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Physico-chemical studies on cellobiose oxidase from Phanerochaete chrysosporiumLiu, Bing-Lan January 1995 (has links)
No description available.
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Implication de la subérine dans la régulation de l'activité cellulolytique des espèces de Streptomyces causant la gale commune de la pomme de terrePadilla Reynaud, Rebeca January 2017 (has links)
L’agent phytopathogène Streptomyces scabiei est une bactérie du phylum actinobactérie présent dans les sols à travers le monde. S. scabiei est l’agent principal responsable de la gale commune de la pomme de terre. Cette maladie est coûteuse pour les producteurs de la pomme de terre. En effet, la pomme de terre va perdre sa valeur commerciale lorsque la bactérie attaque le périderme du tubercule et provoque des lésions liégeuses en surface ou plus ou moins en profondeur. C’est dans le périderme de la pomme de terre que la subérine va se déposer pour protéger le tubercule des agressions biotiques. Des études antérieures ont mis en évidence le rôle de la subérine comme inducteur de la production de thaxtomine A (phytotoxine essentielle à la virulence) chez S. scabiei. De même, la subérine va induire chez S. scabiei, la sécrétion d’enzymes dégradant les parois végétales, en particulier des glycosyl hydrolases, dont des cellulases.
Cette thèse vise à élucider les mécanismes impliques dans la production de cellulases chez la souche S. scabiei EF-35. Dans un premier temps, le sécrétome de S. scabiei ayant poussé en présence de subérine et cellulose, soit avec un seul des deux polymères, a été analysé. Ces analyses du sécrétome ont révélé que l’addition de subérine dans un milieu contenant de la cellulose induisait une surproduction de glycosyl hydrolases. L’induction des enzymes cellulolytiques par la subérine, correlait avec la présence d’un inhibiteur de subtilase (SCAB_8801) qui pourrait jouer un rôle dans la différenciation cellulaire et le métabolisme secondaire. Ces résultats ont permis d’avancer un modèle dans lequel la subérine et le cellobiose jouent conjointement un rôle pour activer les mécanismes de virulence de la bactérie.
Dans un deuxième temps, nous avons voulu savoir si le cellobiose (la molécule résultante de la dégradation de la cellulose) induisait des enzymes cellulolytiques chez S. scabiei, mais aussi chez deux autres espèces de Streptomyces pathogènes et une espèce de Streptomyces non pathogène. L’activité cellulolytique de S. scabiei en présence de subérine est beaucoup plus importante que lorsque la bactérie est en présence de cellobiose (de cinq à dix fois supérieure). De même, la présence de subérine dans le milieu de culture de S. scabiei augmente l’expression rélative des gènes des cellulases. Les deux autres Streptomyces pathogènes (S. acidiscabies et S. turgidiscabies) exhibent un profil contraire à S. scabiei. En effet, S. acidiscabies et S. turgidiscabies affichent une activité cellulolytique et une expréssion rélative des gènes de cellulases plus importante dans le milieu supplémenté de cellobiose que dans le milieu supplémenté de subérine. Streptomyces scabiei semble donc mieux adaptée que les autres espèces de Streptomyces à dégrader le matériel cellulosique encastré dans les parois subérisées du périderme.
Les résultats présentés dans cette thèse visent à apporter des éléments de réponse pour mieux comprendre les interactions S. scabiei – pomme de terre. Ainsi, les biopolymères retrouvés dans la pomme de terre (subérine et cellulose) vont jouer un rôle crucial dans la virulence de l’agent phytopathogène. Cependant, il semblerait que S. scabiei s’est spécialisé au cours du temps à coloniser son hôte, la pomme de terre. La subérine induit la production de cellulases chez S. scabiei mais pas chez les autres deux Streptomyces pathogènes testés (S. acidiscabies et S. turgidiscabies). Ceci nous laisse penser que la subérine est impliquée dans des mécanismes qui restent encore à élucider.
