Characterization of the Different Dextransucrase Activities Excreted in Glucose, Fructose, or Sucrose Medium by Leuconostoc mesenteroides NRRL B-1299.

When grown in glucose or fructose medium in the absence of sucrose, Leuconostoc mesenteroides NRRL B-1299 produces two distinct extracellular dextransucrases named glucose glucosyltransferase (GGT) and fructose glucosyltransferase (FGT). The production level of GGT and FGT is 10 to 20 times lower than that of the extracellular dextransucrase sucrose glucosyltransferase (SGT) produced on sucrose medium (traditional culture conditions). GGT and FGT were concentrated by ultrafiltration before sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.

Influence of thermal processing on the allergenicity of peanut proteins.

Peanuts are one of the most common and severe food allergens. Nevertheless, the occurrence of peanut allergy varies between countries and depends on both the exposure and the way peanuts are consumed. Processing is known to influence the allergenicity of peanut proteins.

Role of the two catalytic domains of DSR-E dextransucrase and their involvement in the formation of highly alpha-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.

Physicochemical parameters involved in the interaction of Saccharomyces cerevisiae cells with ion-exchange adsorbents in expanded bed chromatography.

Expanded bed adsorption (EBA) is an interesting primary technology allowing the adsorption of target proteins from unclarified feedstock in order to combine separation, concentration, and purification steps. However, interactions between cells and adsorbent beads during the EBA process can strongly reduce the performance of the separation. So, to minimize these interactions, the mechanisms of cell adsorption on the support were investigated.

Glucosylation of alpha-butyl- and alpha-octyl-D-glucopyranosides by dextransucrase and alternansucrase from Leuconostoc mesenteroides.

For the first time, glucosylation of alpha-butyl- and alpha-octylglucopyranoside was achieved using dextransucrase (DS) of various specificities, and alternansucrase (AS) from Leuconostoc mesenteroides. All the glucansucrases (GS) tested used alpha-butylglucopyranoside as acceptor; in particular, DS produced alpha-D-glucopyranosyl-(1-->6)-O-butyl-alpha-D-glucopyranoside and alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->6)-O-butyl-alpha-D-glucopyranoside. In contrast, alpha-octylglucopyranoside was glucosylated only by AS which was shown to be the most efficient catalyst.

Molecular characterization of DSR-E, an alpha-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.

Reactor optimization for alpha-1,2 glucooligosaccharide synthesis by immobilized dextransucrase.

The immobilization of dextransucrase in Ca-alginate beads relies on the close association between dextran polymer and dextransucrase. However, high amounts of dextran in the enzyme preparation drastically limit the specific activity of the immobilized enzyme (4 U/mL of alginate beads). Moreover, even in the absence of diffusion limitation at the batch conditions used, the enzyme behavior is modified by entrapment so that the dextran yield increases and the alpha-1,2 glucooligosaccharides (GOS) are produced with a lower yield (46.

Factors affecting alpha,-1,2 glucooligosaccharide synthesis by Leuconostoc mesenteroides NRRL B-1299 dextransucrase.

The optimization of alpha-1,2 glucooligosaccharide (GOS) synthesis from maltose and sucrose by Leuconostoc mesenteroides NRRL B-1299 dextransucrase was achieved using experimental design and consecutive analysis of the key parameters. An increase of the pH of the reaction from 5.4 to 6.

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.

Amylosucrase, a glucan-synthesizing enzyme from the alpha-amylase family.

Amylosucrase (E.C. 2.

Enzymatic synthesis of alpha-butylglucoside linoleate in a packed bed reactor for future pilot scale-up.

The enzymatic synthesis of a mixture of unsaturated fatty acid alpha-butylglucoside esters, containing more than 60% alpha-butylglucoside linoleate, was achieved through lipase-catalyzed esterification. The continuous evaporation under reduced pressure of the water produced enabled substrate conversions greater than 95% to be reached. Two immobilized lipases from Candida antarctica (Chirazyme L2, c.

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.

Characterisation of the activator effect of glycogen on amylosucrase from Neisseria polysaccharea.

Amylosucrase produces an insoluble alpha-1,4-linked glucan from sucrose, releasing fructose. In addition to polymerisation, in the presence of sucrose as sole substrate, amylosucrase catalyses sucrose hydrolysis and oligosaccharide synthesis in significant proportions. The effects of both glycogen acceptor and sucrose concentrations on the reactions catalysed by the highly purified amylosucrase from Neisseria polysaccharea were investigated.

Amylosucrase from Neisseria polysaccharea: novel catalytic properties.

Amylosucrase is a glucosyltransferase that synthesises an insoluble alpha-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.

