactose, fructose and rhamnose (De Vuyst &Degeest, 1999), lactic acid bacteria produce homopoly-saccharides which contain only one type of monosac-charide, fructose or glucose, respectively, the fructansand the glucans. In fact, most of these homopolysac-charides share the feature of being synthesized byextracellular glycansucrases using sucrose as the glycosyl(fructose or glucose) donor.

Unlike the majority of polysaccharides, these poly-mers are not generally produced by glycosyltransferaseswhich use nucleotide-sugar precursors, but by transgly-cosylases (glycansucrases) which are able to use theenergy of the osidic bond of sucrose to catalyse thetransfer of a corresponding glycosyl moiety:

In addition to the synthesis of high-molecular-masspolymers, glycansucrases generally catalyse the produc-tion of low-molecular-mass oligosaccharides whenefficient acceptor molecules, such as maltose, are presentin the reaction mixture in addition to sucrose (Koepsellet al., 1952):

*Corresponding author. Tel.: +33-561-55-94-15; fax: +33-561-55-

94-00.

E-mail address: monsan@insa-tlse.fr (P. Monsan)

0958-6946/01/$ - see front matter r 2001 Elsevier Science Ltd. All rights reserved.

PII: S 0 9 5 8 - 6 9 4 6 ( 0 1 ) 0 0 1 1 3 - 3

2. Fructans

Two types of fructose homopolysaccharides areproduced by fructosyltransferases from sucrose: levanand inulin, which contain b-2,6 and b-2,1 osidic bonds,respectively (Fig. 1).Little is known about fructan-synthesizing sucrases.

The only reported 3D structure is that of the levansu-crase from Bacillus subtilis, but at a poor level ofresolution, 0.38 nm (Lebrun & van Rapenbusch, 1980).The proposed mechanism of catalysis for fructosyl-transferases is a two-step mechanism involving bifunc-tional catalysis in which an acidic group and anucleophilic group of the enzymes are involved in thetransfructosylation reaction (Sinnot, 1990).

2.1. Levan

Levansucrase (E.C. 2.4.1.10) catalyses the transfer ofd-fructosyl residues from fructose to yield the b-2,6osidic bonds which characterize levan (Fig. 1). Thesynthesis of this fructan has been studied in most detailin B. subtilis and Zymomonas mobilis, but no significantapplications have as yet been developed involving thispolysaccharide.Among lactic acid bacteria, levansucrase is produced

by strains from the oral flora, such as Streptococcussalivarius and Streptococcus mutans (Giffard, Allen,Milward, Simpson, & Jacques, 1993; Shiroza & Kur-amitsu, 1988). The enzyme from S. salivarius has amolecular mass of 140 kDa. It is bound to the cell wall,

but is partly released into the culture medium in thepresence of sucrose (Milward & Jacques, 1990).Levansucrase is produced in cultures of Leuconostoc

mesenteroides NRRL B-512F in addition to dextran-sucrase. Levansucrase is responsible for at least 25%of the reducing sugars produced when grown in thepresence of sucrose (Robyt & Walseth, 1979). This is asignificant level of glucose and explains why thereis fructose repression of dextransucrase production inLeuc. mesenteroides NRRL B-512F and demonstrationof levansucrase activity (Dols, Remaud-Sim!eon, &Monsan, 1998).The presence of a levansucrase activity in Lactoba-

cillus reuteri LB 121 was also recently reported (VanGeel-Schutten et al., 1999; van Geel-Schutten, 2000). Itwas also associated with a glucansucrase activity, butchemostat cultures of this strain resulted in rapidaccumulation of spontaneous exopolysaccharide-nega-tive mutants without any sucrase activity. Mutants withno levansucrase activity emerged following a pH shift-down (van Geel-Schutten, 2000).

