As enzimas hidrolíticas são consideradas, por plena definição, aquelas com a capacidade de realizar a reação de hidrólise. Devido à sua capacidade de degradar substâncias naturais, as enzimas hidrolíticas são de grande uso industrial e podem ser aplicadas nas indústrias têxtil, alimentar, farmacêutica, biocombustíveis- agrícola, assim como na produção de derivados biotecnológicos de maior valor agregado. Conhecidas como enzimas que liberam arabinose e arabinofuranose, as arabinofuranosidases da família 62 são capazes de quebrar as conexões de arabinoxilanos. A importância biotecnológica de tais proteínas é desde o uso na agricultura à indústria. Elas podem ser utilizadas, por exemplo, em coquetéis na digestão de alimentos para animais, vinhos e para melhorar a eficiência de clarificação de sucos. Além disso, sua ação de deslignificação é promissora na degradação da biomassa para a produção de bicombustíveis. Este projeto procura realizar a clonagem, expressão heteróloga em bactérias e caracterização estrutural de uma α-L-arabinofuranosidase do fungo termofílico Thielavia terrestris. Esta enzima pertence à família 62 das hidrolases de glicosídeos, que é conhecida por melhorar a ação das celulases e, portanto, de interesse na degradação da biomassa lignocelulósica, como o bagaço de cana-de-açúcar. Estudos biofísicos, bioquímicos e estruturais apresentados neste estudo buscam promover a compreensão dos mecanismos de ação desta enzima e sua aplicação na hidrólise do bagaço de cana-de-açúcar. O trabalho destaca uma estrutura tridimensional inédita dentro da família 62 portadora de um fenômeno conhecido como domain swapping, possibilitando maiores investigações dentro do grupo das arabinofuranosidases. / Hydrolytic enzymes are considered, for the full definition, those with the ability to perform the hydrolysis reaction. Because of their ability of degrading natural substances, hydrolytic enzymes are of wide industrial use and can be applied in the textile, food, pharmaceutical, and agricultural industries, in addition to being used in the production of biofuels. Known as enzymes that release arabinose, the arabinofuranosidases from family 62 are able to break arabinoxylans connections. The biotechnological importance of these proteins are used in the agricultural and industry. These enzymes are employed to process as cocktails for animal feed digestion, wines and to improve the efficiency of juices clarification. Moreover, its delignification action is promising in the biomass degradation for biofuel production. This project seeks to carry out cloning, heterologous expression in bacteria and characterization of an α-L-arabinofuranosidase from thermophilic fungus Thielavia terrestris. This enzyme belonging to the glycoside hydrolase family 62, which is known to enhance the action of cellulases, and thus of interest in the lignocellulosic biomass degradation such as sugar cane bagasse. This study will characterize the protein structure and biochemistry in order to understand the mechanism and optimal conditions of action. The biophysical, biochemical and structural tests seek to promote an understanding of the mechanisms of action of this enzyme and its application in the hydrolysis of sugarcane bagasse. The work shows a new three- dimensional structure within the family 62 caused by a phenomenon known as domain swapping. Thus, it is possible to predict further investigations on the arabinofuranosidases group.

La société Adisseo produit un cocktail d’enzymes hydrolytiques appelé Rovabio® Excel sécrété par un champignon filamenteux Talaromyces versatilis. Ce cocktail est utilisé comme additif alimentaire pour augmenter la digestibilité de complexes polysaccharidiques en nutrition animale et ainsi augmenter la valeur nutritionnelle des matières premières agricoles. Une récente étude protéomique de ce champignon (Guais et al., 2008) a révélé la présence d’un grand nombre d’arabinofuranosidases (ABFs) appartenant à différentes familles de glycosides hydrolases : 5 ABFs de la famille GH54, 3 ABFs de la famille GH62 et enfin une de la famille GH51. Un des objectifs de mes travaux de thèse a été le clonage, l’expression hétérologue (hôte lévurien Pichia pastoris) des 9 gènes codant pour ces 9 enzymes et la caractérisation complète de la famille GH62. La caractérisation des capacités d’hydrolyse des ABFs 54 et 62 a également été étudiée grâce à une technique d’empreintes d’hydrolyse enzymatique sur arabinoxylane de blé. Enfin, la dernière partie de mes travaux consistait à confectionner des mélanges d’enzymes hydrolytiques des différentes familles d’ABFs associés à des xylanases et de suivre l’efficacité de la dégradation de l’arabinoxylane insoluble grâce à l’utilisation d’un réacteur torique permettant l’acquisition d’images et l’analyse en ligne de la dégradation. Ces travaux sur le réacteur ont permis de mettre en évidence une synergie entre Abfs et Xylanases. / Adisseo produce and commercialize a hydrolytic enzymatic cocktail termed Rovabio and secreted by a filamentous fungus Talaromyces versatilis. This cocktail is used as feed additive for increased digestibility of complex polysaccharides in animal nutrition. A recent genomic study of this fungus revealed the presence of 5 arabinofuranosidases (Abfs) to family GH 54, 3 of GH 62 and 1 of GH51. The first aim of my thesis works was about cloning, heterologous overexpression (in pichia pastoris yeast) of this 9 genes encoding for this 9 enzymes and characterization of the family GH 62. Mode of action of ABFs 54 and 62s has been characterized by enzymatic fingerprinting analysis on wheat arabinoxylan. Then, last part was to design enzymatic cocktail with differents families of ABFs and Xylanases and test their impact on insoluble arabinoxylan hydrolysis with toric reactor. These works on reactor have bringing to light a synergy between ABFs and Xylanases