Crystallization and preliminary X-ray studies of recombinant amylosucrase from Neisseria polysaccharea.

Recombinant amylosucrase from Neisseria polysaccharea was crystallized by the vapour-diffusion procedure in the presence of polyethylene glycol 6000. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 95.7, b = 117.

Sequence analysis of the gene encoding alternansucrase, a sucrose glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355.

The gene encoding alternansucrase (ASR) from Leuconostoc mesenteroides NRRL B-1355, an original sucrose glucosyltransferase (GTF) specific to alternating alpha-1,3 and alpha-1,6 glucosidic bond synthesis, was cloned, sequenced and expressed into Escherichia coli. Recombinant enzyme catalyzed oligoalternan synthesis from sucrose and maltose acceptor. From sequence comparison, it appears that ASR possesses the same domains as those described for GTFs specific to either contiguous alpha-1,3 osidic bond or contiguous alpha-1,6 osidic bond synthesis.

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%.

Glucansucrases: mechanism of action and structure-function relationships.

Glucansucrases are produced principally by Leuconostoc mesenteroides and oral Streptococcus species, but also by the lactic acid bacteria (Lactococci, Lactobacilli). They catalyse the synthesis of high molecular weight D-glucose polymers, named glucans, from sucrose. In the presence of efficient acceptors, they catalyse the synthesis of low molecular weight oligosaccharides.

Kinetic modeling of oligosaccharide synthesis catalyzed by leuconostoc mesenteroides NRRL B-1299 dextransucrase.

The kinetic behavior of soluble and insoluble forms of dextransucrase from Leuconostoc mesenteroides NRRL B-1299 was investigated with sucrose as substrate and maltose as acceptor. To study the parameters involved, a kinetic model was applied that was previously developed for L. mesenteroides NRRL B-512F dextransucrase.

Enzymatic synthesis of alpha-butylglucoside lactate: a new alpha-hydroxy acid derivative.

An alpha-hydroxy acid derivative, alpha-butylglucoside lactate, was successfully prepared by enzymatic transesterification of alpha-butylglucoside with a lactate alkyl ester in a non-aqueous medium using immobilized lipase as biocatalyst. Ester synthesis in organic solvent was optimized. Solvent choice was made on the basis of substrate solubility and enzyme stability in the medium.

Sequence analysis of the gene encoding amylosucrase from Neisseria polysaccharea and characterization of the recombinant enzyme.

The Neisseria polysaccharea gene encoding amylosucrase was subcloned and expressed in Escherichia coli. Sequencing revealed that the deduced amino acid sequence differs significantly from that previously published. Comparison of the sequence with that of enzymes of the alpha-amylase family predicted a (beta/alpha)8-barrel domain.

Structural characterization of the maltose acceptor-products synthesized by Leuconostoc mesenteroides NRRL B-1299 dextransucrase.

The glucooligosaccharides (GOS), produced by Leuconostoc mesenteroides NRRL B-1299 dextransucrase through an acceptor reaction with maltose and sucrose, were purified by reverse phase chromatography. Logarithmic plots of retention time vs. dp of the GOS gave three parallel lines suggesting the existence of at least three families of homologous molecules.

Effect of Leuconostoc mesenteroides NRRL B-512F dextransucrase carboxy-terminal deletions on dextran and oligosaccharide synthesis.

Dextransucrase (DSR-S) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes synthesis of soluble dextran from sucrose. In the presence of efficient acceptor molecules, such as maltose, the reaction pathway is shifted toward glucooligosaccharide synthesis. Like glucosyltransferases from oral streptococci, DSR-S possesses a C-terminal glucan-binding domain composed of a series of tandem repeats.

Cloning and sequencing of a gene coding for an extracellular dextransucrase (DSRB) from Leuconostoc mesenteroides NRRL B-1299 synthesizing only a alpha (1-6) glucan.

The coding region for a novel Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene (dsrB) was isolated and sequenced. Using degenerate primers homologous to a conserved region present in dextransucrases from Streptococcus (GTFs) and L. mesenteroides NRRL B-512F (DSRS) and conserved amino acid sequences located in the N-terminal catalytic region of these enzymes, about 60% of the DSRB encoding gene was isolated.

Characterization of Leuconostoc mesenteroides NRRL B-512F dextransucrase (DSRS) and identification of amino-acid residues playing a key role in enzyme activity.

Dextransucrase (DSRS) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes the synthesis of soluble dextran from sucrose or oligosaccharides when acceptor molecules, like maltose, are present. The L. mesenteroides NRRL B-512F dextransucrase-encoding gene (dsrS) was amplified by the polymerase chain reaction and cloned in an overexpression plasmid.