Catalyseurs de la dégradation de polysaccharides dans le cadre de diverses applications industrielles, de nombreuses glycoside hydrolases (GH) possèdent également une activité de transglycosylation qui peut être exploitée pour la synthèse d'oligosaccharides. Afin d'augmenter cette activité, minoritaire par rapport à l'hydrolyse, des expériences de mutagenèse rationnelle peuvent être employées. Toutefois, l'ensemble des bases moléculaires régissant l’équilibre entre ces deux activités reste en revanche difficile a élucider. L'étude de quatre GH (Ttβgly, AgaD, TcTS, TrSA) par simulation de dynamique moléculaire a permis la découverte de canaux d'eau internes à leurs structures et connectant le site actif au milieu. Cette observation suggère que les canaux d'eau internes aux GH pourraient être impliqués dans leur activité d'hydrolyse. Plusieurs paires de résidus bordant deux de ces canaux ont été mis en évidence chez Ttβgly et AgaD et semblent contrôler le passage de l'eau du canal vers le site actif. La mutagenèse de ces résidus a été entreprise afin de tenter d'augmenter l'activité de transglycosylation chez ces deux enzymes. Une réduction de l'hydrolyse d'un facteur 7 et 50 au profit de l'activité de transglycosylation a été caractérisée chez les deux meilleurs mutants de Ttβgly et AgaD, respectivement. L'analyse des simulations a révélé que ces résultats étaient corrélés à une augmentation de la dynamique des molécules d'eau internes aux deux canaux étudiés. Cette étude souligne ainsi l'importance fonctionnelle de l'eau interne aux hydrolases et suggère que l'ingénierie de sa dynamique peut constituer une approche originale pour convertir les GH en transglycosidases. / Known for their ability to hydrolyse glycosidic linkages,numerous glycoside hydrolases (GH) are also able tocatalyse transglycosylation reaction which can beharnessed for the synthesis of oligosaccharides. Althoughin the vast majority of cases hydrolysis prevails overtransglycosylation reaction, the latter has already beenincreased through mutagenesis and directed evolutionexperiments. However, little is known about the regulationof the balance between both activities. We discover, viamolecular dynamics (MD) simulations, several potentialwater channels connecting the bulk to the active site infour GH: Ttβgly, AgaD and the pair of homologsTcTS/TrSA. This observation supports the hypothesis thatchannels could be involved in the hydrolytic activity of GH.Amino acid residues forming bottlenecks at the interfacebetween two water channels and the active site in Ttβglyand AgaD were suspected to control water access fromthe bulk to the channel interior and the active site.Mutagenesis of key amino acids in the vicinity of selectedchannels was performed in order to attempt to increasethe transglycosylation/hydrolysis ratio balance.Characterization of the best mutants showed a 7 and 50fold decrease of hydrolysis compared to the wild type forTtβgly and AgaD respectively, while the transglycosylaseactivity was improved. MD simulations showed that thesemodifications were correlated with greater water dynamicsin the corresponding channels. These results highlight theimportance of water dynamics in hydrolases catalysis andsuggest that modifying the protein internal water dynamicscould serve as a generic approach to engineertransglycosylase activity in GH.

No description available.

Glicosídeos essenciais a vida podem ser sintetizados por métodos enzimáticos com altíssima especificidade. O mecanismo de reação de β-glicosidases GH1 por dupla-substituição envolve a formação de intermediário covalente (glicosil-enzima) que pode seguir duas rotas. Uma envolve sua hidrólise na ligação β-glicosídica entre glicone e aglicone do substrato, liberando o monossacarídeo da extremidade não-redutora; enquanto na outra rota, transglicosilação ou síntese por controle cinético, o intermediário é atacado por um aceptor glicosídico (2ª molécula de substrato), gerando um novo glicosídeo. BglB possui resíduos (W e H) que formam um \"canal\" por onde a água ataca o intermediário covalente no sítio ativo, sendo alvos de potenciais mutações que alteram o balanço entre as rotas e ampliando a eficiência de transglicosilação. Para caracterizar as bases moleculares da razão entre essas duas rotas catalíticas, β-glicosidases da família GH1 Spodoptera frugiperda (Sfβglu; AF052729) e Tenebrio molitor (Tmβglu; AF312017) foram produzidas como enzimas recombinantes em leveduras Pichia pastoris GS115, concentradas com 90% (p/v) de sulfato de amônio e diálise reversa com PEG 10000, e dialisadas em tampão CP 100 mM pH 6. A pureza foi confirmada por banda única com tamanho semelhante a 50 kDa (Sfβgly) e 56 kDa (Tmβgly) em SDS-PAGE. A atividade recuperada após purificação é 152% (Sfβgly) e 171% (Tmβgly) e a concentração protéica de 0,134 