Homopolysaccharides from lactic acid bacteria

International Dairy Journal 11 (2001) 675–685

Homopolysaccharides from lactic acid bacteria Pierre Monsan*, Sophie Bozonnet, Ce! cile Albenne, Gilles Joucla, Rene! -Marc Willemot, Magali Remaud-Sime! on ! Centre de Bioingenierie Gilbert Durand, DGBA-INSA, UMR CNRS 5504, UMR INRA 792, INSA, De´pt de Ge´nie 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 extracellular transglycosylases (glycansucrases) using sucrose as glycosyl donor. The energy of the osidic bond of sucrose enables the efficient transfer of a d-fructosyl or d-glucosyl residue via the formation of a covalent glycosyl-enzyme intermediate. In addition to the synthesis 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 enzymatic synthesis of fructans (levan and inulin) is poorly documented at the molecular level, the field of Streptococcus and Leuconostoc glucansucrases (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 2001 Elsevier 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 of exopolysaccharides (van Geel-Schutten, 2000), which are mainly involved in cell adhesion and protection. Until recently, industrial interest has resulted from their physical–chemical properties, but these polysaccharides have now raised new interest due to their potential for nutritional and health applications. In addition to heteropolysaccharides composed of glucose, galactose, fructose and rhamnose (De Vuyst & Degeest, 1999), lactic acid bacteria produce homopolysaccharides which contain only one type of monosaccharide, fructose or glucose, respectively, the fructans and the glucans. In fact, most of these homopolysaccharides share the feature of being synthesized by extracellular glycansucrases using sucrose as the glycosyl (fructose or glucose) donor.

Unlike the majority of polysaccharides, these polymers are not generally produced by glycosyltransferases which use nucleotide-sugar precursors, but by transglycosylases (glycansucrases) which are able to use the energy of the osidic bond of sucrose to catalyse the transfer of a corresponding glycosyl moiety:

In addition to the synthesis of high-molecular-mass polymers, glycansucrases generally catalyse the production of low-molecular-mass oligosaccharides when efficient acceptor molecules, such as maltose, are present in the reaction mixture in addition to sucrose (Koepsell et al., 1952):

*Corresponding author. Tel.: +33-561-55-94-15; fax: +33-561-5594-00. E-mail address: [email protected] (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

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2. Fructans Two types of fructose homopolysaccharides are produced by fructosyltransferases from sucrose: levan and 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 levansucrase from Bacillus subtilis, but at a poor level of resolution, 0.38 nm (Lebrun & van Rapenbusch, 1980). The proposed mechanism of catalysis for fructosyltransferases is a two-step mechanism involving bifunctional catalysis in which an acidic group and a nucleophilic group of the enzymes are involved in the transfructosylation reaction (Sinnot, 1990). 2.1. Levan Levansucrase (E.C. 2.4.1.10) catalyses the transfer of d-fructosyl residues from fructose to yield the b-2,6 osidic bonds which characterize levan (Fig. 1). The synthesis of this fructan has been studied in most detail in B. subtilis and Zymomonas mobilis, but no significant applications have as yet been developed involving this polysaccharide. Among lactic acid bacteria, levansucrase is produced by strains from the oral flora, such as Streptococcus salivarius and Streptococcus mutans (Giffard, Allen, Milward, Simpson, & Jacques, 1993; Shiroza & Kuramitsu, 1988). The enzyme from S. salivarius has a molecular mass of 140 kDa. It is bound to the cell wall,

Fig. 1. Structures of fructans: (A) levan, (B) inulin-type fructan.

but is partly released into the culture medium in the presence of sucrose (Milward & Jacques, 1990). Levansucrase is produced in cultures of Leuconostoc mesenteroides NRRL B-512F in addition to dextransucrase. Levansucrase is responsible for at least 25% of the reducing sugars produced when grown in the presence of sucrose (Robyt & Walseth, 1979). This is a significant level of glucose and explains why there is fructose repression of dextransucrase production in Leuc. mesenteroides NRRL B-512F and demonstration of levansucrase activity (Dols, Remaud-Sime! on, & Monsan, 1998). The presence of a levansucrase activity in Lactobacillus reuteri LB 121 was also recently reported (Van Geel-Schutten et al., 1999; van Geel-Schutten, 2000). It was also associated with a glucansucrase activity, but chemostat cultures of this strain resulted in rapid accumulation of spontaneous exopolysaccharide-negative mutants without any sucrase activity. Mutants with no levansucrase activity emerged following a pH shiftdown (van Geel-Schutten, 2000).

