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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
71

Enrichment of lignocellulosedegrading microorganisms byiterative culturing / Anrikning av lignocellulosanedbrytande mikroorganismer genom iterativ kultivering

Rosenholm, Angelica January 2016 (has links)
No description available.
72

Toward Transition State Analysis of O-Glycoside Hydrolysis by Human Sucrase/Isomaltase

Bakhtiari, Rasa January 2014 (has links)
Type 2 diabetes is a major health concern worldwide. One of its complications is postprandial hyperglycemia, i.e., high blood glucose concentrations, caused by glucose fast release from dietary polysaccharides into the bloodstream after meals. α-Glucosidase inhibitor drugs reduce postprandial hyperglycemia by inhibiting maltase/glucoamylase (MGAM) and sucrase/isomaltase (SI). MGAM and SI transform polysaccharides into absorbable monosaccharides, and inhibiting them delays monosaccharide release into the blood. The three commercially available α-glucosidase inhibitors are limited by their absorption abilities, inhibition efficacies, and side effects, which highlights the need for more specific α-glucosidase inhibitors. Because enzymes catalyze their reactions by tightly binding to their cognate transition states (TS), TS analogs can be powerful inhibitors and potential drugs. The measurement and interpretation of kinetic isotope effects (KIEs) is the only method that can directly determine TS structures on large molecules. In this work, methods to prepare radioisotopically labelled maltose were developed, as well as methods to measure KIEs on acid- and enzyme-catalyzed maltose hydrolysis. However, the methods developed did not achieve the required precision for TS analysis. Also, KIEs were calculated computationally for a model reaction of maltose hydrolysis. / Thesis / Master of Science (MSc)
73

MICROBIAL GLYCOSIDE HYDROLASE MEDIATED MODIFICATION OF HOST CELL SURFACE GLYCANS

Pasupathi, Aarthi January 2023 (has links)
All cells and extracellular matrices of prokaryotes and eukaryotes are made up of glycans, the carbohydrate macromolecules that play a predominant role in cell-to-cell interaction, protection, stabilization, and barrier functions. Glycans are also central to human microbiome-host interactions where bacterial glycans are recognized by innate immune signaling pathways, and host mucins are a major nutrient source for various gut bacteria. Many microorganisms encode glycoside hydrolases (GHs) to utilize the available host cell surface glycans as a nutrient source and to modulate host protein function. The GHs are divided into families having conserved linkage specificity within each family and individual family members can be specific for dramatically divergent macromolecular substrates. In general, within a given GH family very few members have been biochemically characterized and the substrate specificity is poorly understood. GH genes are abundant in the human gut microbiome and culture-enriched metagenomics identified more than 10,000 distinct bacterial GH genes in an individual. The focus of this thesis is endo-β-N-acetylglucosaminidases (ENGases) encoded by GH18 and GH85 families. Bioinformatic analysis shows that the predicted proteins within each of these GH families fell into separate clusters in the Sequence Similarity Networks of each family. The hypothesis of this project is that human microbiome-encoded ENGases from the same GH family differ in their substrate specificities and within the SSN network of the same GH family, enzymes with similar substrate specificity may fall in the same cluster. In this work, I established conditions for overexpression of GH18 and GH85 proteins and investigated the activity of these enzymes on various substrates. / Thesis / Master of Science (MSc) / All the cell surfaces of animals, plants, and microbes are coated with sugars, also known as glycans. These sugars on the cell surface act as a barrier and protect them from the external environment. Glycans on the cells of both microbes and humans are essential for basic interactions between them. Many bacteria produce enzymes such as glycoside hydrolases to obtain nutrients from dietary sugars and alter the sugars on host proteins. There are various families of these enzymes, and they act on specific sugars and cleavage sites. The substrate specificities and characterization of these enzymes from most bacteria found in the human microbiome have not been studied in detail. My work focuses on developing standard enzyme assays for determining specific substrate specificities. This tool can be used to reshape glycans and understand their role in cell processes.
74

