京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20426号 / 農博第2211号 / 新制||農||1048(附属図書館) / 学位論文||H29||N5047(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 加納 健司, 教授 植田 充美, 教授 三上 文三 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20715号 / 農博第2244号 / 新制||農||1053(附属図書館) / 学位論文||H29||N5081(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 加納 健司, 教授 宮川 恒, 教授 三芳 秀人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19022号 / 農博第2100号 / 新制||農||1030(附属図書館) / 学位論文||H27||N4904(農学部図書室) / 31973 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 加納 健司, 教授 阪井 康能, 教授 小川 順 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20425号 / 農博第2210号 / 新制||農||1047(附属図書館) / 学位論文||H29||N5046(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 加納 健司, 教授 宮川 恒, 教授 三芳 秀人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No description available.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24664号 / 農博第2547号 / 新制||農||1098(附属図書館) / 学位論文||R5||N5445(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 白井 理, 教授 菅瀬 謙治, 教授 三芳 秀人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

La corrosion induite par les micro-organismes (CIM) génère des pertes économiques mondiales chiffrées en milliards d’euros par an. Il est communément admis que les bactéries sulfato-réductrices (BSR) jouent un rôle clé dans la CIM anaérobie des aciers. Malgré cette unanimité, les essais en laboratoire peinent à reproduire la corrosion des aciers observées en milieu naturel; bien plus, ils n’expliquent pas quel est l’élément qui déclenche la corrosion, puisque les BSR présentes dans de nombreux environnements naturels n’induisent pas systématiquement de corrosion. L’objectif de ce travail est d’évaluer la pertinence dans le domaine de la CIM de nouveaux mécanismes de transferts électroniques entre aciers et protéines ou cellules microbiennes. La première partie de la thèse évalue l’effet d’une [Fe]-hydrogénase sur les processus de corrosion anaérobie des aciers au carbone. L’hypothèse d’une catalyse directe de la réduction des protons par des hydrogénases adsorbées a souvent été suggérée dans la bibliographie, elle est ici clairement démontrée. L’hydrogénase de Clostridium acetobutylicum, qu’elle soit active, désactivée ou dénaturée accélère la corrosion de l’acier au carbone. La présence de phosphate dans le milieu rend les interprétations plus complexes mais ne modifie pas le mécanisme. Une nouvelle hypothèse est avancée qui donne un rôle essentiel aux centres fer-soufre de la protéine. La catalyse de la corrosion par les hydrogénases pourrait donc être rapprochée des mécanismes bien connus de catalyse par le sulfure de fer. Dans ce cas l’état redox des centres fer-soufre serait une clé essentielle de l’apparition ou non de la corrosion. La deuxième partie élucide le rôle de Geobacter sulfurreducens sur la corrosion anaérobie de trois types de matériaux : aciers au carbone (1145), ferritique (403) et austénitiques (304L et 316L). Les résultats mettent en évidence pour la première fois que des cellules bactériennes adhérées induisent un anoblissement du potentiel libre des aciers et accélèrent la corrosion des aciers faiblement alliés par un mécanisme de transfert direct d’électrons. Suivant les concentrations d’accepteurs et de donneurs d’électrons en solution, G. sulfurreducens peut accentuer la propagation de la corrosion en catalysant directement la réduction cathodique ou, au contraire, en absence d’accepteurs et en excès de donneurs, protéger contre la corrosion. L’apparition de la corrosion ne peut donc être induite que par la conjonction défavorable de plusieurs paramètres. Ces résultats obtenus en laboratoire apportent de nouvelles voies d’investigations des phénomènes de CIM qui doivent maintenant être confrontées aux milieux naturels. / Microbially influenced corrosion (MIC) costs billions of euros per year. It is commonly agreed that sulphate-reducing bacteria (SRB) play a key role in anaerobic MIC of steels. In spite of this, laboratory experiments have difficulty in reproducing the corrosion of steels that is observed in natural environments. Moreover, they do not explain what triggers corrosion since SRB, ubiquitous in natural environments, do not systematically induce corrosion. The aim of this work was to evaluate the relevance of new electron transfer mechanisms between steels and proteins or microbial cells in the domain of MIC. The first part of the thesis evaluates the impact of [Fe]-hydrogenase on the anaerobic corrosion of mild steels. The direct catalysis of proton reduction by hydrogenases has often been suggested in the literature; here, it