µg/µL (Sfβgly) e 0,217 µg/µL (Tmβgly). A razão entre as reações de hidrólise e transglicosilação (vH/vT) foi analisada utilizando os substratos pNPβ-gluco (0,1 mM a 8 mM) e MUβ-gluco (0,1 mM a 40 mM), a partir da velocidade de formação dos produtos pNP ou MU e glicose, respectivamente. Tmβgly catalisa reações de transglicosilação com ambos, mas vH/vT depende da [S] variando de +∞ (vH>>vT) a 1,5 para pNPβ-gluco e +∞ a 1 para MUβ-gluco. Sfβgly, ao contrário, não transglicosila com substrato MUβ-gluco. Além disso, vH/vT para pNPβ-gluco é 2 e independe da [S]. Já Sfβgly K201F conjuga propriedades de ambas, pois não transglicosila com MUβ-gluco, mas para pNPβ-gluco há ocorrência de transglicosilação dependente da [S], variando de +∞ (vH>>vT) a 1,25. Os parâmetros cinéticos (Vmax e Km) foram ajustados no Enzifitter e por simulação numérica na equação do modelo cinético ping-pong para dois substratos, e mostram maior (Km2) em Tmβgly e Sfβgly K201F do que em Sfβgly. Também foi avaliado o efeito da adição de aceptores alternativos que substituem a água em ensaios com pNPβ-gluco 4mM na presença de alcóois. Para as três enzimas a adição de metanol torna a vglc menor do que de vpNP, indicando ocorrência de transglicosilação. Com adição de n-propanol vH/vT diminui cerca de 7 vezes em Sfβgly e 2 vezes em Tmβgly. Os efeitos destes alcoóis sobre Sfβgly são maiores do que para Tmβgly e Sfβgly K201F, sugerindo acesso mais fácil destes ao intermediário covalente em Sfβgly, indicando diferenças entre as enzimas na configuração desta região. Portanto, notamos que vH/vT não está ligada à afinidade pela segunda molécula de substrato, podendo ser modulada por mutação do resíduo K201 de Sfβgly, posição relacionada ao acesso da água ou aceptor alternativo ao sítio ativo. / Glycosides are essential to life and can be synthesized by enzymatic methods with high specificity. The reaction mechanism of GH1 β-glucosidases by double-substitution involves the formation of covalent intermediate (glycosyl-enzyme) which may follow two routes. One of them involves hydrolysis in β-glycosidic bond between aglycone glucone and the substrate, releasing a monosaccharide from the non-reducing end, whereas in the other route, transglycosylation or synthesis by kinetic control, the intermediate is attacked by a glucosyl acceptor (second substrate molecule), generating a new glucoside. BglB has two residues (W and H) that form a \"channel\" through which water attacks the covalent intermediate on the active site. Thus, the residues are potential targets for mutations that alter the balance between routes, increasing the efficiency of transglycosylation. To characterize the molecular basis of the ratio between these two catalytic routes, two β-glycosidases from GH1 family, Spodoptera frugiperda (Sfβgly; AF052729) and Tenebrio molitor (Tmβgly; AF312017) were produced as recombinant enzymes in Pichia pastoris GS115, concentrated using 90% (w/v) ammonium sulfate and reverse dialysis with PEG 10000, and dialyzed in 100 mM CP buffer pH 6. SDS-PAGE confirmed that Sfβgly (~50 kDa) and Tmβgly (~56 kDa) were pure after that procedure. The activity recovered after purification were 152% (Sfβgly) and 171% (Tmβgly) and protein concentration were 0.134 mg/mL (Sfβgly) and 0.217 mg/mL (Tmβgly). The ratio between the hydrolysis and transglycosylation (vH/vT) was analyzed using pNPβ-gluco substrates (0.1 mM to 8 mM) and MUβ-gluco (0.1 mM to 40 mM), the rate of formation of pNP or MU and glucose. Tmβgly catalyzes transglycosylation reactions with both substrates, but vH/vT depends on [S] ranging from +∞ (vH>>vT) to 1.5 for pNPβ-gluco and + ∞ to 1 for MUβ-gluco. In contrast, Sfβgly did not catalyses transglycosilation with MUβ-gluco. Moreover, vH/vT for pNPβ-gluco-is 2 and is independent of [S]. Sfβgly K201F combines properties of both, because it does not catalyse transglycosylation with MUβ-gluco, but for pNPβ-gluco the occurrence of transglycosylation is dependent on [S], ranging from + ∞ (vH>>vT) to 1.25. The kinetic parameters (Vmax e Km) were adjusted in Enzfitter and numerical simulation showing that Km2is higher for Tmβgly and Sfβgly K201F than Sfβgly. We also evaluated the effect of adding alternative acceptors that replace the water in pNPβ gluco-4 mM. For the three enzymes the addition of methanol makes vglc less than pNP, indicating the occurrence of transglycosylation. Addition of n-propanol decreases vH/vT about 7 times in Sfβgly and 2 times Tmβgly. The effects on Sfβgly are higher than on Tmβgly and Sfβgly K201F, suggesting easier access to the covalent intermediate in Sfβgly. We observe that vH/vT is not related to affinity for the second substrate molecule and may be modulated by mutation of residue K201 Sfβgly, position related to the access of water or alternative acceptor to the active site.

