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Dissertations / Theses on the topic 'Electrochemistry of enzymes'
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Whitaker, Richard George. "The electrochemistry of redox enzymes." Thesis, University of Warwick, 1989. http://wrap.warwick.ac.uk/4235/.
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The work presented in this thesis is of two types. Firstly methods for the electrochemical immobilisation of redox enzymes in organic polymers are described. The electrochemical monitoring of the immobilised enzyme reaction by detection of one of the enzyme's products is discussed, and the results obtained for such a system under a variety of experimental conditions are presented. A good understanding of the way in which such a system operates' was obtained by using a specially developed kinetic model., This model is explained fully in the theory chapter of this thesis. A variety of organic polymers were used in the electrochemical immobilisation process, with varying degrees of success. The flexibility of this approach is demonstrated by the use of a variety of immobilisation matrices and also by the development of bienzyme and bilayer devices. The final experimental chapter presents work on the covalent modification of redox enzymes with a variety of, redox centres based. on ferrocene. Although attempts to electrochemically immobilise a modified enzyme were not successful, some interesting information about the kinetic behaviour and stability of a series Of modified enzymes was obtained. An indication of possible work forming an extension to this thesis is given in the final part of this thesis. The electrochemical immobilisation techniques and the procedure for covalently modifying, enzymes using electroactive, groups are relatively recent ideas. Much work remains to be done before a better understanding of these systems is gained.
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Hunt, Nicholas Imber. "Biological electrochemistry." Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386592.
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De, Oliveira Pedro M. A. "Studies of enzymes by electrochemistry and atomic force microscopy." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298717.
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Xu, Lang. "Investigating the current/voltage/power/stability capabilities of enzyme-based membrane-less hydrogen fuel cells." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:efef7124-3444-4531-872b-2ee8868e0aa0.
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Fuel cell is a device that can directly convert chemical energy into electrical energy. For low-temperature fuel cells, catalysts are required. Fuel cells using Pt-based or other non-biological materials as catalysts are known as conventional fuel cells. Inspired from Nature, enzymes can be used as catalysts in fuel cells known as enzyme-based fuel cells. The conventional and enzymatic fuel cells share the same underlying electrochemical principles, while enzyme-based fuel cells have their intrinsic advantages and disadvantages due to enzyme properties. The objective of this thesis is to investigate the current/voltage/ power/stability capabilities of enzyme-based membrane-less H2 fuel cells in order to design the enzymatic fuel cells with improved performance. This thesis presents a facile, effective method for the construction of 3D porous carbon electrodes. The 3D porous carbon electrodes are constructed by compacting suitable carbon nanomaterials into discs. The 3D porous carbon electrodes, with large roughness, high specific surface area, and optimized pore size distribution, are able to increase the loading density of enzymes, that is, reaction sites per unit geometric electrode area. The high loading density of enzymes can result in the high current/power density of the enzyme-based membrane-less H2 fuel cells. Moreover, the large enzyme loading can bring about the improvement in fuel cell stability because current becomes limited by mass transport of dissolved gases rather than enzyme immobilization so that neither inactivation nor desorption of enzymes would influence the current output. Based on one type of 3D porous carbon electrodes, the maximum power density of enzyme-based membrane-less H2 fuel cells has increased to the mW•cm2 level by at least one order of magnitude and the half-life has also increased from several hours to one week. This thesis presents a method for the increase in power density otherwise limited by low cathodic currents due to meagre O2 in non-explosive H2-rich H2-air mixtures. The power density of enzyme-based membrane-less H2 fuel cells can be increased by re-proportioning cathode/anode geometric area ratio to balance the cathodic and anodic currents under such an unusual H2-air mixture. This thesis also demonstrates that the 3D porous carbon electrode can improve the apparent O2 tolerance of anodic catalysts – hydrogenases, which are very important for the fuel cell performance. The degrees of apparent O2 tolerance for both O2-tolerant and O2-sensitive [NiFe]-hydrogenases are greatly increased based on the 3D porous carbon electrodes, so that even an O2-sensitive [NiFe]-hydrogenase can be used as an anodic catalyst in the enzyme-based membrane-less H2 fuel cell under a non-explosive H2-rich H2-air mixture. This thesis presents a design of a test bed in which series and parallel connections of sandwich-like electrode stacks can be varied. The fuel cell test bed has demonstrated low-loss interconnects and efficient stack configuration. Operated under a non-explosive H2-air mixture containing only 4.6% O2 at 20 °C, the maximum volume power density of the fuel cell test bed exceeds 2 mW•cm3, capable of powering electronic gadgets, which is a good demonstration of electricity that originates from the buried active sites of enzymes and is transmitted by long-range electron hopping in accordance with Marcus theory.
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Owens, Zachary J. "The purification and electrochemistry of his-tagged photosystem II." [Denver, Colo.] : Regis University, 2009. http://165.236.235.140/lib/ZOwens2009.pdf.
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Goldet, Gabrielle. "Electrochemical investigations of H2-producing enzymes." Thesis, University of Oxford, 2009. http://ora.ox.ac.uk/objects/uuid:696e5b9d-a80f-493e-85d4-0954be499b72.
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Hydrogenases are a family of enzyme that catalyses the bidirectional interconversion of H+ and H2. There are two major classes of hydrogenases: the [NiFe(Se)]- and [FeFe]-hydrogenases. Both of these benefit from characteristics which would be advantageous to their use in technological devices for H2 evolution and the generation of energy. These features are explored in detail in this thesis, with a particular emphasis placed on defining the conditions that limit the activity of hydrogenases when reducing H+ to produce H2. Electrochemistry can be used as a direct measure of enzymatic activity; thus, Protein Film Electrochemistry, in which the protein is adsorbed directly onto the electrode, has been employed to probe catalysis by hydrogenases. Various characteristics of hydrogenases were probed. The catalytic bias for H2 production was interrogated and the inhibition of H2 evolution by H2 itself (a major drawback to the use of some hydrogenases in technological devices to produce H2) was quantified for a number of different hydrogenase. Aerobic inactivation of hydrogenases is also a substantial technological limitation; thus, inactivation of both H2 production and H2 oxidation by O2 was studied in detail. This was compared to inhibition of hydrogenases by CO so as to elucidate the mechanism of binding of diatomic molecules and determine the factors limiting inactivation. This allows for a preliminary proposal for the genetic redesigning of hydrogenases for biotechnological purposes to be made. Finally, preliminary investigation of the binding of formaldehyde, potentially at a site integral to proton transfer, opens the field for further research into proton transfer pathways, the structural implications thereof and their importance in catalysis.
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Yorke, Jake. "Engineering cytochrome P450BM3 into a drug metabolising enzyme." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:92dcddfe-b3fc-46e8-9e5e-77910fb03783.
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Directed evolution studies by Whitehouse et al. identified several variants of P450BM3 (CYP102A1) with enhanced substrate oxidation rates across a range of substrates. This thesis describes the use of these ‘generic accelerator’ variants, in combination with selectivity altering mutations to engineer P450BM3¬ for the oxidation of pharmaceuticals. Using engineered variants the non-steroidal anti-inflammatory drug diclofenac was metabolised to the primary human metabolites 4′- and 5-hydroxydiclofenac, with total conversion of 2 mM substrate by 5 μM enzyme. The local-anaesthetic lidocaine and the steroid testosterone were similarly metabolised to human metabolites. This is the first report of a drug compound being totally converted to the human metabolites by a P450BM3 variant, and is also the first report of lidocaine metabolism by a P450¬BM3 variant. The engineered variants are akin to CYP3A4, the primary human drug metabolising enzyme, as they show activity towards a range of compounds including anionic, cationic and neutral drugs. This range of activity is at the expense of NADPH coupling, which remains low with these substrates. In order to more fully understand the origin of the rate enhancing properties of the generic accelerator variants, spectroelectrochemical, stopped-flow and kinetic studies were performed. A custom optically transparent thin layer electrode system was designed and fabricated for use in spectroelectrochemical titrations. A spectroelectrochemical cell and gold mesh electrode were designed and used in spectroelectrochemical investigations of P450BM3 variants, as well as other P450s and their redox partners. These spectroelectrochemical, stopped-flow and kinetic studies, in combination with X-ray crystal structures provided insight into the origin of the rate enhancing properties of these enzymes and supplied the first example of the complete characterization of the thermodynamic and kinetic properties of WT and mutant P450BM3 for the oxidation of a non-natural substrate. The generic accelerator variants are, in the resting state, in a more catalytically ready conformation than the WT enzyme, and reorganization energy barriers appear to be lowered, so that fewer substrate-induced structural changes are required to promote electron transfer and initiate the catalytic cycle.
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Jarrar, Haytem. "Bioélectrodes enzymatiques pour des applications en biocapteurs et en biopiles." Thesis, Montpellier, Ecole nationale supérieure de chimie, 2011. http://www.theses.fr/2011ENCM0017/document.
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La principale originalité de ce travail est la mise en œuvre de deux voies d'immobilisation du biorécepteur sur différents matériaux d'électrodes. Dans un premier temps, nous avons démontré que le polyneutral red (PNR) représente une bonne matrice de rétention pour les enzymes. De plus, de part ses propriétés de médiation vis-à-vis des enzymes et principalement de leur cofacteur (NAD/FAD), ce polymère permet une connexion intime entre le site actif de l'enzyme et l'électrode. L'ensemble de ces caractéristiques nous a permis de mettre en œuvre une bioélectrode applicable en tant qu'anode d'un biocapteur à glucose et d'une cellule de biopile à combustible. Dans un second temps, la glocose oxydase a été immobilisée de façon covalente sur une électrode. L'électro-oxydation de l'éthylène a été menée sur les électrodes de carbone vitreux pour obtenir des fonctions amines. La voie proposée est simple, rapide et efficace. Puis, la glucose oxydase a été greffée avec succès par la méthode EDC / NHS sur les fonctions amines après optimisation des conditions de pH. Ces bioélectrodes ont ensuite été testées en tant que biocapteur à glucose montrant une bonne sensibilité de glucose avec une bonne stabilité sur une période de 4 semaines ce qui prouve l'efficacité de la méthode de greffage pour des applications de détection et dosageThe main originality of this work is the development of two-way to immobilize a bioreceptor on different electrode materials. Initially, we demonstrated that the polyneutral red (PNR) is a good matrix for retaining enzymes. In addition, its properties of mediation towards enzymes and mainly their cofactor (NAD / FAD), this polymer provides an intimate connection between the active site of the enzyme and the electrode. All these features allowed us to develop an bioelectrodes as the anode of a biosensor for glucose and a fuel cell biopile. In a second step, the glocose oxidase was covalently immobilized on an electrode. The electro-oxidation of ethylene diamine was carried out on glassy carbon electrodes to obtain amine functions. This proposed way is simple, fast and efficient. Then, glucose oxidase was successfully grafted by the method EDC / NHS on amine functions after the optimization of pH conditions. These bioelectrodes were then tested as glucose biosensor and showed good sensitivity with good stability over a period of 4 weeks which proves the effectiveness of the grafting method for detection and assay applications
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Wang, Vincent Cho-Chien. "New insights into enzymatic CO₂ reduction using protein film electrochemistry." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:f1061854-f6b8-4562-81e0-968c80e1da3a.
