京都大学 / 新制・課程博士 / 博士(農学) / 甲第23243号 / 農博第2450号 / 新制||農||1084(附属図書館) / 学位論文||R3||N5333(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 三芳 秀人, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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

Ce mémoire est consacré à l’optimisation de la connexion enzymatique pour l’oxydation du glucose et la réduction du dioxygène dans une matrice de nanotubes de carbone (CNTs) sous forme de compression dans les biopiles à glucose, et à l’assemblage de biopiles dans un système à flux. Dans un premier temps, le transfert électronique indirect de la glucose oxydase (GOx) et de la glucose déshydrogénase FAD-dépendante (FADGDH) est optimisé dans une matrice nanostructurée de CNTs contenant différents médiateurs rédox. Ces bioanodes ont pu être combinées avec des biocathodes similaires à bases d’enzymes à cuivre, la laccase (Lac) et la bilirubine oxydase (BOD). La biopile GOx-NQ/Lac présente une puissance de l’ordre de 150 µW sous 150 mmol.L-1 de glucose et la biopile GOx-NQ/BOD orientée par la PP IX, quant à elle, possède une puissance de l’ordre de 0,5 mW sous 5 mmol.L-1 de glucose. Cette biopile présente une très bonne alternative à l’implantable ou à l’alimentation d’un appareil électronique à faible demande énergétique. La partie suivante concerne l’élaboration d’un design de biopile à flux optimisant la diffusion du substrat à l’intérieur de la bioélectrode. De ce fait, plusieurs systèmes de biopiles GOx-NQ/BOD à flux de substrat ont été étudiés. La configuration de flux traversant a permis d’obtenir une puissance de l’ordre de 1 mW sous 5 mmol.L-1 de glucose et oxygène dissous. La possibilité d’utiliser cette pile en décharge continue ou en cycle de charge/décharge a été étudiée. Ce système de biopile à flux de glucose a permis également d’associer plusieurs biopiles en série ou en parallèle. Ainsi, l’alimentation d’un minuteur et d’un test d’ovulation a pu être réalisée à l’aide de biopiles associées en série. D’autre part, l’utilisation d’un circuit de gestion de l’énergie a permis d’alimenter un capteur de température en stockant l’énergie produite par deux biopiles connectées en série. Cette partie se consacre également à une biopile basée sur l’association de la HRP à la cathode et la GOx-NQ à l’anode. Ce système est très intéressant puisque grâce à la maitrise du sens du flux de notre substrat, le peroxyde d’hydrogène formé par l’anode peut être alors consommé par la cathode. Cette pile s’est montrée parfaitement opérationnelle en condition physiologique et a abouti à l’obtention de puissances de l’ordre de 0,8 mW. / This work is devoted to the optimization of the enzymatic connection for the oxidation of glucose and the reduction of dioxygen in a matrix of carbon nanotubes (CNTs) in the form of compression in glucose biofuel cells, and the assembly of biofuel cells in a flow system.First, mediated electron transfer of glucose oxidase (GOx) and FAD-dependent glucose dehydrogenase (FADGDH) is optimized in a nanostructured CNTs matrix containing different redox mediators. These bioanodes could be combined with similar biocathodes with copper enzyme bases, laccase (Lac) and bilirubin oxidase (BOD). The GOx-NQ/Lac biofuel cell has a power of the order of 150 μW under 150 mmol L-1 of glucose and the biofuel cell GOx-NQ/BOD oriented by the PP IX, order of 0.5 mW under 5 mmol L-1 of glucose. This biofuel cell presents a very good alternative to the implantable or to the supply of an electronic device with low energy demand.The next part concerns the development of a biofuel cell design with flux optimizing the diffusion of the substrate inside the bioelectrode. As a result, several GOx-NQ/BOD flow systems have been studied. The flow-through configuration made it possible to obtain a power of the order of 1 mW under 5 mmol L-1 of glucose and dissolved oxygen. The possibility of using this battery in continuous discharge or in charge/discharge cycle has been studied. This biofuel cell system with a glucose flow has also made it possible to associate several biofuel cells in series or in parallel. Thus, the power supply of a timer and an ovulation test could be realised using associated biofuel cells in series. The use of an energy management circuit made it possible to supply a temperature sensor by storing the energy produced by two biofuel cells connected in series.Moreover, this part is about another biofuel cell based on the association of HRP with the cathode and the GOx-NQ at the anode. This system is very interesting because, thanks to the control of the flow direction of our substrate, the hydrogen peroxide formed by the anode can then be consumed by the cathode. This stack was perfectly operational in physiological condition and led to the achievement of powers of the order of 0.8 mW.

