Soutenance de thèse Hiu Mun Man

07/12/2022 - 14:30 - G. Morin
Hiu-Mun MAN,

Characterisation of enzymatic catalysis by microscopy and electrochemistry: application to H2/O2 bio-fuel cells

Amid the global energy crisis, enzymatic biofuel cells, which utilise biocatalysts (enzymes) to convert chemical energy to electricity, are presenting to be one of the most promising alternative and clean energy resources. This involves the immobilization of redox enzymes on an electrode. Nevertheless, achieving efficient enzyme electrochemistry still raises many challenges. We can obtain the kinetic and thermodynamic information regarding the enzymatic reaction using electrochemical methods. However, an electrochemical measurement is a summation of currents over the entire electrode surface, giving us only global information. This brings the need of coupling electrochemistry to microscopy techniques to explore the spatially resolved information in situ . In this case, fluorescence confocal microscopy coupled with electrochemistry allows to probe the local heterogeneities as well as the diffusion layer of the enzymatic electrode during catalysis.

In this thesis, the coupling of confocal laser scanning fluorescence microscopy with electrochemistry has been demonstrated for the characterization of electro-enzymatic catalysis. The main reaction investigated was oxygen reduction reaction catalysed by the enzyme bilirubin oxidase from Myrothecium verrucaria (MvBOD). Graphite and gold (micro)electrodes were fabricated especially for the electrochemical-microscopy setup. The adsorption behaviour of MvBOD on these electrodes was investigated by electrochemistry. The enzymatic O2-reduction involves proton consumption coupled with the electron transfer reaction. Using the in-situ analysis method, local pH variations occurring at the vicinity of the bioelectrode throughout enzymatic catalysis are visualised via a pH-dependent fluorophore, fluorescein . We have shown that the fluorescence intensity recorded is directly proportional to the catalytic current values. The influence of ionic strength on enzymatic catalysis has then been specifically explored. The influence of ionic strength on enzyme electroactivity and enzyme specific activity were probed using electrochemistry and UV-vis spectroscopy, respectively. Then, proton depletion profiles at the electrochemical interface in buffered and unbuffered electrolytes were reconstructed, in order to determine the influence of ionic strength on the local environment of enzymes. Finally, the enzymes have been labelled with fluorescent dyes (Alexa Fluor dyes 430 and 647), making it possible to reveal the local heterogeneities of the enzyme distribution at the electrode surface.

Keywords : enzyme electrochemistry, in-situ fluorescence microscopy, metalloenzymes, bilirubin oxidase

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