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Carbon monoxide dehydrogenase (CODH) is known to catalyze CO oxidation and CO₂ reduction reversibly with the minimal overpotential. A great advantage of protein film electrochemistry (PFE) is its ability to probe catalysis over a wide range of potentials, especially in the low potential region required for CO₂ reduction. CODH I and CODH II from Carboxydothermus hydrogenoformans(Ch) and the composite enzyme acetyl-CoA synthase/carbon monoxide dehydrogenase (ACS/CODH) from Moorella thermoacetica(Mt) are intensively studied throughout this thesis. The different catalytic redox-states in CODH, Cox (inactive state), Cred1 (for CO oxidation) and Cred2 (for CO₂ reduction) as characterized by spectroscopy, are studied by PFE in the presence of substrate-mimic inhibitors. Cyanide, isoelectronic with CO, mainly inhibits CO oxidation, whereas cyanate, isoelectronic with CO₂, mainly targets CO₂ reduction. Sulfide inhibits CODH rapidly when the potential is more positive than −50 mV, which suggests that sulfide reacts to form a state at the oxidation level of Cox in CODH and is not an activator for CODH catalysis as suggested before. Thiocyanate only partially inhibits CO oxidation. No inhibition of CODH by azide is detected, which is in contrast with previous studies with ACS/CODHMt. The main differences between CODH ICh and CODH IICh are the stronger CO product inhibition and inhibition of CODH IICh by cyanide. These discoveries might shed light on the possible role of CODH IICh,/sub> in biological systems. In comparison with bidirectional (reversible) electrocatalysis by CODH ICh and CODH IICh, only unidirectional electrocatalysis for CO oxidation by ACS/CODHMt is observed with an overpotential of 0.1 V and the electrocatalytic current is much smaller. In order to identify whether ACS influences the performance of CODH, several chemical reagents, such as sodium dodecyl sulfate (which separates CODH and ACS partially), 1, 10-phenanthroline, (which inhibits the active site in ACS) and acetyl-CoA (the product of the reaction carried out by ACS/CODHMt) are added. However, we have yet to observe any electrocatalytic current from CO₂ reduction. Inhibition of ACS/CODHMt by cyanide, cyanate and azide is consistent with previous studies by spectroscopy. Oxygen attack toward the active site in CODH is proved by cyanide protection. The inactive state, Cox can prevent oxygen attack and reductive reactivation restores CODH activity. In contrast, oxygen damages the active site irreversibly when CODH is in the Cred1 state. The new substrate, nitrous oxide (N₂O), isoelectronic with CO₂, is reduced by CODH and acts as the suicide substrate. Finally, hydrogen formation in the direction of CO oxidation and formate formation in the direction of CO₂ reduction by CODH are detected. The small solvent kinetic isotope effect is observed in CO oxidation. These findings suggest metal-hydride should play a role in CODH catalysis, which might provide a new direction to design better catalysts for CO₂ reduction.
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Maerten, Clément. "Bio-inspired self-construction and self-assembly of organic films triggered by electrochemistry." Thesis, Strasbourg, 2016. http://www.theses.fr/2016STRAE045.
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Les architectures moléculaires qui se forment exclusivement sur une surface sont encore rares. L’électrodéposition est un procédé exploitant des « signaux » électriques afin de déclencher et contrôler l’assemblage de films. Récemment, une nouvelle méthode : l’autoconstruction de films en « une étape » par l’utilisation d’un morphogène (un gradient de catalyseur généré depuis une électrode), a attiré l’attention de la communauté scientifique. En effet, elle permet l’auto-assemblage rapide de films polymériques robustes. Cependant, cette technique était limitée à des systèmes basés sur la chimie click du Cu (I). Le but de ce travail était d’étendre cette stratégie à d’autres systèmes en utilisant une approche bio-inspirée. Le concept du morphogène a été appliqué pour développer deux nouveaux systèmes d’autoconstruction déclenchées par électrochimie. Le premier système est basé sur l’autoconstruction covalente de films polymériques induite par l’oxydation d’une molécule organique, inspirée de la moule. Le deuxième est basé sur l’auto-assemblage de films de polyphénols par électro-assemblage par liaisons de coordinations. Enfin, nous avons appliqué ces deux concepts pour immobiliser électrochimiquement une enzyme sur une électrode afin de créer un biosenseurMolecular architectures that spontaneously grow exclusively near a surface are rare. Electrodeposition is a process in which imposed electrical « signals » are employed to direct the assembly of thin films. Recently, a new method based on the one-pot self-construction of films by means of a morphogen (a catalyst gradient generated from a surface) has attracted attention since it allows the quick self-assembly of robust films. Nevertheless, this technique was quite limited to systems based on click chemistry.The purpose of this work was to extend this strategy to other systems using a bio-inspired approach. The one-pot morphogen concept was applied to design two new electro-triggered self-construction concepts. The first one is based on the self-construction of covalent polymer films triggered by mussel-inspired molecule oxidation. The second one is based on the electro-self-assembly of polyphenols films based on ionic bonds coordination. Finally, we tried to apply these concepts in order to electrochemically immobilize an enzyme on an electrode to create a biosensor
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Idris, Zulkifli. "Electrocatalytic cycling of nicotinamide cofactors by Ralstonia eutropha soluble hydrogenase." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:3d458a13-ce61-4ae4-bc93-5a7db3bb371d.
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Nicotinamide cofactors in their reduced and oxidised forms are important redox agents in biology. Of about 3000 dehydrogenases available to date, many require these cofactors for their activity. Dehydrogenases are of interest to chemists as they offer asymmetric catalysis to yield chiral products. The requirement of dehydrogenases for nicotinamide cofactors necessitates research into finding the best way of recycling the oxidised or reduced forms of these cofactors. Electrocatalytic NAD(P)H oxidation and NAD(P)⁺ reduction on standard electrodes is problematic due to unwanted side reactions and high overpotential requirements, but in Nature efficient enzyme catalysts are available to facilitate these reactions. The focus of this Thesis, the Soluble Hydrogenase of R. eutropha (SH) is a multimeric bidirectional hydrogenase that couples H2 oxidation to the reduction of NAD⁺ to NADH. Protein Film Electrochemistry (PFE) has been employed to study NAD⁺-reducing catalytic moieties of the SH for the first time. It is shown that SH subunits on an electrode are able to catalyse NADH oxidation and NAD⁺ reduction efficiently with minimal overpotential, which is significant because in vivo, NAD(H) cycling is coupled to 2H⁺/H₂ cycling and these reactions are closely spaced in potential. Substrate affinities and inhibition constants for the SH, determined using PFE are discussed in the context of the SH function and the related catalytic domains of respiratory Complex I. A range of molecules that are known to inhibit the related Complex I have been investigated for their ability to inhibit the SH moieties: the similarity between inhibition constants is consistent with structural and functional similarity between the SH and Complex I. The ability of the SH moieties to sustain NAD(H) catalysis in the presence of O₂ is also demonstrated and is consistent with the requirement for the SH to function under aerobic conditions and to reactivate the inactivated hydrogenase moiety by supplying low potential electrons from NADH. Engineered variants of the SH, designed to enhance the affinity towards NADP⁺, were investigated for the first time, using PFE. Electrochemical characterisation of the variants is presented and results are discussed alongside findings on the wild type SH. The variants are shown to exhibit NADP⁺ reduction, and to have higher affinity towards NADP⁺ than the wild type SH. The first efficient NADP⁺ reduction and NADPH oxidation is observed for one of the variants on a graphite electrode and the best variant showed a KM of 1.7 mM for NADP⁺. This Thesis also provides evidence for the ability of moieties of the SH to be used in cofactor regeneration systems. Two novel systems are demonstrated. The first involves H₂ driven NADH recycling based on the NAD⁺-reducing moiety of the SH immobilised on graphite particles together with a hydrogenase or platinum, with electrons from H₂ passed from the hydrogenase through the graphite to the NAD⁺-reducing moiety. The second involves an electrode modified with the NAD⁺-reducing moiety of the SH, and is demonstrated as an electrochemical NADH recycling system coupled with NADH-dependent pyruvate reduction to lactate by lactate dehydrogenase. The ability of variants of the SH to catalyse NADP⁺ reduction suggests that it may also be possible to use these systems for recycling NADPH for catalysis of important biotransformation reactions by NADPH-dependent dehydrogenases.
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Hexter, Suzannah Victoria. "Principles of electrocatalysis by hydrogen activating metalloenzymes." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:60aeee02-a16c-4c86-bf48-61306512fa86.
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Hydrogenases catalyse the interconversion of H2 and H+. Protein Film Electrochemistry (PFE), a technique in which a redox enzyme is adsorbed directly onto an electrode, enables a detailed description of the catalytic function of these metalloenzymes to be obtained. Unlike small-molecule electrocatalysts, the hydrogenase active site is surrounded by a protein structure ensuring that it is relatively unperturbed by the electrode surface. In this thesis, PFE is used alongside mathematical modelling to explain differences between [NiFe]- and [FeFe]-hydrogenases, highlighting some important considerations for efficient, reversible electrocatalysis. This thesis probes the unusual reaction between [NiFe]-hydrogenases and cyanide. Through a detailed study utilising PFE, Electron Paramagnetic Resonance (EPR) and Attenuated Total Reflection Infrared spectroelectrochemistry (ATR-IR), it is demonstrated that cyanide promotes the formation of the inactive Ni-B state. Preferred formation of the Ni-B state over more slowly reactivating Unready states is considered an important characteristic of the O2-tolerant class of [NiFe]-hydrogenases. The nature of the Ni-L state, commonly thought to be an artefact formed when a [NiFe]-hydrogenase is exposed to visible light, is probed via EPR and ATR-IR. In this thesis, the Ni-L state is shown to occur in samples of Hydrogenase-1 from Escherichia coli that have not been exposed to visible light, calling into question the true nature of this state. Finally, this thesis details the first study in which PFE is used to investigate the spontaneous incorporation of a synthetic active site mimic complex into apo-hydrogenase. Incorporation into apo-hydrogenase from Chlamydomonas reinhardtii and Clostridium pasteurianum is discussed, in both cases resulting in fully functional [FeFe]-hydrogenase, electrochemically indistinguishable from the native enzyme.
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Roessler, Maxie M. "EPR investigations of iron-sulfur cluster relays in enzymes." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:ac6fa892-f54a-490d-927b-161231f00777.
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Electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for obtaining structural information about chemical centres with unpaired electrons. In complex biological systems, EPR spectroscopy can be used to probe these paramagnetic centres and the long-range interactions between them. This thesis investigates two important types of enzymes, and in particular the role of the iron-sulfur electron-transfer centres they contain, with a variety of EPR techniques. Complex I (NADH:Ubiquinone Oxidoreductase) plays a key role in the electron transfer chain essential to the formation of ATP, and its malfunction has been related to numerous human diseases. It is a giant enzyme that contains the longest relay of iron-sulfur clusters known. EPR experiments conducted on complex I from bovine mitochondria yield crucial insight into the mechanism of efficient long-range electron transfer and bring us a step closer to understanding the functioning of this important complex. Hydrogenases are produced by micro-organisms and catalyse the reversible oxidation of H2. Most hydrogenases, including Hyd-2 from Escherichia coli, are very air-sensitive, but some, including E. coli Hyd-1 and Salmonella Hyd-5, are able to function in the presence of atmospheric levels of O2. Understanding the origins of this 'O2-tolerance' is of paramount importance if hydrogenases are to be exploited in future energy technologies. In this thesis, native E. coli Hyd-1 and Hyd-2, Salmonella Hyd-5, as well as O2-tolerant and O2-sensitive variants of E. coli Hyd-1 are characterised using EPR. The EPR investigations elucidate properties of the active site and the electron-transfer relay and, in conjunction with other techniques, reveal structural and mechanistic details of how a highly unusual iron-sulfur cluster in the electron-transfer chain enables some hydrogenases to sustain catalytic activity in the presence of O2.
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Giroud, Fabien. "Biomatériaux d'électrode appliqués à la réalisation et à la caractérisation d'un biocapteur immunologique et de biopiles enzymatiques." Phd thesis, Université de Grenoble, 2011. http://tel.archives-ouvertes.fr/tel-00690367.
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Ce mémoire est consacré au développement d'un immunocapteur impédancemétrique et de deux biopiles enzymatiques. Premièrement, le poly(pyrrole-NHS) est utilisé pour l'immobilisation successive d'un modèle de la ciprofloxacine (CF) et de l'anticorps dirigé spécifiquement contre CF. La détection est réalisée par la spectroscopie d'impédance électrochimique. Elle détecte le déplacement en solution de l'anticorps. Le seuil de détection est de 1.10-12 g.mL-1. Deuxièmement, la production énergétique est abordée suivant deux approches. La première se base sur l'apparition d'un gradient de pH produit par deux enzymes (la GOx et l'uréase) et converti en f.e.m. en utilisant un couple rédox sensible au pH. La seconde, repose sur les propriétés biocatalytiques de la GOx d'oxyder le glucose et de la polyphénol oxydase de réduire le dioxygène. Cette pile est capable de fonctionner aussi bien in vitro que in vivo. Une fois optimisée, la pile affiche une f.e.m. de 315 mV et une puissance de 27 μW.
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Heath, Rachel Sarah. "Studies of a 'blue' copper oxidase electrocatalyst." Thesis, University of Oxford, 2008. http://ora.ox.ac.uk/objects/uuid:e8359408-d3d4-4fe3-910a-cc69265a1546.
