Coupling of electrochemical and optical measurements in a microtiter plate for the fast development of electro enzymatic processes with P450s

Abstract

Electro enzymatic processes offer novel opportunities in catalysis by combining advantages of enzyme catalysis and electrochemistry. An efficient electrochemical cofactor substitution system can help to overcome economical hurdles for the technical use of cofactor dependent enzymes. The in vitro biocatalysis with P450 BM-3 was investigated aiming for the substitution of the expensive natural cofactor NADPH by electrochemistry as “electron source”. An electrochemical 24-well microtiter plate (eMTP) was developed, which can be employed in a standard microtiter plate reader and enables parallelized electrochemical experiments in combination with simultaneous optical measurements. The eMTP was applied to screen a P450 monooxygenase BM-3 mutein library and determine the behavior of P450 BM-3 muteins in an electrochemically driven surrogate assay with the mediator cobalt sepulchrate. Besides determining reaction rates also the influence of single reaction parameters e.g. applied potential, enzyme and mediator concentration were measured. Additionally the developed eMTP based screening system allows a fast development of an electro enzymatic process.