2.2. Inulin-type

Fructooligosaccharides containing b-2,1 osidic bonds(Fig. 1) are of nutritional interest, as they are non-digestible and potentially present very interesting pre-biotic properties for both humans and animals (Toku-naga, Nakada, Tashiro, Hirayama, & Hidaka, 1993;Bouhnik et al., 1999; Diplock et al., 1999). They areobtained either by enzymatic synthesis from sucrose,using a fungal fructosyltransferase, or by controlledhydrolysis of inulin polymers. But, as yet, suchfructooligosaccharides have not been synthesized usingfructosyltransferases from lactic acid bacteria, eventhough this group of organisms has this type ofenzymatic activity.

S. mutans Ingbritt A strain produces a fructan whichonly contains b-2,1 linked fructosyl units (Baird, Long-year, & Ellwood, 1973), while S. mutans JC-2 producesan inulin-type fructan consisting mainly of b-2,1linked fructosyl units with 5% b-2,6 branches(Rosell & Birkhed, 1974; Ebisu, Kato, Kotani, &Misaki, 1975).Very surprisingly, L. reuteri LB 121 which is known to

produce a linear levan (see above), also contains a ftfAgene (2400 bp) which was isolated with PCR techniques(van Geel-Schutten, 2000). In the presence of sucrose,the corresponding fructosyltransferase, FTFA, producesfructooligosaccharides with a degree of polymerisationof 3–4, and a high molecular mass polysaccharide withb-2,1 linked fructosyl units only. FTFA contains aputative N-terminal secretion signal peptide. It shows90% similarity with SACB of S. mutans, which alsosynthesizes a fructan containing only b-2,1 osidic bonds.Fig. 1. Structures of fructans: (A) levan, (B) inulin-type fructan.

P. Monsan et al. / International Dairy Journal 11 (2001) 675–685676

3. Glucans

Glucansucrases catalyse the synthesis of a variety ofglucans containing mostly a-1,6, a-1,3, a-1,4 and a-1,2linked d-glucosyl units (Sidebotham, 1974; Monchois,Willemot, & Monsan, 1999). The structure (Fig. 2) ofthese glucans has been elucidated using a wide range ofmethods: periodic oxidation, polymer methylation,acetolysis, Smith degradation, enzymatic hydrolysisand 13C NMR (Jeanes et al., 1954; Seymour, Knapp,& Bishop, 1976; Seymour, Slodki, Plattner, & Jeanes,

1977; Seymour & Knapp, 1980; Misaki, Torii, Savai, &Goldstein, 1980; C #ot!e & Robyt, 1982a).Extracellular glucansucrases are mostly produced by

lactic acid bacteria belonging to the genera: Leucono-stoc, Streptococcus and Lactobacillus (Sidebotham,1974; Mooser, 1992). The only exception is the produc-tion of amylosucrase (E.C. 2.4.1.4), which catalyses thesynthesis of a-1,4-d-glucosyl linkages from sucrose, byNeisseria sp. (MacKenzie, McDonald, & Johnson, 1978;Potocki de Montalk, Remaud-Sim!eon, Willemot, Plan-chot, & Monsan, 1999). Amylosucrase is the only

Fig. 2. Structures of glucans: (A) dextran, (B) mutan and, (C) alternan.

P. Monsan et al. / International Dairy Journal 11 (2001) 675–685 677

glucansucrase belonging to family 13 of glycosidehydrolases, while all the other glucansucrases belongto family 70 (Henrissat & Davies, 1997).Various strains produce more than one glucansucrase:

three distinct enzymes are produced by S. mutans 6715(serotype g) (Shimamura, Tsumori, & Mukasa, 1983),four are produced by S. sobrinus (Walker, Cheetham,Taylor, Pearce, & Slodki, 1990), three by L. mesente-roides NRRL B-1355 (Smith, Zahnley, Wong, Lundin,& Ahlgren, 1998).The glucansucrases from Streptococcus sp. are gen-

erally produced constitutively, whereas those fromLeuconostoc sp. are specifically induced by sucrose,except for some constitutive mutants (Mizutani, Yama-da, Takayama, & Shoda, 1994; Kim & Robyt, 1995).Streptococcal glucansucrases (generally named gluco-

