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21132号 / 農博第2258号 / 新制||農||1056(附属図書館) / 学位論文||H30||N5106(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 小川 順, 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

Biofuel cells (BFCs) that utilize enzymes as catalysts represent a new sustainable and renewable energy technology. Numerous efforts have been directed to improve the performance of the enzymatic BFCs (EBFCs) with respect to power output and operational stability for further applications in portable power sources, self-powered electrochemical sensing, implantable medical devices, etc. This concept article details the latest advances about the EBFCs based on porous nanoarchitectures over the past 5 years. Porous matrices from carbon, noble metal, and polymer promote the development of EBFCs through the electron transfer and mass transport benefits. We will also discuss some key issues on how these nanostructured porous media improve the performance of EBFCs in the end.

Para a bioeletrocatálise de oxidação de etanol, a alteração da superfície eletródica e a otimização do processo de imobilização enzimática se fazem necessárias. Neste cenário, as fibras flexíveis de carbono (FFC) merecem destaque, pois além de sua superfície ser facilmente modificada devido à presença de carbono sp2, as mesmas possuem alta resistência mecânica e elasticidade, combinadas com a alta condutividade elétrica e térmica. Nesta tese de doutorado, apresenta-se como obter bioeletrodos de FFC modificadas com a enzima álcool desidrogenase (ADH) NAD-dependente, visando também aprimorar a oxidação da coenzima NADH (dinucleotídeo de nicotinamida e adenina). Os resultados mostram que quando as FCF são previamente submetidas a um tratamento oxidativo em meio ácido (KMnO4/H2SO4), obtém-se bioeletrodos estáveis, robustos e com alta área superficial. Além disso, observou-se que esses eletrodos possuem grupos funcionais contendo oxigênio que auxiliam na bioeletrocatálise de oxidação do etanol. Presume-se que presença de grupos quinonas seja responsável por facilitar a regeneração da coenzima, ou seja, estes grupos atuam decisivamente na oxidação do NADH. A alta qualidade dos bioeletrodos possibilitou manter a atividade catalítica da ADH por longo prazo, propriedade essa crucial para o estudo da oxidação do etanol acoplada à espectrometria de massas (DEMS). Devido a este estudo, foi possível observar concomitantemente a regeneração da coenzima (NADH -> NAD+) e a geração de acetaldeído como produto de bioeletroxidação do etanol, ambos em estado estacionário. Em suma, o estudo aqui apresentado introduz uma abordagem que combina não só o desenvolvimento de fibras de carbono tratadas quimicamente para aplicação em bioeletrocatálise, mas também um foco inédito no acoplamento entre a espectrometria de massas e a bioeletroquímica para a resolução de mecanismos enzimáticos. / Regarding the bioelectrocatalysis of the ethanol oxidation, the electrodic surface modification and the optimization of enzymatic immobilization are necessary. In this scenario, the flexible carbon fibers (FCF) are noteworthy, because besides their surface can be modified in an easy way due the presence of carbon sp2, they have high mechanical resistance and elasticity, combined with high electrical and thermal conductivity. In this doctoral thesis, it is presented how to obtain bioelectrodes of FFC modified with the enzyme alcohol dehydrogenase (ADH) NAD-dependent, as well as to improve the oxidation of the coenzyme NADH (nicotinamide adenine dinucleotide). The results show that when FCF is previously submitted to an oxidative treatment in acidic medium (KMnO4/H2SO4), stable, robust and high surface area bioelectrodes are obtained. In addition, it was observed that these electrodes have oxygen-containing functional groups that improve the bioelectrocatalysis of ethanol oxidation. There is proposed that the presence of quinone groups is responsible for facilitating the regeneration of the coenzyme, i. e., these groups act decisively in the oxidation of NADH. The high quality of the bioelectrodes allowed it to maintain the catalytic activity of the ADH for long term, property crucial for the study of the oxidation of ethanol coupled to mass spectrometry (DEMS). By using DEMS, there were possible to observe coenzyme regeneration and the generation of acetaldehyde as a bioelectrooxidation product of ethanol, both at steady state, which were simultaneously observed. In summary, the present study introduces an to an approach that combines not only the development of chemically treated carbon fibers for application in bioelectrocatalysis, but also an unprecedented focus on the coupling between mass spectrometry and bioelectrochemistry for the resolution of enzymatic mechanisms.

