Francois Bernard Paul

Microbial polysaccharides with actual potential industrial applications

The microbial polysaccharides reviewed include xanthan gum, scleroglucan, PS-10, PS-21 and PS-53 gums, polysaccharides from Alcaligenes sp., PS-7 gum, gellan gum, curdlan, bacterial alginate, dextran, pullulan, Bakers Yeast Glycan, 6-deoxy-hexose-containing polysaccharides and bacterial cellulose. Factors limiting the commercial potential of certain microbial polysaccharides such as availability, rheological properties, and polyvalency are outlined. The polysaccharides are classified according to their uses as viscosity-increasing agents and as gelling agents. A third category includes polysaccharides with specific applications such as tailor-made dextran and pullulan and polysaccharides used as substrates for the preparation of rare sugars. The difficulties encountered in development of a polysaccharide at the industrial level are pointed out.

Acceptor reaction of a highly purified dextransucrase with maltose and oligosaccharides. Application to the synthesis of controlled-molecular-weight dextrans☆

Abstract Kinetic studies and product characterization of the acceptor reaction of a highly purified dextransucrase isolated from cultures of Leuconostoc mesenteroides suggested that the introduction of increasing amounts of maltose in the medium of reaction produced an increase of the kinetic constants, K′m and Vmax, of the enzyme for sucrose. A Box-Hunter mathematical optimization concerning the products of the reaction with maltose showed that the sucrose-to-maltose ratio is of prime influence upon the yield of the oligosaccharides produced and that their weight-average molecular weight (Mw) is a linear function of that ratio. In view of directly synthesizing controlled-molecular-weight dextrans, various populations of oligosaccharides were produced and used as glucosyl acceptors in a second-step reaction with sucrose. The molecular weight and polydispersity of the second-step products were related to the sucrose-to-acceptor ratio and to the characteristics of the oligosaccharide acceptor.

Production and purification of alternansucrase, a glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355, for the synthesis of oligoalternans

Alternansucrase, a glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355, was produced by fed-batch cultures and purified by two-phase partition with polyethylene glycol. The contaminating dextransucrase activity was eliminated by heat treatment. Some properties of the enzyme were examined, in particular its transferase activity in the presence of maltose. A general procedure for oligoalternan production and purification is defined.

Characterization of α-(1→3) Branched Oligosaccharides Synthesized by Acceptor Reaction with the Extracellular Glucosyltransferases from L. Mesenteroides NRRL B-742

Abstract The glucosyltransferases from L. mesenteroides are known to catalyze the transfer of the D-glucosyl group of sucrose onto sugars, now commonly named acceptors. We investigated in the present work, the acceptor reaction catalyzed by the extracellular glucosyltransferases from L. mesenteroides NRRL B-742. The enzymes of the culture supernatant, purified by aqueous two-phase partition between dextran and polyethylene glycol solutions, were found to efficiently transfer the glucose moiety of sucrose onto maltose acceptor. By increasing the sucrose/maltose ratio (S/M), it was possible to catalyze the synthesis of oligosaccharides of increasing degree of polymerisation (d.p.). For an S/M ratio of 7, both linear oligosaccharides (only composed of α-(1→6) linkages and a maltose residue at the reducing end) and branched oligosaccharides were produced. A glucanase treatment permitted isolation of the branched products which were then analyzed by carbon 13 NMR spectroscopy. The chemical shifts arising from ...

Phosphorylation coupled to H2 oxidation by chromatophores from Rhodopseudomonas capsulata.

The phototrophic bacterium, Rhodopseudomonas capsulata can use Hz as electron donor for either photoautotrophic or chemoautotrophic growth [ 1,2]. When grown in the presence of Ha, cells contain a relatively high hydrogenase activity of the Hz -uptake type [3]. This hydrogenase is located in the membrane [3], which also contains the redox components of the respiratory chain. Cells of Rps. capsulata grown anaerobically in the light have been shown to have respiratory activities [4] similar to those found in aerobic cells [5]. The purpose of the experiments reported here was to show that hydrogenase formed in the membranes of photosynthetically grown cells can feed electrons to the respiratory chain and that Hz oxidation is linked to ATP synthesis.