The objectives of the doctoral thesis are following: (i) to design carbohydrate/oxygen enzymatic biofuel cells (EBFCs); (ii) to determine the factors limiting the performance of EBFCs; (iii) to characterise the bioelectrochemical properties of the enzymes adsorbed at conductive nanostructures and evaluate the viscoelasticity of these nanostructures. In this work 5-amino-1,10-phenanthroline (5AP) has been found to be the best redox mediator for glucose oxidase (GOx) enzyme among five studied phenanthroline derivatives with different functional groups. Later the 5AP cross-linked with GOx enzyme on a graphite rod electrode (GRE) was employed as an anode while GRE with co-immobilised horseradish peroxidase (HRP) and GOx was exploited as a cathode in order to design a glucose powered EBFC. A positively charged bi-functional thiol, N-(6-mercapto)hexylpyridinium (MHP), was exploited to electrostatically attach the cellobiose dehydrogenase (CDH) enzymes from Corynascus thermophilus (CtCDH) and Humicola insolens (HiCDH) to the gold nanoparticle (AuNP) surface. This coupling enabled a sufficient direct electron transfer between the enzymes and the AuNP-modified gold surface. Therefore, the HiCDH enzyme, showing better performance characteristics, was employed as an anodic biocatalyst in the designing of a mediatorless carbohydrate (glucose or lactose)/oxygen EBFC. The biocathode of the EBFC was based on bilirubin oxidase from Myrothecium verrucaria directly immobilised on the surface... [to full text] / Disertacinio darbo tikslai: (i) sukonstruoti fermentinius angliavandenių/deguonies biokuro elementus (FBKE); (ii) nustatyti FBKE veikimą ribojančius faktorius; (iii) apibūdinti fermentų, adsorbuotų laidžiose nanostruktūrose, bioelektrokatalizines charakteristikas ir įvertinti šių nanostruktūrų viskoelastines savybes. 5-amino-1,10-fenantrolino (5AF) junginys, iš penkių šiame darbe tirtų fenantrolinų junginių besiskiriančių funkcinėmis grupėmis, įvertintas kaip geriausias elektronų pernašos (EP) tarpininkas gliukozės oksidazės (GO) katalizuojamoje heterogeninėje reakcijoje. 5AF junginys kartu su GO fermentu (5AF/GO) buvo panaudotas anodinio elektrodo konstrukcijoje, o atitinkamai bifermentinė krienų peroksidazės (KP) ir GO sistema (KP/GO) – katodinio elektrodo konstrukcijoje. Šie elektrodai panaudoti gliukozės FBKE kūrimui. Teigiamą krūvį turintis bifunkcinis tiolinis N-(6-merkapto)heksilopiridinio (MHP) junginys panaudotas fermentų imobilizacijai aukso nanodalelių (AuND) paviršiuje elektrostatinės sąveikos būdu. AuND paviršiuje imobilizuoti celiobiozės dehidrogenazės (CDH) fermentai, išskirti iš Corynascus thermophilus (CtCDH) ir Humicola insolens (HiCDH) kamienų, sudarė fermentas-AuND sąsają įgalinančią tiesioginę EP. HiCDH fermentas kaip biokatalizatorius pritaikytas anodinio elektrodo konstrukcijoje AuND/MHP/HiCDH kuriant tiesiogine EP paremtus angliavandenių (gliukozės, laktozės)/deguonies FBKE. Bilirubino oksidazė (BO), tiesiogiai imobilizuota AuND paviršiuje (AuND/BO)... [toliau žr. visą tekstą]

Disertacinio darbo tikslai: (i) sukonstruoti fermentinius angliavandenių/deguonies biokuro elementus (FBKE); (ii) nustatyti FBKE veikimą ribojančius veiksnius; (iii) apibūdinti fermentų, adsorbuotų laidžiose nanostruktūrose, bioelektrokatalizines charakteristikas ir įvertinti šių nanostruktūrų viskoelastines savybes. 5-amino-1,10-fenantrolino (5AF) junginys, iš penkių šiame darbe tirtų fenantrolinų junginių besiskiriančių funkcinėmis grupėmis, įvertintas kaip geriausias elektronų pernašos (EP) tarpininkas gliukozės oksidazės (GO) katalizuojamoje heterogeninėje reakcijoje. 5AF junginys kartu su GO fermentu (5AF/GO) buvo panaudotas anodinio elektrodo konstrukcijoje, o atitinkamai bifermentinė krienų peroksidazės (KP) ir GO sistema (KP/GO) – katodinio elektrodo konstrukcijoje. Šie elektrodai panaudoti gliukozės FBKE kūrimui. Teigiamą krūvį turintis bifunkcinis tiolinis N-(6-merkapto)heksilopiridinio (MHP) junginys panaudotas fermentų imobilizacijai aukso nanodalelių (AuND) paviršiuje elektrostatinės sąveikos būdu. AuND paviršiuje imobilizuoti celiobiozės dehidrogenazės (CDH) fermentai, išskirti iš Corynascus thermophilus (CtCDH) ir Humicola insolens (HiCDH) kamienų, sudarė fermentas-AuND sąsają įgalinančią tiesioginę EP. HiCDH fermentas kaip biokatalizatorius pritaikytas anodinio elektrodo konstrukcijoje AuND/MHP/HiCDH kuriant tiesiogine EP paremtus angliavandenių (gliukozės, laktozės)/deguonies FBKE. Bilirubino oksidazė (BO, tiesiogiai imobilizuota AuND paviršiuje (AuND/BO)... [toliau žr. visą tekstą] / The objectives of the doctoral thesis are following: (i) to design carbohydrate/oxygen enzymatic biofuel cells (EBFCs); (ii) to determine the factors limiting the performance of EBFCs; (iii) to characterise the bioelectrochemical properties of the enzymes adsorbed at conductive nanostructures and evaluate the viscoelasticity of these nanostructures. In this work 5-amino-1,10-phenanthroline (5AP) has been found to be the best redox mediator for glucose oxidase (GOx) enzyme among five studied phenanthroline derivatives with different functional groups. Later the 5AP cross-linked with GOx enzyme on a graphite rod electrode (GRE) was employed as an anode while GRE with co-immobilised horseradish peroxidase (HRP) and GOx was exploited as a cathode in order to design a glucose powered EBFC. A positively charged bi-functional thiol, N-(6-mercapto)hexylpyridinium (MHP), was exploited to electrostatically attach the cellobiose dehydrogenase (CDH) enzymes from Corynascus thermophilus (CtCDH) and Humicola insolens (HiCDH) to the gold nanoparticle (AuNP) surface. This coupling enabled a sufficient direct electron transfer between the enzymes and the AuNP-modified gold surface. Therefore, the HiCDH enzyme, showing better performance characteristics, was employed as an anodic biocatalyst in the designing of a mediatorless carbohydrate (glucose or lactose)/oxygen EBFC. The biocathode of the EBFC was based on bilirubin oxidase from Myrothecium verrucaria directly immobilised on the surface... [to full text]

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.

Diferentes estratégias têm sido propostas a fim de melhorar o desempenho dos bioeletrodos utilizados nas biocélulas a combustíveis e nos biossensores. Por examplo, a funcionalização de eletrodos de carbono tem sido feita para esse fim. Neste estudo, propomos o desenvolvimento de fibras flexíveis de carbono (FFCs) funcionalizadas com grupos quinona e modificados com álcool desidrogenase (ADH) NAD-dependente para obter bioeletrodos para uma bio-eletrocatálise eficiente de etanol. Grupos quinona na superfície das FFCs foram obtidas utilizando o tratamento oxidativo com permanganato e também pelo ancoramento eletroquímico de antraquinona: ambas metodologias resultaram em bioeletrodos para a eletro-oxidação de NADH que pode aumentar a bio-eletrocatálise do etanol. De acordo dados espectroscópicos, microscópicos, e eletroquímicos, defeitos contendo grupos C=O nos eletrodos de FFCs são atribuídos à melhora na oxidação do NADH, aumentando a bio-eletrocatálise do etanol. Para se investigar o papel dos grupos quinona na eletro-oxidação do NADH, propomos uma configuração experimental baseado na espectroscopia de ressonância paramagnética de elétrons em modo operando (operando EPR). Com essa técnica, fomos capaz de mostrar a correlação entre o número de elétrons livres desemparelhados, a concentração superficial de quinonas e a oxidação do NADH com controle eletroquímico. Correlação para a concentração de spins revela um aumento no número de elétrons desemparelhados livres com o aumento do sobrepotencial aplicado e a oxidação do NADH, o que corrabora com a hipótese de que grupos quinona podem afetar a eletrocatálise rumo à oxidação do NADH a NAD+. É vislumbrado que operando EPR pode fornecer infromação útil para provar a dinâmica da transferência de elétrons em superfície de carbono e possa ser extendida a outros sistemas bioeletroquímicos. / There are several strategies to improve the performance of bioelectrodes applied in biosensors and biofuel cells. For instance, surface functionalization of the carbon-based electrodes has been used to this intend. Herein, we propose the development of flexible carbon fibers (FCFs) functionalized with quinone groups and modified with NAD-dependent alcohol dehydrogenase (ADH) to obtain bioelectrodes for efficient ethanol bio-electrocatalysis. Quinones groups on FCFs surfaces were obtained by using oxidative treatment with permanganate, and also by electrochemical grafting of anthraquinone: both these methodologies result in bioelectrodes for the electro-oxidation of NADH that can improve the ethanol bio-electrocatalysis. Based on spectroscopic, microscopic and electrochemical data, defects containing C=O groups on FCFs electrodes are