title: Directed evolution of a Baeyer-Villiger monooxygenase to enhance enantioselectivity.
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 approximately 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 approximately 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 approximately 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 approximately 86).
authors: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Str. 4, 17487, Greifswald, Germany.
authors: Anett
authors: A
authors: Kirschner
authors: Uwe T
authors: UT
authors: Bornscheuer
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019020:Directed Molecular Evolution; D007659:Ketones / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011551:Pseudomonas fluorescens / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D007659:Ketones; D006899:Mixed Function Oxygenases