, Volume 70, Issue 1, pp 53-59
Date: 07 Jul 2005

Biotransformation of β-ionone by engineered cytochrome P450 BM-3

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Abstract

Wild-type cytochrome P450 monooxygenase from Bacillus megaterium (P450 BM-3) has a low hydroxylation activity for β-ionone (<1 min−1). Substitution of phenylalanine by valine at position 87 led to a more than 100-fold increase in β-ionone hydroxylation activity (115 min−1). Enzyme activity could be further increased by both site-directed and random mutagenesis. The mutant R47L Y51F F87V, designed by site-directed mutagenesis, and the mutant A74E F87V P386S, obtained after two rounds of error-prone polymerase chain reaction, exhibited an increase in activity of up to 300-fold compared to the wild-type enzyme. The triple mutant R47 LY51F F87V exhibited moderate enantioselectivity, forming (R)-4-hydroxy-β-ionone with an optical purity of 39%. All mutants regioselectively converted β-ionone into 4-hydroxy-β-ionone. The regioselectivity is determined amongst others by the absolute configuration of the substrate.