attributed to improve the NADH oxidation, enhancing the ethanol bio-electrocatalysis. In order to investigate the role of quinone groups on the NADH electro-oxidation, we propose an experimental setup based on operando electron paramagnetic resonance spectroscopy (operando EPR). With this technique, we are able to show a correlation among the number of free unpaired electrons, surface concentration of quinones and NADH oxidation under electrochemical control. Correlation for the spin concentration reveals an increasing number of free unpaired electrons with increasing applied overpotential and NADH oxidation, which corroborates the hypothesis that quinone groups can act as electrocatalysts towards the oxidation of NADH to NAD+. It is glimpsed that operando EPR can provide useful information in probing the electron transfer dynamics on a carbon surface and may be extended to others bioelectrochemical systems.

Biocélulas a combustível (BFCs) são definidas como dispositivos bioeletroquímicos que utilizam componentes biológicos, como enzimas ou microrganismos, para converter energia química em energia elétrica. Neste estudo, reporta-se o desenvolvimento de um bioeletrodo composto por fibras flexíveis de carbono (FFC) modificadas com a enzima álcool desidrogenase (ADH), o qual foi utilizado juntamente com a levedura Saccharomyces cerevisiae , sendo enzima e microrganismos usados como biocatalisadores cooperativos para bioprodução e oxidação de etanol. A glicose é oxidada pelas células de levedura sob condições anaeróbias, e o etanol formado pela fermentação alcóolica é, em seguida, oxidado a acetaldeído pela enzima ADH. A oxidação de etanol pela ADH resulta ainda, na redução da molécula de nicotinamida adenina dinucleotídeo, NAD+ a NADH. Posteriormente, o NADH formado nessa reação é eletroquimicamente oxidado a NAD+ na superfície do bioeletrodo de FFC baseado em ADH (FFC-ADH). Avaliou-se a influência da temperatura e do pH na bioeletrocatálise de etanol pela ADH e a melhor resposta obtida foi em 40 ºC e pH 8,5. Além disso, obteve-se uma excelente correlação linear entre os valores de concentração de etanol e densidade de corrente, indicando que a resposta bioeletrocatalítica da ADH é diretamente proporcional à concentração de etanol produzido a partir da fermentação. O conceito de que microrganismos e enzimas podem trabalhar cooperativamente para produzir uma nova classe de bioeletrodos, foi introduzido nesse trabalho. Por fim, demonstrou-se, que o bioeletrodo cooperativo pode ser aplicado com sucesso em uma BFC, utilizando o biocátodo de difusão de gás contendo a enzima bilirrubina oxidase (BOx) imobilizada em sua superfície. / Biofuel cells (BFCs) are defined as bioelectrochemical devices that use biological components, such as enzymes or microorganisms, to convert chemical energy into electric energy. In this study, we report the development of a bioelectrode composed of flexible carbon fibers (FCF) modified with the enzyme alcohol dehydrogenase (ADH) together with Saccharomyces cerevisiae yeast, being enzyme and microorganisms used as cooperative biocatalysts for bioproduction and oxidation of ethanol. Glucose is oxidized by the yeast cells in anaerobic conditions, and ethanol is produced through alcoholic fermentation and then it is oxidized to acetaldehyde by the ADH enzyme. The ethanol oxidation by ADH also results in the reduction of the nicotinamide adenine dinucleotide molecule, NAD+ to NADH. Subsequently, the NADH produced in this reaction is electrochemically oxidized to NAD+ on the surface of the FCF bioelectrode based on ADH (FCF-ADH). The influence of temperature and pH on the bioelectrocatalysis of ethanol was evaluated and the best performance was found at 40 ºC and pH 8.5. Additionally, the results demonstrated an excellent linear correlation between the ethanol concentration and the current generated, which indicates that the bioelectrocatalytic response of ADH is directly proportional to concentration of ethanol produced from the fermentation. The present study has introduced the concept that microorganisms and enzymes can work cooperatively to produce a new class of bioelectrodes. Finally, it has been demonstrated that the cooperative bioelectrode can be applied successfully to BFC using a gas-diffusion biocathode containing the bilirubin oxidase enzyme (BOx) immobilized on its 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.

