Etude de l'implication de cellulases dans la voie de biosynthèse de cellulose chez les bactéries

Abstract

By definition, cellulases are enzymes that catalyze the degradation of cellulose. However, their involvement in cellulose biosynthesis by bacteria and plants has been reported, although their exact contribution remains unclear. In the present study, we have investigated the involvement of cellulases from glycoside hydrolase family 5 (GH5) in cellulose synthesis. In particular, we have functionally and structurally characterized the Ps_Cel5A cellulase from Pseudomonas stutzeri and its metagenome-derived homolog RBcel1, which both belong to the GH5 family and are suspected to be involved in cellulose biosynthesis. In addition, we have also compared these enzymes with the well-characterized Ta_Cel5A cellulase from the cellulolytic fungus Thermoascus aurantiacus. The first part of the work was devoted to the description of the tridimensional structure of RBcel1, in comparison with other glycoside hydrolases from GH5. In the second part of the study, we focused on the functional and structural comparison between RBcel1, Ps_CelA and Ta_Cel5A. Biochemical analysis has highlighted that, besides their hydrolytic activity, RBcel1 and Ps_Cel5A were able to catalyze transglycosylation in vitro. This synthesis reaction was not detected for Ta_Cel5A, which seemed to remain hydrolytic only. Determination of the structure of RBcel1 in complex with cellobiose has revealed distinct features in the aglycone substrate binding sites compared to Ta_Cel5A which could potentially explain the observed differences in their activities in vitro. Finally, the involvement of Ps_Cel5A in cellulose production by P. stutzeri was confirmed, by analysis the ability of P. stutzeri-ΔPs_Cel5A to produce the polymer. Complementation of this mutant strain by the three cellulases was also performed and discussed.Caractérisation de la synthèse de cellulose par deux cellulases d'origine bactérienn

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