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This thesis concerns the electrochemical investigation of high-potential laccases. These multicopper oxidases are efficient electrocatalysts for the dioxygen reduction reaction. A method for stabilising laccase on a graphite electrode was established. The method involved modification of the graphite surface by diazonium coupling of a 2-anthracene molecule. A laccase ‘film’ adsorbed on this modified surface remained stable for over two months and, typically, the current density for dioxygen reduction was doubled compared to a laccase ‘film’ on an unmodified surface. Protein film voltammetry was used to investigate thermodynamic and kinetic aspects of the electrochemical behaviour of laccase. The effect of inhibitors on the magnitude of reduction current and the position of the wave (related to the overpotential for the reaction) was also studied. Fluoride, chloride and azide showed different modes of inhibition and inhibition constants ranged from micromolar for azide to millimolar for chloride. In cyclic voltammetry experiments it was only in the presence of high concentrations of the inhibitors fluoride, chloride and azide that a non-turnover signal, corresponding to a one electron transfer process, was revealed. The evidence suggested that the non-turnover signal arose from interfacial electron transfer between the electrode and the type 1 or ‘blue’ copper. Evaluation of the peak areas allowed determination of the catalytic rate constant, kcat, as 300 s–1, and the electroactive surface coverage as four pmol cm–2. The rate of interfacial electron transfer was rapid enough to not limit catalysis at high overpotentials. A spectroelectrochemical cell was designed to investigate the behaviour of the type 1 copper in the presence of inhibitors and at different pH values. The inhibitors fluoride, chloride and azide had little effect on the reduction potential of the type 1 copper, but at higher pH values the reduction potential of the type 1 copper was decreased.
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Lonsdale, Thomas. "Dihydrogen driven cofactor recycling for use in bio-catalysed asymmetric organic synthesis." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:a0407748-e34f-410a-9c78-a8316b7a3d4d.
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Asymmetric reductions are used to produce chiral molecules, which are important precursors for the pharmaceutical industry. Bio catalytic reductions often display high enantioselectivity without the cost and toxicity associated with metal catalysis. However, unlike metal catalysts which use H2 directly, many useful redox-enzymes require the hydride donor NADH. NADH is expensive; therefore for a bio-catalytic process to be viable it must be recycled, usually by using a sacrificial carbon based substrate, generating super-stoichiometric amounts of waste. Two different methods for H2-driven NADH recycling are explored in this project: using soluble hydrogenases (SH) and, carbon particles modified with a hydrogenase and an NAD+-reductase moiety. The conductive carbon particles allow electrons from H2-oxidation to be channelled from the hydrogenase to the NAD+ reductase for reduction of NAD+. This project focuses on four main areas. The first looks at using the enzyme-modified particles for the production of high value chiral amines. A yield of >98% was achieved using the enzyme-modified particles with an L alanine dehydrogenase for H2 driven conversion of pyruvate to L-alanine. Moreover, a faster rate of reaction was demonstrated with the L-alanine dehydrogenase immobilised on particles versus with the L-alanine dehydrogenase in solution. The second section focuses on elevated temperature NADH recycling: an SH and an NAD+-reductase from a thermophilic organism were characterised. The NAD+-reductase was subsequently used as part of a system for recycling NADH at >35 °C. When demonstrated in combination with an enoate-reductase a 62 % yield was obtained for the reduction of 2 methyl 2 cyclopentenone. In the third strand SHs and enzyme-modified particles were investigated as recycling systems for NADH analogues. In summary, this thesis expands the scope for application of H2-driven biocatalytic reduction reactions.
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Foster, Carina Elizabeth. "Reactions of [FeFe]-hydrogenase with carbon monoxide and formaldehyde." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:a1d43c4a-861a-4bb0-88ce-2edf4c334f79.
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The use of H2 as an energy carrier has in recent years been identified as a promising future solution to the current energy crisis. Hydrogenases are metalloenzymes found in many microorganisms and are used to catalyse the reversible inter-conversion of protons and H2. These enzymes and their synthetic analogues have been recognised as a way to facilitate the use of H2 as a fuel. A major challenge to the future use of these catalysts is their reactions with small molecule inhibitors, such as oxygen and carbon monoxide. Detailed understanding of the structure and catalytic mechanism of these highly efficient catalysts is vital for the design of bio-inspired functional analogues for use in technological applications. In this thesis electrochemical studies of three [FeFe]-hydrogenases are presented, performed using the technique of protein film electrochemistry. The strong potential dependence of the reaction of these hydrogenases with carbon monoxide and formaldehyde is characterised and rationalised. These studies provide compelling evidence for the formation of a previously ambiguous super-reduced state of [FeFe]-hydrogenase at low potential. This state is shown to be active and stable, and it is proposed that this state is involved in catalytic H2 production. This super-reduced state is shown to have a high affinity for the reversible binding of formaldehyde, but a very low affinity for both carbon monoxide and oxygen. Activation of carbon monoxide inhibited [FeFe]-hydrogenase can be very rapidly induced by the application of a sufficiently reducing potential. These enzymes, considered to be oxygen sensitive, are shown to be extremely tolerant to irreversible oxygen damage at very reducing potentials where the super-reduced state is formed.
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Sabuncu, Sinan. "Investigation of enzymes from the respiratory chain by using electrochemical and spectroscopic techniques." Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAF017/document.
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Le présent travail porte sur l’étude de deux protéines de la famille des oxydases à hème-fer par des techniques de spectroscopie et d’électrochimie. Le premier chapitre décrit l’étude du cytochrome bo3 oxydase issue d’E. coli. Nous nous sommes intéressés à l’étude des interactions enzyme-quinone par l’utilisation de quinones avec des longueurs chaines isoprenyl différentes. Notre but est de mieux comprendre le rôle de la longueur de la chaine des quinones sur l’activité catalytique de l’enzyme et sur les propriétés redox des cofacteurs à hème. Dans l’étape suivante, on a étudié les résidus impliqués dans le site de liaison des quinones (haute affinité, QH). Plusieurs mutations de ces résidus sont étudiées pour mieux comprendre l’importance de chacun des résidus dans cette liaison. Dans la dernière partie de ce premier chapitre, la spectroscopie SEIRAS «spectroscopie d’absorption infrarouge exaltée de surface» est introduite comme une technique alternative pour l’étude des protéines membranaires. Dans le second chapitre, la protéine membranaire cNOR issue de P. denitrificans est étudiée. Nous nous sommes focalisés sur l’effet de différents environnements (pH, présence de protéo-liposomes) sur la stabilité de la cNOR. Pour ce faire, trois valeurs de pH (6.5, 7.5 et 8.5) sont choisies et quelques échantillons de cNOR sont reconstitués dans des protéo-liposomes. Enfin, le donneur de proton terminal (au centre binucléaire) dans la protéine cNOR était étudié. De plus, nous avons étudié les ligands des ions Ca2+ puisqu’il est proposé que le donneur de proton est situé proche de cette régionThis thesis is focused on the study of two members of the heme-copper oxidase family by using spectroscopic and electrochemical techniques. In the first chapter cytochrome bo3 oxidase from E. coli was studied. We focused on the quinone-enzyme interactions by using quinones with different isoprenyl chains. Our aim was to better understand the role of isoprenyl chain on the catalytic activity of the enzyme and the redox properties of the heme cofactors. In the next step we studied the residues that are suggested to be in the high-affinity (QH) quinone binding site. Several site-directed mutants of these residues were investigated in order to better understand the position of QH binding site and the importance of each residue. In the last part of this chapter surface-enhanced infrared absorption spectroscopy (SEIRAS) was introduced as an alternative technique to study the membrane proteins. In the second chapter cytochrome c dependent nitric oxide reducates (cNOR) from P. denitrificans was studied. We focused on the effect of different environment (pH, proteoliposomes) on the stability of cNOR. For that purpose three pH values (6.5, 7.5 and 8.5) was selected and some of the cNOR samples were reconstituted in liposomes. Finally, the terminal proton donor (to the binuclear center) in cNOR was investigated. We studied the ligands of the Ca2+ site in cNOR since it was suggested that the proton donor may be close to this area
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De, poulpiquet de Brescanvel Anne. "Biopiles enzymatiques H2-O2 : nanostructuration de l'interface électrochimique pour l'immobilisation des enzymes redox." Thesis, Aix-Marseille, 2014. http://www.theses.fr/2014AIXM4752/document.
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Dans la nature, la réduction de l'oxygène et l'oxydation de l'hydrogène sont catalysées par des enzymes oxydoréductases. Ces catalyseurs spécifiques, efficaces, renouvelables et biodégradables constituent une alternative séduisante au platine dans les piles à combustible. L'immobilisation à des interfaces nanostructurées de l'hydrogénase membranaire tolérante à l'oxygène de la bactérie hyperthermophile Aquifex aeolicus, et de la bilirubine oxydase thermostable de la bactérie Bacillus pumilus, a été étudiée dans ce sens.L'électrochimie et la dynamique moléculaire ont permis d'affiner le modèle d'orientation de l'hydrogénase sur les surfaces planes. L'efficacité de l'immobilisation de l'hydrogénase sur différents nanomatériaux carbonés (nano-particules, tubes et fibres de carbone) structurant la surface de l'électrode a été évaluée. Les nanofibres de carbone (CNFs) ont permis de former une bioanode efficace pour l'oxydation de l'H2 en l'absence de médiateurs redox. L'étude a souligné l'importance d'un transport efficace du substrat dans le film carboné mésoporeux. Les CNFs ont également été utilisées comme matériau d'électrode pour réaliser la 1ère connexion directe de la bilirubine oxydase. L'existence d'une forme resting alternative de l'enzyme, influencée par les ions chlorures, le pH et la température, a été mise en évidence. Une biocathode efficace pour la réduction de l'oxygène a été développée.Les deux électrodes thermostables ont permis le développement de la 1ère biopile H2/O2 qui délivre des densités de puissance supérieures au mW.cm-2 sur une large gamme de température. Ce résultat ouvre la voie à l'alimentation électrique de dispositifs de faibles puissancesThe oxygen reduction and the hydrogen oxidation reactions are realized in nature by oxidoreductase enzymes. These highly efficient, specific, renewable and biodegradable catalysts appear as a seducing alternative to platinum in fuel cell devices. The immobilization at nanostructured interfaces of the membrane-bound oxygen-tolerant hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus, and of the thermostable bilirubin oxidase from Bacillus pumilus, has been studied within this objective.Electrochemistry and molecular dynamics have been used to validate the orientation model of the hydrogenase at planar electrodes. Hydrogenase immobilisation in 3D-networks based on various carbon materials (nanoparticles, nanotubes and nanofibers) has been especially studied. Fishbone carbon nanofibers were demonstrated to provide an efficient platform for mediatorless H2 oxidation. Mass transport inside the carbon mesoporous film has been especially studied and demonstrated to be one of the limitations of the catalytic efficiency. Direct electrical connection of bilirubin oxidase has also been realized for the first time thanks to its immobilization on carbon nanofiber films. An alternative resting form of the enzyme, influenced by chlorides, pH and temperature, has been evidenced. An efficient biocathode for the oxygen reduction reaction has been developed. Thanks to the two thermostable electrodes, the first H2-O2 bio fuel cell able to deliver power densities over 1 mW.cm-2 over a large temperature range has been developed. This result paves the way for the electrical alimentation of low-power devices
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Artigues, Cladera Margalida Esmeralda. "Estudio de biosensores electroquímicos basados en inmovilización enzimática." Doctoral thesis, Universitat Ramon Llull, 2019. http://hdl.handle.net/10803/667847.
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Els biosensors electroquímics són dispositius d'anàlisi que combinen l'especificitat de les reaccions bioquímiques amb la capacitat analítica de les tècniques electroquímiques. Gràcies a aquesta combinació, és possible determinar de forma ràpida, sensible i fiable diferents analits en mostres amb matrius complexes. Per tot això, l'ús de biosensors és una alternativa als mètodes clàssics d'anàlisi per a realitzar processos de control de qualitat en diferents sectors industrials.
En la present Tesi, s'han desenvolupat biosensors amperomètrics enzimàtics basats en la immobilització d’oxidases sobre una interfase electroquímica de nanotubs d'òxid de titani (IV) altament ordenats (TiO2NTAs). Per a això, s'han estudiat diferents processos d'immobilització d'enzims basats en captura polimèrica i en immobilització covalent, i s'han avaluat els paràmetres analítics dels biosensors desenvolupats.
