Optimization of operation conditions for improved cytochrome P450BM3 enzymatic reaction yield

Abstract

The P450 cytochrome monooxygenase CYP102A1 from Bacillus megaterium, better known as P450BM3, is a heme‐thiolate enzyme that catalyzes the hydroxylation of numerous substrates. Many of the resulting products are of commercial interest to the pharmaceutical and fine chemical industries. Unlike most other P450 cytochromes, P450BM3 is both soluble and fused to its natural redox partner, which supplies the necessary electrons from NADPH to drive its reaction forward. However, the industrial use of this enzyme is limited by its poor stability and its expensive cofactor. In this work, we explore the effects of buffer formulation and temperature on the stability of wildtype and P450BM3 mutant R966D/W1046S as well as on the stability of nicotinamide cofactors NADPH, NADH, and the biomimetic cofactor N‐benzyl‐1,4‐dihydronicotinamide. We demonstrate that cofactor stability is more important to increase product yield than that of the enzyme. We also demonstrate that low temperatures enhance oxido‐reduction reactions coupling, thus resulting in an increase in the molar ratio of p‐nitrophenolate produced from 10‐pNCA per oxidized cofactor. Overall, the optimized reaction conditions lead to a 2 to 2.6‐fold increase in total product output when wildtype P450BM3 or R966D/W1046S mutant is used with either of these three aforementioned cofactors.

Issue Highlights

“…The optimized reaction conditions lead to a 2.6-fold increase in total product output with P450BM3 variant R966D/W1046S. [2] The dynamics of the flow in packed beds is complex and the classical methods often fail to resolve the conservation equations of the continuum model. Pseudocontinuum models are utilized instead by superimposing an extrinsic velocity on the continuum model and adopting empirically correlated dispersion parameters.…”

“…Secondly, we demonstrate that low temperatures enhance oxido‐reduction reactions coupling, thus further increasing product output. The optimized reaction conditions lead to a 2.6‐fold increase in total product output with P450BM3 variant R966D/W1046S [2] …”