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Contribution à la compréhension du mécanisme de formation de dextranes ou gluco-oligosaccharides ramifiés en alpha-1,2 par l'enzyme GBD-CD2 : études cinétique et structurale / Contribution to the understanding of the alpha-(1→2) branching mechanism of dextrans and gluco-oligosaccharides by GBD-CD2 enzyme : kinetic and structural studiesBrison, Yoann 20 September 2010 (has links)
Issue de la troncature de la dextrane-saccharase DSR-E, l’alpha-(1→2) transglucosidase recombinante GBD-CD2 catalyse à partir de saccharose le branchement de molécules acceptrices tels que les dextranes, les isomalto-oligosaccharides ou les gluco-oligosaccharides (GOS ; [6)-alpha-D-Glcp-(1→]n-alpha-D-Glcp-(1→4)-D-Glcp, avec 1<n<9). L’objet de cette étude a porté sur la compréhension des relations structure-activité de GBD-CD2 afin d’investiguer les facteurs structuraux responsables de la synthèse des liaisons osidiques de type alpha-(1→2). La troncature rationnelle du domaine de liaison au glucane (GBD) de l’enzyme GBD-CD2 (192 kDa) a abouti à l’isolement de trois formes tronquées actives, de masses moléculaires égales à 180, 147 et 123 kDa. Après purification de GBD-CD2 et de delta N123-GBD-CD2 (123 kDa), des études cinétiques ont permis de mettre en évidence que les enzymes présentent la même régiospécificité. L’activité d’hydrolyse du saccharose peut être modélisée par le modèle de Michaelis – Menten (kcat respectifs de 109 et 76 s-1). En présence de dextrane accepteur, ces enzymes sont activées. L’activité d’alpha-(1→2) glucosylation suit un modèle Ping Pong Bi Bi (kcat respectifs de 970 et 947 s-1). En modulant le ratio molaire entre le donneur d’unités glucosyle et l’accepteur de ces unités ([saccharose]/[dextrane]), il est possible de synthétiser des dextranes dont le pourcentage de liaisons alpha-(1→2) est contrôlé et varie de 10% à 40%. La caractérisation des produits de la réaction menée en présence de saccharose et de GOS a permis d’isoler et de caractériser pour la première fois des GOS arborant des unités glucosyle branchées en alpha-(1→2) sur les unités glucosyle adjacentes de la chaîne principale. Enfin, la résolution de la structure de delta N123-GBD-CD2 à 3,2 Å révèle que cette enzyme adopte le repliement original « en U » similaire à celui décrit pour GTF180-delta N. La comparaison des gorges catalytiques des deux dextrane-saccharases cristallisées apporte des éléments pouvant expliquer la régiospécificité singulière de delta N123-GBD-CD2, et ouvre la voie à des travaux de mutagenèse visant à investiguer le rôle de résidus potentiellement clés / GBD-CD2 is a recombinant alpha-(1→2) transglucosidase constructed by truncation of the DSR-E dextransucrase from Leuconostoc mesenteroides NRRL B-1299. From sucrose, GBD-CD2 catalyses the alpha-(1→2) branching reaction onto acceptor molecules such as dextrans, isomalto-oligosaccharides or gluco-oligosaccharides (GOS; [6)-alpha-D-Glcp-(1→]n-alpha-D-Glcp-(1→4)-D-Glcp, 1<n<9). This work has been focused on structure activity relationship studies. Rational truncations of the glucan binding domain (GBD) led to the expression in E. coli of three active enzymes, showing molecular masses of 180, 147 and 123 kDa. After purification of the recombinant GBD-CD2 and delta N123-GBD-CD2, we showed that both enzymes display the same regiospecificity. Steady-state kinetics revealed that the activity of sucrose hydrolysis displays a Michaelis Menten type of kinetics (kcat 109 s-1 and 76 s-1, respectively). In the presence of dextran acceptor, these enzymes are activated. The alpha-(1→2) transglucosidase activity from sucrose onto dextrans was modelled by a Ping Pong Bi Bi mechanism (kcat 970 s-1 and 947 s-1, respectively). When varying the molar ratio between the glucosyl donor and the acceptor ([sucrose]/[dextran]), the percentage of alpha-(1→2) linkages in dextrans can be controlled from 10% to 40%. Additionally, from reactions in the presence of GOS and sucrose, we isolated and characterized new alpha-(1→2) branched GOS with contiguous alpha-(1→2) branchings along linear GOS chains. Finally, the X-ray structure of delta N123-GBD-CD2 at 3.2 Å resolution revealed that this enzyme has a very original “U folding” similar to that described for GTF180-delta N. Study of the residues lining the catalytic gorges of the two crystallized enzymes revealed the structural determinants possibly involved in the singular regiospecificity of delta N123-GBD-CD2. Our work opens the way to mutagenesis work for discovering key structural determinants of delta N123-GBD-CD2
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Strategies for the Discovery of Carbohydrate-Active Enzymes from Environmental BacteriaLarsbrink, Johan January 2013 (has links)
The focus of this thesis is a comparative study of approaches in discovery of carbohydrate-active enzymes (CAZymes). CAZymes synthesise, bind to, and degrade all the multitude of carbohydrates found in nature. As such, when aiming for sustainable methods to degrade plant biomass for the generation of biofuels, for which there is a strong drive in society, CAZymes are a natural source of environmentally friendly molecular tools. In nature, microorganisms are the principal degraders of carbohydrates. Not only do they degrade plant matter in forests and aquatic habitats, but also break down the majority of carbohydrates ingested by animals. These symbiotic microorganisms, known as the microbiota, reside in animal digestive tracts in immense quantities, where one of the key nutrient sources is complex carbohydrates. Thus, microorganisms are a plentiful source of CAZymes, and strategies in the discovery of new enzymes from bacterial sources have been the basis for the work presented here, combined with biochemical characterisation of several enzymes. Novel enzymatic activities for the glycoside hydrolase family 31 have been described as a result of the initial projects of the thesis. These later evolved into projects studying bacterial multi-gene systems for the partial or complete degradation of the heterogeneous plant polysaccharide xyloglucan. These systems contain, in addition to various hydrolytic CAZymes, necessary binding-, transport-, and regulatory proteins. The results presented here show, in detail, how very complex carbohydrates can efficiently be degraded by bacterial enzymes of industrial relevance. / <p>QC 20130826</p>
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