En als processos de captura polimèrica, s'ha estudiat l'ús de kappa-carragenina, 2-hidroxietilmetacrilat (HEMA) i quitosà. Aquests hidrogels s'han utilitzat per a la immobilització de l'enzim glucosa oxidasa (GOx) i s'ha observat que tant el HEMA com el quitosà generen un microentorn favorable per a la conservació de l'activitat biològica de l'enzim.
banda Per a la immobilització covalent, s'ha utilitzat pentafluorofenilmetacrilat (PFM) generació amb l’objectiu de generar enllaços entre les molècules d'enzim i la superfície del transductor. Per a això, s'ha modificat la superfície de la interfase electroquímica TiO2NTAs mitjançant dues tècniques de plasma: polimerització de PFM i sembrat del mateix polímer. S'ha observat que la superfície polimeritzada de PFM (ppPFM) presenta major hidrofobicitat que la superfície en la qual s'ha realitzat el sembrat de PFM (pgPFM) i que això té influència en la conformació que adopten les molècules d'enzim. Mentre que en la superfície ppPFM predominen conformacions amb baixa activitat, en la superfície pgPFM la major part de la població de les molècules de GOx adopten conformacions amb activitat catalítica. Per aquests motius, els biosensors amb sembrat per plasma de PFM presenten major sensibilitat enfront de la presència de glucosa que els biosensors basats en la polimerització de PFM.
Finalment, s'han desenvolupat biosensors amperomètrics de glucosa i de glutamat amb matrius d'immobilització polimèriques i covalents: Ti/TiO2NTAs/GOx/Quitosà, Ti/TiO2NTAs/HEMA-co-EGDA/pgPFM/GOx/Quitosà i Ti/TiO2NTAs/GmOx/Quitosà. S'han realitzat determinacions sobre mostres alimentàries emprant aquests biosensors i els resultats s'han comparat amb els obtinguts amb tècniques de referència.Los biosensores electroquímicos son dispositivos de análisis que combinan la especificidad de las reacciones bioquímicas con la capacidad analítica de las técnicas electroquímicas. Gracias a esta combinación, es posible determinar de forma rápida, sensible y fiable distintos analitos en muestras con matrices complejas. Por ello, el uso de biosensores es una alternativa a los métodos clásicos de análisis para realizar procesos de control de calidad en distintos sectores industriales. En la presente Tesis, se han desarrollado biosensores amperométricos enzimáticos basados en la inmovilización de oxidasas sobre una interfase electroquímica de nanotubos de óxido de titanio (IV) altamente ordenados (TiO2NTAs). Para ello, se han estudiado diferentes procesos de inmovilización de enzimas basados en captura polimérica y en inmovilización covalente, y se han evaluado los parámetros analíticos de los biosensores desarrollados. En los procesos de captura polimérica, se ha estudiado el uso de kappa-carragenina, 2-hidroxietilmetacrilato (HEMA) y quitosano. Estos hidrogeles se han utilizado para la inmovilización del enzima glucosa oxidasa (GOx) y se ha observado que tanto HEMA como quitosano generan un microentorno favorable para la conservación de la actividad del enzima. Para la inmovilización covalente, se ha utilizado pentafluorofenilmetacrilato (PFM) con el objetivo de generar enlaces entre las moléculas de enzima y la superficie del transductor. Para ello, se ha modificado la superficie de la interfase electroquímica TiO2NTAs mediante dos técnicas de plasma: polimerización de PFM y sembrado del mismo polímero. Se ha observado que la superficie polimerizada de PFM (ppPFM) presenta mayor hidrofobicidad que la superficie en la que se ha realizado el sembrado de PFM (pgPFM) y que ello tiene influencia en la conformación que adoptan las moléculas de enzima. Mientras que en la superficie ppPFM predominan conformaciones con baja actividad, en la superficie pgPFM la mayor parte de la población de las moléculas de GOx adoptan conformaciones con actividad catalítica. Por estos motivos, los biosensores con sembrado por plasma de PFM presentan mayor sensibilidad frente a la presencia de glucosa que los biosensores basados en la polimerización de PFM. Finalmente, se han desarrollado biosensores amperométricos de glucosa y de glutamato con matrices de inmovilización poliméricas y covalentes: Ti/TiO2NTAs/GOx/Quitosano, Ti/TiO2NTAs/HEMA-co-EGDA/pgPFM/GOx/Quitosano y Ti/TiO2NTAs/GmOx/Quitosano. Se han realizado determinaciones sobre muestras alimentarias empleando estos biosensores y los resultados se han comparado con los obtenidos con técnicas de referencia.
Electrochemical biosensors are analytical devices that combine the specificity of biochemical recognition processes with the analytical power of electrochemical techniques. Consequently, it is possible to perform rapid, sensitive and reliable determinations of different analytes present in complex samples. For this reason, the use of biosensors is an alternative to classical analytical methods to perform quality control processes in different industrial sectors. In this work, we have developed enzymatic amperometric biosensors based on the immobilization of oxidases on an electrochemical interface of highly ordered titanium dioxide nanotubes array (TiO2NTAs). Thus, processes of enzyme immobilization based on polymeric entrapment and covalent immobilization have been studied. The analytical parameters of these biosensors have been evaluated. For polymeric entrapment processes, kappa-carrageenan, 2-hydroxyethyl methacrylate (HEMA) and chitosan have been studied as immobilization matrices. These hydrogels have been used for the immobilization of the enzyme glucose oxidase (GOx) and it has been observed that both, HEMA and chitosan, generate a favorable microenvironment for the conservation of the activity of the enzyme. For covalent immobilization, pentafluorophenylmethacrylate (PFM) has been used in order to generate bonds between the enzyme molecules and the surface of the transducer. Thus, the electrochemical interface TiO2NTAs has been modified by two plasma techniques: polymerization of PFM and grafting of the same polymer. It has been observed that the polymerized surface of PFM (ppPFM) has a higher hydrophobicity than the surface in which the PFM has been grafted (pgPFM). Hydrophobicity has influence on the adopted enzyme molecules conformation. On the ppPFM surface, conformations with low activity predominate, and on the pgPFM surface most of the population of GOx molecules adopt conformations with catalytic activity. For these reasons, the biosensors with plasma grafted PFM show higher sensitivity in presence of glucose than the biosensors based on the PFM polymerization. Finally, amperometric glucose and glutamate biosensors with polymeric and covalent immobilization matrices have been developed: Ti/TiO2NTAs/GOx/Chitosan, Ti/TiO2NTAs/HEMA-co-EGDA/pgPFM/GOx/Chitosan and Ti/TiO2NTAs/GmOx/Chitosan. These biosensors have been used to determine the glucose and glutamate content in different food samples. The results have been compared with those obtained with reference techniques.
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Jacques, Julien. "Réactivité de la nitrate réductase périplasmique étudiée par spectroscopie RPE et électrochimie directe." Thesis, Aix-Marseille, 2014. http://www.theses.fr/2014AIXM4710/document.
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La nitrate réductase périplasmique de Rhodobacter sphaeroides catalyse la réduction du nitrate en nitrite. C'est une métalloenzyme qui comprend un cofacteur à molybdène, un centre fer - soufre et deux hèmes.La réactivité du cofacteur à molybdène reste mal comprise pour plusieurs raisons. Entre autres : l'hétérogénéité des signatures RPE Mo(V), état semi-réduit du site actif, et l'existence d'états inactifs de l'enzyme selon les conditions.Pour comprendre la réactivité et la pertinence catalytique des principales espèces Mo(V), nous avons entrepris une caractérisation des processus d'activation et d'inactivation par électrochimie sur film de protéines, et une étude de leur structure par spectroscopies RPE et HYSCORE.Nos observations cinétiques suggèrent que l'activation irréversible de l'enzyme implique un réarrangement d'une des ptérines du cofacteur à Mo.Ceci est mis en évidence par la modification des couplages magnétiques intercentres du fait de l'activation, et par des modifications de structure au delà de la première sphère de coordination du Mo.Enfin, l'étude de l'inactivation réversible de l'enzyme par électrochimie montre l'implication des différents états redox du site actif dans le mécanisme d'inhibition, et donne les conditions nécessaires au piégeage de formes Mo(V) activesRhodobacter sphaeroides periplasmic nitrate reductase catalyses the reduction of nitrate into nitrite. It is a metalloenzyme containing a molybdenum cofactor, an iron - sulfur cluster, and two haems.The reactivity of the molybdenum cofactor remains elusive for many reasons. Among others : the heterogeneity of the EPR signatures of Mo(V), the semi-reduced state of the active site, and the existence of inactive states of the enzyme, depending on conditions.In order to understand the reactivity and the catalytic relevance of the major Mo(V) species, we have undertaken a characterisation of the activation and inactivation processes by protein-film-electrochemistry, and a study of their structure by EPR and HYSCORE spectroscopies.Our kinetic observations suggest that the irreversible activation of the enzyme involves a rearrangement of one of the pterins of the Mo cofactor.This is evidenced by the modification of intercentre magnetic couplings due to the activation, and by structural modifications beyond the first coordination sphere of Mo.Finally, the study of enzyme reversible inactivation by electrochemistry shows the involvement of the different redox states of the active site in the inhibition mechanism, and yields the necessary conditions to trapping active Mo(V) forms
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Reeve, Holly A. "New approaches for cofactor recycling : application to chemical synthesis and electrochemical devices." Thesis, University of Oxford, 2015. http://ora.ox.ac.uk/objects/uuid:514ec071-36aa-45a9-9f03-15bb22f967c4.
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The work in this Thesis addresses the challenges associated with using redox enzymes for chemical synthesis. The use of enzymes as catalysts in the synthesis of fine chemicals is becoming more wide spread, in part due their ability to catalyse reactions with incredible selectivity under relatively mild conditions. In particular, enzymes are useful for selective reduction of ketones to enantiomerically pure alcohols or amines, and partial oxidations of alkanes to alcohols. However, a key limitation to exploiting redox enzymes in these reaction pathways is the requirement for a specialised electron source, usually the expensive nicotinamide cofactors NADH or NADPH. Existing cofactor regeneration methods use a second enzyme with a sacrificial substrate which is oxidised to generate a stoichiometric waste product; this complicates isolation of the desired product and prevents the environmental benefits of biocatalysis from being fully realised. In order to provide clean and efficient biocatalytic routes, improved recycling methods for these cofactors are crucial. This Thesis develops two novel methods for in situ cofactor recycling. The first is an electro-enzymatic system; an NAD+-reductase enzyme is shown to use electrons directly from an electrode for supply of NADH to a co-immobilised cofactor-dependent enzyme. The second uses a hydrogenase, NAD+ reductase and cofactor-dependent enzyme immobilised on conducting particles for H2-driven NADH regeneration. This relies on the thermodynamically favourable reduction of NAD+ by H2 when the hydrogenase and NAD+-reductase are in electronic contact, provided by the conducting particle. The electro-enzymatic approach to NAD+ reduction is then adapted for electrochemical devices; an enzyme catalysed fuel cell and a self-powered biosensor were considered.
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Dochter, Alexandre. "Polymer films and brushes self-construction by electrochemically triggered morphogens." Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAE039/document.
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Les multicouches de polyelectrolytes, systèmes auto-assemblés par adsorption successive de polycations et de polyanions, constituent un matériau d’intérêt pour la fonctionnalisation de surface. Ce type de revêtement possède toutefois quelques limitations majeures : leur construction est lente et fastidieuse ; leur tenue mécanique et chimique est faible. Récemment, une méthode basée sur l’auto-construction de films par l’utilisation d’un morphogène, i.e. un gradient de catalyseur généré depuis une surface, a été développé permettant ainsi une rapide construction de revêtements robustes. Cette technique reste toutefois limitée à des systèmes particuliers basés sur la chimie click ou sur des interactions hôtes-invités. Nos travaux ont permis de diversifier cette approche de construction tout-en-un par l’utilisation de morphogènes. Dans un premier temps, des brosses de polymères ont été construites en une étape depuis une surface par la réaction de polymérisation ATRP. Cette réaction a été catalysée par la formation d’ions Cu(I) (le morphogène) par électrochimie depuis la surface de travail. L’approche morphogénique a également été utilisée afin de construire des films de polyélectrolytes et de polyampholytes en une étape par la génération électrochimique d’un gradient de protons (le morphogène) depuis la surface de travail. Ces films ont été fonctionnalisés pour présenter une activité enzymatiquePolyelectrolyte multilayers, i.e. self-assembled systems based on successive polycation and polyanion adsorptions, constitute interesting materials for surface functionalization. These coatings possess several limitations: they are weak towards chemical and mechanical constraint and their buildup is long and tedious. Recently, a new method based on the self-construction of films by the means of a morphogen (a catalyst gradient generated from a surface) has attracted attention since it allows the quick self-assembly of robust films. Nevertheless, this technique was quite limited to peculiar systems based on click chemistry or on host-guest interactions.This present work generalize the one-pot morphogenic approach to other systems. In the first place, polymer brushes were built up from a surface by ATRP polymerization. The Cu(I) catalyst (the morphogen) was electrochemically generated at the interface.The morphogenic approach was later used to buildup polyelectrolytes and polyampholyte films in a one-pot manner by electrochemically generating protons (the morphogens) at the interface. These films exhibited an enzymatic activity
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Pescador, Álvarez Paula. "Colloidal and molecular assemblies for bioengineering applications." Doctoral thesis, Universitat Rovira i Virgili, 2007. http://hdl.handle.net/10803/8557.
