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Directed evolution of a Baeyer–Villiger monooxygenase to enhance enantioselectivity

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Abstract

The Baeyer–Villiger monooxygenase (BVMO) BmoF1 from Pseudomonas fluorescens DSM 50106 was shown before to enantioselectively oxidize different 4-hydroxy-2-ketones to the corresponding hydroxyalkyl acetates, being the first example of a BVMO-catalyzed kinetic resolution of aliphatic acyclic ketones. However, the wild-type enzyme exhibited only moderate E values (E ∼ 55). Thus, the enantioselectivity was enhanced by means of directed evolution and optimization of reaction conditions since it was found that higher E values (E ∼ 70 for wild-type BmoF1) could already be obtained when performing biotransformations in shake flasks rather than small tubes. In a first step, random mutations were introduced by error-prone polymerase chain reaction, and BmoF1 mutants (>3,500 clones) were screened for improved activity and enantioselectivity using a microtiter-plate-based screening method. Mutations S136L and L252Q were found to increase conversion compared to wild type, while several mutations (H51L, F225Y, S305C, and E308V) were identified enhancing the enantioselectivity to a varying extent (E ∼ 75–90). In a second step, beneficial mutations were recombined by consecutive cycles of QuikChange® site-directed mutagenesis resulting in a double mutant (H51L/S136L) showing both improved conversion and enantioselectivity (E ∼ 86).

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Acknowledgements

We thank the Fonds der Chemischen Industrie (Frankfurt, Germany) and the Studienstiftung des Deutschen Volkes (Bonn, Germany) for stipends to Anett Kirschner.

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  1. Anett Kirschner

    Present address: Biochemistry Laboratory, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands

Authors and Affiliations

  1. Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Str. 4, 17487, Greifswald, Germany

    Anett Kirschner & Uwe T. Bornscheuer

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  1. Anett Kirschner
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  2. Uwe T. Bornscheuer
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Correspondence to Uwe T. Bornscheuer.

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Kirschner, A., Bornscheuer, U.T. Directed evolution of a Baeyer–Villiger monooxygenase to enhance enantioselectivity. Appl Microbiol Biotechnol 81, 465–472 (2008). https://doi.org/10.1007/s00253-008-1646-4

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  • DOI: https://doi.org/10.1007/s00253-008-1646-4

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