Glicosídeos essenciais a vida podem ser sintetizados por métodos enzimáticos com altíssima especificidade. O mecanismo de reação de β-glicosidases GH1 por dupla-substituição envolve a formação de intermediário covalente (glicosil-enzima) que pode seguir duas rotas. Uma envolve sua hidrólise na ligação β-glicosídica entre glicone e aglicone do substrato, liberando o monossacarídeo da extremidade não-redutora; enquanto na outra rota, transglicosilação ou síntese por controle cinético, o intermediário é atacado por um aceptor glicosídico (2ª molécula de substrato), gerando um novo glicosídeo. BglB possui resíduos (W e H) que formam um \"canal\" por onde a água ataca o intermediário covalente no sítio ativo, sendo alvos de potenciais mutações que alteram o balanço entre as rotas e ampliando a eficiência de transglicosilação. Para caracterizar as bases moleculares da razão entre essas duas rotas catalíticas, β-glicosidases da família GH1 Spodoptera frugiperda (Sfβglu; AF052729) e Tenebrio molitor (Tmβglu; AF312017) foram produzidas como enzimas recombinantes em leveduras Pichia pastoris GS115, concentradas com 90% (p/v) de sulfato de amônio e diálise reversa com PEG 10000, e dialisadas em tampão CP 100 mM pH 6. A pureza foi confirmada por banda única com tamanho semelhante a 50 kDa (Sfβgly) e 56 kDa (Tmβgly) em SDS-PAGE. A atividade recuperada após purificação é 152% (Sfβgly) e 171% (Tmβgly) e a concentração protéica de 0,134 µg/µL (Sfβgly) e 0,217 µg/µL (Tmβgly). A razão entre as reações de hidrólise e transglicosilação (vH/vT) foi analisada utilizando os substratos pNPβ-gluco (0,1 mM a 8 mM) e MUβ-gluco (0,1 mM a 40 mM), a partir da velocidade de formação dos produtos pNP ou MU e glicose, respectivamente. Tmβgly catalisa reações de transglicosilação com ambos, mas vH/vT depende da [S] variando de +∞ (vH>>vT) a 1,5 para pNPβ-gluco e +∞ a 1 para MUβ-gluco. Sfβgly, ao contrário, não transglicosila com substrato MUβ-gluco. Além disso, vH/vT para pNPβ-gluco é 2 e independe da [S]. Já Sfβgly K201F conjuga propriedades de ambas, pois não transglicosila com MUβ-gluco, mas para pNPβ-gluco há ocorrência de transglicosilação dependente da [S], variando de +∞ (vH>>vT) a 1,25. Os parâmetros cinéticos (Vmax e Km) foram ajustados no Enzifitter e por simulação numérica na equação do modelo cinético ping-pong para dois substratos, e mostram maior (Km2) em Tmβgly e Sfβgly K201F do que em Sfβgly. Também foi avaliado o efeito da adição de aceptores alternativos que substituem a água em ensaios com pNPβ-gluco 4mM na presença de alcóois. Para as três enzimas a adição de metanol torna a vglc menor do que de vpNP, indicando ocorrência de transglicosilação. Com adição de n-propanol vH/vT diminui cerca de 7 vezes em Sfβgly e 2 vezes em Tmβgly. Os efeitos destes alcoóis sobre Sfβgly são maiores do que para Tmβgly e Sfβgly K201F, sugerindo acesso mais fácil destes ao intermediário covalente em Sfβgly, indicando diferenças entre as enzimas na configuração desta região. Portanto, notamos que vH/vT não está ligada à afinidade pela segunda molécula de substrato, podendo ser modulada por mutação do resíduo K201 de Sfβgly, posição relacionada ao acesso da água ou aceptor alternativo ao sítio ativo. / Glycosides are essential to life and can be synthesized by enzymatic methods with high specificity. The reaction mechanism of GH1 β-glucosidases by double-substitution involves the formation of covalent intermediate (glycosyl-enzyme) which may follow two routes. One of them involves hydrolysis in β-glycosidic bond between aglycone glucone and the substrate, releasing a monosaccharide from the non-reducing end, whereas in the other route, transglycosylation or synthesis by kinetic control, the intermediate is attacked by a glucosyl acceptor (second substrate molecule), generating a new glucoside. BglB has two residues (W and H) that form a \"channel\" through which water attacks the covalent intermediate on the active site. Thus, the residues are potential targets for mutations that alter the balance between routes, increasing the efficiency of transglycosylation. To characterize the molecular basis of the ratio between these two catalytic routes, two β-glycosidases from GH1 family, Spodoptera frugiperda (Sfβgly; AF052729) and Tenebrio molitor (Tmβgly; AF312017) were produced as recombinant enzymes in Pichia pastoris GS115, concentrated using 90% (w/v) ammonium sulfate and reverse dialysis with PEG 10000, and dialyzed in 100 mM CP buffer pH 6. SDS-PAGE confirmed that Sfβgly (~50 kDa) and Tmβgly (~56 kDa) were pure after that procedure. The activity recovered after purification were 152% (Sfβgly) and 171% (Tmβgly) and protein concentration were 0.134 mg/mL (Sfβgly) and 0.217 mg/mL (Tmβgly). The ratio between the hydrolysis and transglycosylation (vH/vT) was analyzed using pNPβ-gluco substrates (0.1 mM to 8 mM) and MUβ-gluco (0.1 mM to 40 mM), the rate of formation of pNP or MU and glucose. Tmβgly catalyzes transglycosylation reactions with both substrates, but vH/vT depends on [S] ranging from +∞ (vH>>vT) to 1.5 for pNPβ-gluco and + ∞ to 1 for MUβ-gluco. In contrast, Sfβgly did not catalyses transglycosilation with MUβ-gluco. Moreover, vH/vT for pNPβ-gluco-is 2 and is independent of [S]. Sfβgly K201F combines properties of both, because it does not catalyse transglycosylation with MUβ-gluco, but for pNPβ-gluco the occurrence of transglycosylation is dependent on [S], ranging from + ∞ (vH>>vT) to 1.25. The kinetic parameters (Vmax e Km) were adjusted in Enzfitter and numerical simulation showing that Km2is higher for Tmβgly and Sfβgly K201F than Sfβgly. We also evaluated the effect of adding alternative acceptors that replace the water in pNPβ gluco-4 mM. For the three enzymes the addition of methanol makes vglc less than pNP, indicating the occurrence of transglycosylation. Addition of n-propanol decreases vH/vT about 7 times in Sfβgly and 2 times Tmβgly. The effects on Sfβgly are higher than on Tmβgly and Sfβgly K201F, suggesting easier access to the covalent intermediate in Sfβgly. We observe that vH/vT is not related to affinity for the second substrate molecule and may be modulated by mutation of residue K201 Sfβgly, position related to the access of water or alternative acceptor to the active site.