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La técnica de recubrimiento "capa por capa' (layer-by-layer, LbL) de superficies cargadas mediante materiales con carga opuesta es una herramienta versátil para la fabricación de ensamblados moleculares e interfaces funcionales. Sus principales ventajas sobre otras metodologías, como las monocapas autoensambladas (SAM) o la técnica de Langmuir-Blodgett, son una enorme flexibilidad combinada con su gran simplicidad y bajo coste. Con instrumentación sencilla y protocolos de preparación simples, es posible ensamblar estructuras complejas y estables con un control nanométrico sobre su composición y estructura.La metodología LbL es especialmente adecuada para la integración de biomoléculas (proteínas, lípidos, DNA) en multicapas funcionales, ya que el proceso de ensamblaje se lleva a cabo en condiciones suaves. Las propiedades biológicas de estos materiales se mantienen o incluso mejoran tras su incorporación en los films.
Otra ventaja de esta técnica es que permite recubrir sustratos de virtualmente cualquier tamaño y forma con films funcionales de manera sencilla y controlada. De particular importancia ha sido la extensión del método a la modificación de partículas coloidales. Aparte de su interés desde un punto de vista fundamental y aplicado, los coloides constituyen herramientas de gran potencial para la creación de estructuras de diseño específico en los campos de la bio y nanotecnología. La funcionalización vía LbL de partículas coloidales permite integrar múltiples funcionalidades en las partículas, y proporciona además una ruta para la creación de estructuras tridimensionales que facilitan la transición desde la nano- a la micro- y macroescala.
La técnica LbL ha supuesto también un gran avance en el desarrollo de sistemas electroquímicos. Diversos materiales electroactivos pueden ser incorporados en las multicapas, junto con otras especies que proporcionan funcionalidades adicionales. Además, parámetros críticos como el grosor del film, el transporte de materia y la conductividad pueden ajustarse con precisión en estas estructuras, incrementando la capacidad de control sobre el funcionamiento final del sistema. En particular, las estructuras LbL han encontrado numerosas aplicaciones en el área de los biosensores electroquímicos. Estos dispositivos proporcionan una interfaz entre funciones biológicas específicas y procesos de transducción electrónicos, y ofrecen una alternativa con gran potencial para el desarrollo de plataformas de biodetección integradas.
En el presente trabajo, la técnica LbL se emplea para ensamblar films multicapa de enzimas y polielectrolitos en la superficie de micropartículas de sílice. Dos enzimas diferentes, glucosa oxidasa (GOx) y peroxidasa (HRP) son co-inmovilizadas junto con capas precursoras e intermedias de polielectrolitos. En los films resultantes se desarrolla una reacción enzimática secuencial, con la conversión inicial de glucosa en acido glucónico y peróxido de hidrógeno, catalizada por GOx, y la posterior reducción del peróxido de hidrógeno a agua por acción de la HRP. El enfoque secuencial LbL permite explorar la influencia de diferentes combinaciones de polielectrolitos sobre la inmovilización y funcionalidad de los enzimas. Técnicas como la citometría de flujo, microscopía confocal y electrónica y medidas espectrofotométricas proporcionan información sobre la interacción entre los diferentes componentes de las capas, así como sobre la estabilidad de las suspensiones coloidales y el comportamiento de los films en presencia de los diferentes sustratos enzimáticos.
Una funcionalidad electroquímica se integra adicionalmente en estos films mediante la incorporación de un polímero redox a la estructura. De este modo, los eventos específicos que tienen lugar durante la catálisis enzimática se transducen en una señal eléctrica. Las partículas nanoestructuradas asumen un doble papel en el sistema final. Por una parte, actúan como sustratos de alta área superficial para la fabricación de microreactores enzimáticos. Además, los coloides se incorporan en un film de polímero redox y se inmovilizan en la superficie de electrodos de oro, actuando como elementos de construcción para la fabricación de un biosensor electroquímico que permite la detección de glucosa y peróxido de hidrógeno.
The layer-by-layer (LbL) coating of charged surfaces with oppositely charged materials is a powerful and versatile approach for the fabrication of functional molecular assemblies and interfaces. The key advantages of this technique over other methodologies such as self-assembled monolayers (SAM) or Langmuir-Blodgett (LB) are its unparalleled flexibility in combination with its simplicity and inexpensiveness. With simple instrumentation and easy preparation steps it is possible to assemble highly complex and stable architectures with nanoscale control over their composition and structure.
The LbL approach is particularly suitable for the integration of biomolecules (proteins, lipids, DNA) into functional multilayers, since layer buildup is carried out under mild conditions. Many biologically relevant species can be incorporated into the films while maintaining or even improving their biological functions. A further advantage of this technique is that substrates of virtually any size and shape can be coated with functional films in a simple and controlled fashion. Of particular interest has been the extension of the LbL method to the modification of colloidal particles. Apart from their interest from a fundamental and applied point of view, colloids have emerged as powerful tools particularly suited to meet the challenge of creating tailored building blocks for the rapidly evolving fields of bio- and nanotechnology. The LbL functionalisation of colloidal particles provides a new route for the creation of composite architectures which allow the integration of nanoscale-defined materials into two- and three-dimensional structures. This ultimately opens the way not only to functional microsystems but also to the fabrication of macroscopic devices.
One of the fields in which the LbL technique has represented a major advance is the development of electrochemical systems. Electrochemically active materials can be readily incorporated into multilayer films, together with other species which provide additional functionalities. Furthermore, critical parameters such as film thickness, mass transport and conductivity can be precisely tuned, allowing an increased control over the performance of the system. In particular, LbL assemblies have found many successful applications in the area of electrochemical biosensors. These devices provide an interface between biological functions and electronic signal-transduction processes, and offer great potential for the development of new miniaturised, low-cost, integrated biodetection platforms.
In the present work, the LbL technique is employed to assemble multilayer films of enzymes and polyelectrolytes on the surface of silica microparticles. Two different enzymes, glucose oxidase (GOx) and horseradish peroxidase (HRP) are co-immobilised together with precursor and intermediate polyelectrolyte layers. In the resulting multilayer films a sequential reaction takes place, with the conversion of glucose to gluconic acid and hydrogen peroxide catalysed by GOx and the subsequent reduction of hydrogen peroxide to water catalysed by HRP. The sequential LbL approach allows to explore the influence of different polyelectrolyte combinations on the immobilisation and functionality of the enzymes. Flow cytometry, confocal and electron microscopy and spectrophotometric measurements provide information about the interaction between the different layer components, as well as the stability of the colloidal substrates and the behaviour of the multilayer films in the presence of the different enzyme substrates.
An electrochemical functionality is further added to these films with the incorporation of an osmium-based redox polymer to the structure. In this way the specific chemical events taking place at the redox centers of the enzymes are transduced into an electrical signal. The nanostructured particles assume a multiple role in the final system. On one hand, they act as immobilisation substrates and high surface area carriers for the creation of enzymatic microreactors. Moreover, the LbL-coated colloids are embedded in a redox polymer film and immobilised on the surface of gold electrodes, acting as building blocks fo the fabrication of an electrochemical biosensor able to detect glucose and hydrogen peroxide.
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Woolerton, Thomas William. "Development of enzymatic H2 production and CO2 reduction systems." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:393741ac-94b1-4d56-b680-d9a434db77e2.
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One of today’s most pressing scientific challenges is the conception, development and deployment of renewable energy technologies that will meet the demands of a rapidly increasing population. The motivation is not only dwindling fossil fuel reserves, but also the necessary curtailment of emissions of the greenhouse gas carbon dioxide (a product of burning fossil fuels). The sun provides a vast amount of energy (120,000 TW globally), and one major challenge is the conversion of a fraction of this energy into chemical energy, thereby allowing it to be stored. Dihydrogen (H₂) that is produced from water is an attractive candidate to store solar energy (a ‘solar fuel’), as are high energy carbon-containing molecules (such as CO) that are formed directly from carbon dioxide. One key aspect is the development of catalysts that are able to offer high rates and efficiencies. In biology, some microbes acquire energy from the metabolism of H₂ and CO. The biological catalysts - enzymes - that are responsible are hydrogenases (for the oxidation of H₂ to protons); and carbon monoxide dehydrogenases (CODHs, for the oxidation of CO to CO₂). These redox enzymes, containing nickel and iron as the only metals, are extraordinary in terms of their catalytic characteristics: many are fully reversible catalysts and offer very high turnover frequencies (thousands per second are common), with only tiny energy input requirements. This Thesis uses a hydrogenase from the bacterium Escherichia coli, and two CODHs from the bacterium Carboxydothermus hydrogenoformans, as the catalysts in H2 production and CO₂ reduction systems. Chapter 3 describes the concept and development not of a solar fuel system, but of a device that catalyses the water-gas shift reaction (the reaction between CO and water to form H₂ and CO₂) - a process of major industrial importance for the production of high purity H₂. Chapters 4, 5 and 6 detail photochemical CO₂ reduction systems that are driven by visible light. These systems, operating under mild, aqueous conditions, involve CODHs attached either to TiO₂ nanoparticles that are sensitised to visible light by the co-attachment of a ruthenium-based dye complex, or to cadmium sulfide nanomaterials that, having a narrow band gap, are inherently photoexcitable by visible light. The motivation here is not the construction of technological devices; indeed, the enzymes that are used are fragile, highly sensitive to oxygen, and impossible to scale to industrial levels. Rather, the drivers are those of scientific curiosity (can the incorporation of these remarkable biological catalysts enable the creation of outstanding solar fuel devices?), and of producing systems that serve as benchmarks and inspiration for the development of fully synthetic systems that are robust and scalable.
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Birkin, Peter Robert. "Microelectrochemical enzyme transistors." Thesis, University of Southampton, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240628.
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Siritanaratkul, Bhavin. "Enzyme-material composites for solar-driven reactions." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:55df8993-254b-4960-8ef4-fd9624206f3b.
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Using sunlight to drive chemical reactions has long been one of the goals in developing sustainable processes. Previous research has focused on solar fuel production in the form of H2, but this thesis demonstrates that solar-to-chemicals processes can be constructed to produce more complex compounds, using hybrid systems composed of enzymes and inorganic materials. Tetrachloroethene reductive dehalogenase (PceA), an enzyme that catalyzes the conversion of tetrachloroethene (PCE) to trichloroethene (TCE) and subsequently to cis-dichloroethene (cDCE), was shown to accept electrons from both graphite and TiO2 electrodes. Irradiation by UV light onto PceA-adsorbed TiO2 particles led to the selective production of TCE and cDCE, which was not possible without PceA as a catalyst. Ferredoxin-NADP+ reductase (FNR) is a key enzyme in photosynthesis, as it receives energetic electrons from Photosystem I and produces NADPH as an energy carrier for downstream 'Dark' reactions involving CO2 assimilation. This thesis presents the discovery of FNR activity on indium tin oxide (ITO) electrodes which led to direct electrochemical investigation of the properties of FNR, both in the absence and presence of its substrate, NADP+. The FNR-adsorbed electrode, termed 'the electrochemical leaf', rapidly interconverts NADP+/NADPH, and this was coupled to a downstream NADPH-dependent enzyme, thus demonstrating a new approach to cofactor regeneration for enzyme-catalyzed organic synthesis. The NADP+ reduction by FNR was also driven by light using a photoanode made of visible-light responsive semiconductors.
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Wulff, Philip. "Principles of hydrogen catalysis in the presence of oxygen by a [NiFe] hydrogenase from E. coli." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:9e434467-d50b-484a-a17e-ef3091636269.