is clearly demonstrated. Hydrogenase from Clostridium acetobutylicum, whether it is active, deactivated on denatured, can accelerate the corrosion of mild steel. The presence of a phosphate medium makes the interpretations more complex without modifying the mechanism. A new hypothesis implying the crucial role of iron-sulphur clusters contained in the protein is brought to light. Corrosion catalysis by hydrogenases could be compared with well-known mechanisms of corrosion catalysis by iron sulphide. In this case, the redox state of iron-sulphur clusters would play a key role in the occurrence of corrosion. The second part elucidates the role of Geobacter sulfurreducens in anaerobic corrosion of three types of steels: mild steel (1145), ferritic (403) and austenitic steels (304L and 316L). Results show, for the first time, that adherent bacterial cells induce open circuit potential ennoblement of steels and accelerate the corrosion of slightly alloyed steels by a direct electron transfer mechanism. Depending on the concentrations of the electron acceptors and donors in the medium, G. sulfurreducens could either enhance corrosion propagation by direct catalysis of proton reduction or, in the absence of acceptors and with an excess of donors, protect against corrosion. Thus the occurrence of corrosion relies on the unfavourable conjunction of many parameters. These results obtained in laboratory conditions open new paths for investigating MIC in natural environments.

No description available.

A eletroquímica direta de enzimas redox depende da distância entre os sítios redox da proteína e a superfície do eletrodo e também da eficiência na imobilização dessas enzimas na superfície eletródica. Dessa forma, a obtenção de enzimas mais hidrofóbicas possibilita a melhora na interação entre elas e a superfície de eletrodos sólidos, como os de carbono. Neste estudo, foi desenvolvida uma rota para a obtenção da glicose oxidase oligomerizada (Ol-GOx) com o objetivo de melhorar a interação entre a enzima e a superfície de fibras de carbono, uma vez que enzimas oligomerizadas contêm suas porções hidrofóbicas expostas.Para tanto, diferentes ácidos de Brønsted foram utilizados, sendo que a enzima obtida a partir da reação com o ácido trifluorometanosulfônico (TFMS) foi a que se manteve ativa cataliticamente. A Ol-GOx se mostrou um biocatalisador promissor devido a sua hidrofobicidade e seu tamanho, os quais permitiram uma imobilização mais eficiente em superfícies de carbono. Após a caracterização estrutural, concluiu-se que a Ol-GOx é formada por um oligômero composto por 10 unidades de GOx nativa com raio hidrodinâmico de aproximadamente 96 nm. Por voltametria cíclica estudou-se a transferência direta de elétrons (TDE) entre o cofator dinucleotídeo de flavina e adenina (FAD) e a superfície das fibras de carbono, sendo observado um aumento de 7 vezes nas correntes faradaicas em relação ao obtido para a GOx nativa. Além disso, as propriedades bioeletrocatalíticas foram melhoradas em 30% quando analisada a oxidação da glicose. Concluiu-se ainda que quanto maior a quantidade de folhas-β presente na estrutura proteica, maior a TDE observada entre a enzima e a superfície das fibras de carbono. / The direct electrochemistry of redox enzymes is dependent on the distance between the active centers of the protein and the electrode surface, and also on the efficiency in the immobilization of these enzymes on the electrodic surface. Thus, the synthesis of more hydrophobic enzymes could lead to better interaction between the redox enzymes and the solid electrode surfaces, such as carbon electrodes. In this study, it was proposed a chemical route to obtain oligomerized glucose oxidase (Ol-GOx), aiming to improve the interaction between the enzyme and the surface of carbon fibers, since oligomerized proteins have their hydrophobic chains exposed. After structural characterization, it was concluded that Ol-GOx is formed by 10 dimeric units of native GOx with a hydrodynamic radius corresponding to approximately 96 nm. By cyclic voltammetry, it was studied the direct electron transfer (DET) between the flavin adenine dinucleotide (FAD) cofactor and the surface of carbon fibers, where it was observed an increase of 7-fold in the faradaic currents in comparison to that observed for native GOx. Besides, bioelectrocatalytic properties are 30% improved, when analyzed the glucose oxidation by cyclic voltammetry. It was also concluded that the greater the β-sheet content in protein structure, the higher the DET observed between the enzyme and the carbon fibers surface.