Biochemical characterizations and food applications of carbohydrate active enzymes secreted from microorganisms / 微生物が分泌する糖質関連酵素の生化学的解析と産業利用

Sakai, Kiyota 24 July 2023 (has links)
京都大学 / 新制・論文博士 / 博士(農学) / 乙第13567号 / 論農博第2913号 / 新制||農||1101(附属図書館) / (主査)教授 小川, 順, 教授 阪井, 康能, 教授 栗原, 達夫 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM
75

Effect of surface modifications on biodegradation of nanocellulose and microbial response

Singh, Gargi 22 September 2015 (has links)
History teaches us that novel materials, such as chlorofluorocarbon and asbestos, can have dire unintended consequences to human and environmental health. The exponential growth of the field of nanotechnology and the products developed along the way provide the opportunity for a new paradigm of design thinking, in which human and environmental impacts are considered early on in product development. In particular, nanocellulose is touted as a promising green nanomaterial, as it is sourced from an effectively inexhaustible feedstock of wood-based cellulose and is assumed to be harmless to the environment since it is derived from a natural material and assumed to be biodegradable. The various forms of nanocellulose possess an impressive diversity of properties, making it suitable for a wide variety of applications such as drug delivery, reinforcement, food additives, and iridescent make-up. However, as nanomaterials can have different properties relative to their bulk form, it is questionable whether they are truly environmentally friendly, particularly in terms of their biodegradability and potential impacts to receiving environments. Given the projected mass-scale application of nanocellulose and the inevitability of its subsequent release into environment, the purpose of this study was to determine the biodegradability of nanocellulose and the response of environmentally-relevant microbial communities. Specifically, it was hypothesized that cellulose in the nano size range would display distinct biodegradation patterns and rates, relative to larger forms of cellulose. Further, it was hypothesized that modification of nanocellulose, in terms of morphology and surface properties (e.g., charge), would further influence its biodegradability. Wetlands and anaerobic digesters were selected as two environmentally-relevant receiving environments that also play critical roles in global carbon turnover. To examine the biodegradability of nanocellulose, two distinct microbial consortia were enriched from wetland (W) and anaerobic digester (AD) inocula and applied in parallel experiments. The consortia were grown under anaerobic conditions with microcrystalline cellulose as the sole carbon substrate over a period of 246 days before being aliquoted to microcosms for subsequent biodegradation assays. Various forms of nanocellulose were spiked into the microcosms and compared with microcrystalline cellulose as a non nano reference. Microcosms were sacrificed in triplicate with time to monitor cellulose degradation as well as various measures of microbial community response. Microbial communities were characterized in terms of gene markers for total bacteria (16S rRNA genes) and anaerobic cellulose degraders (glycoside hydrolase family 48 genes, i.e., cel48) as well as high throughput amplicon sequencing of 16S rRNA genes (V4 region). A series of three studies examined: 1) the effect of nanocrystalline versus microcrystalline cellulose; 2) the effects of nanocellulose morphology (crystalline rod versus filament) and surface functionalization (cationic and anionic); and 3) metagenomic characterization of cellulose degrading communities using next-generation DNA sequencing. It was found that the nano- size range did not hinder cellulose degradation, in fact, nanocrystalline cellulose degraded slightly faster than microcrystalline cellulose according to 1st order kinetics (1st order decay constants: 0.62±0.08 wk-1 for anionic nanocrystalline cellulose versus 0.39±0.05 wk-1 for microcrystalline cellulose exposed to AD culture; 0.69±0.04 wk-1 for anionic nanocrystalline cellulose versus 0.58±0.05 wk-1 for microcrystalline cellulose exposed to W). Experiments comparing the effects of surface functionalization indicated that anionic nanocellulose degraded faster than cationic cellulose (1st order decay constants for cationic nanocrystalline cellulose: 0.48±0.06 wk-1 and 0.58±0.07 wk-1 on exposure to AD and W cultures respectively). Measurements of 16S rRNA and cel48 genes were consistent with this trend of greater biological growth and cellulose-degrading potential in the anionic nanocellulose condition, suggesting that surface properties can influence biodegradation patterns. Taxonomic characterization of 16S rRNA gene amplicons suggested that taxa known to contain anaerobic cellulose degraders were enriched in both W and AD consortia, which shifted in a distinct manner in response to exposure to the different cellulosic materials. This suggests that distinct groups of microbes may drive the biodegradation of different forms of cellulose. Further, metagenomic investigation provided new insight into taxonomic and functional aspects of anaerobic cellulose degradation, including identification of enzymatic families associated with degradation of the various forms of cellulose. Overall, the findings of this study advance understanding of anaerobic cellulose degradation and indicate that nanocellulose is likely to readily degrade in receiving environments and not pose an environmental concern. / Ph. D.
76