2.2. Inulin-type Fructooligosaccharides containing b-2,1 osidic bonds (Fig. 1) are of nutritional interest, as they are nondigestible and potentially present very interesting prebiotic properties for both humans and animals (Tokunaga, Nakada, Tashiro, Hirayama, & Hidaka, 1993; Bouhnik et al., 1999; Diplock et al., 1999). They are obtained either by enzymatic synthesis from sucrose, using a fungal fructosyltransferase, or by controlled hydrolysis of inulin polymers. But, as yet, such fructooligosaccharides have not been synthesized using fructosyltransferases from lactic acid bacteria, even though this group of organisms has this type of enzymatic activity. S. mutans Ingbritt A strain produces a fructan which only contains b-2,1 linked fructosyl units (Baird, Longyear, & Ellwood, 1973), while S. mutans JC-2 produces an inulin-type fructan consisting mainly of b-2,1 linked 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 ftfA gene (2400 bp) which was isolated with PCR techniques (van Geel-Schutten, 2000). In the presence of sucrose, the corresponding fructosyltransferase, FTFA, produces fructooligosaccharides with a degree of polymerisation of 3–4, and a high molecular mass polysaccharide with b-2,1 linked fructosyl units only. FTFA contains a putative N-terminal secretion signal peptide. It shows 90% similarity with SACB of S. mutans, which also synthesizes a fructan containing only b-2,1 osidic bonds.

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

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1977; Seymour & Knapp, 1980; Misaki, Torii, Savai, & # e! & Robyt, 1982a). Goldstein, 1980; Cot Extracellular glucansucrases are mostly produced by lactic acid bacteria belonging to the genera: Leuconostoc, Streptococcus and Lactobacillus (Sidebotham, 1974; Mooser, 1992). The only exception is the production of amylosucrase (E.C. 2.4.1.4), which catalyses the synthesis of a-1,4-d-glucosyl linkages from sucrose, by Neisseria sp. (MacKenzie, McDonald, & Johnson, 1978; Potocki de Montalk, Remaud-Sime! on, Willemot, Planchot, & Monsan, 1999). Amylosucrase is the only

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

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glucansucrase belonging to family 13 of glycoside hydrolases, while all the other glucansucrases belong to 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. mesenteroides NRRL B-1355 (Smith, Zahnley, Wong, Lundin, & Ahlgren, 1998). The glucansucrases from Streptococcus sp. are generally produced constitutively, whereas those from Leuconostoc sp. are specifically induced by sucrose, except for some constitutive mutants (Mizutani, Yamada, Takayama, & Shoda, 1994; Kim & Robyt, 1995). Streptococcal glucansucrases (generally named glucosyltransferases), particularly from S. mutans, are involved in cariogenesis phenomena. In fact, the glucan polymers synthesized by these enzymes play a key role in the adhesion of bacteria to the tooth surface to form dental 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 been totally elucidated. The key central step of the transfer of the d-glucosyl unit is the formation of a covalent glucosyl-enzyme intermediate. This step involves, as in the case of the enzymes of family 13 of glucoside hydrolases (Henrissat & Davies, 1997), in particular a-amylases, a catalytic triad consisting of two aspartic acids and one glutamic acid residue (MacGregor, Jesperen, & Svensson, 1996; Devulapalle, Goodman, Gao, Hemsley, & Mooser, 1997). Such a catalytic site was clearly identified by Mooser and Iwakoa (1989) who isolated a covalent glucosyl-enzyme complex from a quenched reaction of the glucosyltransferase from S. sobrinus with radiolabelled sucrose. An aspartic acid residue 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 of acceptors (Koepsell et al., 1952; Ebert & Schenk, 1968) as follows: *