A eletroquímica direta de enzimas redox depende da distância entre os sítios redox da proteína e a superfície do eletrodo e também da eficiência na imobilização dessas enzimas na superfície eletródica. Dessa forma, a obtenção de enzimas mais hidrofóbicas possibilita a melhora na interação entre elas e a superfície de eletrodos sólidos, como os de carbono. Neste estudo, foi desenvolvida uma rota para a obtenção da glicose oxidase oligomerizada (Ol-GOx) com o objetivo de melhorar a interação entre a enzima e a superfície de fibras de carbono, uma vez que enzimas oligomerizadas contêm suas porções hidrofóbicas expostas.Para tanto, diferentes ácidos de Brønsted foram utilizados, sendo que a enzima obtida a partir da reação com o ácido trifluorometanosulfônico (TFMS) foi a que se manteve ativa cataliticamente. A Ol-GOx se mostrou um biocatalisador promissor devido a sua hidrofobicidade e seu tamanho, os quais permitiram uma imobilização mais eficiente em superfícies de carbono. Após a caracterização estrutural, concluiu-se que a Ol-GOx é formada por um oligômero composto por 10 unidades de GOx nativa com raio hidrodinâmico de aproximadamente 96 nm. Por voltametria cíclica estudou-se a transferência direta de elétrons (TDE) entre o cofator dinucleotídeo de flavina e adenina (FAD) e a superfície das fibras de carbono, sendo observado um aumento de 7 vezes nas correntes faradaicas em relação ao obtido para a GOx nativa. Além disso, as propriedades bioeletrocatalíticas foram melhoradas em 30% quando analisada a oxidação da glicose. Concluiu-se ainda que quanto maior a quantidade de folhas-β presente na estrutura proteica, maior a TDE observada entre a enzima e a superfície das fibras de carbono. / The direct electrochemistry of redox enzymes is dependent on the distance between the active centers of the protein and the electrode surface, and also on the efficiency in the immobilization of these enzymes on the electrodic surface. Thus, the synthesis of more hydrophobic enzymes could lead to better interaction between the redox enzymes and the solid electrode surfaces, such as carbon electrodes. In this study, it was proposed a chemical route to obtain oligomerized glucose oxidase (Ol-GOx), aiming to improve the interaction between the enzyme and the surface of carbon fibers, since oligomerized proteins have their hydrophobic chains exposed. After structural characterization, it was concluded that Ol-GOx is formed by 10 dimeric units of native GOx with a hydrodynamic radius corresponding to approximately 96 nm. By cyclic voltammetry, it was studied the direct electron transfer (DET) between the flavin adenine dinucleotide (FAD) cofactor and the surface of carbon fibers, where it was observed an increase of 7-fold in the faradaic currents in comparison to that observed for native GOx. Besides, bioelectrocatalytic properties are 30% improved, when analyzed the glucose oxidation by cyclic voltammetry. It was also concluded that the greater the β-sheet content in protein structure, the higher the DET observed between the enzyme and the carbon fibers surface.