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[NiFe] hydrogenases are metalloenzymes that act as highly efficient molecular electrocatalysts for the interconversion of protons and molecular hydrogen. Unlike any other known molecular electrocatalyst, the members of a subgroup of respiratory membrane-bound [NiFe] hydrogenases are able to maintain H2 catalysis in the sustained presence of O2. This O2-tolerance depends on the ability to respond to oxidative inactivation by O2 by exclusively forming rapidly reactivated active site states, thus implying a catalytic cycle in which O2 acts as a competing substrate to H2. Using isotope ratio mass spectrometry it is proven that the O2-tolerant Escherichia coli Hydrogenase 1 responds to O2 attack by acting as a four-electron oxidoreductase, catalysing the reaction 2 H2 + O2 → 2 H2O, equivalent to hydrogen combustion. Special features of the enzyme’s electron relay system enable delivery of the required electrons. A small fraction of the H2O produced arises from side reactions proceeding via reactive oxygen species, an unavoidable consequence of the presence of low-potential relay centres that release electrons from H2 oxidation. While the ability to fully reduce O2 to harmless H2O at the active site to generate the rapidly reactivated state Ni-B, determines if a hydrogenase is O2-tolerant, the ratio of oxidative inactivation to reductive reactivation rates determines how tolerant the enzyme is. It is shown by protein film electrochemistry that the (αβ)2 dimeric assembly of Hyd-1 plays an important role in O2-tolerance by aiding reactivation of one catalytic unit through electron transfer from the other. The teamwork between two redundant partners implicates a new role for dimerisation and represents a new example of cooperativity in biology. Finally, the non-natural amino acid p-azido-L-phenylalanine was synthesised and incorporated into Hyd-1, testing the possibility of introducing labels at specific sites.
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Singh, Kulveer. "Structure-function studies of the oxidoreductase bilirubin oxidase from Myrothecium verrucaria using an electrochemical quartz crystal microbalance with dissipation." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:0376cc7e-f572-4e0c-96f0-43b0b4b91d99.
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This thesis presents the development and redesign of a commercial electrochemical quartz crystal microbalance with dissipation (E–QCM–D). This was used to study factors affecting the efficiency of the four electron reduction catalysed by the fuel cell enzyme bilirubin oxidase from Myrothecium verrucaria immobilised on thiol modified gold surfaces. Within this thesis, the E–QCM–D was used to show that application of a constant potential to bilirubin oxidase adsorbed to thiol-modified gold surfaces causes activity loss that can be attributed to a change in structural arrangement. Varying the load by potential cycling distorts the enzyme by inducing rapid mass loss and denaturation. Attaching the enzyme covalently reduces the mass loss caused by potential cycling but does not mitigate activity loss. Covalent attachment also changes the orientation of the surface bound enzyme as verified by the position of the catalytic wave (related to the overpotential for catalysis) and reactive labelling followed by mass spectrometry analysis. The E–QCM–D was used to show how electrostatic interactions affect enzyme conformation where high pH causes a reduction in both mass loading at the electrode and a reduction in activity. At pH lower than the enzyme isoelectric point, there is a build up of multilayers in a clustered adsorption. When enzyme adsorbs to hydrophobic surfaces there is a rapid denaturation which completely inactivates the enzyme. Changing the surface chemistry from carboxyl groups to hydroxyl and acetamido groups shows that catalysis is shifted to more negative potentials as a result of an enzyme misorientation. Further to this, increasing the chain length of the thiol modifier indicates that an increased distance between surface and enzyme reduces activity, enzyme loading and results in a conformational rearrangement that permits electron transfer over longer distances.
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Keeley, Deborah Michelle. "Electrochemical studies of biologically important materials." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325879.
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Mizzon, Giulia. "Bioelectrochemistry by fluorescent cyclic voltammetry." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:6a1134dd-c24d-4e60-ac83-936a6918131f.
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Understanding the factors influencing the ET characteristics of redox proteins confined at an electrochemical interface is of fundamental importance from both pure (fundamental science) and applied (biosensory) perspectives. This thesis reports on progress made in the emerging field of coupled electrochemical characterization and optical imaging in moving the analysis of redox-active films to molecular scales. More specifically the combination of cyclic voltammetry and wide-field Total Internal Reflection (TIRF) microscopy, here named ‘Fluorescent Cyclic Voltammetry’ (FCV), was applied to monitoring the response of surface-confined redox active proteins at submonolayer concentrations. The combined submicrometre spatial resolution and photon capture efficiency of an inverted TIRF configuration enabled the redox reactions of localized populations of proteins to be directly imaged at scales down to a few hundreds of molecules. This represents a 6-9 orders of magnitude enhancement in sensitivity with respect to classical current signals observed in bioelectrochemical analysis. Importantly, measurements of redox potentials at this scale could be achieved from both natural and artificially designed bioelectrochemical fluorescent switches and shed fundamental light on the thermodynamic and kinetic dispersion within a population of surface confined metalloproteins. The first three chapters of this thesis provide an overview of the relevant literature and a theoretical background to both the rapidly expanding fields of electroactive monolayers bioelectrochemistry and TIRF imaging. The initial design and construction of a robust electrochemically and optically addressable fluorescent switch, crucial to the applicability of FCV is reported in chapter 5. The generation of optically transparent, and chemically modifiable electrode surfaces suitable for FCV are also described. Chapter 6 describes the response of the surface confined azurin-based switch. Analysis of the spatially-resolved redox reaction of zeptomole samples in various conditions enables the mapping of thermodynamic dispersion across the sampled areas. In chapter 7 the newly developed FCV detection method was extended to investigate more complex bioelectrochemical systems containing multiple electron transferring redox centres and responding optically at different wavelengths. This approach provides a platform for spectral resolution of different electrochemical processes on the same sample. Finally in chapter 8 an electrochemical procedure is proposed for investigating the kinetic response of redox proteins using a fundamentally new methodology based on interfacial capacitance. In using variations in the surface chemistry to tune the rate of electron transfer, the approach was shown to be a robust and facile means of characterising redox active films in considerably more detail than possible through standard electrochemical methodologies. Ultimately, it can be applied to probe dispersion within protein populations and represents a powerful means of analysing molecular films more generally.
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Mashazi, Philani Nkosinathi. "Study of metallophthalocyanines attached onto pre-modified gold surfaces." Thesis, Rhodes University, 2007. http://eprints.ru.ac.za/868/.
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Ho, Wah On. "Direct electron transfer peroxidase enzyme electrodes and their application to electrochemical immunoassay." Thesis, University of Newcastle Upon Tyne, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.384829.
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Murphy, Bonnie J. "The importance of electron transfer in determining properties of [NiFe]-hydrogenases." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:42258640-0fa6-4c48-a0f6-d6c8b9d7b3e0.
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[NiFe] hydrogenases are microbial metalloenzymes that catalyse the reversible interconversion between molecular hydrogen and protons with high selectivity and efficiency. The catalytic properties of different [NiFe] hydrogenases vary according to the physiological roles they each play, yet all seem to be based upon an almost identical catalytic site architecture. Through efforts to understand the structural and mechanistic basis for the differing properties of [NiFe] hydrogenases, it has become increasingly evident that electron transfer to and from the active site, mediated by a set of Iron-Sulphur clusters, influences to a significant extent the observed catalytic properties of different hydrogenases. Here we present a comprehensive study of E. coli Hyd-1, an O2-tolerant hydrogenase, by PFE with a focus on the properties that are characteristic of O2-tolerant enzymes: overpotential requirement, lack of H2 production, low KH2M, and high Eswitch. We show that Hyd-1 catalysis can be made reversible by increasing the equilibrium potential for the reaction through changes in substrate concentration, and that electron transfer into and out of the enzyme molecule, rather than active site properties, is responsible for the characteristics of overpotential and bias in Hyd-1. We present a set of experiments with Hyd-2 from E. coli in which surface-exposed cysteine residues are specifically introduced near the distal and medial Iron-Sulphur clusters to act as points of attachment for photosensitizer molecules, and a study of the kinetics of electron injection from photoexcited molecules to the enzyme and subsequent absorbance changes attributed to transient redox changes at the active site. We are able to show lightdependent H2 production from a Hyd-2 + photosensitizer system. Finally, we present the first purification of the formate-hydrogen lyase (FHL) complex from E. coli, the complex responsible for H2-production by this organism during fermentation, and we provide a characterisation of the complex by EPR and PFE. The properties of Hyd-3, the hydrogenase subunit of the FHL, seem to differ from those observed previously for other [NiFe] hydrogenases.
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Mashazi, Philani Nkosinathi. "Electrochemical sensing and immunosensing using metallophthalocyanines and biomolecular modified surfaces." Thesis, Rhodes University, 2012. http://hdl.handle.net/10962/d1018248.
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The synthesis of cobalt and manganese phthalocyanine complexes bearing eight hexylthio and four amino substituents was carried out. The formation of thin films of these complexes using different modification methods was also studied. Hexylthio functionalized metallophthalocyanine complexes were immobilized onto gold electrode surfaces using the self-assembly techniques. Surface modifications using cobalt and manganese tetraamino phthalocyanine as polymers, monolayers (onto electrografted surfaces) and as carbon nanotube – metallophthalocyanine conjugates was also carried out. The new method of modifying gold electrodes with metal tetraamino phthalocyanine complexes was investigated. The modified electrode surfaces were studied for their electrocatalytic properties and as potential electrochemical sensors for the detection of hydrogen peroxide (H₂O₂). The limits of detection for the H₂O₂ were of the orders of ~10⁻⁷ M for all the modified electrodes. The modified electrodes gave very good analytical parameters; such as good sensitivity, linearity at studied concentration range and well-defined analytical peaks with increased current densities. The modification methods were reproducible, highly conducting thin films were formed and the modified electrodes were very stable. The design of electrochemical immunosensors for the detection of measles-specific antibodies was also carried out. The modified surface with measles-antigen as sensing element was accomplished using covalent immobilization for an intimate connection of the measles-antigen as a sensing layer onto an electrode surface. Two methods of detecting measles-specific antibodies were investigated and these methods were based on electrochemical impedance, i.e. label-free detection, and voltammetric method using horse-radish peroxidase (HRP) labeled antibody as a reporter. The detection of measles-specific antibodies was accomplished using both these methods. The potential applications of the designed immunosensor were evaluated in real samples (human and newborn calf serum) and the electrodes could detect the antibodies in the complex sample matrix with ease.
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Paengnakorn, Pathinan. "Electrochemical and IR spectroelectrochemical studies of ligand binding to the metal centres of nitrogenase." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:83ea2ef0-c4a4-4014-8ba1-63ff11fbbbc4.
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Nitrogenase is a metalloenzyme that plays a key role in biological nitrogen fixation by catalysing the reduction of dinitrogen to ammonia. Study of nitrogenase is particularly challenging because of its unique electron transfer and catalytic components. This Thesis describes the development of a mediated electron transfer system for the MoFe protein of nitrogenase, in order to overcome the complexity of electron transfer by the native reductant Fe protein coupled to hydrolysis of ATP. A series of redox mediators was employed including EuIII/II-polyaminocarboxylate complexes, which have reduction potentials in a very negative range. In the presence of the redox mediators, the wild type MoFe protein exhibits a catalytic current due to protein-catalysed proton reduction. With this mediated electron transfer method, the potential of proton reduction by nitrogenase was determined for the first time. The redox mediator system was also applied in an infrared (IR) spectroelectrochemical study of CO binding to the wild type and β-98His variant MoFe protein. The first IR evidence was provided for ATP-independent CO binding to the active site of the MoFe protein, in both the wild type and the variant. The peak wavenumbers and time-dependent changes in intensity found in this study are consistent with the result of previous CO coordination with nitrogenase obtained by electron transfer from the Fe protein driven by ATP. This strongly suggests that this mediated electron transfer approach can deliver low potential electrons into the MoFe protein and reduce the active site FeMoco to the substrate binding level. Moreover, this technique allows electrocatalytic activity of the protein to be monitored and the change in redox activity can be correlated directly to the potential. With the same technique, a study of cyanide binding was performed on different variant MoFe proteins of nitrogenase. The redox properties of the isolated cofactor of Mo- and V-dependent nitrogenase were investigated in parallel to the study of the protein-bound cofactors. It was found that FeVco, the active site from V-nitrogenase, exhibited different redox properties compared to that of Mo-nitrogenase. This might account for the unexpected activity in CO reduction that was reported previously for V-nitrogenase.