Ruminococcus gnavus E1 appartient au groupe des Firmicutes, l’un des deux groupes dominants du microbiote intestinal humain. Les a-galactosidases sont des glycosides hydrolase (GH) actives sur des substrats contenant des galactoses liés en a. Elles sont très largement distribuées dans tous les domaines du vivant, bactéries, champignons, plantes et animaux, mais sont absentes du tractus digestif humain. Ces travaux portent sur les caractéristiques enzymatiques et la régulation de l’expression de deux -galactosidase, Aga1 et Aga2, de R. gnavus E1. L’analyse bioinformatique de leur environnement génétique respectif indique une organisation simple pour Aga1 tandis qu’Aga2 est organisée en opéron. Elles ont été exprimées en système hétérologue chez E. coli, purifiées et leurs propriétés biochimiques ainsi que leurs spécificités de substrat ont été analysées. Le profil de croissance de la souche indique une préférence pour des substrats complexes (raffinose et mélbiose) faisant intervenir les a-galactosidase pour leurs utilisations ainsi que leur assimilation. / Ruminococcus gnavus E1 belongs to the Firmicutes, one of the two dominant groups in the human gut microbiota. a-galactosidases are glycoside hydrolases (GH) active on a-galactoside containing substrates. They are widely distributed through all the domains of life: bacteria, fungi, plants, and animals, but are absent from the human gastro-intestinal tract.Here we report the enzymatic characteristics and regulation of expression for two GH36 -galactosidases, Aga1 and Aga2, from R. gnavus E1. Bioinformatics analysis of their respective genetic environment showed a different organisation, Aga1 having a simple organisation while Aga2 is organised as part of an operon. They were heterologously expressed in Escherichia coli, purified to homogeneity and their biochemical properties and substrate preferences comparatively analysed. The growth pattern of the strain in minimum media demonstrates a preference for complex substrates (melibiose and raffinose) that require the expression of the a-galactosidases for their utilisation and assimilation

Élargir le répertoire de composés accessibles dans le domaine des Glycosciences est d’un intérêt majeur pour la communauté des biologistes du fait que ces composés, oligosaccharides et glyco-conjugués, sont impliqués dans diverses fonctions biologiques, aussi bien au niveau structurel, qu’énergétique voire même signalétique jouant un rôle primordial dans les interactions inter- ou intracellulaires. L’assemblage, la modification ou la déconstruction de ces glyco-structures complexes est possible grâce à l’action d’enzymes, parmi lesquelles l’on retrouve les CAZymes (Carbohydrate Active enZymes). Ces enzymes font partie du répertoire de la base de données CAZy, incluant les Glycoside Hydrolases (GHs) qui représentent le groupe le plus important et ayant pour fonction biologique principale l’hydrolyse des liens glycosidiques. Cependant, un certain nombre de GHs possède aussi la capacité de catalyser des réactions de synthèse (transglycosylation) en tant qu’activité secondaire mineure, voire en tant qu’activité principale pour un nombre restreint d’entre elles, qui sont alors appelées transglycosylases. Sachant que ces deux types de comportements peuvent se retrouver au sein d’une même famille de GH (donc étroitement liés sur le plan évolutif), la découverte et la compréhension des déterminants moléculaires qui ont été développés par les GHs au cours de leur évolution pour permettre cette partition d’activité, entre hydrolyse et transglycosylation, est d’une importance capitale pour le domaine de la synthèse chimio-enzymatique et des Glycosciences de manière plus générale.Ce travail de thèse décrit une proposition de synthèse pour apporter une réponse à cette question fondamentale via une revue critique de la littérature sur le sujet. Sur le plan expérimental, a été réalisée l’évolution moléculaire d’une enzyme spécifique des pentoses, l’α-L-arabinofuranosidase de Thermobacillus xylanilyticus (TxAbf) de la famille GH51, vers les premières transarabinofuranosylases de type ‘non-Leloir’. Cette évolution itérative a été développée en utilisant un panel d’outils d’ingénierie enzymatique combinant des approches aléatoire, semi-rationnelle, de prédiction in silico suivie de recombinaison dans un processus d’évolution dirigée global. Une analyse fine des mutants générés sur le plan mécanistique en lien avec la partition hydrolyse/transglycosylation mène à des conclusions en accord avec la proposition de synthèse issue de la revue de la littérature sur le sujet. Sur un plan plus appliqué, ces nouveaux biocatalyseurs