Open Access

Claudia Ley

• Cytochrome P450 monooxygenases catalyze a variety of reactions, but biotechnological approaches with isolated enzymes are still rare. One of the main challenges is their cofactor dependency hampering use in industrial processes. A promising possibility is the substitution of the cofactor by electrochemical means. For this strategy a technology is needed to parallelize electroenzymatic experiments and to accelerate research efforts. An electrochemical microtiter plate (eMTP), which is described in the first part of this work, enables parallelized electrochemical experiments with simultaneous optical data acquisition in up to eight wells of a microtiter plate. This is the first example for parallel spectroelectrochemistry in commercially available microtiter plates, wherein each well contains a set of independent electrodes. The well known redox probe potassium ferrocyanide and the mediator ABTS were characterized in the eMTP to proof the functionality and reliability of the system. Furthermore a mutant library of P450 BM3, a well studied fatty acid monooxygenase with high catalytic activity, was screened for its performance in the surrogate pNCA assay via the mediator cobalt sepulchrate. The eMTP was used to find optimized reaction conditions for the electroenzymatic process. The second part of the thesis focused on the conducting polymer polypyrrole. Due to its biocompatibility, conductivity and structural versatility polypyrrole provides all requirements needed for the efficient immobilization and electrochemical actuation of redox molecules at electrodes. Direct electron transfer between redox enzymes and electrodes is of high interest, since it represents an approach for heterogeneous process design with integrated enzyme retention and simultaneous control of the redox state of the enzymés active site. Pyrrole and two carboxylic acid modified derivatives were introduced as flexible immobilization matrices. A set of redox active iron compounds and small heme type redox proteins was screened towards improved direct electron transfer via the conducting polymer matrix. Based on the commercially available monomers several immobilization methods were employed, which were in addition applied to P450 BM3.