Novel 96-well quantitative bioelectrocatalytic analysis platform reveals highly efficient direct electrode regeneration of cytochrome P450 BM3 on indium tin oxide

Abstract

Enzymes are the most effective catalysts for a broad range of difficult chemical reactions e.g. hydroxylation of non-activated C-H Bonds and stereoselective synthesis. Nevertheless, a lot of enzymes are not accessible for the biotechnological applications or industrial use. One reason is the prerequisite of expensive cofactors.In this context, we developed a bioelectrocatalytic analysis platform for the electrochemical and photonic quantification of the direct electron transfer from the electrode to redox enzymes and therefore, bypass the need of soluble cofactors that had to be continuously exchanged or regenerated. As reference enzyme, we chose cytochrome P450 BM3 that is restricted by NADPH dependence. We optimized the substrate spectrum for aromatic compounds by introduction of the triple mutation A74G/F87V/L188Q and established a sensitive fluorimetric product formation assay to monitor the enzymatic conversion of 7-ethoxycoumarine to 7-hydroxycoumarine. Gold and indium tin oxide electrodes were characterized with respect to surface morphology, charge-transfer resistance and P450 BM3 immobilization as well as activity. Using gold electrodes, no significant product formation by electrode mediated direct electron transfer could be detected. In contrast, P450 BM3 adsorbed on unmodified indium tin oxide electrodes revealed 36% activity by electrode mediated direct electron transfer in comparison to enzyme regeneration by NADPH. Since the reaction volumes are in the microliter range and upscaling of the measurement system is easily possible, our analysis platform is a useful tool for bioelectrocatalytic enzyme characterization and library screening based optimization for applications in the field of enzyme catalyzed chemical synthesis but also enzyme based fuel cells.