ont ensuite été mis en oeuvre dans des voies de synthèse chimio-enzymatiques pour la préparation de composés furanosylés de structure contrôlée. Le transfert d’L-arabinofuranosyles permet la génération d’arabinoxylo-oligosaccharides (AXOS) ainsi que la conception d’oligosaccharides non naturels, tel que des galactofuranoxylo-oligosaccharides ou des arabinofuranogluco-oligosaccharides. Dans son ensemble, ce travail de recherche constitue les premières étapes clés du développement de méthodes de synthèse chimio-enzymatique plus élaborées pour la conception d’arabinoxylanes artificiels. Dans le contexte actuel de transition vers une bio-économie, reposant sur des concepts tels que ceux de la bioraffinerie ou de la chimie verte, nous espérons que les outils de glycosynthèse développés au cours de ces travaux trouveront leur application dans la valorisation des pentoses issus de la biomasse. La synthèse à-façon d’arabinoxylooligo- et polysaccharides présente nombre de valorisations possibles allant de la préparation de prébiotiques à la conception de matériaux bio-inspirés en passant par la synthèse de modèles de parois végétales. / Widening the spectrum of available compounds in the field of Glycosciences is of utmost importance for the entire biology community, because carbohydrates are determinants of a myriad of life-sustaining or threatening processes. The assembly, modification or deconstruction of complex carbohydrate-based structures mainly involves the action of enzymes, among which one can identify Carbohydrate Active enZymes (CAZymes). These enzymes form part of the CAZy database repertoire and include Glycoside Hydrolases (GHs), which are the biggest group of CAZymes, whose main role is to hydrolyze glycosidic linkages. However, some GHs also display the ability to perform synthesis (transglycosylation), an activity that mostly manifests itself as a minor one alongside hydrolysis, but which is the only activity displayed by a rather select group of GHs that are often called transglycosylases. Understanding how transglycosylases have resulted from the process of evolution is both intringuing and crucial, because it holds the key to the creation of tailored glycosynthetic enzymes that will revolutionize the field of glycosciences.In this thesis, an extensive review of relevant scientific literature that treats the different aspects of GH-catalyzed transglycosylation and glycosynthesis is presented, along with experimental results of work that has been performed on a family GH-51 α-L-arabinofuranosidase, a pentose-acting enzyme from Thermobacillus xylanilyticus (TxAbf). The conclusions of the literature are presented in the form of a hypothesis, which describes the molecular basis of the hydrolysis/transglycosylation partition and thus provides a proposal on how to engineer dominant transglycosylation activity in a GH. Afterwards, using a directed evolution approach, including random mutagenesis, semi-rational approaches, in silico predictions and recombination it has been experimentally possible to create the very first ‘non-Leloir’ transarabinofuranosylases. The mechanistic analysis of the resultant TxAbf mutants notably focusing on the hydrolysis/transglycosylation partition reveals that the results obtained are consistent with the initial hypothesis that was formulated on the basis of the literature review.To demonstrate the applicative value of the experimental work performed in this study, the TxAbf mutants were used to develop a chemo-enzymatic methodology that has procured a panel of well-defined furanosylated compounds. Enzyme-catalyzed transfer of arabinofuranosyl moities can be used to generate arabinoxylo-oligosaccharides (AXOS), but the design of non-natural oligosaccharides, such as galactofuranoxylo-oligosaccharides or arabinofuranogluco-oligosaccharides is also possible. Overall, the work presented constitutes the first steps towards the development of more sophiscated methodologies that will procure the means to synthesize artificial arabinoxylans, with a first proof of concept being presented at the very end of this manuscript.In the present context of the bioeconomy transition, which relies on technologies such as biorefining and green chemistry, it is expected that the glycosynthetic tools that have been developed in this work will be useful for the conversion of pentose sugars obtained from biomass. The synthesis of tailor-made arabinoxylo-oligo- and polysaccharides may lead to a variety of potential applications including the production of prebiotics, surfactants or bio-inspired materials and, more fundamentally, the synthesis of artificial models of plant cell wall.

