Electrochemical study of redox enzymes and their utilization on modified electrodes

Abstract

The present work focuses on the extraction of thermodynamic and kinetic information of redox enzymes and molecules they interact with, through electrochemical techniques. In the introduction, a quick overview is made on redox enzymes in general and how they can be exploited electrochemically together with the challenges faced. As one of the most prominent problems when using cyclic voltammetry for the study of redox enzymes is the existence capacitance currents, a major part of this work focuses on exploring an alternative, large amplitude fast Fourier transform alternating current voltammetry (FTacV), so as to overcome this obstacle. The investigation of FTacV takes place for both free in a solution and immobilized on an electrode surface electroactive species. The respective results for cyclic voltammetry are presented as well for comparison. Starting with the free species, the chief observables, i.e. peak height, peak width and peak potential of the principal peaks, are determined for FTacV for a reversible reaction of free electroactive species. The values of the chief observables are derived from a semi-analytic solution as well as from the solution of the full initial-boundary value problem. A condition under which FTacV is independent of scan rate, up to the fifth harmonic, is introduced. The dependence of the chief observables on the parameters of the system is shown. Then, the case of quasi reversible and irreversible kinetics is studied correlating the chief observables with the electrode kinetics. Moreover two step electron reactions are examined in order to get a visual representation of the resulting voltammograms. Two mechanisms are also explored when coupling an electroactive species with a homogeneous reaction. The first is the EC mechanism and the second the homogeneous catalytic reaction scheme. Convolution voltammetry is also introduced in this work as some of its aspects would of use in the experimental part of this work. The analysis that comes right after that concerns the immobilized species. Start- ing from an one step reaction of an electroactive speceis doing a similar study to the one conducted for the free species, moving on to two step reactions. As a next step, the coupling of the redox reaction of the immobilized species with a reaction that concerns a species free in the bulk solution shall follow. More specifically the Michaelis- Menten kinetics of an immobilized enzyme shall be thoroughly analyzed as the case of an electrochemical-chemical (EC) mechanism. Methodologies regarding the extraction of kinetic constants are also introduced. Moving on to the experimental section of this work, Lytic polysaccharide monooxy- genases (LPMOs) are introduced and the expression process of three LPMOs with two different expression vectors is analyzed. These expressed LPMOs are then immobilized with two different methodologies and kinetic information is obtained regarding both the interaction of the immobilized enzyme with the electrode and the interaction of the en- zyme with phosphoric acid swollen cellulose (PASC) using FTacV. The methodologies that are introduced earlier are utilized in this case. The next step is to examine possible electron donors that work with the LPMOs electrochemically using cyclic voltammetry, FTacV and convolution voltammetry. Var- ious phenolic compounds and lingin from wheat straw are first examined as electron donors with an LPMO analyzing their reaction products with high-performance anion- exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Then, based on the elution patterns and the presence and intensity of the oxidized products, in combination with the voltammetric data, an interpretation was given to the interaction of these substrates as reducing agents for the LPMOs. In the last part of the work, a laccase like multi copper oxidase from the fungus Thermothelomyces Thermophila is examined immoblized on an electrode surface and its interaction with epinephrine using FTacV. The application of the theory of probing an immobilized redox enzyme reacting with a free in the solution substrate following a Michaelis-Menten kinetic scheme is thoroughly examined.