Ce mémoire est consacré à l'optimisation de la connexion enzymatique d'enzymes pour l'oxydation du glucose et la réduction de O2 sur matrices de nanotube de carbone (CNT) dans les biopiles à glucose.Premièrement, le transfert électronique indirect de la glucose oxydase (GOx) est optimisé dans une matrice nanostructurée de CNT contenant la 1,4-naphtoquinone comme médiateur rédox. Cette bioanode a ensuite été combinée avec des biocathodes similaires à bases d'enzymes à cuivre (laccase et tyrosinase). La biopile GOx-NQ/Lac a permis d'obtenir des puissances maximales de l'ordre de 1,5 mW.cm-2. Les utilisations de cette pile en décharge courte, longue et sa stabilité dans le temps ont également été étudiées. La seconde partie présente la préparation d'une autre anode basée sur la connexion indirecte d'une glucose déshydrogènase NAD+-dépendante (GDH-NAD+) comme alternative pour l'oxydation du glucose. La GDH-NAD+ a été combinée avec un catalyseur d'oxydation de NADH par différentes méthodes. Tout d'abord, elle a été encapsulée au sein du métallopolymère rédox, puis, la modification supramoléculaire a dans un second temps permis d'immobiliser le catalyseur moléculaire et l'enzyme à la surface des CNTs. Ces deux bioanodes ont permis respectivement l'obtention de courants catalytiques d'oxydation du glucose de 1,04 et 6 mA.cm-2. La seconde bioanode a été combinée avec une biocathode à base de BOD et a permis l'obtention de densités de courants maximales de l'ordre de 140 µW.cm-2 La dernière partie concerne l'élaboration d'une biocathode bienzymatique pour la réduction de O2. Le DET de la HRP sur CNTs a dans un premier temps été optimisé par modification de la surface par différents dérivés pyrène. Ensuite, la combinaison de la GOx et de la HRP sur la même électrode a permis de réduire efficacement O2 en 2 étapes. La biocathode est capable de délivrer une densité de courant maximale de l'ordre de 200 µA.cm-2. Cette dernière, combinée avec la bioanode GDH présentée précédemment a permis d'obtenir une biopile opérationnelle en conditions physiologiques et 10 mM de NAD+, en étant capable de débiter une densité de puissance maximale de l'ordre de 57 µW.cm-2. / This work focuses on the optimization of the electrical wiring of glucose oxidizing and dioxygen reducing enzymes on carbon nanotube (CNT) matrixes for glucose biofuel cells.In the first part, glucose oxidase (GOx) mediated electron transfer (MET) is optimized in nanostructured CNTs matrixes by mechanical compression of a CNTs/GOx composite containing 1,4-naphtoquinone as redox mediator. This bioanode was then combined with MCOs (laccase and tyrosinase) based biocathodes. The GOx-NQ/Lac biofuel cell was able to deliver a maximum power density of 1.5 mW.cm-2. The use of this biofuel cell in short/long time discharge and in storage has also been studied. The second part presents the preparation of another bioanode based on the indirect wiring of a NAD+-dependant glucose dehydrogenase (GDH-NAD+) as an alternative for glucose oxidation. The GDH-NAD+ has been combined with an NADH oxidation catalyst by two different techniques. The first one involves the encapsulation of the protein in the metallopolymer redox film, whereas the second one relies on the supramolecular modification of the CNTs by the molecular catalyst and the enzyme. Both bioanodes showed good catalytic properties toward glucose oxidation in presence of NAD+ with respectively 1.04 mA cm-2 and 6 mA cm-2. The latter has been combined with a BOD based biocathode to form a biofuel cell exhibiting maximum power densities of 140 µW cm-2. The last part of this work focuses on the design of a bienzymatic biocathode for O2 reduction. The DET of horseradish peroxidase (HRP) was first investigated and optimized by modification of the CNTs with pyrenes derivatives. The combination of the HRP with the GOx on the same electrode enables an efficient reduction of O2 in a 2-step process. The biocathode could exhibit maximum currents densities of 200 µA cm-2. This cathode along with the previous GDH bioanode formed a biofuel cell functional in physiological conditions and 10 mM NAD+ showing maximum power densities of 57 µW cm-2.