La valorisation non alimentaire de la biomasse végétale représente un enjeu actuel majeur dans le contexte du développement de la bioraffinerie végétale. En Champagne-Ardenne, la transformation des co-produits d'origine agricole fait l'objet d'études importantes. Le son et la paille, co-produits abondants de la filière blé, sont riches en hémicelluloses, polysaccharides constitués essentiellement de pentoses, majoritairement de D-xylose. La principale voie de valorisation des pentoses tels que le xylose est leur fonctionnalisation par voie chimique, mais l'utilisation d'enzymes telles que des xylanases et des β-xylosidases représente une voie de synthèse plus respectueuse de l'environnement et permettant, en outre, d'accéder à de nouvelles molécules inaccessibles par la voie chimique classique (DP plus importants). L'étude conduite dans le cadre de la thèse a concerné l'évaluation et l'amélioration de la synthèse enzymatique par transglycosylation d'alkyl xylosides et oligoxylosides catalysée par une β-xylosidase et deux xylanases. Une seconde voie de valorisation enzymatique a été étudiée, à savoir la synthèse de xylosides et oligoxylosides présentant des propriétés biologiques pouvant notamment être exploitées pour des applications cosmétiques. A travers la mutagénèse dirigée au niveau du sous-site aglycone d'une β-xylosidase de Bacillus halodurans, nous avons montré l'importance de résidus aromatiques de ce sous-site sur les capacités d'hydrolyse et de transglycosylation de l'enzyme. Les résultats obtenus nous ont permis de dégager quelques pistes d'ingénierie du sous-site aglycone de la xylosidase pour optimiser les rendements de transglycosylation en présence d'alcools à longues chaînes carbonées. L'étude de l'effet des conditions réactionnelles sur la synthèse d'alkyl oligoxylosides catalysée par deux xylanases (l'une de Thermobacillus xylanilyticus, l'autre commerciale) a montré la possibilité de produire les alkyl oligoxylosides par voie enzymatique avec des rendements satisfaisants, notamment en réduisant les problèmes physiques d'accessibilité de l'alcool au site actif de l'enzyme. Nous avons également mis en évidence des profils de synthèse différents d'une enzyme à l'autre mais également en fonction des conditions réactionnelles, notamment en ce qui concerne le DP moyen. Notre étude montre également que la synthèse d'alkyl oligoxylosides est possible directement à partir de son de blé prétraité par voie hydrothermale. En ce qui concerne les propriétés physico-chimiques des produits de transglycosylation, nous avons montré que, tout en étant non purifiés, les pentyl et octyl β-D-oligoxylosides obtenus par synthèse enzymatique présentent des propriétés comparables à celles de produits commerciaux utilisés pour leurs propriétés tensio-actives. L'étude de méthodes de synthèse enzymatiques et chimio-enzymatique de xylosides et d'oligoxylosides ciblés pour certaines propriétés biologiques a montré que les deux xylanases étudiées précédemment pouvaient utiliser en tant qu'accepteur, en plus des alcools linéaires, des alcools aromatiques, des diols ou encore un ester ou un alcyne, ce qui a permis d'accéder à des produits originaux. Des tests biologiques réalisés sur des fibroblastes humains, ont montré que les xylosides et oligoxylosides ainsi produits ne sont pas cytotoxiques. Des tests sur l'initiation de la biosynthèse de glycosaminoglycanes, menés avec certains produits, ont conduit à des résultats positifs pour la biosynthèse d'acide hyaluronique. / Non-food valorization of plant biomass currently represents a major challenge in the context of plant biorefinery. In Champagne-Ardenne, the processing of agricultural by-products is the subject of numerous studies. Wheat bran and straw are abundant agricultural by-products and are rich in hemicelluloses, which are polysaccharides essentially constituted by pentoses, mainly D-xylose. The major route for the valorization of pentoses such as xylose is their functionalization by chemical means. Nevertheless, the use of enzymes, such as xylanases and β-xylosidases, represents a more environmentally friendly way and can also lead to the synthesis of new molecules inaccessible through conventional chemistry. The study conducted during the PhD concerned the evaluation and the improvement of the enzymatic synthesis by transglycosylation of alkyl xylosides and oligoxylosides catalyzed by a β-xylosidase and two xylanases. The second pathway for enzymatic valorization investigated was the synthesis of xylosides and oligoxylosides with biological properties that could be applied for cosmetic use.By site-directed mutagenesis in the aglycone subsite of a β-xylosidase from Bacillus halodurans, we have shown the role of aromatic residues on the hydrolysis and transglycosylation capabilities of the enzyme. The results obtained allowed us to identify some engineering of the aglycone subsite of the β-xylosidase to optimize the transglycosylation yields in the presence of alcohols with long carbon chains.The study of the effect of the reaction conditions on the synthesis of alkyl oligoxylosides catalyzed by two xylanases (one from Thermobacillus xylanilyticus and other is commercially available) has shown the ability to produce alkyl oligoxylosides by enzymatic synthesis with acceptable yields. We also pointed out that, depending on the reaction conditions, the products obtained were different particularly as regards the average DP. Our study has also shown that the synthesis of alkyl oligoxylosides is possible directly from hydrothermally pretreated wheat bran. Concerning the physico-chemical properties of transglycosylation products, we have demonstrated that non purified pentyl and octyl β-D-oligoxylosides obtained by enzymatic transglycosylation exhibit similar properties than commercial products used for their surface-active properties. The study of enzymatic an chemo-enzymatic methods for the synthesis of xylosides and oligoxylosides targeted for their biological properties showed that both xylanases previously studied could use linear alcohols but also aromatic alcohols, diols, ester or alkyne alcohols as acceptors for the preparation of original compounds. Biological tests performed on human fibroblasts pointed out that the xylosides and oligoxylosides produced are not cytotoxic. Initiation of the biosynthesis of glycosaminoglycans tested with some of the products has led to positive results for the synthesis of hyaluronic acid by the fibroblasts.