Dans la nature, la réduction du dioxygène est catalysée par des enzymes de la famille des oxydoréductases. A l’heure actuelle, ces protéines spécifiques et efficaces sont envisagés comme biocatalyseurs au sein de biopile enzymatique. Dans ce contexte, l’optimisation de l’orientation et de la connexion d’oxydases multi-cuivre (MCOs) pour la réduction d’O2 sur des matrices de nanotubes carbone (CNTs) fonctionnalisées a été étudiée. Dans un premier temps, le transfert électronique direct de la laccase est optimisé par la fonctionnalisation non covalente de CNTs par divers dérivés hydrophobes. La dynamique moléculaire ainsi que la modélisation électrochimique ont permis la rationalisation des performances des différentes biocathodes développées. Dans une seconde approche, la modification spécifique par des groupements pyrène de la surface de laccases modifiées par mutagénèse a également été envisagée. La fonctionnalisation supramoléculaire de CNTs par des feuillets de graphène fonctionnalisés d’une part, et par des nanoparticules d’or d’autre part, a également permis de favoriser la connexion de laccases. La seconde partie présente l’élaboration d’autres types de biocathodes basées sur la connexion directe de bilirubines oxydases. Plusieurs stratégies de fonctionnalisation covalente et non covalente de CNTs ont été envisagées. Les différentes biocathodes élaborées par l’assemblage supramoléculaire de MCOs et de matériaux nanostructurés délivrent des densités de courant de réduction du dioxygène de plusieurs mA cm-2. Ces nouvelles bioélectrodes combinées à une bioanode qui catalyse l’oxydation du glucose ont permis le développement de biopiles enzymatiques glucose/O2 délivrant des densités maximales de puissances allant de 250 µW cm-2 à 750 µW cm-2 selon les conditions expérimentales. Enfin une bioanode à base d’une hydrogénase hyperthermophile a été développée et associée à une biocathode à base de bilirubine oxydase pour former un nouveau design de biopile H2/O2. Au sein de ce dispositif, la biocathode à diffusion de gaz réduit directement l’oxygène provenant de l’air, ce qui permet de s’affranchir de l’utilisation d’une membrane séparatrice tout en protégeant l’hydrogénase de sa désactivation en présence d’oxygène. Cette nouvelle biopile délivre une densité maximale de puissance de 750 µW cm-2. / The reduction of oxygen is realized in nature by oxidoreductase enzymes. Currently, these highly specific and efficient proteins are considered as biocatalysts for the development of biofuel cells. In this context, optimizing the orientation and the connection of multicopper oxidase (MCOs) for the reduction of O2 on functionalized carbon nanotubes was studied. In the first part of this manuscript, direct electron transfer of laccase is assessed and optimized by the non-covalent functionalization of CNTs by various hydrophobic derivatives. Electrochemical modeling and molecular dynamics enabled the rationalization of the developed biocathodes efficiency. In a second approach, the specific modification by pyrene moieties of laccases surface modified by protein engineered has also been considered. Additionally, supramolecular functionalization of CNTs by modified graphene sheets and gold nanoparticles also helped to promote laccase connection. The second part presents the development of other types of biocathodes based on the direct connection of bilirubin oxidase. Several strategies of covalent and non-covalent CNTs functionalization have been considered. The different biocathodes developed by the supramolecular assembly of nanostructured materials and MCOs delivered current density of several mA cm-2 for oxygen reduction. These new bioelectrodes combined with a bioanode which catalyzes the glucose oxidation have enabled the development of glucose/O2 enzymatic biofuel cells; delivering maximum power densities from 250 µW cm-2 to 750 µW cm-2 depending on the experimental conditions. Finally a hyperthermophilic hydrogenase based bioanode was developed and associated with a bilirubin oxidase-based biocathode to form a new design of H2/O2 biofuel cell. Within this device, the gas diffusion biocathode directly reduces oxygen from the air, which eliminates the use of a separation membrane while protecting the hydrogenase from its deactivation in the presence oxygen. This new biofuel cell delivers a maximum power density of 750 µW cm-2.