syltransferases), particularly from S. mutans, are in-volved in cariogenesis phenomena. In fact, the glucanpolymers synthesized by these enzymes play a key role inthe adhesion of bacteria to the tooth surface to formdental plaque (Muzaka & Slade, 1973; Hamada & Slade,1979; Hamada & Slade, 1980).Despite a large amount of scientific work, the

catalytic mechanism of glucansucrases has still not beentotally elucidated. The key central step of the transfer ofthe d-glucosyl unit is the formation of a covalentglucosyl-enzyme intermediate. This step involves, as inthe case of the enzymes of family 13 of glucosidehydrolases (Henrissat & Davies, 1997), in particulara-amylases, a catalytic triad consisting of two asparticacids and one glutamic acid residue (MacGregor,Jesperen, & Svensson, 1996; Devulapalle, Goodman,Gao, Hemsley, & Mooser, 1997). Such a catalytic sitewas clearly identified by Mooser and Iwakoa (1989) whoisolated a covalent glucosyl-enzyme complex from aquenched reaction of the glucosyltransferase from S.sobrinus with radiolabelled sucrose. An aspartic acidresidue was identified at the catalytic site of two S.sobrinus glucosyltransferases (Mooser, Hefta, Paxton, &Lee, 1991).From this covalent glucosyl-enzyme intermediate, the

glucosyl residue can be transferred into a series ofacceptors (Koepsell et al., 1952; Ebert & Schenk, 1968)as follows:

* to the growing dextran chain to extend it by onecarbohydrate unit;

* to a carbohydrate or a non-carbohydrate acceptor toyield an oligosaccharide or a gluco-conjugate;

* to a water molecule: this corresponds to the hydro-lysis of the sucrose molecule (Robyt & Corrrigan,1977; Robyt & Walseth, 1979; Luzio, Parnaik, &Mayer, 1983; Yokoyama, Kobayashi, & Matsuda,1985);

* to a fructose molecule to give either neo-synthesis of asucrose molecule, i.e. ‘‘isotopic exchange’’ (Mayer,

Matthews, Futerman, Parnaik, & Jung, 1981; Jung &Mayer, 1981) or the production of leucrose: a-d-glucopyranosyl-1,5-d-fructopyranose (Stodola,Koepsell, & Sharpe, 1956; Schwengers, 1991).

In addition, in the absence of residual sucrose in thereaction medium, glucansucrases can catalyse dispro-portionation reactions involving oligosaccharides assubstrates (Binder, C #ot!e, & Robyt, 1983; Lopez-Munguia et al., 1993).Glucan polymer synthesis follows a processive me-

chanism. This is deduced from the observations thatintermediate oligosaccharides cannot be detected in thereaction medium during the synthesis, and high mole-cular weight polysaccharides are obtained at earlyreaction times ( Tsuchiya, Hellman, & Koepsell, 1953;Bovey, 1959; Ebert et al., 1968).Two alternative mechanisms have been proposed for

the glucan chain growth (Monchois et al., 1999):

* Non-reducing end elongation: Only one covalentglucosyl-enzyme intermediate is involved (Mooser,1992). In the case of dextransucrase, the initialacceptor could be the dextran when associated tothe enzyme (Hehre, 1941), but polymer synthesisoccurs even in the absence of dextran (Ebert &Schenk, 1968; Robyt & Corrrigan, 1977; Miller,Ecklund, & Robyt, 1986). Sucrose has been suggestedas the initial acceptor by Neely (1960), in disagree-ment with the results of Parnaik and Mayer (1982)and Su and Robyt (1994). But Cheethman, Slodki,and Walker (1991) have shown that a sucrose residueoccurs at the end of the dextran chain produced bythe glucosyltransferase GTF-S3 from S. sobrinus.

