Emmanuel Petit

Extraction and characterization of an alginate from the brown seaweed Sargassum turbinarioides Grunow

Matrix polysaccharide from the brown algae Sargassum turbinarioides collected in the coastal waters of Nosy Be (Madagascar) in the Indian Ocean was isolated and its structure was studied by 1H-NMR spectroscopy, FT-IR, SEC-MALLS and HPAEC. An alginate with a molecular weight of 5.528u2009×u2009105xa0g mol−1 was identified as sole polysaccharide. Values of the M/G ratio, FGG, FMM and FGM (or FGM) blocks were measured at respectively 0.94, 0.39, 0.36 and 0.25 and compared with those of alginates from other Sargassum species. This sodium alginate appeared similar to some of the other Sargassum alginates with M/Gu2009<u20091, high values of homopolymeric blocks (ηu2009<u20091) and significant polyguluronic block content.

Separation and fractionation of exopolysaccharides from Porphyridium cruentum

In this work the extraction of EPSs from culture media of Porphyridium cruentum, by dialysis, solvent-precipitation with 3 polar alcohols (methanol, ethanol and isopropanol) and membrane separation techniques has been studied. Diafiltration (DF) using a membrane with a 300 kDa molecular weight cut off was the most efficient technique compared to solvent-extraction and dialysis methods. After extraction, EPS fraction was characterized in terms of rheological properties and biochemical content. The product exhibited shear thinning behavior and a critical overlap concentration equal to 0.6 g/L. The monosaccharide composition was investigated after acidic hydrolysis. Xylose, galactose, glucose and glucuronic acid were identified as the main constitutive monomers.

Production of oligoglucuronans using a monolithic enzymatic microreactor

A glucuronan lyase (EC 4.2.2.14) was immobilized on a monolithic Convective Interaction Media (CIM) disk. The immobilization yield was equal to 29% of the initial activity and 35% of the initial protein amount. Degradations of three glucuronans with various O-acetylation degrees were investigated and compared with degradations using free enzyme. The immobilized glucuronan lyase was inhibited by the O-acetylation degree like the free enzyme. (1)H NMR analyses were used to study the O-acetylation degree of oligoglucuronans and demonstrated that the average degrees of polymerization were inclusive between 4 and 13 after 24h of degradation. This first immobilization of a glucuronan lyase constitutes a new tool to produce oligoglucuronans.

Purification and Properties of a Glucuronan Lyase from Sinorhizobium meliloti M5N1CS (NCIMB 40472)

ABSTRACT A glucuronan lyase extracted from Sinorhizobium meliloti strain M5N1CS was purified to homogeneity by anion-exchange chromatography. The purified enzyme corresponds to a monomer with a molecular mass of 20 kDa and a pI of 4.9. A specific activity was found only for polyglucuronates leading to the production of 4,5-unsaturated oligoglucuronates. The enzyme activity was optimal at pH 6.5 and 50°C. Zn2+, Cu2+, and Hg2+ (1 mM) inhibited the enzyme activity. No homology of the enzyme N-terminal amino acid sequence was found with any of the previously published protein sequences. This enzyme purified fromS. meliloti strain M5N1CS corresponding to a new lyase was classified as an endopolyglucuronate lyase.

Enzymatic degradation and bioactivity evaluation of C-6 oxidized chitosan

C-6 oxidized chitosan was produced from chitosan by performing selective oxidation with NaOCl and NaBr using 2,2,6,6-tetramethylpiperidine-1-oxy radical (TEMPO) as catalyst. Endocellulase, Celluclast 1.5 L, Glucanex(®), Macerozyme R-10, hyaluronidase, hyaluronate lyase, red scorpionfish chitinase, glucuronan lyase and a protein mix from Trichoderma reesei were used to degrade the C-6 oxidized chitosan. Glucanex(®), the crude extract from T. reesei IHEM 4122 and Macerozyme R-10 validated the enzymatic degradation through final hydrolysis yields of the derivative respectively close to 36.4, 20.3 and 12.9% (w/w). The best initial reaction velocity (2.41 U/mL) was observed for Glucanex(®). The antileishmanial activity of the derivative was evaluated against Leishmania infantum LIPA 137. The antibacterial activities against Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were also tested. Results showed an antileishmanial activity (IC50: 125 μg/mL) of the obtained derivatives against L. infantum LIPA 137.

