Josiane Courtois

Oligosaccharides from land plants and algae: production and applications in therapeutics and biotechnology.

Since the past decades, oligosaccharides are considered for their potential biological activities. To exploit them, it was essential to obtain pure molecules in large amounts. Several strategies were developed to produce specific sugar sequences with specific substitution patterns from land plants and algae polysaccharides. Then, pure oligosaccharides were analyzed for their potential biological activities and relations between oligomers structure and function were tackled. First they can be health beneficial molecules when they are added to the diet to enhance the growth of probiotic bacteria, in that case, oligomers that resist to the digestive process are used as specific substrate for the growth of health beneficial bacteria. In other cases, oligomers have to interact with receptors on cells. In this instance, a specific conformation is needed to allow the sugar sequence to establish specific linkages with the receptor. So, to be adapted to the receptor, the oligosaccharides have to present specific groups to the receptor, there, the polymerization degree of oligosaccharides as well as the flexibility of the glycosidic linkages has to be considered.

Structure of a polysaccharide from a Rhizobium species containing 2-deoxy-β-d-arabino-hexuronic acid

The structure of the extracellular polysaccharide (EPS) produced by the Rhizobium sp. B strain isolated from atypical nodules on alfalfa has been determined using a combination of chemical and physical techniques (methylation analysis, high pH-anion exchange chromatography (HPAEC), mass spectrometry and 1-D and 2-D NMR spectroscopy). As opposed to the EPS from other strains of Rhizobium, the EPS from the sp. B strain contains D-Glc together with L-Rha and 2-deoxy-D-arabino-hexuronic acid. It is a polymer of a repeating unit having the following structure: --> 4)-beta-D-Glcp-(1 --> 4)-alpha-L-Rhap -(1 --> 3)-beta-D-Glcp-(1 --> 4)-2-deoxy-beta-D-GlcpA-(1 -->. The polysaccharide also contains 0.6 O-acetyl groups per sugar which have not been located.

Structural characterization and rheological properties of an extracellular glucuronan produced by a Rhizobium meliloti M5N1 mutant strain

The mutant strain M5N1 C.S. (NCIMB 40472) of Rhizobium meliloti M5N1 is able to produce during fermentation a partially acetylated extracellular (1-->4)-beta-D-glucuronan. At low concentration (1 g.l-1), in the presence of monovalent cations, this new glucuronate behaves as a thickening agent, whereas at higher concentration a thermoreversible gel is obtained. With such divalent cations as Ca2+, a thermally stable gel can be formed.

Polysaccharide Lyases: Recent Developments as Biotechnological Tools

Polysaccharide lyases, which are polysaccharide cleavage enzymes, act mainly on anionic polysaccharides. Produced by prokaryote and eukaryote organisms, these enzymes degrade (1,4) glycosidic bond by a beta elimination mechanism and have unsaturated oligosaccharides as major products. New polysaccharides are cleaved only by their specific polysaccharide lyases. From anionic polysaccharides controlled degradations, various biotechnological applications were investigated. This review catalogues the degradation of bacterial, plant and animal polysaccharides (neutral and anionic) by this family of carbohydrate acting enzymes.

1H NMR spectroscopic determination of poly 3-hydroxybutyrate extracted from microbial biomass

Abstract 1 H NMR analysis was performed in order to identify the nature of the poly 3-hydroxyalkanoate (PHA) accumulated by Rhizobium meliloti M5N1 and determine its concentration within the cells. Since the PHA was identified as being poly 3-hydroxybutyrate (PHB), the method of quantification was previously tested on standard PHB. The use of either methanol or benzene as the internal reference showed good accuracy. The extraction of PHB was carried out using dispersions of sodium hypochlorite and chloroform. A treatment time of 1 h with a 10% concentration of hypochlorite allowed for total recovery of PHB. After extraction of different amounts of PHB, aliquots were submitted to 1 H NMR analysis. Methanol used as the internal reference gave an overvaluation of the PHB content. However, benzene met all the requirements of an internal reference due to its chemical inertia in our experimental conditions. NMR data obtained using benzene as the internal reference are in good agreement with corresponding data obtained by gas chromatographic (GC) analysis.

Influence of fermentation conditions and microfiltration processes on membrane fouling during recovery of glucuronane polysaccharides from fermentation broths

We have investigated the recovery of exopolysaccharides produced by Sinorhizobium meliloti M5N1 CS bacteria from fermentation broths using different membrane filtration processes: cross-flow filtration with a 7 mm i.d. tubular ceramic membrane of 0.5-microm pores under fixed transmembrane pressure or fixed permeate flux and dynamic filtration with a 0.2 microm nylon membrane using a 16-cm rotating disc filter. With the tubular membrane, the polysaccharide mass flux was mainly limited by polymer transmission that decayed to 10% after 90 min. The mass flux of polymer produced under standard fermentation conditions (70 h at 30 degrees C) stabilized after 70 min to 15 g/h/m(2). This mass flux rises to 36 g/h/m(2) when the mean stirring speed during fermentation is increased and to 123 g/h/m(2) when fermentation is extended to 120 h. In both cases, the mean molecular weight of polysaccharides drops from 4.0 10(5) g/mol under standard conditions to 2.7 10(5) g/mol. A similar reduction in molecular weight was observed when the fermentation temperature was raised to 36 degrees C without benefit to the mass flux. These changes in fermentation conditions have little effect on stabilized permeate flux, but raise significantly the sieving coefficient, due probably to molecular weight reduction and the filamentous aspect of the polymer as observed from SEM photographs. The polymer-mass flux was also increased by reducing transmembrane pressure (TMP) and raising the shear rate by inserting a rod in the membrane lumen. Operation under fixed permeate flux instead of constant TMP inhibited fouling during the first 4 h, resulting in higher sieving coefficients and polymer mass fluxes. The most interesting results were obtained with dynamic filtration because it allows operation at high-shear rates and low TMP. Sieving coefficients remained between 90 and 100%. With a smooth disc, the polysaccharide mass flux remained close to 180 g/h/m(2) at 1500 rpm and cell concentrations from 1 to 3 g/L. When radial rods were glued to the disc to increase wall shear stress and turbulence, the mass flux rose to 275 g/h/m(2) at the same speed and cell concentration.

