Catalytic hydroxylation in biphasic systems using CYP102A1 mutants

Cytochrome P450 monooxygenases are biocatalysts that hydroxylate or epoxidise a wide range of hydrophobic organic substrates. Their technical application is, however, limited to a small number of whole-cell processes. The use of the isolated P450 enzymes is believed to be impractical due to their low stability, stoichiometric need of the expensive cofactor NAD(P)H and low solubility of most substrates in aqueous media. We investigated the behaviour of an isolated bacterial monooxygenase (mutants of CYP102A1) in a biphasic reaction system supported by cofactor recycling with the NADP(+)-dependent formate dehydrogenase from Pseudomonas sp 101. Using this experimental set-up cyclohexane, octane and myristic acid were hydroxylated. To reduce the process costs a novel NADH-dependent mutant of CYP102A1 was designed. For recycling of NADH an NAD(+)-dependent FDH was used. The stability of the monooxygenase mutants under the reaction conditions in the biphasic system was quite high as revealed by total turnover numbers of up to 12,850 in the NADPH-dependent cyclohexane hydroxylation and up to 30,000 in the NADH-dependent myristic acid oxidation.