Process Optimization for the Electroenzymatic Hydroxylation of 3-Phenoxytoluene Catalyzed by P450 BM-3

Abstract

Bioelectrochemical hydroxylation of 3-phenoxyroluene has been carried out as a model system in which redesigned P450 BM-3 accepting electrons from electrode surface modified with a conducting polymer, poly(3,4-ethylenedioxythiophene) doped with polystyrene sulfonic acid (PEDOT/PSS) was used. Experimental runs were performed in accordance with central composite design and conducted batch-wise to visualize the effects of independent factors (thickness of enzyme-polymer layer, applied potential and substrate concentration). Process was optimized with response surface methodology. Optimum conditions at 25ºC were estimated as - 0.70 V (vs. SCE) applied potential, 1.35 mM substrate concentration and ~60.35 nm thickness of enzyme-polymer layer to achieve 330.49 μM of product formation after 6 h of reaction in the presence of catalase. Kinetic studies reveal that electrochemical system can be an alternative and cost-effective electron donor compared to natural cofactor with further improvement of protein engineering strategies.