Extracellular endo-1,4-b-mannanase (EC 3.2.1.78) gene of Aspergillus fumigatus IMI 385708 (formerly known as Thermomyces lanuginosus IMI 158749) was cloned and transformed into Aspergillus sojae (ATCC 11906) and Pichia pastoris GS115. High level of expression was achieved in both expression systems. Attempts to produce heterologous mannanase in Arabidopsis thaliana, suitable for large scale production, were not successful. Comparison of the expression levels of heterologous mannanase reveals that A. sojae is a better expression system than P. pastoris with respect to extracellular mannanase activity. The production of mannanase in A. sojae (AsT1) after 3 days of incubation reached 204 U/ml in YpSs containing 1 % glucose. In P. pastoris (PpT1), highest production was observed after 10 hrs of induction with methanol (61 U/ml). Expressed enzymes were purified and analyzed. Both enzymes have specific activity c. 349 U/mg protein with pH and temperature optimum of c. 4.5 and c. 60 &deg / C for mannanases from AsT1 and c. 5.2-5.6 and c. 45 &deg / C for mannanases from PpT1. A truncated form of mannanase (MAN-S) deleted at amino acids from P291 to P368, which still displayed hydrolytic activity was also isolated and characterized. MAN-S has pH and temperature optimum of c. 6.5-8.0 and c. 60 &deg / C. During incubation of the mannanase on locust bean gum, transglycosylation reactions, in which longer or rare prebiotic oligosaccharides could be produced catalyzed by glycolysis, was detected. The products of hydrolytic activity of the enzyme on various carbohydrates were analyzed by PACE and MALDI-TOF. Accordingly, hexamannose and smaller oligosaccharides were characterized.

Made available in DSpace on 2014-06-11T19:32:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-03-04Bitstream added on 2014-06-13T20:04:38Z : No. of bitstreams: 1 carvalho_afa_dr_rcla.pdf: 997227 bytes, checksum: 92775cbd61cca9ee00b5f1a54feef2bf (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As α-glicosidases (EC 3.2.1.20) são exoamilases que atuam nas ligações glicosídicas α-1,4 da extremidade nãoredutora, preferencialmente em pequenos oligossacarídeos para liberar α-glicose. Os objetivos desse trabalho foram purificar a α-glicosidase produzida, em fermentação submersa, pelo fungo Thermoascus aurantiacus CBMAI 756; caracterizar a enzima purificada; avaliar a ação sobre os derivados de amido e substratos sintéticos e construir um biblioteca de cDNA de Thermoascus aurantiacus para isolar o gene codificando para esta característica. As estratégias adotadas para isolar a α-glicosidase foram: a) concentração do extrato enzimático bruto usando uma membrana de ultrafiltração, b) por precipitação com sulfatodeamônio, c) cromatografias detroca aniônica (Q-Sepharose)e a filtração em gel(Sephacryl S-200 e Superose 12). Após o último passo, filtração em gel na coluna Superose 12, obteve-se uma atividade específica final de 404 (U/mg proteína) e um fator de purificação de 173 vezes. A eletroforese em SDS-PAGE usando amostra semi-desnaturada possibilitou aobservação de uma única banda protéica, a qual correspondeu à banda observada em gel de atividade e no gel específico para glicoproteína. A aparente massa molecular da enzima em SDS-PAGE foi83 kDa. Essa enzima apresentou 42% de carboidrato ligado à estrutura protéica. A atividade máxima foi observada em pH 4,5 e a 70°C. A enzima mostrou-se estável nas faixas de pH 3,0-9,0 e perdeu 40% da atividade original nas temperaturas de 40, 50 e 60°C, incubadas por 1hora. Na presença dos íons Na+, Ba2+, Co2+,Ni2+, Mg2+, Mn2+,Al3+, Zn2+ e Ca2+ a enzima manteve 90-105% da atividade máxima e foi inibida por Cr3+, Ag+ eHg2+. Baseados no cálculo do IC50, a hidrólise da maltose foi mais sensível a castanospermina (0,015 mM) do que a acarbose (0,11 mM) e voglibose (0,06 mM). OKm foi 0,07 μM e o Vmax... / α-Glucosidase (EC 3.2.1.20) are exoamylase that act on α-1,4 bonds from nonreducing end, preferentially short oligosaccharides to release α-glucose. The objective of this work was to purify the -glucosidase produced in submerged fermentation, with the fungus Thermoascus aurantiacus CBMAI 756, characterize the purified enzyme and to evaluate its action on starch derivatives and synthetic substrates, and construction of cDNA library of Thermoascus aurantiacus. The strategies adopted to isolate the -glucosidase were: a) concentration of crude enzyme using a membrane ultra filtration b) by ammonium sulphate precipitation, c) anion exchange chromatography (Q-Sepharose) and gel filtration (Sephacryl S-200 and Superose). After the last step, gel filtration on Superose 12 column, it was obtained a final specific activity of 404 (U/mg protein) and 173-fold enzyme purification. The electrophoresis in SDS-PAGE using semi-denatured sample allowed the observation of a single protein band, which corresponded to the bands obtained in gel of -glucosidase activity and