* Reducing end elongation: This mechanism was sug-gested by Ebert and Schenk (1968) and demonstratedby Robyt, Kimble, and Walseth (1974) in the case ofimmobilized dextransucrase from Leuc. mesenteroidesNRRL B-512F using a pulse and chase reaction with14C-labelled sucrose. Similar results have beenobtained with the glycosyltransferases from S. mutans6715 (Robyt & Martin, 1983) and S. sanguis ATCC10558 (Ditson & Mayer, 1984). This mechanisminvolves two identical nucleophilic sites able to yieldtwo covalent glucosyl-enzyme intermediates fromtwo sucrose molecules (Robyt et al., 1974). The C-6hydroxyl from one of these two glucosyl residuesmakes a nucleophilic attack on the C-1 of the secondglucosyl intermediate to give an a-1,6 glucosidiclinkage between the two glucosyl moieties. Thereleased nucleophilic site then attacks another glucosemolecule to give a new glucosyl-enzyme intermediate.This symmetrical and alternative role of the twonucleophilic sites results in the growth of the glucanchain by its reducing end, without the need for aprimer and without the release of the glucan chainfrom the enzyme before the addition of the next

P. Monsan et al. / International Dairy Journal 11 (2001) 675–685678

glucosyl residue. Branching of the glucan chain doesnot necessarily involve any additional enzyme, butcan occur when such a chain acts as an acceptor ofglucosyl units.

It is difficult to make a clear choice between these twomechanisms, but until now sequence analysis andstructure prediction of known glucansucrases has onlyresulted in the identification of a single active site(MacGregor et al., 1996; Devulapalle et al., 1997).In the case of the acceptor reaction, glucosyl units are

clearly added at the non-reducing end of the acceptoroligosaccharides (Koepsell et al., 1952; Robyt &Walseth, 1978). Robyt and Martin (1983) have sug-gested an oligosaccharide synthesis mechanism invol-ving independently the two nucleophilic sitesparticipating in the polysaccharide synthesis. But thesuppression of one of the potential nucleophilic sites bysite-directed mutagenesis, i.e. the replacement of aspar-tic acid residue 551 of Leuc. mesenteroides NRRL B-512F dextransucrase by an asparagine residue, totallysuppresses the ability of the enzyme to catalyse thesynthesis of oligosaccharides in the presence of maltoseas acceptor (Monchois, Remaud-Sim!eon, Russell, Mon-san, & Willemot, 1997). This result is not compatiblewith an identical role for the two nucleophilic sites.More than thirty genes encoding glucansucrases have

been isolated and sequenced. The enzymes are closelyrelated and share a common structure (Fig. 3). They arecomposed of four distinct structural domains (Monchoiset al., 1999):

* The N-terminal domain begins with a well conservedsignal peptide (Monchois, Remaud-Sim!eon, Monsan,& Willemot, 1998a), as these enzymes are extracel-lular. The only exception is the dextransucrase geneDSR-A from Leuc. mesenteroides NRRL B-1299(Monchois, Willemot, Remaud-Sim!eon, Croux, &Monsan, 1996).

* A highly variable stretch of 123–129 amino acids,which does not seem to play an important role inenzyme activity (Abo et al., 1991), and is not presentin DSR-A from Leuc. mesenteroides NRRL B-1299(Monchois et al., 1996).

* A highly conserved core region of about 1000 aminoacid containing the active site, and more particularlythe catalytic triad, as demonstrated for the glucosyl-transferases GTF-I and GTF-S from S. sobrinus

(Mooser & Iwakoa, 1989; Mooser, Hefta, Paxton, &Lee, 1991). This domain presents a permutated (b=a)8barrel structure, when compared with family 13 of theglycoside hydrolases (Monchois et al., 1999).

* A C-terminal domain composed of a series ofrepeating units (Ferretti, Gilpin, & Russell, 1987;Gilmore, Russell, & Ferretti, 1990; Abo et al., 1991),which is responsible for the binding of the synthesizedglucan (Monchois et al., 1999). The presence of theC-terminal domain is generally necessary to keep anenzyme active. In the case of Leuc. mesenteroidesNRRL B-512F dextransucrase (DSR-S), C-terminaldomain truncation results in a strong decrease ofactivity, both for polysaccharide and oligosaccharidesynthesis (Monchois, Reverte, Remaud-Sim!eon,Monsan, & Willemot, 1998b).