Para a bioeletrocatálise de oxidação de etanol, a alteração da superfície eletródica e a otimização do processo de imobilização enzimática se fazem necessárias. Neste cenário, as fibras flexíveis de carbono (FFC) merecem destaque, pois além de sua superfície ser facilmente modificada devido à presença de carbono sp2, as mesmas possuem alta resistência mecânica e elasticidade, combinadas com a alta condutividade elétrica e térmica. Nesta tese de doutorado, apresenta-se como obter bioeletrodos de FFC modificadas com a enzima álcool desidrogenase (ADH) NAD-dependente, visando também aprimorar a oxidação da coenzima NADH (dinucleotídeo de nicotinamida e adenina). Os resultados mostram que quando as FCF são previamente submetidas a um tratamento oxidativo em meio ácido (KMnO4/H2SO4), obtém-se bioeletrodos estáveis, robustos e com alta área superficial. Além disso, observou-se que esses eletrodos possuem grupos funcionais contendo oxigênio que auxiliam na bioeletrocatálise de oxidação do etanol. Presume-se que presença de grupos quinonas seja responsável por facilitar a regeneração da coenzima, ou seja, estes grupos atuam decisivamente na oxidação do NADH. A alta qualidade dos bioeletrodos possibilitou manter a atividade catalítica da ADH por longo prazo, propriedade essa crucial para o estudo da oxidação do etanol acoplada à espectrometria de massas (DEMS). Devido a este estudo, foi possível observar concomitantemente a regeneração da coenzima (NADH -> NAD+) e a geração de acetaldeído como produto de bioeletroxidação do etanol, ambos em estado estacionário. Em suma, o estudo aqui apresentado introduz uma abordagem que combina não só o desenvolvimento de fibras de carbono tratadas quimicamente para aplicação em bioeletrocatálise, mas também um foco inédito no acoplamento entre a espectrometria de massas e a bioeletroquímica para a resolução de mecanismos enzimáticos. / Regarding the bioelectrocatalysis of the ethanol oxidation, the electrodic surface modification and the optimization of enzymatic immobilization are necessary. In this scenario, the flexible carbon fibers (FCF) are noteworthy, because besides their surface can be modified in an easy way due the presence of carbon sp2, they have high mechanical resistance and elasticity, combined with high electrical and thermal conductivity. In this doctoral thesis, it is presented how to obtain bioelectrodes of FFC modified with the enzyme alcohol dehydrogenase (ADH) NAD-dependent, as well as to improve the oxidation of the coenzyme NADH (nicotinamide adenine dinucleotide). The results show that when FCF is previously submitted to an oxidative treatment in acidic medium (KMnO4/H2SO4), stable, robust and high surface area bioelectrodes are obtained. In addition, it was observed that these electrodes have oxygen-containing functional groups that improve the bioelectrocatalysis of ethanol oxidation. There is proposed that the presence of quinone groups is responsible for facilitating the regeneration of the coenzyme, i. e., these groups act decisively in the oxidation of NADH. The high quality of the bioelectrodes allowed it to maintain the catalytic activity of the ADH for long term, property crucial for the study of the oxidation of ethanol coupled to mass spectrometry (DEMS). By using DEMS, there were possible to observe coenzyme regeneration and the generation of acetaldehyde as a bioelectrooxidation product of ethanol, both at steady state, which were simultaneously observed. In summary, the present study introduces an to an approach that combines not only the development of chemically treated carbon fibers for application in bioelectrocatalysis, but also an unprecedented focus on the coupling between mass spectrometry and bioelectrochemistry for the resolution of enzymatic mechanisms.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21836号 / 農博第2349号 / 新制||農||1068(附属図書館) / 学位論文||H31||N5208(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 三芳 秀人, 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