*

*

*

to the growing dextran chain to extend it by one carbohydrate unit; to a carbohydrate or a non-carbohydrate acceptor to yield an oligosaccharide or a gluco-conjugate; to a water molecule: this corresponds to the hydrolysis 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 a sucrose molecule, i.e. ‘‘isotopic exchange’’ (Mayer,

Matthews, Futerman, Parnaik, & Jung, 1981; Jung & Mayer, 1981) or the production of leucrose: a-dglucopyranosyl-1,5-d-fructopyranose (Stodola, Koepsell, & Sharpe, 1956; Schwengers, 1991). In addition, in the absence of residual sucrose in the reaction medium, glucansucrases can catalyse disproportionation reactions involving oligosaccharides as # e! , & Robyt, 1983; Lopezsubstrates (Binder, Cot Munguia et al., 1993). Glucan polymer synthesis follows a processive mechanism. This is deduced from the observations that intermediate oligosaccharides cannot be detected in the reaction medium during the synthesis, and high molecular weight polysaccharides are obtained at early reaction 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 covalent glucosyl-enzyme intermediate is involved (Mooser, 1992). In the case of dextransucrase, the initial acceptor could be the dextran when associated to the enzyme (Hehre, 1941), but polymer synthesis occurs even in the absence of dextran (Ebert & Schenk, 1968; Robyt & Corrrigan, 1977; Miller, Ecklund, & Robyt, 1986). Sucrose has been suggested as the initial acceptor by Neely (1960), in disagreement with the results of Parnaik and Mayer (1982) and Su and Robyt (1994). But Cheethman, Slodki, and Walker (1991) have shown that a sucrose residue occurs at the end of the dextran chain produced by the glucosyltransferase GTF-S3 from S. sobrinus. Reducing end elongation: This mechanism was suggested by Ebert and Schenk (1968) and demonstrated by Robyt, Kimble, and Walseth (1974) in the case of immobilized dextransucrase from Leuc. mesenteroides NRRL B-512F using a pulse and chase reaction with 14 C-labelled sucrose. Similar results have been obtained with the glycosyltransferases from S. mutans 6715 (Robyt & Martin, 1983) and S. sanguis ATCC 10558 (Ditson & Mayer, 1984). This mechanism involves two identical nucleophilic sites able to yield two covalent glucosyl-enzyme intermediates from two sucrose molecules (Robyt et al., 1974). The C-6 hydroxyl from one of these two glucosyl residues makes a nucleophilic attack on the C-1 of the second glucosyl intermediate to give an a-1,6 glucosidic linkage between the two glucosyl moieties. The released nucleophilic site then attacks another glucose molecule to give a new glucosyl-enzyme intermediate. This symmetrical and alternative role of the two nucleophilic sites results in the growth of the glucan chain by its reducing end, without the need for a primer and without the release of the glucan chain from the enzyme before the addition of the next

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

glucosyl residue. Branching of the glucan chain does not necessarily involve any additional enzyme, but can occur when such a chain acts as an acceptor of glucosyl units. *

It is difficult to make a clear choice between these two mechanisms, but until now sequence analysis and structure prediction of known glucansucrases has only resulted 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 acceptor oligosaccharides (Koepsell et al., 1952; Robyt & Walseth, 1978). Robyt and Martin (1983) have suggested an oligosaccharide synthesis mechanism involving independently the two nucleophilic sites participating in the polysaccharide synthesis. But the suppression of one of the potential nucleophilic sites by site-directed mutagenesis, i.e. the replacement of aspartic acid residue 551 of Leuc. mesenteroides NRRL B512F dextransucrase by an asparagine residue, totally suppresses the ability of the enzyme to catalyse the synthesis of oligosaccharides in the presence of maltose as acceptor (Monchois, Remaud-Sime! on, Russell, Monsan, & Willemot, 1997). This result is not compatible with an identical role for the two nucleophilic sites. More than thirty genes encoding glucansucrases have been isolated and sequenced. The enzymes are closely related and share a common structure (Fig. 3). They are composed of four distinct structural domains (Monchois et al., 1999): *