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Petro, Benjamin J. "Preparation and Characterization of Hydrogenase Enzyme Active Site-inspired Catalysts: The Effects of Alkyl Bulk and Conformer Strain as Studied by Photoelectron Spectroscopy, Electrochemistry and Computational Methods." Diss., The University of Arizona, 2009. http://hdl.handle.net/10150/194329.
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A series of alkyldithiolatodiironhexacarbonyl complexes of the form &mu:-(RS2)Fe2(CO)6, where RS2 is: 1,2-ethanedithiolate (eth-cat), cis-1,2-cyclopentanedithiolate (pent-cat), cis-1,2-cyclohexanedithiolate (hex-cat), and 2-exo,3-exo-bicyclo[2.2.1]heptanedithiolate (norbor-cat), are reported. These complexes display structures and catalytic behavior toward production of molecular hydrogen with similarities to the active site of the diiron hydrogenase enzymes. Hydrogen production is desirable as an alternative fuel source and these catalysts are capable of producing H2 in the presence of weak acid under electrochemical conditions. Through understanding of the factors which control the catalytic activity of these catalysts it may be possible to contribute to the development of a hydrogen fuel economy.Significant scan-rate dependence under electrochemical conditions is observed, resulting in an initial 1-to-2 electron reduction depending on how quickly the singly reduced species can reorganize. The rate of this reorganization directly corresponds to the internal strain within the system and can be ranked in the following order of increasing rate of reorganization: pent-cat < norbor-cat < eth-cat < hex-cat. Additionally, these catalysts all successfully catalyze protons to molecular hydrogen under electrochemical conditions in the presence of acetic acid via an ECEC catalytic mechanism, where, E is an electrochemical step (reduction) and C is a chemical step (protonation).Density functional theory computations support the reported catalytic processes by calculating physically observable quantities, such as: pKa values, reduction potentials, adiabatic ionization energies and carbonyl stretching frequencies in the infrared (IR) region. These quantities were used to suggest reasonable reactive intermediates within the catalytic cycle. The electronic structure of each catalyst was examined using photoelectron spectroscopy and the global minimum cationic structure, in all cases, involves a structure with a bridging carbonyl ligand, akin to that of the enzyme active site.The most significant outcome of this work is the unprecedented diiron center rotation upon reduction. As conformational strain involving the dithiolate ligand increases, the rate of reorganization of the anion increases leading to cleavage of an iron-sulfur bond to provide an alternative protonation site, a key step toward molecular hydrogen formation. This site is less basic than the unrotated form and helps evolve H2 with thermodynamic favorability.
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Song, Qingsheng. "Development of Dual Gas Diffusion-Type Biofuel Cells on the Basis of Electrochemical Understanding of Enzyme-Modified Electrodes." 京都大学 (Kyoto University), 2017. http://hdl.handle.net/2433/225650.
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Kyoto University (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
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Kriegel, Sébastien. "Transformation of a membrane protein from the respiratory chain into a sensor for the analysis of its interaction with substrates, inhibitors and lipids." Phd thesis, Université de Strasbourg, 2013. http://tel.archives-ouvertes.fr/tel-01017392.
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The field of bioenergetics deals with the flow and transformation of energy within and between living organisms and their environment. The work presented in this thesis report focuses on cellular respiration and more specifically on the first enzyme of the respiratory chain, NADH:ubiquinone oxidoreductase (Complex I). This was done to clarify details about its function and its implication in disease. First, the creation of a sensor involving the biomimetically immobilized enzyme is presented and probed through a combination of surface enhanced infrared absorption spectroscopy (SEIRAS) and electrochemistry. This sensor is then tested against different substrates and inhibitors. In a second part, the interaction of Complex I with lipids, inhibitors (Zn2+ and NADH-OH) and the role of a Tyrosine residue situated in the NADH binding pocket are investigated through electrochemically induced UV-Vis and FTIR difference spectroscopies. The results gathered through these experiments are then explored under a structural perspective and a coupling mechanism between quinone reduction and proton translocation by Complex I is proposed.
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Hernández, Ibáñez Naiara. "Exploration of novel materials in (bio)electrocatalysis: sensing in complex media and biocathodes for the CO2 reduction." Doctoral thesis, Universidad de Alicante, 2018. http://hdl.handle.net/10045/88207.
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Las etapas de transferencia electrónica o transferencia de carga involucradas en reacciones electroquímicas juegan un papel muy importante en un gran número de procesos biológicos y bioquímicos. Hoy en día, el interés de la comunidad científica se centra en explorar y entender exhaustivamente la naturaleza biológica y química de los fenómenos bioelectroquímicos que ocurren en los seres vivos, con el objeto de mimetizarlos en el laboratorio. Los procesos bioelectrocatalíticos presentan un amplio abanico de aplicaciones dirigidas al: (i) desarrollo de biorreactores electroquímicos para la mitigación de las emisiones de gases de efecto invernadero, la eliminación de contaminantes presentes en aguas residuales y urbanas, o la síntesis de productos con alto valor añadido para la industria, (ii) el desarrollo de biopilas y biobaterías, y (iii) el desarrollo de (bio)sensores electroquímicos con fines analíticos. Sin embargo, la implantación en el mercado de dispositivos basados en procesos biocatalíticos aún se enfrenta a varios desafíos, como son la robustez, la estabilidad a largo plazo, la reproducibilidad y la rentabilidad de producción en términos de materiales y fabricación de los dispositivos electroquímicos. La motivación de esta tesis doctoral es la de enfrentarse a algunos de los desafíos con los que se encuentra hoy en día la bioelectrocatálisis, para ello esta tesis doctoral se centra, principalmente en el estudio de nuevos materiales y mejora de rutas y estrategias bioelectrocatalíticas, con la finalidad de desarrollar dispositivos electroquímicos con aplicaciones analíticas y en la obtención de productos de valor añadido. En primer lugar esta tesis doctoral recoge el estudio y desarrollo de (bio)sensores electroquímicos para la determinación de lactato, L-cisteína, peróxido de hidrógeno y pH en medios biológicos complejos, y en segundo lugar estudia la bioelectrosíntesis de ácido fórmico a través de la reducción bioelectroquímica de dióxido de carbono.
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Tye, Jesse Wayne. "Explorations of iron-iron hydrogenase active site models by experiment and theory." [College Station, Tex. : Texas A&M University, 2006. http://hdl.handle.net/1969.1/ETD-TAMU-1014.
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Gerey, Bertrand. "Complexes homo- et hétéro-nucléaires de manganèse et de métaux alcalino-terreux : vers des modèles du centre de dégagement d'oxygène du photosystème II." Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GREAV041/document.
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Ce mémoire de thèse est consacré au développement et à la caractérisation de nouveaux complexes homo- et hétéronucléaires de manganèse et de métaux alcalino-terreux dans le cadre de la modélisation du cluster inorganique Mn4CaO5 de l’OEC du photosystème II.De nouveaux ligands incluant un nombre variable de groupements pyridine-carboxylates basés sur les architectures tris-(2-picolyl)amine et bis-(2-picolyl)ethylamine ont été synthétisés. Ces ligands ont permis d’isoler de nouveaux complexes homonucléaires avec Ca2+ et Sr2+ et ainsi que des complexes hétéronucléaires MnII–Ca. L’analyse par spectroscopie XAS sur le calcium de certaines de ces espèces (parmi d’autres modèles) a permis de calibrer cette technique et ainsi montrer que le XAS pourrait être utilisé pour sonder le cluster naturel dans les différents états du cycle de Kok.Par la suite, une nouvelle famille de complexes tétranucléaires MnII3M’ (M’ = Li+, Ca2+, Sr2+, Mn2+) a été isolée, reposant sur une base métallomacrocyclique trinucléaire accueillant un cation M’ dans la cavité formée. Ces complexes ont révélé un comportement électrochimique original présentant trois systèmes redox successifs et réversibles dont le potentiel varie avec M’. Ces espèces ont démontré une excellente stabilité en solution, y compris à l’état d’oxydation du manganèse +III. Des complexes homonucléaires M3M (M = Fe2+, Co2+) similaires ont aussi été étudiés.Enfin, des complexes binucléaires de Mn3+ pontés par des ligands oxo ont été isolés, ainsi que leurs précurseurs de Mn2+. Par ailleurs, un exemple de cluster métallomacrocyclique à valence mixte Mn2+/Mn3+ et incorporant du calcium a été obtenu, qui représente le premier exemple d’un manganèse(II) ponté par un ligand hydroxo à deux ions Ca2+This thesis is focused on the development and characterization of new homo- and heteronuclear complexes of manganese and alkaline-earth metals for the modelization of the Mn4CaO5 inorganic cluster of the OEC of photosystem II.New ligands incorporating a varied number of pyridine-carboxylate groups and based on the tris-(2-picolyl)amine and bis-(2-picolyl)ethylamine architectures have been synthesized. These ligands enabled the isolation of Ca2+ and Sr2+ homonuclear complexes as well as heteronuclear MnII–Ca complexes. Part of these species have been characterized (among other models) by Ca XAS spectroscopy, enabling the calibration of this technique for the study of more complex systems such as the OEC.Furthermore, a new family of MnII3M’ (M’ = Li+, Ca2+, Sr2+, Mn2+) tetranuclear complexes has been isolated, based on a trinuclear Mn2+ metallamacrocyclic architecture hosting a M’ cation in the formed cavity. These complexes exhibited an original electrochemical behavior, displaying three successive reversible redox processes in oxidation whose potentials vary depending on the metal M’. These species demonstrated an excellent stability in solution, even at the +III oxidation state of manganese. Similar homonuclear M3M (M = Fe2+, Co2+) complexes have been isolated.Finally, binuclear Mn3+ complexes bridged by oxo ligands have been isolated, as well as their Mn2+ precursors. Moreover, a mixed-valence Mn2+/Mn3+ metallamacrocyclic cluster incorporating calcium has been synthesized, revealing the first example of a manganese(II) bridged by an hydroxo ligand to Ca2+ ions
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Esnault, Charles. "Modification électrochimique de surface pour la mesure des interactions ADN/Protéines (HsRad51 - Transposase)." Phd thesis, Université du Maine, 2012. http://tel.archives-ouvertes.fr/tel-00752894.
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Depuis l'apparition du terme "biosensor" à travers un article de Lyons et Clark en 1962, les biocapteurs ont connu un véritable essor tant au niveau académique qu'industriel. Le principal objectif de ce travail de thèse était de créer une surface permettant l'immobilisation spécifique par liaison covalente de simple ou double brin d'ADN puis d'étudier les interactions pouvant exister entre une protéine donnée et l'ADN. Pour préparer la surface à cette immobilisation, nous avons opéré une réduction électrochimique de sel d'aryldiazoniums. Ce type de modification nous a permis de fixer de manière covalente sur la surface conductrice des fonctions de type Ar-SO2Cl. Par l'utilisation de la QCM et de l'AFM, nous avons pu par la suite détailler les mécanismes de fonctionnement de protéines (HsRad51 et Transposase) en interaction avec l'ADN simple ou double brin fixé, que ce soit d'un point de vue cinétique ou bien structural.
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Nxusani, Ezo. "Synthesis and analysis of Novel Platinum group Metal Chalcogenide Metal Quantum Dot and Electrochemical Markers." University of the Western Cape, 2018. http://hdl.handle.net/11394/6424.
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Magister Scientiae - MSc (Chemistry)Although cadmium and lead chalcogenide quantum dot have excellent optical and photoluminescent properties that are highly favorable for biological applications, there still exists increasing concerns due to the toxicity of these metals. We, therefore, report the synthesis of new aqueous soluble IrSe quantum dot at room temperature utilizing a bottom-up wet chemistry approach. NaHSe and H2IrCl6 were utilized as the Se and Ir source, respectively. High-resolution transmission electron microscopy reveals that the synthesized 3MPA-IrSe Qd are 3 nm in diameter. The characteristics and properties of the IrSe Qd are investigated utilizing, Selected Area electron diffraction, ATR- Fourier Transform Infra-Red Spectroscopy, Energy Dispersive X-ray spectroscopy, Photoluminescence, Cyclic Voltammetry and chronocoulometry. A 3 fold increase in the optical band gap of IrSe quantum dot in comparison to reported bulk IrSe is observed consistent with the effective mass approximation theory for semiconductor materials of particles sizes < 10 nm. The PL emission of the IrSe quantum dot is at 519 nm. Their electro-activity is studied on gold electrodes and exhibit reduction and oxidation at - 107 mV and +641 mV, with lowered reductive potentials. The synthesized quantum dot are suitable for low energy requiring electrochemical applications such as biological sensors and candidates for further investigation as photoluminescent biological labels.