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A computational study of acidic Ionic Liquids for cellobiose hydrolysis in ionic liquidsNel, Jessica Lisé 08 May 2020 (has links)
The current environmental situation, with respect to global warming and the ever– approaching depletion of fossil fuel sources, places significance on the development of green fuel and platform chemical production methods. In this context, processes that utilise biomass sources as feedstock, are of great interest. Cellulose, which is the most abundant biopolymer in nature, is a renewable low–cost carbon resource derived from harvest residues and sources like wood and straw. Glucose generation from cellulose requires a saccharide conversion, whereby the β-(1,4)-glycosidic bond linkages in the cellobiose polymer repeating units are cleaved. Problems arise in the hydrolysis of cellulose as experimental and theoretical studies have shown cellulose to have very low solubility in water and most other general molecular solvents. This results in the use of harsh pretreatments at high temperatures and pressures to extract cellulose from lignocellulosic material and strong acids catalysts (pKa < −3.2). Room temperature ionic liquids (RTILs) provide potentially environmentally friendly alternative. It has been shown that ILs can dissolve cellulose under relatively benign conditions and can possibly be adapted into a one-pot-like process of hydrolysis using acid-functionalised IL catalysts. This dissertation investigated the effect of various ionic liquids on the thermodynamics of cellobiose acid hydrolysis, as both a catalyst and as a solvent, using computational means. An appropriate thermodynamic cycle protocol, a DLPNO-CCSD(T)/ccpVTZ//TPSS/def2-TZVP [M05-2X/6-31+G** (SMD)] proton exchange cycle, was established through benchmarking for the prediction of Brønsted acid-functionalised ionic liquid pKa values in ionic liquids. The sulfonyl-functionalised acidic IL was shown to be the most acidic IL resulting in a lower protonation free energy. Solvation in ionic liquids resulted in higher protonation and barrier height free energies relative to solvation in water. The current environmental situation, with respect to global warming and the ever– approaching depletion of fossil fuel sources, places significance on the development of green fuel and platform chemical production methods. In this context, processes that utilise biomass sources as feedstock, are of great interest. Cellulose, which is the most abundant biopolymer in nature, is a renewable low–cost carbon resource derived from harvest residues and sources like wood and straw. Glucose generation from cellulose requires a saccharide conversion, whereby the β-(1,4)-glycosidic bond linkages in the cellobiose polymer repeating units are cleaved. Problems arise in the hydrolysis of cellulose as experimental and theoretical studies have shown cellulose to have very low solubility in water and most other general molecular solvents. This results in the use of harsh pretreatments at high temperatures and pressures to extract cellulose from lignocellulosic material and strong acids catalysts (pKa < −3.2). Room temperature ionic liquids (RTILs) provide potentially environmentally friendly alternative. It has been shown that ILs can dissolve cellulose under relatively benign conditions and can possibly be adapted into a one-pot-like process of hydrolysis using acid-functionalised IL catalysts. This dissertation investigated the effect of various ionic liquids on the thermodynamics of cellobiose acid hydrolysis, as both a catalyst and as a solvent, using computational means. An appropriate thermodynamic cycle protocol, a DLPNO-CCSD(T)/ccpVTZ//TPSS/def2-TZVP [M05-2X/6-31+G** (SMD)] proton exchange cycle, was established through benchmarking for the prediction of Brønsted acid-functionalised ionic liquid pKa values in ionic liquids. The sulfonyl-functionalised acidic IL was shown to be the most acidic IL resulting in a lower protonation free energy. Solvation in ionic liquids resulted in higher protonation and barrier height free energies relative to solvation in water.
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Etude d’une CDH et de glycosyl hydrolases de la famille 61 : Implication dans les processus de dégradation des lignocellulosesBey, Mathieu 12 December 2012 (has links)
En réponse aux préoccupations environnementales, les procédés industriels comme la production de bioéthanol de deuxième génération sont apparus. Basés sur la conversion enzymatique de la cellulose, ces processus font face à un problème majeur, la réticence de la biomasse lignocellulosique à l'hydrolyse. Afin de résoudre ce problème et celui lié aux coûts d'utilisation de cocktails de cellulases, les recherches se sont axées sur diverses méthodes permettant d'augmenter l'hydrolyse de la cellulose. Les champignons filamenteux sont connus pour être des dégradeurs naturels du bois et, par conséquent, sont utilisés dans de nombreuses applications biotechnologiques. Récemment, quelques études ont révélé l'importance d'enzymes fongiques telles que la CDH et les GH61 dans la dégradation oxydative de la lignocellulose. Les travaux réalisés au cours de cette thèse ont permis de démontrer l'importance de ces enzymes oxydatives dans les phénomènes de déconstruction de la lignocellulose. L'utilisation de ces enzymes oxydatives offre de réelles voies d'amélioration de la production de bioéthanol et de compréhension de la dégradation in vivo des lignocelluloses par les champignons. / In response to environmental concerns, industrial processes such as second generation bioethanol production have emerged. Based on enzymatic cellulose conversion, these processes are confronted with a major problem, the recalcitrance of lignocellulosic biomass. To solve the problem caused by substrate recalcitrance and high cost of cellulase cocktails, research has focused on various methods to enhance cellulose hydrolysis. Fungi are known to be natural degraders of wood and consequently are used in derived biotechnological applications. Recently, several studies have revealed the importance of fungal enzymes such as GH61 and CDH in the oxidative degradation of lignocellulose. During the work done on this thesis, we demonstrated implication of these oxidative enzymes in lignocellulose deconstruction to enhance hydrolysis performed by more classical cellulases. Utilization of oxidative enzymes offers a suitable way for bioethanol processing enhancement and comprehension of the in vivo lignocellulosic degradation by fungi.
