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Title: Structure-function studies of the oxidoreductase bilirubin oxidase from Myrothecium verrucaria using an electrochemical quartz crystal microbalance with dissipation
Author: Singh, Kulveer
ISNI:       0000 0004 5369 4122
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2014
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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.
Supervisor: Blanford, Christopher ; Wong, Luet Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Chemistry & allied sciences ; Biophysical chemistry ; Catalysis ; Chemical kinetics ; Electrochemistry and electrolysis ; Enzymes ; Inorganic chemistry ; Organic chemistry ; Physical & theoretical chemistry ; Surface chemistry ; Surface analysis ; Computer aided molecular and material design ; Biochemistry ; Materials Sciences ; Surfaces ; Materials engineering ; Quartz-crystal microbalance ; Electrochemical quartz-crystal microbalance ; electrochemistry ; voltammetry ; gold electrodes ; enzyme catalysis ; redox enzyme ; reduction ; adsorption ; bilirubin oxidase ; laccase ; oxygen reduction ; fuel cell