Molecular Design and Mechanistic Characterization of Glycoside Hydrolases using Computational and Experimental Techniques

Badieyan, Somayesadat 05 April 2012 (has links)
Cellulase activity is due to the activity of multiple enzymes, including endoglucanases, cellobiohydrolases and glucosidases that work synergistically to solubilize crystalline cellulose efficiently. The dependence of hydrolysis reaction rate on temperature predicts that large increases in performance and decreased enzyme cost would be achieved if the enzymatic degradation could be operated at elevated temperatures. However there is always a tradeoff between the activity and stability of enzymes. So obtaining cellulases with high thermostability and simultaneously enhanced activity is a great challenge in the field of bioethanol production. In the studies presented in this dissertation, different computational techniques, such as Molecular Dynamics (MD), Molecular Docking, Quantum Mechanics (QM) and hybrid Quantum Mechanics and Molecular Mechanics (QM/MM), along with several site-directed mutagenesis and in vitro assays have been applied to the study and design of the activity and stability of cellulases. Using molecular dynamics to investigate the thermal unfolding of endoglucanases of family 5 of glycoside hydrolases (GH5), a good correlation between the optimum activity temperatures of cellulases and their structural fluctuations was revealed. These data led us to hypothesize that cellulase stability could be enhanced by redesign of enzyme dynamics through altering the amino acid composition in the highly flexible regions of an endoglucanase that would increase its local or global rigidity. Cellulase C, a GH5 member, was stabilized thermally and chemically by cross linking its highly flexible subdomain. Family 1 of glycoside hydrolases were investigated by QM and hybrid QM/MM methods to analyze the role of non-catalytic polar residues at the active site of GH1 glucosidases that make hydrogen bonds to the glucose moiety at subsite -1. A tyrosine residue in simultaneous interaction with O5 of the glucose ring and the carboxylate group of the nucleophilic glutamate was found to play a significant role in the energy profile along the hydrolysis reaction coordinates. It was shown to reduce the energy barrier of the deglycosylation step by ~12 Kcal/mol. Exclusion of this tyrosine from QM calculation substantially influenced the preactivated structure of the glucose moiety in the enzyme-substrate complex and affected the structural distortion and charge distribution in transition states. / Ph. D.
77

Etudes structures/Fonctions et Ingénierie de l'alpha-L-arabinofuranosidase de Thermobacillus Xylanilyticus / Structure/functions studies and engineering of the Thermobacillus xylanilyticus alpha-L-arabinofuranosidase