in a specific gel for glycoprotein. The apparent molecular mass of the enzyme in SDSPAGE was 83 kDa. This enzyme exhibited a carbohydrate content of 42%. Maximum activity was observed at pH 4.5 and at 70°C. It was stable in the pH range 3.0-9.0 and the enzyme lost 40% of its initial activity at 40, 50 and 60 °C, incubated for 1 h. In the presence of ions Na+, Ba2+, Co2+, Ni2+, Mg2+, Mn2+, Al3+, Zn2+, Ca2+ the enzyme maintained 90-105% of its maximum activity and was inhibited by Cr3+, Ag+ and Hg2+. Based on IC50 values, hydrolysis of maltose was more sensitive to castanospermine (0.015 mM) than acarbose (0.11 mM) and voglibose (0.06 mM). The Km was 0.07 μM, and the Vmax was 318.0 μmol/min/mg. The - glucosidase showed a transglycosylation property, by the release of oligosaccharides after 3h of incubation with maltose... (Complete abstract click electronic access below)

Orientador: Eleni Gomes / Banca: Marta Cristina Teixeira Duarte / Banca: Suraia Said / Banca: Valéria Marta Gomes de Lima / Banca: Heloiza Ferreira Alves do Prado / Resumo: As α-glicosidases (EC 3.2.1.20) são exoamilases que atuam nas ligações glicosídicas α-1,4 da extremidade nãoredutora, preferencialmente em pequenos oligossacarídeos para liberar α-glicose. Os objetivos desse trabalho foram purificar a α-glicosidase produzida, em fermentação submersa, pelo fungo Thermoascus aurantiacus CBMAI 756; caracterizar a enzima purificada; avaliar a ação sobre os derivados de amido e substratos sintéticos e construir um biblioteca de cDNA de Thermoascus aurantiacus para isolar o gene codificando para esta característica. As estratégias adotadas para isolar a α-glicosidase foram: a) concentração do extrato enzimático bruto usando uma membrana de ultrafiltração, b) por precipitação com sulfatodeamônio, c) cromatografias detroca aniônica (Q-Sepharose)e a filtração em gel(Sephacryl S-200 e Superose 12). Após o último passo, filtração em gel na coluna Superose 12, obteve-se uma atividade específica final de 404 (U/mg proteína) e um fator de purificação de 173 vezes. A eletroforese em SDS-PAGE usando amostra semi-desnaturada possibilitou aobservação de uma única banda protéica, a qual correspondeu à banda observada em gel de atividade e no gel específico para glicoproteína. A aparente massa molecular da enzima em SDS-PAGE foi83 kDa. Essa enzima apresentou 42% de carboidrato ligado à estrutura protéica. A atividade máxima foi observada em pH 4,5 e a 70°C. A enzima mostrou-se estável nas faixas de pH 3,0-9,0 e perdeu 40% da atividade original nas temperaturas de 40, 50 e 60°C, incubadas por 1hora. Na presença dos íons Na+, Ba2+, Co2+,Ni2+, Mg2+, Mn2+,Al3+, Zn2+ e Ca2+ a enzima manteve 90-105% da atividade máxima e foi inibida por Cr3+, Ag+ eHg2+. Baseados no cálculo do IC50, a hidrólise da maltose foi mais sensível a castanospermina (0,015 mM) do que a acarbose (0,11 mM) e voglibose (0,06 mM). OKm foi 0,07 μM e o Vmax... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: α-Glucosidase (EC 3.2.1.20) are exoamylase that act on α-1,4 bonds from nonreducing end, preferentially short oligosaccharides to release α-glucose. The objective of this work was to purify the -glucosidase produced in submerged fermentation, with the fungus Thermoascus aurantiacus CBMAI 756, characterize the purified enzyme and to evaluate its action on starch derivatives and synthetic substrates, and construction of cDNA library of Thermoascus aurantiacus. The strategies adopted to isolate the -glucosidase were: a) concentration of crude enzyme using a membrane ultra filtration b) by ammonium sulphate precipitation, c) anion exchange chromatography (Q-Sepharose) and gel filtration (Sephacryl S-200 and Superose). After the last step, gel filtration on Superose 12 column, it was obtained a final specific activity of 404 (U/mg protein) and 173-fold enzyme purification. The electrophoresis in SDS-PAGE using semi-denatured sample allowed the observation of a single protein band, which corresponded to the bands obtained in gel of -glucosidase activity and in a specific gel for glycoprotein. The apparent molecular mass of the enzyme in SDSPAGE was 83 kDa. This enzyme exhibited a carbohydrate content of 42%. Maximum activity was observed at pH 4.5 and at 70°C. It was stable in the pH range 3.0-9.0 and the enzyme lost 40% of its initial activity at 40, 50 and 60 °C, incubated for 1 h. In the presence of ions Na+, Ba2+, Co2+, Ni2+, Mg2+, Mn2+, Al3+, Zn2+, Ca2+ the enzyme maintained 90-105% of its maximum activity and was inhibited by Cr3+, Ag+ and Hg2+. Based on IC50 values, hydrolysis of maltose was more sensitive to castanospermine (0.015 mM) than acarbose (0.11 mM) and voglibose (0.06 mM). The Km was 0.07 μM, and the Vmax was 318.0 μmol/min/mg. The - glucosidase showed a transglycosylation property, by the release of oligosaccharides after 3h of incubation with maltose... (Complete abstract click electronic access below) / Doutor