Cytochrome P450 biocatalysis is an attractive potential alternative to traditional chemical synthesis for achieving asymmetric hydroxylations, and the general application of these enzymes in combinatorial biocatalytic schemes would generate greater product diversity. Unfortunately, many factors limit the large-scale integration of P450s into synthetic schemes. In particular, the poor water solubility of many potential P450 substrates and the expense of the required cofactor, NAD(P)H, are major obstacles for the use of isolated P450 preparations.

To address the solubility limitations of some P450 substrates, we have examined P450cam activity in two-phase hexane/water emulsions with and without the anionic surfactant, bis(2-ethylhexyl) sulfosuccinate sodium salt (AOT). Hydroxylation of camphor to hydroxycamphor by the three-component P450cam system was chosen as the model reaction, and NADH regenerated with yeast alcohol dehydrogenase (YADH). P450cam was activated in the surfactant-free emulsions, and AOT improved the activity even further, at least over the range of camphor concentrations for which initial rates were readily measurable in all media. The highly-uncoupled oxidation of hydroxycamphor to 2,5-diketocamphane was also observed, and limited the yield of hydroxycamphor in the two-phase emulsions. These results indicate that a surfactant-stabilized two-phase emulsion is a promising reaction medium for practical P450 biocatalysis, although its effectiveness for a given P450/substrate combination can depend on several factors, including competitive or sequential reactions and NAD(P)H uncoupling.

To improve the cost-efficiency of P450 reactions, we replaced the expensive natural cofactor, NAD(P)H, with two biomimetic NADH analogs for P450cam and P450 BM-3 reactions. We also used the catalyst precursor, [Cp*Rh(bpy)(H ₂O)]OTf₂ and sodium formate to generate and regenerate the NADH analogs in situ with P450 reactions. Wild-type (WT) P450cam oxidation reactions could be supported by the NADH analogs, but WT P450 BM-3 was not active with the analogs. Protein engineering was used to improve the activity of both P450s with the NADH analogs. While mutations of a key residue in the reductase (PdR) for P450cam afforded only a small increase in initial rate, mutations in the P450 BM-3 reductase domain enabled the use of NADH analogs at rates comparable to that of the natural cofactor. Protein engineering in combination with chemically-catalyzed regeneration is thus a promising strategy for utilizing economically advantageous NAD(P)H analogs in P450-catalyzed oxidation reactions.