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Surface interactions of biomass derived oxygenates with heterogeneous catalystsFoo, Guo Shiou 07 January 2016 (has links)
Energy demand is projected to increase by 56% before 2040 and this will lead to the fast depletion of fossil fuels. Currently, biomass is the only sustainable source of organic carbon and liquid fuels. One major method of converting biomass involves the utilization of heterogeneous catalysts. However, there is still a lack of understanding in the reaction mechanisms and surface interactions between biomass-derived oxygenates and catalysts. Specifically, three important reactions are investigated: i) dehydration of glycerol, ii) hydrolysis of cellulose and cellobiose, and iii) hydrodeoxygenation of bio-oil. Some important concepts are gathered and provide insight into the most attractive conversion strategies. These concepts include the role of Lewis and Brønsted acid sites, synergistic effect between defect sites and functional groups, the advantage of weak acid sites, steric effect imposed by aromatic substituents, and the evolution of surface species in catalyst deactivation. These studies show that a deep understanding of surface chemistry can help to elucidate elementary reaction steps, and there is great potential in using heterogeneous catalysts for the conversion of biomass into targeted fuels and chemicals.
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A study of some reaction rates in the homogeneous system water-sodium hydroxide-cellobioseMacLaurin, Donald James 01 January 1969 (has links)
The broad objective of the study was to gain further knowledge of the reaction
rates and mechanisms by which carbohydrates, particularly 3(l-4) glucans, are transformed
and degraded in aqueous alkaline solutions.
While the isomerization, epimerization, and degradation of carbohydrates has
been extensively studied and reviewed, there are practically no kinetic data available
on these important reactions due apparently to a lack of reasonable procedures
for assay of the reaction systems.
Because of the important theoretical, physiological, and industrial implications
of these reactions, it appeared useful to have kinetic data on them and concomitantly
thus to develop a method for obtaining such data. The specific problem selected for study from this broad area was the measurement
of reaction rates prevailing in the homogeneous system: cellobiose-l molar
sodium hydroxide-water at 22°C. and to derive-the related rate constants from the
reaction rate expressions and then to assess current understanding of these reactions
in light of the kinetic data obtained.
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La fermentation de la cellulose par Clostridium cellulolyticum métabolisme modèle d'un Clostridium cellulolytique mésophile /Desvaux, Mickaël Petitdemange, Henri January 2001 (has links) (PDF)
Thèse de doctorat : Biologie structurale, moléculaire et cellulaire : Nancy 1 : 2001. / Thèse : 2001NAN10174. Titre provenant de l'écran-titre.
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Molecular aspects of cellobiose dehydrogenase produced by Trametes versicolorDumonceaux, Timothy J. January 1998 (has links)
Under cellulolytic conditions, the white-rot fungus Trametes versicolor produces cellobiose dehydrogenase (CDH), an enzyme with a number of biochemical properties that are potentially relevant to the degradation of lignin and cellulose. To clarify its biochemical properties, CDH was purified from cultures of T. versicolor. Two isoforms of CDH were found: a 97 kDa isoform with both heme and flavin cofactors, and an 81 kDa isoform with a flavin cofactor. Both isoforms of CDH were found to be quite non-specific in their reductive half reactions. The flavin enzyme catalyzed many of the same reactions as the heme/flavin enzyme, but less efficiently. The flavin isoform reduced Fe(III) and Cu(II) only at concentrations well above those found physiologically. Thus the heme/flavin enzyme, but not the flavin enzyme, could be involved in promoting and sustaining the generation of hydroxyl radicals (·OH) by Fenton's chemistry. / To characterize further the structural features of CDH, a genomic clone was isolated and sequenced. CDH was found to consist of 748 amino acids, without its predicted 19 amino acid signal peptide. Consistent with the domain structure of other CDHs, T. versicolor CDH appeared to be divided into an amino terminal heme domain and a carboxy terminal flavin domain, connected by a hydroxyamino acid-rich linker. Within the flavin domain, a putative cellulose-binding domain (CBD) was found by alignment to the hypothesized CBD of P. chrysosporium CDH. The CBD of CDH appeared to be structurally unrelated to other CBDs which have been reported. / A cDNA clone encoding T. versicolor CDH was isolated by RT-PCR. Using this clone, three vectors for the heterologous expression in Aspergillus oryzae of CDH were prepared. These vectors were built by performing in-frame fusions of the cDNA to control sequences from the highly expressed A. oryzae amylase gene. These vectors were transformed into A. oryzae and one strain was isolated which contained the expression construct DNA. / A rapid method for cloning cdh-like genes was developed. Using short stretches of amino acids completely conserved within T. versicolor and P. chrysosporium CDH, PCR primers were designed to amplify a homologous gene from other fungi. The primers were tested using genomic DNA of Pycnoporus cinnabarinus. A 1.8-kb fragment of P. cinnabarinus cdh was thereby amplified and cloned, and its sequence was determined. The three CDHs displayed very high homology at the amino acid level. / Finally, to probe the role of CDH in lignocellulose degradation by T. versicolor, a "knockout" vector was constructed consisting of a phleomycin-resistance cassette inserted into the protein coding sequence of cloned T. versicolor cdh. T. versicolor was transformed with the knockout vector and the transformants were analyzed for their CDH-producing phenotype. Three isolates were found that produced no detectable CDH. Biobleaching and delignification by the CDH(-) strains appeared to be unaffected, suggesting that CDH does not play an important role in these processes.
