René-Marc Willemot

Homopolysaccharides from lactic acid bacteria

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.

Amylosucrase from Neisseria polysaccharea: novel catalytic properties.

Amylosucrase is a glucosyltransferase that synthesises an insoluble α‐glucan from sucrose. The catalytic properties of the highly purified amylosucrase from Neisseria polysaccharea were characterised. Contrary to previously published results, it was demonstrated that in the presence of sucrose alone, several reactions are catalysed, in addition to polymer synthesis: sucrose hydrolysis, maltose and maltotriose synthesis by successive transfers of the glucosyl moiety of sucrose onto the released glucose, and finally turanose and trehalulose synthesis – these two sucrose isomers being obtained by glucosyl transfer onto fructose. The effect of initial sucrose concentration on initial activity demonstrated a non‐Michaelian profile never previously described.

Glucansucrases: molecular engineering and oligosaccharide synthesis

Due to their informative role in biological systems, the potential development of oligosaccharide utilization is very important. Today, their industrial application is increasing rapidly especially for their capability of specific stimulation of beneficial bacteria. Future development will require the access to specific oligosaccharides synthesized via processes compatible with technical and economical industrial constraints. In this context, glucansucrases are attractive tools allowing the production of different glucooligosaccharides (GOS) from simple substrates such as sucrose and maltose. These bacterial enzymes are responsible for the synthesis of glucan polymers. They can also synthesize oligomers when an acceptor molecule is introduced into the medium. A large variety of glucosidic bonds are formed corresponding to variable regiospecificities dependent on the enzyme origin. More than 30 glucansucrase-encoding genes have been cloned and sequenced. Many data were provided from studies on the structure/function relationship on these sucrose-converting glucosyltransferases (GTF). Sequence alignment analysis allowed identification of essential amino acids and clearly showed analogies with enzymes from the large α-amylase family. It is now possible to list some determinants possibly involved in the glucansucrase specificity, but many additional investigations and data, especially about the three-dimensional structure, will be necessary for the rational design of specific enzymatic tools for GOS synthesis.

Molecular Characterization of DSR-E, an α-1,2 Linkage-Synthesizing Dextransucrase with Two Catalytic Domains

A novel Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene, dsrE, was isolated, sequenced, and cloned in Escherichia coli, and the recombinant enzyme was shown to be an original glucansucrase which catalyses the synthesis of alpha-1,6 and alpha-1,2 linkages. The nucleotide sequence of the dsrE gene consists of an open reading frame of 8,508 bp coding for a 2,835-amino-acid protein with a molecular mass of 313,267 Da. This is twice the average mass of the glucosyltransferases (GTFs) known so far, which is consistent with the presence of an additional catalytic domain located at the carboxy terminus of the protein and of a central glucan-binding domain, which is also significantly longer than in other glucansucrases. From sequence comparison with family 70 and alpha-amylase enzymes, crucial amino acids involved in the catalytic mechanism were identified, and several original sequences located at some highly conserved regions in GTFs were observed in the second catalytic domain.

Cloning and sequencing of a gene coding for a novel dextransucrase from Leuconostoc mesenteroides NRRL B-1299 synthesizing only α(1–6) and α(1–3) linkages

Abstract The coding region for a Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene ( dsrA ) was isolated and sequenced. Using a pair of primers designed on the basis of two highly conserved amino-acid (aa) sequences in L. mesenteroides NRRL B-512F dextransucrase and streptococcal glucosyltransferases (GTFs), a fragment of dsrA was amplified by the polymerase chain reaction (PCR). This PCR product was used as an hybridization probe to isolate a 1.8-kb fragment identified as the central region of dsrA . Cleavage by Sac I of this fragment allowed two probes to be obtained to isolate the 5′ and the 3′ ends of dsrA . The nucleotide sequence of the dsrA gene was determined and found to consist of an open reading frame (ORF) of 4870 base pairs (bp) coding for a 1290-aa protein with an M r of 145 590. The aa sequence exhibited a high similarity with other GTFs. The two domains previously described in GTFs are conserved in DSRA: an N-terminal conserved domain and a C-terminal domain composed of a series of repeats. Surprisingly, the expected signal peptide was not detected. The entire gene was reconstructed and the activity of DSRA was investigated. The dextran produced appeared to be composed of 85% α(1–6) and 15% α(1–3) linkages and the oligosaccharides synthesized in the presence of maltose were mainly composed of α(1–6) linkages. This enzyme is a novel dextransucrase from L. mesenteroides NRRL B-1299 producing no α(1–2) linkages and is the first glucosyltransferase having no signal peptide described.