Identification of glucuronan lyase from a mutant strain of Rhizobium meliloti

The Rhizobium meliloti M5N1CS (NCIMB 40472) mutant strain wich induces nodule formation on alfalfa roots, produces a (1 --> 4)-beta-D-glucuronan partially acetylated. During fermentation under specific conditions, the molecular weight of the polymer decrease, the presence of polysaccharide degrading enzyme was suspected. A glucuronan lyase was identified, this new bacterial lyase produces d.p. 4 oligoglucuronans, substituents (acetates) present on the substrate reduced the enzyme activity.

A new tool to detect high viscous exopolymers from microalgae.

Microalgae are microorganisms often surrounded by a slime layer made of secreted polymeric substances sometimes including polysaccharides. These polysaccharides, weakly described in the literature, can constitute value-added molecules in several industrial areas. The aim of this article is to show that a new tool, the BioFilm Ring Test®, can be used to detect viscous microalgal exopolymers. Two red microalgal strains (Rhodella violacea and Porphyridium purpureum), one cyanobacterium (Arthrospira platensis) and their excreted polymeric fractions were studied. R. violacea and P. purpureum induced a positive response with the BioFilm Ring Test® contrary to A. platensis. Finally, the understanding of the fractions viscosity involvement in the BRT response was performed by a rheological study.

A new method to screen polysaccharide cleavage enzymes

The activity of polysaccharide cleavage enzymes has usually been evaluated by qualitative plate screening methods and quantitative colorimetric or chromatographic assays. The recent development of protein engineering has shown the limits of these techniques when applied to high throughput screening. Here we propose a microplate method to measure the activity of polysaccharide cleavage enzymes through small variations in viscosity. Polysaccharide solutions are co-incubated with magnetic particles in enzyme buffers. The cleavage action of polymer-degrading enzymes increases the mobility of the particles in a magnetic field, even at low levels of enzyme activities. This reproducible, sensitive technique was used to evaluate enzymatic specificity towards substrates. BioFilm indices (BFI) determined by associated software were used to follow enzyme kinetics and measure the usual variables.

Osmotic stress alters the balance between organic and inorganic solutes in flax (Linum usitatissimum).

Flax (Linum usitatissimum) is grown for its oil and its fiber. This crop, cultivated in temperate regions, has seen a renewed interest due to the presence of abundant molecules of interest for many applications. Little information is available about the behavior of flax during osmotic stress; yet this is considered a major stress that causes significant yield losses in most crops. To control the presence of this stress better, flax behavior was investigated following the application of osmotic stress and the response was examined by applying increasing concentrations of PEG 8000. This resulted in the reorganization of 32 metabolites and 6 mineral ions in the leaves. The analysis of these two types of solute highlighted the contrasting behavior between a higher metabolite content (particularly fructose, glucose and proline) and a decrease in mineral ions (especially nitrate and potassium) following PEG treatment. However, this reorganization did not lead to a greater accumulation of solutes, with the total amount remaining unchanged in leaves during osmotic stress.

Trehalose determination in linseed subjected to osmotic stress. HPAEC-PAD analysis: an inappropriate method

Trehalose is a non-reducing disaccharide involved in stress tolerance in plants. To understand better the role of trehalose in the osmotic stress response in linseed (Linum usitatissimum), trehalose content in leaves was studied. First, the method commonly used for sugar determination, high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), gave unsatisfactory results and the separation efficiency could not be improved by varying the elution conditions. The same problem was also found in the model plant: Arabidopsis thaliana. After clearly highlighting a co-elution of trehalose in these two species by a trehalase assay and liquid chromatography-high resolution mass spectrometry analysis, gas chromatography-mass spectrometry (GC-MS) was used as the analytical method instead. These results confirmed that trehalose content is currently overestimated by HPAEC-PAD analysis, approximately 7 and 13 times for A. thaliana and linseed respectively. Thus GC-MS gave more satisfactory results for trehalose quantification in plants. With this method, trehalose accumulation was observed in linseed during an osmotic stress (-0.30u2009MPa), the quantity (31.49u2009nmolu2009g(-1) dry weight after 48u2009h) appears too low to assign an osmoprotector or osmoregulator role to trehalose in stressed linseed.