A (1→4)-β-D-glucuronan excreted by a mutant of the Rhizobium meliloti M5N1 strain

Abstract A mutant of the R. meliloti M5N1 strain has been selected. This strain, R. meliloti M5N1 CS (NCIMB 40472), excretes an extracellular material composed of 2-O-Ac-β-GlcpA, 3-O-Ac-β-GlcpA, 2,3-di-O-Ac-β-GlcpA and three species of β-GlcpA residues 1→4 linked. For the culture conditions used, the weight average molecular weight of the polymer varied in the range of 6 × 104 < Mw < 4 × 105. High molecular weight glucuronate forms thermoreversible gels at 5 g L−l. In the presence of divalent cation such as Ca2+ or trivalent cations such as Cr3+ or Fe3 +, cross linking of the polymer occurs. This polysaccharide is the first exocellular (1→4)-β-D-glucuronan produced by a R. meliloti strain.

Production of Arabinoxylan-oligosaccharides from Flaxseed (Linum usitatissimum)

Flaxseed mucilage from Linum usitatissimum L. species was constituted by arabinoxylan (about 75%) and pectin (about 25%). A new procedure was developed to obtain only arabinoxylans which implicated treatment of the pectin fraction by enzymatic hydrolysis with pectinase. Then three processes of depolymerization were evaluated on arabinoxylans. First, a thermic hydrolysis in mild acid conditions was performed and an ultrafiltration process was used as purification method. Second, the potential of xylanases from different glycoside hydrolase families for arabinoxylan-oligosaccharides (AXOS) production was tested, and finally a radical depolymerization was conducted. Average molecular weights were determined by high pressure size exclusion chromatography coupled with multiple angle laser light scattering (MALLS), and carbohydrate compositions were determined by high pH anion exchange chromatography pulse amperometric detector (HPAEC-PAD). Both chemical and enzymatic treatments were inefficient to convert arabinoxylans from flaxseed mucilage into AXOS. Only radical depolymerization process was allowed to obtain arabinoxylan-oligosaccharides presenting different molecular weights (11.9 x 10(3) to 1.9 x 10(3) g mol(-1)) with satisfactory yields (75% to 35%).

Distinct Defenses Induced in Wheat Against Powdery Mildew by Acetylated and Nonacetylated Oligogalacturonides

In wheat, little is known about disease resistance inducers and, more specifically, about the biological activities from those derived from endogenous elicitors, such as oligogalacturonides (OGAs). Therefore, we tested the ability of two fractions of OGAs, with polymerization degrees (DPs) of 2-25, to induce resistance to Blumeria graminis f. sp. tritici and defense responses in wheat. One fraction was unacetylated (OGAs-Ac) whereas the second one was 30% chemically acetylated (OGAs+Ac). Infection level was reduced to 57 and 58% relative to controls when OGAs-Ac and OGAs+Ac, respectively, were sprayed 48 h before inoculation. Activities of various defense-related enzymes were then assayed in noninoculated wheat leaves infiltrated with OGAs. Oxalate oxidase, peroxidase, and lipoxygenase were responsive to both OGAs-Ac and OGAs+Ac, which suggests involvement of reactive oxygen species and oxilipins in OGAs-mediated responses in wheat. In inoculated leaves, both fractions induced a similar increase in H₂O₂ accumulation at the site of fungal penetration. However, only OGAs+Ac led to an increase in papilla-associated fluorescence and to a reduction of formed fungal haustoria. Our work provides the first evidence for elicitation and protection effects of preventive treatments with OGAs in wheat and for new properties of acetylated OGAs.

Rapid quantification of O-acetyl and O-methyl residues in pectin extracts

A rapid method for the determination of the degrees of methylation (DM) and acetylation (DA) of pectins was developed. The polymer substitution degree as determined after saponification at 80 degrees C with NaOD during 1H NMR analysis. Under alkaline conditions, the cleavage of O-acetyl and O-methyl linkages allows the detection and the integration of the H-4 signal from galacturonic acid residues in the newly unesterified pectins. So, after a 10-min NMR recording, sodium acetate and sodium methanolate can be easily quantified relative to the clearly identified H-4 signal in galacturonic acid residues. Protons signals from pectin neutral sugars do not interfere with H-4. During the analysis, a limited (<3%) methanol evaporation leading to a weak reduced signal from the methanolate protons was observed. The proposed method allows in few minutes an accurate simultaneous quantification of DM and DA from few mg of pectin extracts, without the need of external standards.