*

*

The N-terminal domain begins with a well conserved signal peptide (Monchois, Remaud-Sime! on, Monsan, & Willemot, 1998a), as these enzymes are extracellular. The only exception is the dextransucrase gene DSR-A from Leuc. mesenteroides NRRL B-1299 (Monchois, Willemot, Remaud-Sime! on, Croux, & Monsan, 1996). A highly variable stretch of 123–129 amino acids, which does not seem to play an important role in enzyme activity (Abo et al., 1991), and is not present in DSR-A from Leuc. mesenteroides NRRL B-1299 (Monchois et al., 1996). A highly conserved core region of about 1000 amino acid containing the active site, and more particularly the catalytic triad, as demonstrated for the glucosyltransferases GTF-I and GTF-S from S. sobrinus

Fig. 3. Schematic general structure of glucansucrases, showing: (A) Nterminal signal-sequence, (B) variable region, (C) catalytic domain and, (D) glucan binding domain (GBD).

679

(Mooser & Iwakoa, 1989; Mooser, Hefta, Paxton, & Lee, 1991). This domain presents a permutated (b=a)8 barrel structure, when compared with family 13 of the glycoside hydrolases (Monchois et al., 1999). A C-terminal domain composed of a series of repeating units (Ferretti, Gilpin, & Russell, 1987; Gilmore, Russell, & Ferretti, 1990; Abo et al., 1991), which is responsible for the binding of the synthesized glucan (Monchois et al., 1999). The presence of the C-terminal domain is generally necessary to keep an enzyme active. In the case of Leuc. mesenteroides NRRL B-512F dextransucrase (DSR-S), C-terminal domain truncation results in a strong decrease of activity, both for polysaccharide and oligosaccharide synthesis (Monchois, Reverte, Remaud-Sime! on, Monsan, & Willemot, 1998b).

Even if the structural characterization of an increasing number of glucansucrase encoding genes, coupled with a site-directed mutagenesis approach, allows a better insight into the role of the various parts of the glucansucrases, additional research is still necessary to reach a full understanding of the catalytic mechanisms of both polysaccharide and oligosaccharide synthesis. Obviously, the elucidation of the 3D structure of this type of enzyme will be a key step in such an understanding, as well as in the possibility to control glucansucrase selectivity. In fact, it is already possible to modify the structure of the products by site-directed mutagenesis of the glucansucrases: the presence of a carboxylic amino acid instead of a threonine at position 589 of the amino acid sequence of a glucansucrase from S. mutans GS-5 synthesising an a-1,6 linked glucan (GTF-S), results in a 30% increase in the synthesis of a1,3 glucosidic bonds (Shimamura, Nakano, Musaka, & Kuramitsu, 1994). Similar results have been obtained with the dextransucrase from Leuc. mesenteroides NRRL B-512F, DSR-S: when the threonine residue at position 667 is replaced by an arginine, the dextran obtained contains 13% of a-1,3 glucosidic bonds instead of 5% with the native enzyme (Remaud-Sime! on, Willemot, Sarc-abal, 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 the gelification of cane sugar syrups. In 1874, the corresponding product was named ‘‘dextran’’, due to its positive rotatory power. The microorganism responsible of the gelification was isolated by Van Tieghem (1878) and named it Leuc. mesenteroides. In 1941, Hehre demonstrated that dextran could be synthesized from sucrose by a cell-free filtrate. The corresponding

