Colette Brassart
Centre national de la recherche scientifique
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Featured researches published by Colette Brassart.
Biotechnology and Applied Biochemistry | 1999
Danielle Derensy‐Dron; Frédéric Krzewinski; Colette Brassart; Stéphane Bouquelet
A new enzyme has been characterized in a cell‐free extract of Bifidobacterium bifidum that catalysed the reversible phosphorolytic cleavage of β‐1,3‐galacto‐oligosaccharides. In the presence of Pi, the phosphorolysis reaction was favoured and was accompanied by a Walden reaction. Cleavage of the β‐glycosidic linkage gave an α‐galactoside derivative (α‐D‐galactose 1‐phosphate). The enzyme possesses a high specificity for β‐D‐galactosido‐(1,3)‐N‐acetylglucosamine and β‐D‐galactosido‐(1,3)‐N‐acetylgalactosamine. This purified intracellular enzyme had an estimated molecular mass of 140 kDa. The galactophosphorolytic activity on disaccharides was optimal at pH 6–6.5 and the reverse reaction was optimal at pH 5.5–6. The temperature optimum for phosphorolysis and the reverse reaction was approx. 50–55 °C. This enzyme is of particular interest in degrading some β‐D‐Gal(1,3) linkages and should be classified as EC 2.4.1.‐.
Current Microbiology | 1996
Frédéric Krzewinski; Colette Brassart; Françoise Gavini; Stéphane Bouquelet
Abstract. Lactose was fermented but not assimilated by the strain Bifidobacterium bifidum DSM 20082. The sugar uptake was measured with lactose 14C. Km and Vmax values were respectively 2.6 mM and 12.11 nmol/min/mg of cell protein. The lactose transport system and the β-D-galactosidase were stimulated when the cells were grown with lactose, but isopropyl-β-D-thiogalactopyranoside had no effect. Lactose uptake was inhibited by compounds which interfered with proton and metal ionophore. Na+, Li+, or K+ did not affect incorporation of lactose. Furthermore, the lactose uptake decreased when an inhibitor of ATP synthesis was used. From the results of this study, the strain contained an active lactose transport system, probably a proton symport as described for Escherichia coli but with a different regulation system.
Current Microbiology | 1997
Frédéric Krzewinski; Colette Brassart; Françoise Gavini; Stéphane Bouquelet
Abstract. Sugar uptake was measured with 3H-galactose and 14C-glucose. Galactose transport system was not modified by inhibitors of known translocases and did not present a saturation kinetic with high concentration of galactose. Glucose incorporation was inhibited by lasalocid (cation symport inhibitor) and increased by KCl. The kinetic parameters KM and Vmax were respectively 9.16 mM and 26.56 nmol/min/mg cell protein. On the basis of this study, galactose crossed through the membrane by diffusion, and glucose was incorporated by a cation symport which is regulated by K+ ions.
Archives of Microbiology | 2008
Sophie Foley; Emilie Stolarczyk; Fadoua Mouni; Colette Brassart; Olivier Vidal; Eliane Aïssi; Stéphane Bouquelet; Frédéric Krzewinski
Bifidobacterium bifidum, in contrast to other bifidobacterial species, is auxotrophic for N-acetylglucosamine. Growth experiments revealed assimilation of radiolabelled N-acetylglucosamine in bacterial cell walls and in acetate, an end-product of central metabolism via the bifidobacterial d-fructose-6-phosphate shunt. While supplementation with fructose led to reduced N-acetylglucosamine assimilation via the d-fructose-6-phosphate shunt, no significant difference was observed in levels of radiolabelled N-acetylglucosamine incorporated into cell walls. Considering the central role played by glutamine fructose-6-phosphate transaminase (GlmS) in linking the biosynthetic pathway for N-acetylglucosamine to hexose metabolism, the GlmS of Bifidobacterium was characterized. The genes encoding the putative GlmS of B. longum DSM20219 and B. bifidum DSM20082 were cloned and sequenced. Bioinformatic analyses of the predicted proteins revealed 43% amino acid identity with the Escherichia coli GlmS, with conservation of key amino acids in the catalytic domain. The B. longum GlmS was over-produced as a histidine-tagged fusion protein. The purified C-terminal His-tagged GlmS possessed glutamine fructose-6-phosphate amidotransferase activity as demonstrated by synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. It also possesses an independent glutaminase activity, converting glutamine to glutamate in the absence of fructose-6-phosphate. This is of interest considering the apparently reduced coding potential in bifidobacteria for enzymes associated with glutamine metabolism.