Role of the Two Catalytic Domains of DSR-E Dextransucrase and Their Involvement in the Formation of Highly α-1,2 Branched Dextran

The dsrE gene from Leuconostoc mesenteroides NRRL B-1299 was shown to encode a very large protein with two potentially active catalytic domains (CD1 and CD2) separated by a glucan binding domain (GBD). From sequence analysis, DSR-E was classified in glucoside hydrolase family 70, where it is the only enzyme to have two catalytic domains. The recombinant protein DSR-E synthesizes both alpha-1,6 and alpha-1,2 glucosidic linkages in transglucosylation reactions using sucrose as the donor and maltose as the acceptor. To investigate the specific roles of CD1 and CD2 in the catalytic mechanism, truncated forms of dsrE were cloned and expressed in Escherichia coli. Gene products were then small-scale purified to isolate the various corresponding enzymes. Dextran and oligosaccharide syntheses were performed. Structural characterization by (13)C nuclear magnetic resonance and/or high-performance liquid chromatography showed that enzymes devoid of CD2 synthesized products containing only alpha-1,6 linkages. On the other hand, enzymes devoid of CD1 modified alpha-1,6 linear oligosaccharides and dextran acceptors through the formation of alpha-1,2 linkages. Therefore, each domain is highly regiospecific, CD1 being specific for the synthesis of alpha-1,6 glucosidic bonds and CD2 only catalyzing the formation of alpha-1,2 linkages. This finding permitted us to elucidate the mechanism of alpha-1,2 branching formation and to engineer a novel transglucosidase specific for the formation of alpha-1,2 linkages. This enzyme will be very useful to control the rate of alpha-1,2 linkage synthesis in dextran or oligosaccharide production.

Identification of key amino acid residues in Neisseria polysaccharea amylosucrase

Amylosucrase from Neisseria polysaccharea catalyzes the synthesis of an amylose‐like polymer from sucrose. Sequence alignment revealed that it belongs to the glycoside hydrolase family 13. Site‐directed mutagenesis enabled the identification of functionally important amino acid residues located at the active center. Asp‐294 is proposed to act as the catalytic nucleophile and Glu‐336 as general acid base catalyst in amylosucrase. The conserved Asp‐401, His‐195 and His‐400 residues are critical for the enzymatic activity. These results provide strong support for the predicted close structural and functional relationship between the sucrose‐glucosyltransferases and enzymes of the α‐amylase family.

Novel oligosaccharides synthesized from sucrose donor and cellobiose acceptor by alternansucrase

Cellobiose was tested as acceptor in the reaction catalyzed by alternansucrase (EC 2.4.1.140) from Leuconostoc mesenteroides NRRL B-23192. The oligosaccharides synthesized were compared to those obtained with dextransucrase from L. mesenteroides NRRL B-512F. With alternansucrase and dextransucrase, overall oligosaccharide synthesis yield reached 30 and 14%, respectively, showing that alternansucrase is more efficient than dextransucrase for cellobiose glucosylation. Interestingly, alternansucrase produced a series of oligosaccharides from cellobiose. Their structure was determined by mass spectrometry and [13C-1H] NMR spectroscopy. Two trisaccharides are first produced: alpha-D-glucopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->4)]-D-glucopyranose (compound A) and alpha-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-(1-->4)-D-glucopyranose (compound B). Then, compound B can in turn be glucosylated leading to the synthesis of a tetrasaccharide with an additional alpha-(1-->6) linkage at the non-reducing end (compound D). The presence of the alpha-(1-->3) linkage occurred only in the pentasaccharides (compounds C1 and C2) formed from tetrasaccharide D. Compounds B, C1, C2 and D were never described before. They were produced efficiently only by alternansucrase. Their presence emphasizes the difference existing in the acceptor reaction selectivity of the various glucansucrases.

Enzymatic synthesis of unsaturated fatty acid glucoside esters for dermo-cosmetic applications.

Unsaturated fatty acid alpha-butylglucoside esters were prepared by enzymatic esterification of alpha-butylglucoside in nonaqueous media. Conditions were firstly optimized using oleic acid as acyl group. Synthesis was possible in several solvents but the presence of water co-product in the medium limited the reaction to a thermodynamic equilibrium corresponding to a maximal conversion yield of 62%. In pure molten substrates, the removal of water under reduced pressure enabled yields superior to 95% to be obtained. Product profiles depended on enzyme origin : whatever the support, immobilized lipase B from Candida antarctica proved to be far more regioselective for the primary hydroxyl group of glucose than immobilized lipase from Rhizomucor miehei. Results obtained could be easily transposed to the acylation of alpha-butylglucoside with a commercial mixture of unsaturated fatty acids containing more than 60% of linoleic acid. The biocatalyst could be recycled more than ten times without any significant activity loss.

Glucosylation of α-butyl- and α-octyl-d-glucopyranosides by dextransucrase and alternansucrase from Leuconostoc mesenteroides

Abstract For the first time, glucosylation of α-butyl- and α-octylglucopyranoside was achieved using dextransucrase (DS) of various specificities, and alternansucrase (AS) from Leuconostoc mesenteroides . All the glucansucrases (GS) tested used α-butylglucopyranoside as acceptor; in particular, DS produced α- d -glucopyranosyl-(1→6)- O -butyl-α- d -glucopyranoside and α- d -glucopyranosyl-(1→6)-α- d -glucopyranosyl-(1→6)- O -butyl-α- d -glucopyranoside. In contrast, α-octylglucopyranoside was glucosylated only by AS which was shown to be the most efficient catalyst. The conversion rates, obtained with this enzyme at sucrose to acceptor molar ratio of 2:1 reached 81 and 61% for α-butylglucopyranoside and α-octylglucopyranoside, respectively. Analyses obtained from liquid chromatography coupled with mass spectrometry revealed that different series of α-alkylpolyglucopyranosides regioisomers of increasing polymerization degree can be formed depending on the specificity of the catalyst.