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"The Investigation and Characterization of Redox Enzymes Using Protein Film Electrochemistry." Doctoral diss., 2014. http://hdl.handle.net/2286/R.I.26871.
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abstract: Redox reactions are crucial to energy transduction in biology. Protein film electrochemistry (PFE) is a technique for studying redox proteins in which the protein is immobilized at an electrode surface so as to allow direct exchange of electrons. Establishing a direct electronic connection eliminates the need for redoxactive mediators, thus allowing for interrogation of the redox protein of interest. PFE has proven a versatile tool that has been used to elucidate the properties of many technologically relevant redox proteins including hydrogenases, laccases, and glucose oxidase.
This dissertation is comprised of two parts: extension of PFE to a novel electrode material and application of PFE to the investigation of a new type of hydrogenase. In the first part, mesoporous antimony-doped tin oxide (ATO) is employed for the first time as an electrode material for protein film electrochemistry. Taking advantage of the excellent optical transparency of ATO, spectroelectrochemistry of cytochrome c is demonstrated. The electrochemical and spectroscopic properties of the protein are analogous to those measured for the native protein in solution, and the immobilized protein is stable for weeks at high loadings. In the second part, PFE is used to characterize the catalytic properties of the soluble hydrogenase I from Dissertation/Thesis
Doctoral Dissertation Biochemistry 2014
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Ortiz, Luis Angel. "High-throughput functional screening of oxidase enzymes." Thesis, 2020. https://hdl.handle.net/2144/42066.
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Our ability to sense small molecules with high specificity, over a broad range of concentrations, is limited and difficult to accomplish in a way that is inexpensive and continuous. The most commercially successful biosensor is the enzyme-based blood glucose electrochemical biosensor, yet for nearly all other biomolecules, detection and monitoring require specialized equipment, trained personnel, and long lead times, and are not amenable to continuous monitoring. Industries in need of enzyme-based small-molecule biosensors, including medical diagnostics, industrial production, environmental monitoring, food safety analysis, and international security, would benefit greatly from the development of new devices capable of measuring biomolecules of interest.
Environmental microbes have been gaining attention because of the vast array of biomolecules that they are capable of sensing and degrading. These microbes do so, in part, through redox enzymes with diverse substrate specificities that represent an immense resource for developing electrochemical biosensors. However, the development of new enzyme biosensors has largely been limited by the lack of a general high-throughput method to identify these redox enzymes, making discovery slow, laborious, and ad hoc.
To address this need, a high-throughput functional screening approach has been developed to isolate microbial oxidase enzymes from complex metagenomic DNA libraries based solely on the enzyme-mediated degradation of any target analyte. The approach can be applied to DNA isolated from any complex microbial sample, including unidentified or unculturable bacteria. In this research, I first describe the development of a general assay to capture the activity of oxidase enzymes expressed in E. coli cells. I then demonstrate how the assay can be used to screen for the nicotine degrading oxidase NicA2 from a genomic DNA library generated from the microbe P. putida. Lastly, I describe the use of this screen to identify a new hydrocortisone-responsive oxidase from a pooled genomic DNA library of eight microbes, representing over 43 Mb of DNA sequence space. This hydrocortisone oxidase represents the first of many new enzymes that can be discovered leveraging our screening platform, which is poised to revolutionize the electrochemical biosensing field and substantially broaden the number of molecules these electrochemical biosensors can detect continuously and quantitatively.2023-02-17T00:00:00Z
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Leote, Ricardo José Branco. "Desenvolvimento e caracterização de biossensores eletroquímicos enzimáticos com enzimas oxidase." Master's thesis, 2017. http://hdl.handle.net/10316/83242.
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Dissertação de Mestrado em Química apresentada à Faculdade de Ciências e TecnologiaNeste trabalho foram desenvolvidos biossensores enzimáticos a partir da modificação de eléctrodos de carbono vítreo com materiais nanoestruturados, nomeadamente nanotubos de carbono e mediadores redox com imobilização de enzimas oxidase. Esta modificação dos eléctrodos convencionais visa melhorar o desempenho dos biossensores aprimorando os parâmetros analíticos.O estudo está dividido em quatro capítulos.O primeiro capítulo trata aspectos gerais sobre a definição, classificação, e componentes dos biossensores, transdutores electroquímicos e materiais de eléctrodo (nanotubos de carbono e nanopartículas), mediadores redox, enzimas e a sua imobilização, bem como os fundamentos de electroquímica e uma breve descrição das técnicas utilizadas.O segundo capítulo inclui informações sobre os reagentes e instrumentação utilizada, estão descritas as condições experimentais em que foram efectuadas as medidas electroquímicas, e uma descrição detalhada da modificação efectuada para preparação de cada biossensor desenvolvido neste trabalho.No terceiro capítulo descrevem-se e discutem-se os resultados obtidos. Este capítulo está dividido em três secções, uma para cada enzima utilizada: glucose oxidase (GOx), lactato oxidase (LOx) e piruvato oxidase (PyOx). A GOx e a LOx foram imobilizadas directamente no eléctrodo não modificado ou em cima do eléctrodo modificado com nanotubos de carbono e um mediador redox, poli(vermelho neutro) para GOx e ferroceno para LOx. A voltametria cíclica foi usado para caracterizar o comportamento electroquímico. Relativamente ao biossensor para piruvato com PyOx foi realizado um estudo mais pormenorizado. Primeiro, descreve-se o biossensor construído sem mediador, a semelhança dos outros dois anteriores; em segundo lugar um biossensor preparado com nanotubos de carbono (CNT). A seguir foi adicionado, junto com os CNT, um mediador redox, azul de Prússia, ou na forma de um filme depositado electroquimicamente ou na forma de nanoparticulas depositadas quimicamente. Cada biossensor foi avaliado através de amperometria a potencial fixo, sendo os seus parâmetros análiticos retirados de curvas de calibração. No caso do biossensor com PyOx, foi feito um estudo para optimização de vários factores experimentais e foi efectuada uma comparação do desempenho obtido com o de outros biossensores da literatura.O capítulo quatro inclui conclusões que podem ser tiradas do trabalho efectuado sobre cada um dos biossensores desenvolvidos, bem como perspectivas futuras.
In this work, enzymatic biosensors have been developed using glassy carbon electrode modified with different nanostructured materials, namely carbon nanotubes and redox mediators with immobilized enzyme oxidases. This modification of conventional electrodes is meant to improve the biosensors performance taking into account their analytical parameters.The study is divided in four chapters.The first chapter is focused on general aspects about definition, classification and biosensors components, electrochemical transducers and electrode materials (carbon nanotubes and nanoparticles), redox mediators, enzymes and their modification, as well as electrochemistry principles and a short description of the techniques used.The second chapter includes information about reagents and instruments used, the experimental conditions for electrochemical experiments are described, and a detailed description for the preparation of each biosensor developed.In the third chapter the results achieved are described and discussed. This chapter is divided in three sections, one for each enzyme used: glucose oxidase (GOx), lactate oxidase (LOx) and pyruvate oxidase (PyOx). GOx and Lox were directly immobilized on the unmodified electrode, or at the electrode modified with carbon nanotubes and a redox mediator, which was poly(neutral red) for GOx and ferrocene for LOx. Cyclic voltammetry was used in order to characterize the electrochemical behavior. As regarding the pyruvate biosensor with PyOx, a more detailed study was performed. First, the biosensor without mediator is described, similarly with the previous two; secondly a biosensor prepared with carbon nanotubes. Further, a redox mediator, Prussian blue was added together with CNT, or as a film, electrochemically deposited, either as nanoparticles, chemically deposited. Each biosensor was evaluated by fixed potential amperometry, their analytical parameters being calculated from calibration curves. For the PyOx biosensor an optimisation of the experimental conditions has been carried out, as well as a comparison of its performance with other biosensors from literature.The fourth chapter includes conclusions, which can be withdrawn from the work about each of the developed biosensors, as well as future perspectives.
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Goran, Jacob Michael. "The bioelectrochemistry of enzymes and their cofactors at carbon nanotube and nitrogen-doped carbon nanotube electrodes." Thesis, 2014. http://hdl.handle.net/2152/30491.
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This dissertation explores the electrochemical behavior of enzymes and their cofactors at carbon nanotube (CNT) and nitrogen-doped carbon nanotube (N-CNT) electrodes. Two common types of oxidoreductases are considered: flavin adenine dinucleotide (FAD)-dependent oxidases and nicotinamide adenine dinucleotide-dependent (NAD⁺)-dehydrogenases. Chapter 1 presents the oxygen reduction reaction (ORR) at N-CNT electrodes as a way to electrochemically measure enzymatic turnover at the electrode surface. The unique peroxide pathway at N-CNT electrodes, which catalytically disproportionates hydrogen peroxide (H₂O₂) back into oxygen, provides an increased ORR current directly proportional to the rate of enzymatic turnover for H₂O₂ producing enzymes, even in an oxygen saturated solution. Biosensing of L-lactate using the increased ORR current is demonstrated using L-lactate oxidase. Chapter 2 explores the surface bound electrochemical signal of FAD when FAD-dependent enzyme or free FAD is allowed to spontaneously adsorb onto the CNT/N-CNT surface. Specifically, the origin of the enzymatically generated FAD signal and the rate constant of the electron transfer are elucidated. Chapter 3 continues the discussion of the cofactor FAD by demonstrating its use as an informative surface specific redox probe for graphitic carbon surfaces. Primarily, FAD can be used to determine the electroactive surface area and the relative hydrophobicity/hydrophilicity of graphitic surfaces. Chapter 4 changes gears to NAD⁺-dependent dehydrogenases by investigating the electrocatalytic oxidation of NADH at N-CNTs in comparison with conventional carbon electrodes or nondoped CNTs. Biosensing of glucose through the oxidation of NADH is demonstrated using glucose dehydrogenase adsorbed onto the N-CNT surface. Chapter 5 continues the discussion of NAD⁺-dependent dehydrogenases by addressing the reaction kinetics of NADH oxidation at N-CNTs as a tool to measure the enzymatic reduction of NAD⁺.
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Maiocco, Stephanie Jane. "Biophysical characterization of electron transfer proteins containing multiple metallocofactors: investigation of the AdoMet radical and cytochrome c peroxidase enzyme superfamilies." Thesis, 2016. https://hdl.handle.net/2144/17706.
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Metallocofactors are ubiquitous in nature, serving multiple purposes in proteins. These metallocofactors typically act as the site of catalysis or as an electron relay to move electrons within the protein, or within the cell, and are very energetically costly to manufacture. Yet, in nature it can appear that supernumerary, or ‘auxiliary’ cofactors are apparent, with no clear function. In this thesis, I address the question of what roles additional cofactors play, and why they are retained.
The radical S-adenosylmethionine (AdoMet) enzyme superfamily has displayed great diversity in the cofactor requirements for its members. Some members of this family contain only the canonical [4Fe-4S] cluster, which reductively cleaves AdoMet to initiate chemistry, while others have additional [2Fe-2S] or [4Fe-4S] clusters. Even greater cofactor complexity is seen with the B12-dependent subclass, featuring a cobalamin-binding domain in addition to the canonical FeS cluster. The majority of this thesis has focused on using the technique of protein film electrochemistry (PFE) to study members of various subclasses of this superfamily: a dehydrogenase: BtrN, two methylthiotransferases: MiaB and RimO, as well as OxsB and TsrM, two B12-dependent enzymes. By evaluating the redox properties of members of different subclasses, we have been able to shed light on the redox properties of this superfamily, in general, and observed that the redox properties of auxiliary clusters can differ widely between subclasses (e.g. BtrN versus MiaB). PFE has also been used to evaluate five ferredoxins that are possible electron donors for MiaB from Thermotoga maritima.
Additionally, bacterial cytochrome c peroxidases (bCCPs) are diheme enzymes catalyzing the detoxification of hydrogen peroxide; however, a novel subclass of bCCPs containing a third heme-binding motif has been identified in enteric pathogens. Protein film electrochemistry has been used to study the redox properties of Escherichia coli YhjA, a member of this subgroup. Further characterization of this novel bCCP was achieved with electron paramagnetic resonance, optical spectroscopy, and steady-state kinetics. Through characterizing YhjA and members of the AdoMet radical enzyme superfamily, we have shed light on the role these additional cofactors play in the mechanism and how these enzymes are tuned for their specific chemistries.2018-08-11T00:00:00Z