Novel enzymatic synthesis of oligosaccharides

Abstract Industrial applications of enzyme catalysts are mostly hydrolysis reactions. But increasing attention is given to enzymatic synthesis reactions. The possibility of obtaining efficient synthesis reactions without involving costly cofactors is illustrated by examples taken in the field of oligosaccharide production. Three main approaches are considered: reversion of hydrolysis reactions, by shifting the position of the reaction equilibrium, “transhydrolytic”; reactions, using the transferase activity of hydrolytic enzymes, transfer reactions, catalyzed by transferase enzymes. The respective advantages and drawbacks of these three approaches are compared. The oligosaccharides thus obtained are of interest in different fields of application: food and feed ingredients, pharmaceutical and immunological products, cosmetic additives, for example.

Enzymatic synthesis of phosphoric monoesters with alkaline phosphatase in reverse hydrolysis conditions

Abstract Title compounds were synthesized on a preparative scale using alkaline phosphatase, orthophosphoric monoester phosphohydrolase B.C. 3.1.3.1, in reverse hydrolysis conditions. Optimization for one of the 25 phosphoryl acceptors investigated (glycerol) shows that up to 55% synthesis yield can be obtained using a large excess of substrate, conditions in which the enzymatic activity remains high. From the results obtained with different phosphoryl group donors, phosphate, pyrophosphate and polyphosphates and with enzymes of different sources, it comes up that the best results are obtained with pyrophosphate and with the weakly purified calf intestine alkaline phosphatase. The extent of enzymatic hydrolysis of the donor can be reduced owing to the existence of two different pH optima for the two reactions, phosphorylation and hydrolysis. The synthesis can be also performed using inert co-solvents which allow to reduce the amount of acceptor used, as long as Zn++ is added to the reaction medium. The results are discussed in terms of the catalytic mechanism of alkaline phosphatase.

Molecular Weight Characterization and Structural Properties of Controlled Molecular Weight Dextrans Synthesized by Acceptor Reaction using Highly Purified Dextransucrase

ABSTRACT Controlled molecular weight dextrans were synthesized using a highly purified dextransucrase from Leuconostoc mesenteroides NRRL B-512F in a multi-step process. Maltose was used as acceptor for the first reaction step. The purified product obtained at a given reaction step was used as acceptor for the next reaction step. Dextrans of molecular weights ranging from 1,000 to 100,000 were thus obtained with a good yield (80 %). After purification, the molecular weight distribution of the products was characterized using size exclusion chromatography coupled with low angle laser light scattering (LALLS). Polydispersity of the products was shown to be similar to that of commerical dextrans. 13C NMR spectra and enzymatic hydrolysis data are consistent with the fact that the enzymatically synthesized dextrans are essentially composed of α(1->6) linkages. It was observed that controlled molecular weight dextrans were less branched than commercial products obtained by acidic hydrolysis of high molecular we...

Versatile enzymatic diacid ester synthesis of butyl α-D-glucopyranoside

Abstract The enzymatic esterification of butyl α-D-glucopyranoside by diacids was investigated with pig pancreatic lipase in acetone. The nature of the acylating agent influenced the regiospecificity of the reaction. A chemoenzymatic reaction scheme was developed to obtain diacid esters using an activated diacid as acyl donor, in a solvent-free process with the immobilized lipase of Mucor miehei (Lipozyme®).

Large-scale enzymatic synthesis of glycerol 1-phosphate

A large-scale synthesis of glycerol 1-phosphate (75 g 1−1; 41%) has been developed using alkaline phosphatase immobilized on corn grits in reverse hydrolysis conditions. The reaction is highly regioselective at position 1 on glycerol but leads to a racemic product. This result is obtained by taking advantage of the difference in pH optima between forward and reverse reactions and the fact that the immobilized enzyme is only weakly inhibited by large concentrations of the two substrates, phosphate and glycerol, and by the product glycerol phosphate. The use of the immobilized enzyme in an embedded form in a flow system also contributes to this significant yield.