Arab-Jaziri, Faten 23 October 2012 (has links)
Dans ce projet de thèse, une variété de techniques a été employée pour étudier l’alpha-L-arabinofuranosidase de Thermobacillus xylanilyticus (TxAbf), notamment en ce qui concerne les relations structure/fonctions et son activité de transglycosylation. Nos travaux ont eu pour objectif d’apporter un éclairage quant au rôle de la dynamique dans l’activité catalytique de la TxAbf, en se focalisant sur le mouvement de la boucle bêta2alpha2, et d’explorer la spécificité du sous-site [+1], un élément du site actif qui est particulièrement pertinent pour l’activité de transglycosylation. Enfin, nous avons entrepris des travaux d’ingénierie visant la création de transarabinofuranosylases performantes. Nos résultats confirment le rôle important de la boucle bêta2alpha2 et suggèrent que le mouvement de celle-ci permet de relocaliser les résidus His98 et Trp99 de manière à créer un site actif opérationnel. Le résidu Trp99 apparaît comme un élément clé du sous-site [-1] de la TxAbf, alors que le résidu His98, qui n’est pas conservé dans l’ensemble des enzymes de la famille GH51, participerait à la formation d’un sous-site [+2’]. Concernant le sous-site [+1], nos résultats confirment la large spécificité celui-ci et montrent clairement que l’encombrement stérique à la position C-5 des glycosides accepteurs est défavorable à la réaction de transglycosylation. Par ailleurs, nous avons pu réaliser pour la première fois la synthèse de trisaccharides, utilisant comme accepteur l’alpha-D-xylobioside de benzyle et comme donneur le β-D-galactofuranoside de para-nitrophényle. Enfin, nos travaux de mutagenèse aléatoire et le criblage de banques a permis d’identifier deux mutations Phe26Leu et Trp178Arg, qui se situent au niveau des sous-sites [-1] et [+1], respectivement. Selon nos premières analyses, les mutants correspondants rendraient moins favorable la déglycosylation de l’intermédiaire glycosyl-enzyme par une molécule d’eau, réduisant ainsi l'hydrolyse secondaire et stabilisant par la même occasion le produit de synthèse. En employant une deuxième méthode de criblage plus sophistiquée, impliquant l’utilisation d’accepteurs xylo-oligosaccharidiques, nous avons pu obtenir des enzymes mutées qui (i) catalysent des réactions de transglycosylation en présence de xylobiose (l’enzyme sauvage ne catalysant que très faiblement cette réaction) (ii) se caractérisent par une absence quasi-totale d’hydrolyse secondaire et (iii) comportent des mutations situées à différentes positions (e.g. au niveau des sous-sites [-1], [+1] et [+2’]) et qui semblent moduler le ratio Transglycosylation/Hydrolyse en faveur de la synthèse / In this investigation, a variety of techniques to study the Thermobacillus xylanilyticus alpha-L-arabinofuranosidase (TxAbf) have been employed, especially with regard to structure-functions relations and the enzyme’s ability to catalyze transglycosylation reactions. The aim of our work was to better understand the dynamic role of the bêta2alpha2 loop and to explore the substrate specificity of the subsite [+1], an important active site element with respect to transglycosylation. Finally, this work has focused on the creation of new transarabinofuranosylases using random engineering and screening approaches.Our results confirm the important role of the bêta2alpha2 loop and suggest that its movement during catalysis relocalizes residues His98 and Trp99 and thus permits the formation of a catalytically-viable active site configuration. Trp99 is relocalized from a solvent exposed position into a buried position and forms a critical element of subsite [-1], whereas His98, a residue that is not conserved in all GH51 members, appears to form a part of subsite [+2’]. Regarding subsite [+1], our results confirm its wide specificity and indicate that steric bulkiness at the C-5 position of glycoside acceptors leads to reduced transglycosylation. In this work, we have also demonstrated for the first time the synthesis by TxAbf of trisaccharides, using benzyl alpha-D-xylobioside as the acceptor and para-nitrophenyl β-D-galactofuranoside as the donor. Finally, random mutagenesis and screening has led to the identification of two mutations Phe26Leu and Trp178Arg, which are located in sub-sites [-1] and [+1] respectively, that appear to reduce the water-mediated deglycosylation of the glycosyl-enzyme intermediate. Consequently, the corresponding mutants reduce secondary hydrolysis and favourably affect the operational stability of synthetic products. Using a second more sophisticated screening method that involves the use of xylo-oligosaccharide acceptors, it has been possible to isolate mutant enzymes that (i) catalyze transglycosylation reactions in the presence of xylobioside (a reaction that is poorly catalyzed by wild type TxAbf), (ii) show almost no secondary hydrolysis, (iii) display point mutations at several key locations (e.g. in sub-sites [-1], [+1] and [+2’]) that seem to modulate the Transglycosylation/Hydrolysis ratio in favour of synthesis
78