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An investigation of the mechanism of the Cellulomonas fimi exoglucanaseTull, Dedreia L. January 1991 (has links)
The exoglucanase from Cellulomonas fimi catalyses the hydrolysis of cellobiose units from the non-reducing terminus of cello-oligosaccharides with overall retention of anomeric configuration. Its mechanism of action is therefore thought to involve a double displacement reaction, involving as the first step, formation of a glycosyl-enzyme intermediate (glycosylation) and as a second step, the hydrolysis of this intermediate (deglycosylation). This mechanism is investigated here through the study of the kinetics of hydrolysis of aryl β-glucosides and aryl β-cellobiosides and by employing the mechanism-based irreversible inactivators, 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucoside (2F-DNPG) and 2", 4"-dinitrophenyl 2-deoxy-2-fluoro-β-D-cellobioside (2F-DNPC).
The study with the aryl β-glucosides revealed that this enzyme is indeed active on glucosides, a feature that had previously been undetected. A linear relationship was found to exist between the logarithm of Vmax for hydrolysis and the phenol pKa as well as between the logarithm of Vmax/Krn and me phenol pKa, showing that glycosylation is both the rate determining step and the first irreversible step for all substrates. The reaction constant calculated, ρ = 2.21, indicates a considerable amount of charge build up at the transition state of glycosylation.
The linear free energy relationship study of the aryl β-cellobiosides revealed no significant dependence of the logarithm of Vmax on the pKa of the phenol, indicating that deglycosylation is rate determining. However, the slight downward trend in this Hammett plot at higher pKa values may suggest that the rate determining step is changing from deglycosylation to glycosylation. However, the logarithm of Vmax/Km does correlate with the pKa of the phenol, thus showing that the first irreversible step is glycosylation. The reaction constant (ρ = 0.60) which reflects the development of charge at the glycosylation transition state for the cellobiosides is less than that calculated for the glucosides, thus suggesting a glycosylation transition state with either a greater degree of acid catalysis or less C-O bond cleavage than that for the glucosides. The inactivators, 2F-DNPC and 2F-DNPG, are believed to inactivate the exoglucanase by binding to the enzyme and forming covalent glycosyl-enzyme intermediates. The inactivated-enzyme was stable in buffer but reactivated in the presence of a suitable glycosyl-acceptor such as cellobiose, presumably via a transglycosylation reaction. These results indicate that covalent 2F-glycosyl-exoglucanase intermediates are stable and are catalytically competent to turn over to product, thus supplying further evidence for the Koshland mechanism. The exoglucanase is inactivated more rapidly by 2F-DNPC than by 2F-DNPG. However, both inactivated forms of the enzyme reactivated at comparable rates in the presence of cellobiose, showing that the second glucosyl unit present on the cellobiosides increases the rate of glycosylation relative to that found for the glucosides but not the rate of deglycosylation.
The stable covalent nature of the 2F-glycosyl-enzyme intermediates provided an excellent opportunity to identify the enzymic nucleophile. This was accomplished by radiolabelling the exoglucanase with a tritiated analogue of 2F-DNPG cleaving the protein into peptides and purifying the radiolabelled peptides. Sequencing of this peptide resulted in the identification of the active site nucleophile as glutamic acid residue 274. This residue was found to be highly conserved in this family of β-glycanases, further indicating its importance in catalysis. / Science, Faculty of / Chemistry, Department of / Graduate
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Molecular aspects of cellobiose dehydrogenase produced by Trametes versicolorDumonceaux, Timothy J. January 1998 (has links)
No description available.
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