Glycoconjugate Journal | 1989
Ossarath Kol; Colette Brassart; Geneviève Spik; Jean Montreuil; Stéphane Bouquelet
We have previously shown that an endo-β-N-acetylglucosaminidase (EC 3.2.1.96) named “Endo B”, isolated from culture filtrates of the basidiomyceteSporotrichum dimorphosporum cleaves asialo-, and to some extent, monosialylated bi-antennary glycans of theN-acetyllactosamine type linked to the asparagine residue of peptide or protein moieties [Bouquelet S, Strecker G, Montreuil J, Spik G (1980) Biochimie 62:43–49]. In the present paper, the substrate specificity of the enzyme towards oligomannoside and hybrid type glycans has been analyzed. The results obtained indicate that ovalbumin glycopeptides containing four to seven mannose residues and bovine lactotransferrin glycopeptides containing four to nine mannose residues were completely hydrolyzed by the enzyme. The degree of cleavage was variable among hybrid type structures, since glycopeptides containing the following glycans: (Gal)1(GlcNAc)3(Man)5(GlcNAc)2; (GlcNAc)3(Man)5(GlcNAc)2; (GlcNAc)3(Man)4(GlcNAc)2 were not hydrolyzed by the enzyme while the percentage of hydrolysis of a glycopeptide containing (GlcNAc)2(Man)5(GlcNAc)2 glycan reached 90%. The bovine lactotransferrin was partially deglycosylated (40%) in the absence of non-ionic detergent while native ovalbumin glycoprotein was not hydrolyzed by the enzyme.The oligomannoside-and theN-acetyllactosamine-type degrading activities present in the culture filtrates were not separated at any step of the purification procedure. Both activities were eluted as a single component with an apparent molecular mass of 89 kDa suggesting that they are located on the same enzyme molecule.Endo B represents a powerful tool for removing oligomannoside-andN-acetyllactosamine-type glycans fromN-glycopeptides andN-glycoproteins. Moreover, advantages in the use of Endo B in a soluble form as well as in an immobilized form result in its high activity and in its stability to heat denaturation and storage.
Journal of Proteomics | 2012
Bernadette Coddeville; Sz-Wei Wu; Emeline Fabre; Colette Brassart; Yoann Rombouts; Adeline Burguière; Laurent Kremer; Kay-Hooi Khoo; Elisabeth Elass-Rochard; Yann Guérardel
The 45/47 kDa Apa, an immuno-dominant antigen secreted by Mycobacterium tuberculosis is O-mannosylated at multiple sites. Glycosylation of Apa plays a key role in colonization and invasion of the host cells by M. tuberculosis through interactions of Apa with the host immune system C-type lectins. Mycobacterium marinum (M.ma) a fish pathogen, phylogenetically close to M. tuberculosis, induces a granulomatous response with features similar to those described for M. tuberculosis in human. Although M.ma possesses an Apa homologue, its glycosylation status is unknown, and whether this represents a crucial element in the pathophysiology induced by M.ma remains to be addressed. To this aim, we have identified two concanavalin A-reactive 45/47 kDa proteins from M.ma, which have been further purified by a two-step anion exchange chromatography process. Advanced liquid chromatography-nanoESI mass spectrometry-based proteomic analyses of peptides, derived from either tryptic digestion alone or in combination with the Asp-N endoproteinase, established that M.ma Apa possesses up to seven distinct O-mannosylated sites with mainly single mannose substitutions, which can be further extended at the Ser/Thr/Pro rich region near the N-terminus. This opens the way to further studies focussing on the involvement and biological functions of Apa O-mannosylation using the M.ma/zebrafish model.
Carbohydrate Research | 2005
Emmanuel Maes; Estelle Garenaux; Gérard Strecker; Yves Leroy; Jean-Michel Wieruszeski; Colette Brassart; Yann Guérardel
Comparative Biochemistry and Physiology C-toxicology & Pharmacology | 2006
Frida Soria; Claudia Sierra; Stéphane Bouquelet; Colette Brassart; Concepción Agundis; Edgar Zenteno; Lorena Vázquez
Carbohydrate Research | 2011
Estelle Garenaux; Emmanuel Maes; S. Levêque; Colette Brassart; Yann Guérardel
Glycobiology | 1995
Olivier Cuvillier; Catherine Alonso; Jean-Michel Wieruszeski; Colette Brassart; Gérard Strecker; Stéphane Bouquelet; Jean-Claude Michalski