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Aspergillus oryzae cytochrome P450 monooxygenase (CYP57B3) is capable of catalyzing hydroxylation of genistein to produce 3΄-hydroxygenistein. Because hydroxylated derivatives of genistein, including 3΄-hydroxygenistein, exhibit various pharmacological activities, CYP57B3 would potentially be useful as a biocatalyst in the pharmaceutical field. We therefore performed random mutagenesis of CYP57B3 to improve its catalytic activities for genistein. Random mutations were introduced by error-prone PCR into CYP57B3, resulting in construction of a library of mutants. From 2000 mutants, we isolated one with a triple mutation in CYP57B3, resulting in three amino acid substitutions (V138I, S243N and V463F). The altered protein, designated CYP57B3-mut, had a high level of activity for the production of 3΄-hydroxygenistein from genistein. In bioconversion reactions, Saccharomyces cerevisiae expressing CYP57B3-mut showed 14-fold higher production of 3΄-hydroxygenistein than that of wild-type CYP57B3. In vitro kinetic analysis revealed that Km values of wild-type and CYP57B3-mut were 14.6 × 103 ± 2.7 × 103 μM and 15.7 ± 1.0 μM, respectively. These results suggest that the affinity of CYP57B3 for genistein could be dramatically improved by the alterations of V138, S243 and V463 in CYP57B3.

Impacts of amino acid substitutions in fungal cytochrome P450 monooxygenase (CYP57B3) on the effective production of 3΄-hydroxygenistein