Even if the structural characterization of an increas-ing number of glucansucrase encoding genes, coupledwith a site-directed mutagenesis approach, allows abetter insight into the role of the various parts of theglucansucrases, additional research is still necessary toreach a full understanding of the catalytic mechanismsof both polysaccharide and oligosaccharide synthesis.Obviously, the elucidation of the 3D structure of thistype of enzyme will be a key step in such an under-standing, as well as in the possibility to controlglucansucrase selectivity. In fact, it is already possibleto modify the structure of the products by site-directedmutagenesis of the glucansucrases: the presence of acarboxylic amino acid instead of a threonine at position589 of the amino acid sequence of a glucansucrase fromS. mutans GS-5 synthesising an a-1,6 linked glucan(GTF-S), results in a 30% increase in the synthesis of a-1,3 glucosidic bonds (Shimamura, Nakano, Musaka, &Kuramitsu, 1994). Similar results have been obtainedwith the dextransucrase from Leuc. mesenteroidesNRRL B-512F, DSR-S: when the threonine residue atposition 667 is replaced by an arginine, the dextranobtained contains 13% of a-1,3 glucosidic bonds insteadof 5% with the native enzyme (Remaud-Sim!eon, Will-emot, Sar-cabal, Potocki de Montalk, & Monsan, 2000).Several types of glucans are obtained from the action

of glucansucrase:

3.1. Dextran

Pasteur (1861) discovered the microbial origin of thegelification of cane sugar syrups. In 1874, the corre-sponding product was named ‘‘dextran’’, due to itspositive rotatory power. The microorganism responsibleof the gelification was isolated by Van Tieghem (1878)and named it Leuc. mesenteroides. In 1941, Hehredemonstrated that dextran could be synthesized fromsucrose by a cell-free filtrate. The corresponding

Fig. 3. Schematic general structure of glucansucrases, showing: (A) N-

terminal signal-sequence, (B) variable region, (C) catalytic domain

and, (D) glucan binding domain (GBD).

P. Monsan et al. / International Dairy Journal 11 (2001) 675–685 679

extracellular enzyme was named ‘‘dextransucrase’’ byHestrin, Averini-Shapiro, and Aschner (1943).Dextransucrase (E.C. 2.4.1.5) produces a glucan

(Fig. 2) which contains at least 50% of a-1,6 osidicbonds within the main chain (Buchholz & Monsan,2001). The degree of branching, involving a-1,2, a-1,3and a-1,4 linkages in dextrans A, B and C, respectively,(Seymour & Knapp, 1980) varies according to the originof the dextransucrase (Table 1).The most widely used dextran is produced by the

dextransucrase of the strain Leuc. mesenteroides NRRLB-512F, which synthesizes a very linear polysaccharidecontaining 95% a-1,6 linkages. The controlled chemicalhydrolysis of this high-molecular-mass dextran allowsthe production of fractions with an average molecularmass of 70 kDa. These products are used for theproduction of chromatography supports for gel permea-tion separation (Sephadexs), in the production of bloodplasma substitutes (Groenwall & Ingelman, 1948), andto prepare dextran sulphate for blood coagulationprevention and iron transport (Soetaert, Schwengers,Buchholz, & Vandamme, 1995).The molecular mass of the corresponding dextransu-

crase has been the subject of discussion. Values rangingfrom 65 kDa (Kobayashi & Matsuda, 1980) to 190 kDa(Willemot, Monsan, & Durand, 1988) have beenreported. Finally, the isolation of the gene encodingthis enzyme (Wilke-Douglas, Perchorowicz, Houck, &Thomas, 1989) led to a protein with a total of 1527amino acids (MM: 170 kDa). Lower values are due toproteolytic degradation, while higher values can beattributed to dextran contamination of the proteinpreparation.