Evolution structurale et fonctionnelle des communautés microbiennes digestives sous l'influence de facteurs biotiques et abiotiques. Développement d'une biopuce ADN ciblant les gènes impliqués dans la dégradation des glucides complexes alimentaires / Structural and functional evolution of digestive microbial communities under biotic and abiotic factors. Development of a DNA microarray targeting genes involved in degradation of dietary complex carbohydrates

Comtet-Marre, Sophie 26 June 2014 (has links)
La dégradation des fibres alimentaires est une fonction essentielle des écosystèmes digestifs microbiens. Chez le ruminant, elle est assurée par des bactéries, champignons et protozoaires capables de produire de nombreuses enzymes nécessaires à l’hydrolyse des polysaccharides de paroi végétale. Parmi les facteurs susceptibles d’influencer l’efficacité de dégradation des fibres, qui est une composante importante de la productivité et de la santé animales, des additifs tels que des levures probiotiques apparaissent comme un levier intéressant. Afin d’approfondir les connaissances sur les facteurs de modulation de l’activité fibrolytique, une biopuce ADN fonctionnelle, outil moléculaire haut-débit, ciblant les gènes codant les enzymes clés de la dégradation de la cellulose et des xylanes dans les écosystèmes digestifs a été développée. Aussi, une méthode efficace dédiée à des échantillons ruminaux pour la soustraction des ARNr à partir des ARN totaux a été mise au point afin d’accroitre la sensibilité de l’outil. La biopuce fonctionnelle a été validée sur échantillons de complexité croissante et démontre d’excellents caractères de spécificité et de sensibilité tout en étant exploratoire et quantitative. Des régulations différentielles de l’arsenal des gènes de la fibrolyse de la bactérie du rumen Fibrobacter succinogenes ont pu être montrées. De même, les résultats sur échantillons de rumen suggèrent un rôle des microorganismes eucaryotes dans la fibrolyse pouvant être plus important qu’initialement envisagé. Cette approche métatranscriptomique dirigée pourra in fine continuer d’être appliquée dans l’étude de l’impact de facteurs biotiques et abiotiques sur la fonction fibrolytique microbienne chez les animaux d’élevage. / Dietary fibre degradation is an essential function of microbial digestive ecosystems. In ruminants, this function is ensured by bacteria, fungi and protozoa, producing a large array of enzymes able to degrade plant cell wall polysaccharides. Among factors likely to influence the efficiency of fibre degradation, which is an important component in animal productivity and health, dietary additives such as probiotic yeasts appear as an interesting tool. To provide more insight on factors modulating fibrolytic activity, we designed a functional DNA microarray targeting genes coding for key enzymes involved in cellulose and xylan degradation by digestive microbiota. Also, an efficient method dedicated to rumen samples for removing microorganisms’ rRNA from total RNA samples was developed to increase the sensitivity of the tool. The DNA microarray was validated using targets of increasing complexity and demonstrated sensitivity and specificity as well as explorative and quantitative potential. Differential expression of genes involved in fibrolysis was evidenced in the rumen bacterium Fibrobacter succinogenes. Moreover, results on rumen samples suggest a more important role of eucaryotes in fibre degradation than previously thought. This targeted metatranscriptomic approach will be further applied to the study of the impact of biotic and abiotic factors on the microbial mechanisms of fibre degradation in livestock.
79

Conception et synthèse d’iminosucres di- à tétravalents comme sondes mécanistiques et agents thérapeutiques potentiels / Design and synthesis of di- or tetravalent iminosugars as mechanistic probes and potential therapeutic agents

