Les exopolysaccharides (EPS) sont des biopolymères pouvant être synthétisés par les Eucaryotes, lesArchées et les Procaryotes. Au niveau bactérien les EPS peuvent être impliqués dans la constitution du biofilm (phénomène de biofouling) lors de la colonisation de nouveaux milieux. Ces biopolymères ont des propriétés physico-chimiques et biologiques spécifiques et innovantes à haut potentiel biotechnologique (agroalimentaire, santé, cosmétique, ingénierie environnementale ...). A l'opposé, leurs rôles écologiques lors de l'établissement de biofilms de souches potentiellement pathogènes peuvent rendre leur éradication compliquée.Les processus de dépolymérisation par voie enzymatique sont nécessaires pour réaliser l'élucidation structurale fine des EPS complexes, pour la production de dérivés bio-actifs calibrés à faible poids moléculaire ou pour empêcher la formation de biofilm. La mise en évidence de ces phénomènes enzymatiques sur des microorganismes modèles peut également permettre de mieux cerner les flux de matière au sein de certains compartiments biologiques en particulier en milieu marin. Néanmoins la complexité et grande diversité de structures des EPS rendent la recherche d’enzymes de dépolymérisation spécifiques difficile.Deux stratégies ont été employées pour trouver des sources d'enzymes.1. La voie bactérienne via l’utilisation de bactéries marines productrices d’EPS.2. La voie virale par la recherche de polysaccharidases de bactériophages marins. En plus d’EPS marins déjà connus, de nouveaux substrats (EPS) originaux ont été produits et caractérisés à partir de batéries marines d’intérêts biotechnologiques et/ou écologiques pour les besoins du projet. Un criblage enzymatique sur 11 souches bactériennes du genre Alteromonas a permis de mettre en évidence que 7 d’entre elles présentaient une activité de dépolymérisation endogène vis-à-vis de leur propre EPS. Une bioprospection a été réalisée afin de constituer une virothèque à partir d’hôtes bactériens producteurs d’EPS dans le but de fournir une source de Cazymes virales potentielles. Sur 33 bactériophages, 10 ont été sélectionnés pour leur capacité à rester infectieux lorsque leurs hôtes synthétisent des EPS. Finalement un système hôte/virus a été sélectionné.Les 5 virus (appelés Carin-1 à 5) infectant Cobetia marina DSMZ 4741 ont été étudiés au niveau de leurs traits de vie. Les capacités de dépolymérisation de Carin-1 et Carin-5 sur l'EPS L6 ont été explorés plus en détail. En parallèle, la structure chimique de l'EPS L6 a été intégralement élucidée. / Exopolysaccharides (EPSs) are a class of biopolymer synthesized by Eukarya, Archea and Procarya.Bacterial EPSs are involved in biofilm establishment and biofouling phenomenon. These polymers have physicochemical and biological properties suitable with biotechnological valorization. At the opposite, their involvment in biofouling of pathogenic strains can be problematic.Enzymatic depolymerization process are necessary for EPSs structural elucidation, Bioactive oligosaccharides production or to disrupt polysaccharidic biofilms. The highlight of enzymatic phenomenon can help to understand biogeochimical process in the ocean. Nevertheless the important structural diversity as well as their complexity make the sourcing of specific enzymes difficult.Two strategies were used to find enzymes.1. The bacterial way by using EPS-producing marine strains2. The viral way, with marine bacteriophages.For the need of the study, several EPS-substrates were produced and characterized. The majority of them were totally new. An enzymatic screening on 11 marine Alteromonas strains shown that 6 were able to depolymerize their EPS in an endogenous way. A bioprospection was realized to isolates marine bacteriophages with potential viral Cazymes. 10 out of 33 phages were selectionned for their ability to be infectious with their hosts in EPS production induced. Finally, a host/virus system was chosen. The bacteriophages infecting Cobetia marina DSMZ 4741 (named Carin-1 to 5) were studied. The polysaccharidase activities of Carin-1 and Carin-5 on the L6 EPS were studied more deeply. In parallel, the complete structural elucidation of the L6 EPS was realized.

Agarose and porphyran are related galactans that are only found within red marine algae. As such, marine microorganisms have adapted to using these polysaccharides as carbon sources through the acquisition of unique Carbohydrate Active enZymes (CAZymes). A recent metagenome study of the microbiomes from a Japanese human population identified putative CAZymes in several bacterial species, including Bacteroides plebeius that have significant amino acid sequence similarity with those from marine bacteria. Analysis of one potential CAZyme from B. plebeius (BpGH50) is described here. While displaying up to 30% sequence identity with β-agarases, BpGH50 has no detectable agarase activity. Its crystal structure reveals that the topology of the active site is much different than previously characterized agarases, while containing the same core catalytic machinery. It is unclear whether the enzyme has endo- or exo- activity; the large binding ‘groove’ is typical of an endo-acting enzyme, while a loop at one end of the groove may provide a terminal pocket for the substrate, which is suggestive of exo-activity. Furthermore, the enzyme contains a basic pocket that may dock a sulphated substrate, like porphyran. While no quantifiable porphyran activity was observed, properties of the putative active site suggest that this unusual enzyme may be specific on an unusual substrate, such as a porphyran-agarose hybrid. / Graduate

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>