Lb. reuteri LB 121 cells growing on sucrose synthe-size, besides a fructan polymer, large amounts of aglucan polymer with a molecular mass of 3500 kDa.This glucan has a unique structure consisting of

terminal, 4-substituted, 6-substituted, and 4,6-disubsti-tuted a-glucose in a molar ratio of 1.1 : 2.7 : 1.5 : 1.0 (VanGeel-Schutten et al., 1999). This homopolysaccharide issynthesized from sucrose by a glucansucrase (MM:146 kDa). The corresponding gene (gtfA) has beenisolated and sequenced. It shows a high homology withthe known glucansucrase genes from lactic bacteria,particularly with the gene encoding alternansucrase(ASR) from Leuc. mesenteroides NRRL B-1355 (VanGeel-Schutten, 2000). The glucansucrase ORF has a sizeof approximately 4100 bp (Van Geel-Schutten, 2000).Mutant strains lacking levansucrase activity, but keep-ing dextransucrase activity, were obtained following apH shiftdown (Van Geel-Schutten et al., 1999).The maltose acceptor reaction is used to produce non-

digestible gluco-oligosaccharides (Fig. 4) with the dex-transucrase from Leuc. mesenteroides NRRL B-1299,which is known to catalyse the synthesis of dextranpolymers containing a-1,2 linked branched chains(Table 1). Dextransucrase keeps its selectivity in theacceptor reaction and produces a series of gluco-oligosaccharides containing such a-1,2 glucosidic lin-kages (Paul, Lopez-Munguia, Remaud, Pelenc, &Monsan, 1992; Remaud-Sim!eon, Lopez-Munguia, Pe-lenc, Paul, & Monsan, 1994; Dols, Remaud-Sim!eon,Willemot, & Vignon, 1998), which resist the attack ofdigestive enzymes (Valette et al., 1993). Such gluco-oligosaccharides present interesting prebiotic properties(Monsan & Paul, 1995; Djouzy et al., 1995). Thesegluco-oligosaccharides are presently marketed for hu-man nutritional and dermocosmetic applications (BioE-colias: BioEurope/Solabia).

3.2. Mutan

Mutansucrase (E.C. 2.4.1.5) produces a water-in-soluble glucan containing more than 50% of a-1,3

Table 1

Structure of the different glucans produced by Leuconostoc mesenteroides and Streptococcus sp. glucansucrases

Osidic linkages

a-1,6 (%) a-1,3 (%) a-1,4 (%) a-1,2 (%)

Leuconostoc mesenteroides B-512F 95 5

B-742 87 13

50 50

B-1355 95 5

54 46

B-1299 66 7 27

65 35

Streptococcus downei Mfe28 12 88

90 10

Streptococcus mutans GS5 13 87

15 85

70 30

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glucosidic linkages (Fig. 2), mainly associated with a-1,6linkages. This enzyme is produced by Leuc. mesente-roides NRRL B-523, B-1149 and several Streptococcusstrains (Sidebotham, 1974; Mooser, 1992).Mutan polysaccharides are involved in the adhesion

of oral flora microorganisms on the tooth surface toform the dental plaque (Hamada & Slade, 1980). Nospecific applications of mutan polymers have beendeveloped up till now.

3.3. Alternan

Alternansucrase (E.C. 2.4.1.140) synthesizes the glu-can, alternan, which contains alternating a-1,6 and a-1,3

glucosidic linkages, with some degree of a-1,3 branch-ings. Three Leuc. mesenteroides strains are known toproduce alternansucrase: Leuc. mesenteroides NRRL B-1355, NRRL B-1501 and NRRL B-1498 (Jeanes et al.,1954; Seymour & Knapp, 1980; C #ot!e & Robyt, 1982a).Jeanes et al. (1954) were the first to report the

production of two different glucansucrases by Leuc.mesenteroides NRRL B-1355. Ethanol precipitation hasresulted in the isolation of two polysaccharide fractions:(i) a poorly water-soluble polysaccharide (Fraction L)and (ii) a water-soluble polysaccharide (Fraction S).Fraction L corresponds to a polysaccha-ride containing95% of a-1,6 glucosidic linkages and 5% a-1,3 branch-ing, similar to the dextran produced by the dextransu-

Fig. 4. Maltose acceptor reactions catalysed by Leuc. mesenteroides B-1299 dextransucrase with examples of the different families of gluco-

oligosaccharide products: OD4, containing only a-1,6 osidic bonds (in addition to the a-1,4 bond of the maltose moiety at the reducing end); R5 with

a terminal a-1,2 glucosidic linkage; and R06 with a-1,2 glucosidic branching on the penultimate glucosyl residue of the non-reducing end.

