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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.

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Acknowledgements

We would like to thank BASF AG for providing 3-hydroxy-β-ionone. This work has been supported by the German Research Foundation (DFG; Project SCHM 1240/6-1).

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  1. Akhmadjan Makhsumkhanov

    Present address: Institute of Microbiology, Uzbek Academy of Sciences, A. Kadiry St. 7b, 700128, Tashkent, Uzbekistan

Authors and Affiliations

  1. Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany

    Vlada B. Urlacher, Akhmadjan Makhsumkhanov & Rolf D. Schmid

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  1. Vlada B. Urlacher
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  2. Akhmadjan Makhsumkhanov
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  3. Rolf D. Schmid
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Correspondence to Vlada B. Urlacher.

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Urlacher, V.B., Makhsumkhanov, A. & Schmid, R.D. Biotransformation of β-ionone by engineered cytochrome P450 BM-3. Appl Microbiol Biotechnol 70, 53–59 (2006). https://doi.org/10.1007/s00253-005-0028-4

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  • DOI: https://doi.org/10.1007/s00253-005-0028-4

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