Stauffert, Fabien 27 November 2015 (has links)
Dans un contexte où les iminosucres multivalents représentent, en tant qu’inhibiteurs puissants de glycosidases, des structures privilégiées pour le développement de nouveaux agents thérapeutiques, nous nous sommes intéressés à ce type de composés pour le traitement de deux maladies génétiques rares. Le premier axe de recherche a consisté à synthétiser des iminosucres di- à tétravalents en série 1-désoxymannojirimycine dans le but d’inhiber l’α1,2-mannosidase I du réticulum endoplasmique qui est impliquée dans la destruction de la protéine delF508-CFTR chez les malades atteints de la mucoviscidose. Un effet multivalent fort sur la correction de cette protéine mutée a alors été mis en évidence avec un composé trivalent basé sur le pentaérythritol. Efficace à des concentrations submicromolaires, ce dernier s’est montré 140 fois plus efficace que le modèle monovalent correspondant. Le second axe de recherche a consisté à identifier de nouveaux chaperons pharmacologiques de la β-glucocérébrosidase, l’enzyme lysosomale impliquée dans la maladie de Gaucher. Pour cela, nous avons préparé une série d’iminosucres hétérodivalents conçus pour cibler simultanément le site actif et un site secondaire de cette enzyme. Même si cet objectif n’a pas encore été atteint, nous avons malgré tout mis en évidence des chaperons monovalents capables de quasiment quadrupler l’activité de la β-glucocérébrosidase portant la mutation G202R. En marge de ces deux axes principaux, une sonde mécanistique basée sur un C-glycoside multivalent a également été développée dans le but de préciser les mécanismes à l’origine des effets multivalents puissants observés pour l’inhibition des glycosidases. / Because multivalent iminosugars represent, as potent glycosidase inhibitors, privileged structures for the design of novel drugs, we took a particular interest in this class of compounds for the treatment of two rare genetic diseases. The first research topic was dedicated to the synthesis of di- to tetravalent iminosugars in the 1-deoxymannojirimycin series in order to inhibit the endoplasmic reticulum α1,2-mannosidase I involved in the destruction of delF508-CFTR, the mutant protein responsible of cystic fibrosis. A strong multivalent effect for restoring its activity in cells was reported with a trivalent analogue based on pentaerythritol. This submicromolar corrector was found to be 140-fold more potent than the corresponding monovalent model. The second research topic focused on the identification of novel pharmacological chaperones of the β-glucocerebrosidase, the lysosomal enzyme involved in Gaucher’s disease. For this purpose, we developed a series of heterodivalent iminosugars designed to both bind to the active site and a secondary site of the enzyme. This goal could not be reached yet, nevertheless we identified monovalent chaperones which were able to fourfold increase β-glucocerebrosidase activity in G202R cell lines. Next to these main research topics, a mechanistic probe based on a multivalent C-glycoside was also developed to investigate the multivalent effect of iminosugar clusters in glycosidase inhibition.
80

Discovery and characterization of biomass-degrading enzymes and enzyme sytems in termite gut microbial ecosystems. / Etude de systèmes enzymatiques du microbiome intestinal de termite pour la dégradation de polymères végétaux