P. Monsan et al. / International Dairy Journal 11 (2001) 675–685 681

crase from Leuc. mesenteroidesNRRL B-512F. FractionS contains alternan and is produced by alternansucrase.This latter enzyme activity is mainly (85%) bound to thebacterial cells and is more thermostable than thedextransucrase activity. The association of enzyme tocells allows efficient removal of the contaminatingdextransucrase activity to give a purified alternansucrasepreparation (Lopez-Munguia et al., 1993).In the presence of sucrose and maltose acting as an

acceptor, alternansucrase retains its selectivity, andcatalyses the synthesis of oligosaccharides containingalternating a-1,6 and a-1,3 linkages (Koepsell et al.,1952; C #ot!e & Robyt, 1982b; Pelenc et al., 1991).Mutant strains of Leuc. mesenteroides NRRL B-

1355C producing alternansucrase on a glucose medium,have been obtained by Kim and Robyt (1994), Smith,Zahnley, and Goodman (1994) and by Smith andZahnley (1997). This has resulted in the identificationof a third glucansucrase in this strain, which synthesizesa water-insoluble dextran-type polymer (Zahnley &Smith, 1995).The gene encoding the alternansucrase (ASR) from

Leuc. mesenteroides NRRL B-1355 has been cloned,sequenced and expressed in E. coli (Arg .uello-Moraleset al., 2000). It contains 6171 bp and encodes a proteinof 2057 amino acids (MM: 229 kDa), which correspondsto the largest glucansucrase described to date. This geneproduces an enzyme with a structural organizationsimilar to that of known glucansucrases from lactic acidbacteria. Alternansucrase thus, belongs to family 70 ofglycoside hydrolases (Henrissat & Davies, 1997) as dothe other glucansucrases from Leuc. mesenteroides. Thevariable region and the C-terminal glucan-bindingdomain are longer than in other glucansucrases, 100and 200 amino acids, respectively. The catalytic domainpresents 49% identity with that of the other glucansu-crases from Leuc. mesenteroides. The purified enzymehas been characterized for its ability to synthesizeoligosaccharides from acceptor carbohydrates: its capa-city to use cellobiose as an acceptor is significantlyhigher than that of the dextransucrase from Leuc.mesenteroides NRRL B-512F (Arg .uello-Morales, Re-maud-Sim!eon, Willemot, Vignon, & Monsan, 2001).Such gluco-oligosaccharides are of potential interest asprebiotics.

3.4. b-1,3 glucan

Lactobacillus subsp. G-77 has been reported toproduce two glucose homopolysaccharides when grownon a glucose medium (Duenas-Chasco et al., 1998). Oneof the exopolysaccharides was shown to be a 2-substituted-(1–3)-b-d-glucan identical to that describedfor the exopolysaccharide from Pediococcus damnosus2.6 (Duenas-Chasco et al., 1997). This is the first reportof the production of a b-glucan by lactic bacteria. The

mechanism of synthesis has not as yet been describedbut it does not involve any glucansucrase, becausesucrose, which is the enzyme inducer and the glucosyldonor, was not present in the culture medium. A similarpolysaccharide is produced by P. damnosus strainsisolated from spoiled wine (Walling, Gindreau, &Lonvaud-Funel, 2001). The second homopolysaccharideis a dextran-type polysaccharide (a-1,6 glucosyl lin-kages) with a-1,2 branching of a single d-glucose unitbut its mechanism of synthesis has not been reported.These polysaccharides have undesirable properties for

some industries as they cause thickening and are at theorigin of a cider (Duenas-Chasco et al., 1997) or a wine(Gindreau, Walling, & Lonvaud-Funel, 2001) spoilageproblem known as ‘‘oiliness’’ or ‘‘ropiness’’.

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