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extracellular enzyme was named ‘‘dextransucrase’’ by Hestrin, 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 osidic bonds within the main chain (Buchholz & Monsan, 2001). The degree of branching, involving a-1,2, a-1,3 and a-1,4 linkages in dextrans A, B and C, respectively, (Seymour & Knapp, 1980) varies according to the origin of the dextransucrase (Table 1). The most widely used dextran is produced by the dextransucrase of the strain Leuc. mesenteroides NRRL B-512F, which synthesizes a very linear polysaccharide containing 95% a-1,6 linkages. The controlled chemical hydrolysis of this high-molecular-mass dextran allows the production of fractions with an average molecular mass of 70 kDa. These products are used for the production of chromatography supports for gel permeation separation (Sephadexs), in the production of blood plasma substitutes (Groenwall & Ingelman, 1948), and to prepare dextran sulphate for blood coagulation prevention and iron transport (Soetaert, Schwengers, Buchholz, & Vandamme, 1995). The molecular mass of the corresponding dextransucrase has been the subject of discussion. Values ranging from 65 kDa (Kobayashi & Matsuda, 1980) to 190 kDa (Willemot, Monsan, & Durand, 1988) have been reported. Finally, the isolation of the gene encoding this enzyme (Wilke-Douglas, Perchorowicz, Houck, & Thomas, 1989) led to a protein with a total of 1527 amino acids (MM: 170 kDa). Lower values are due to proteolytic degradation, while higher values can be attributed to dextran contamination of the protein preparation. Lb. reuteri LB 121 cells growing on sucrose synthesize, besides a fructan polymer, large amounts of a glucan polymer with a molecular mass of 3500 kDa. This glucan has a unique structure consisting of

terminal, 4-substituted, 6-substituted, and 4,6-disubstituted a-glucose in a molar ratio of 1.1 : 2.7 : 1.5 : 1.0 (Van Geel-Schutten et al., 1999). This homopolysaccharide is synthesized from sucrose by a glucansucrase (MM: 146 kDa). The corresponding gene (gtfA) has been isolated and sequenced. It shows a high homology with the known glucansucrase genes from lactic bacteria, particularly with the gene encoding alternansucrase (ASR) from Leuc. mesenteroides NRRL B-1355 (Van Geel-Schutten, 2000). The glucansucrase ORF has a size of approximately 4100 bp (Van Geel-Schutten, 2000). Mutant strains lacking levansucrase activity, but keeping dextransucrase activity, were obtained following a pH shiftdown (Van Geel-Schutten et al., 1999). The maltose acceptor reaction is used to produce nondigestible gluco-oligosaccharides (Fig. 4) with the dextransucrase from Leuc. mesenteroides NRRL B-1299, which is known to catalyse the synthesis of dextran polymers containing a-1,2 linked branched chains (Table 1). Dextransucrase keeps its selectivity in the acceptor reaction and produces a series of glucooligosaccharides containing such a-1,2 glucosidic linkages (Paul, Lopez-Munguia, Remaud, Pelenc, & Monsan, 1992; Remaud-Sime! on, Lopez-Munguia, Pelenc, Paul, & Monsan, 1994; Dols, Remaud-Sime! on, Willemot, & Vignon, 1998), which resist the attack of digestive enzymes (Valette et al., 1993). Such glucooligosaccharides present interesting prebiotic properties (Monsan & Paul, 1995; Djouzy et al., 1995). These gluco-oligosaccharides are presently marketed for human nutritional and dermocosmetic applications (BioEcolias: BioEurope/Solabia). 3.2. Mutan Mutansucrase (E.C. 2.4.1.5) produces a waterin-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 (%) Leuconostoc mesenteroides

B-512F B-742 B-1355 B-1299

Streptococcus downei

Mfe28

Streptococcus mutans

GS5

95 87 50 95 54 66 65 12 90 13 15 70

a-1,3 (%)

a-1,4 (%)

a-1,2 (%)