Arnal, Gregory 12 September 2014 (has links)
Cette thèse a été réalisée dans le cadre du projet Futurol, un projet national français qui vise à produire du bioéthanol à partir de biomasses végétales telles que le bois ou la paille de céréale. Pour cela, la biomasse doit être prétraitée puis digérée enzymatiquement pour libérer des sucres fermentescibles. Ma contribution dans ce projet a été de découvrir des enzymes originales pour l’hydrolyse de l’hémicellulose, un hétéropolysaccharide, constituant majeur de la paroi cellulaire des cellules végétales. Afin de rechercher de nouveaux biocatalyseurs, une approche de métagénomique a été adoptée afin de sonder les intestins de deux espèces de termites : N. corniger, un termite xylophage, et T. hispaniolae un termite humivore / xylophage. 30 000 clones métagénomiques ont été criblés sur 10 substrats cellulosiques et hémicellulosique, et 660 hits ont été obtenus. La comparaison phénotypique a montré une différence claire entre ces deux banques, probablement liée au régime alimentaire des deux espèces de termite. Le séquençage de 45 clones N. corniger a révélé 120 séquences codant pour des enzymes originales, de nombreuses étant multimodulaires et / ou organisées en cluster de gènes. Dans un second temps, une approche à haut-débit a été adoptée pour le clonage, l’expression et la caractérisation légère de 104 enzymes entières ou formes tronquées. 45 protéines recombinantes ont été produites de manière soluble, et les activités de 19 enzymes et de 12 modules enzymatiques ont été montrées, permettant la mise au point d’une boite à outil hemicellulolytique. Dans certains cas, l’activité de modules classés « Inconnus » a pu être déterminée. Cette approche a été particulièrement pertinente dans le cas de Pm69, une enzyme multimodulaire GH3-UNK-CBM48-CE1 montrant les 3 activités glucosidase, xylosidase and estérase. Cette étude a permis de poser les bases d’un brevet sur cette enzyme. D’un autre côté, les enzymes ayant montré une activité xylanase ou féruloyle-estérase se sont révélées complémentaires d’un cocktail cellulolytique durant la dégradation de paille de blé prétraitée. Enfin, dans une troisième partie, nous avons étudié un fragment d’ADN provenant la banque P. militaris, codant pour 19 ORFs et appartenant à une espèce du genre Bacteroides. La caractérisation biochimique d’Abn43A, Abn43B, Abf51A et Abf51B-trunc a montré que ces 4 enzymes portent des actions complémentaires sur l’hydrolyse de l’arabinane, et qu’elles peuvent agir de manière synergique pour la dégradation de ce polymère pectique. Enfin, l’étude détaillée des 19 ORFs codées sur ce fragment d’ADN nous a permis de proposer un schéma global de détection, d’hydrolyse et de métabolisation de l’arabinane par cette espèce du genre Bacteroides. / This thesis was performed in the context of the Futurol project, a French national project that aims at producing bioethanol from plant biomass such as wood and cereal straw. To reach that goal, the biomass must be pretreated, and enzymatically degraded to release fermentable simple sugar. My implication in that project was to discover original enzymes that can hydrolyze the hemicellulose, a major heteropolysaccharide found in plant cell wall.To mine for new biocatalysts, the gut microbial communities of two species of termite were investigated by a metagenomic approach : Nasutitermes corniger, a wood-feeder termite, and Termes hispaniolae supposed to be a soil-wood feeder. 30 000 metagenomic clones were screened on an array of 10 cellulosic and hemicellulosic substrates and 660 hits were obtained. Phenotypic comparison showed clear differences between both environments, probably related to the diet of the termite. The sequence of 45 N. corniger metagenomic inserts revealed 120 original sequences encoding for putative enzymes of interest. Original sequences encoding for multimodular enzymes were revealed and many ORFs were organized in clusters, suggesting that these enzymes are encoded on Polysaccharides Utilization Locus. In a second part, a high-throughput approach was used for the cloning, the expression and the slight characterization of 104 full-size and truncated enzymes. Forty five recombinant proteins were produced soluble, and their investigation revealed the activity of 19 enzymes and of 12 enzymatic modules, representing a hemicellulolytic tool-box for endo- and exo-type activities. In some cases, the implication of “Unkown” domains in the activity of multimodular enzymes was demonstrated. This approach was particularly efficient for the study of the GH3-UNKCBM48-CE1 Pm69, and this study triggered the patent process for this multiactive glucosidase, xylosidase and esterase. The xylanases and the feruloyl esterases were shown to be particularly efficient to supplement cellulolytic cocktails on pretreated wheat straw. In a third part, we investigated a DNA fragment belonging to a species of the genus Bacteroides and that encoded 19 ORFs. The biochemical characterization of Abn43A, Abn43B, Abf51A and Abf51B-trunc showed that these four enzymes harbored complementary actions for the hydrolysis of the arabinan, and that they can act synergistically for the hydrolysis of this pectic polymer. We also revealed that Abn43B had an original mode of action that we classified as exo-arabinanase. Finally, the in-depth study of the 19 ORFs allowed us to propose the entire scheme for arabinan detection, hydrolysis and utilization by the Bacteroides species carrying this DNA sequence

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