Improving the cytochrome P450 enzyme system for electrode-driven biocatalysis of styrene epoxidation

Cytochrome P450 enzymes catalyze a vast array of oxidative and reductive bi otransformations that are potentially useful for industrial and pharmaceuti cal syntheses. Factors such as cofactor utilization and slow reaction rates for nonnatural substrates limit their large-scale usefulness. This paper r eports several improvements that make the cytochrome P450cam enzyme system more practical for the epoxidation of styrene. NADH coupling was increased from 14 to 54 mol %, and product turnover rate was increased from 8 to 70 m in(-1) by introducing the Y96F mutation to P450cam. Styrene and styrene oxi de mass balance determinations showed different product profiles at low and high styrene conversion levels. For styrene conversion less than about 25 mol %, the stoichiometry between styrene consumption and styrene oxide form ation was 1:1. At high styrene conversion, a second doubly oxidized product , alpha-hydroxyacetophenone, was formed. This was also the exclusive produc t when Y96F P450cam acted on racemic, commercially available styrene oxide. The cl-hydroxyacetophenone product was suppressed in reactions where styre ne was present at saturating concentrations. Finally, styrene epoxidation w as carried out in an electroenzymatic reactor. In this scheme, the costly N ADH cofactor and one of the three proteins (putidaredoxin reductase) are el iminated from the Y96F P450cam enzyme system.