5 13 50 5 46 7

88 10 87 85 30

27 35

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

glucosidic linkages (Fig. 2), mainly associated with a-1,6 linkages. This enzyme is produced by Leuc. mesenteroides NRRL B-523, B-1149 and several Streptococcus strains (Sidebotham, 1974; Mooser, 1992). Mutan polysaccharides are involved in the adhesion of oral flora microorganisms on the tooth surface to form the dental plaque (Hamada & Slade, 1980). No specific applications of mutan polymers have been developed up till now. 3.3. Alternan Alternansucrase (E.C. 2.4.1.140) synthesizes the glucan, alternan, which contains alternating a-1,6 and a-1,3

glucosidic linkages, with some degree of a-1,3 branchings. Three Leuc. mesenteroides strains are known to produce alternansucrase: Leuc. mesenteroides NRRL B1355, NRRL B-1501 and NRRL B-1498 (Jeanes et al., # e! & Robyt, 1982a). 1954; Seymour & Knapp, 1980; Cot Jeanes et al. (1954) were the first to report the production of two different glucansucrases by Leuc. mesenteroides NRRL B-1355. Ethanol precipitation has resulted 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 containing 95% of a-1,6 glucosidic linkages and 5% a-1,3 branching, similar to the dextran produced by the dextransu-

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crase from Leuc. mesenteroides NRRL B-512F. Fraction S contains alternan and is produced by alternansucrase. This latter enzyme activity is mainly (85%) bound to the bacterial cells and is more thermostable than the dextransucrase activity. The association of enzyme to cells allows efficient removal of the contaminating dextransucrase activity to give a purified alternansucrase preparation (Lopez-Munguia et al., 1993). In the presence of sucrose and maltose acting as an acceptor, alternansucrase retains its selectivity, and catalyses the synthesis of oligosaccharides containing alternating a-1,6 and a-1,3 linkages (Koepsell et al., # e! & Robyt, 1982b; Pelenc et al., 1991). 1952; Cot Mutant strains of Leuc. mesenteroides NRRL B1355C producing alternansucrase on a glucose medium, have been obtained by Kim and Robyt (1994), Smith, Zahnley, and Goodman (1994) and by Smith and Zahnley (1997). This has resulted in the identification of a third glucansucrase in this strain, which synthesizes a 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 (Arguello-Morales . et al., 2000). It contains 6171 bp and encodes a protein of 2057 amino acids (MM: 229 kDa), which corresponds to the largest glucansucrase described to date. This gene produces an enzyme with a structural organization similar to that of known glucansucrases from lactic acid bacteria. Alternansucrase thus, belongs to family 70 of glycoside hydrolases (Henrissat & Davies, 1997) as do the other glucansucrases from Leuc. mesenteroides. The variable region and the C-terminal glucan-binding domain are longer than in other glucansucrases, 100 and 200 amino acids, respectively. The catalytic domain presents 49% identity with that of the other glucansucrases from Leuc. mesenteroides. The purified enzyme has been characterized for its ability to synthesize oligosaccharides from acceptor carbohydrates: its capacity to use cellobiose as an acceptor is significantly higher than that of the dextransucrase from Leuc. mesenteroides NRRL B-512F (Arguello-Morales, . Remaud-Sime! on, Willemot, Vignon, & Monsan, 2001). Such gluco-oligosaccharides are of potential interest as prebiotics. 3.4. b-1,3 glucan Lactobacillus subsp. G-77 has been reported to produce two glucose homopolysaccharides when grown on a glucose medium (Duenas-Chasco et al., 1998). One of the exopolysaccharides was shown to be a 2substituted-(1–3)-b-d-glucan identical to that described for the exopolysaccharide from Pediococcus damnosus 2.6 (Duenas-Chasco et al., 1997). This is the first report of the production of a b-glucan by lactic bacteria. The

mechanism of synthesis has not as yet been described but it does not involve any glucansucrase, because sucrose, which is the enzyme inducer and the glucosyl donor, was not present in the culture medium. A similar polysaccharide is produced by P. damnosus strains isolated from spoiled wine (Walling, Gindreau, & Lonvaud-Funel, 2001). The second homopolysaccharide is a dextran-type polysaccharide (a-1,6 glucosyl linkages) with a-1,2 branching of a single d-glucose unit but its mechanism of synthesis has not been reported. These polysaccharides have undesirable properties for some industries as they cause thickening and are at the origin of a cider (Duenas-Chasco et al., 1997) or a wine (Gindreau, Walling, & Lonvaud-Funel, 2001) spoilage problem known as ‘‘oiliness’’ or ‘‘ropiness’’.

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