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Directed evolution of a Baeyer–Villiger monooxygenase to enhance enantioselectivity


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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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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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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  • Baeyer–Villiger monooxygenase
  • Directed evolution
  • Enzyme catalysis
  • Enantioselectivity
  • β-hydroxyketones