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Cloning, expression, and characterization of a Baeyer–Villiger monooxygenase from Pseudomonas fluorescens DSM 50106 in E. coli

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Abstract

A gene encoding a Baeyer–Villiger monooxygenase (BVMO) identified in Pseudomonas fluorescens DSM 50106 was cloned and functionally expressed in Escherichia coli JM109. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis showed an estimated 56 kDa-size protein band corresponding to the recombinant enzyme. Expression in BL21 (DE3) resulted mainly in the formation of inclusion bodies. This could be overcome by coexpression of molecular chaperones, especially the DnaK/DnaJ/GrpE complex, leading to increased production of soluble BVMO enzyme in recombinant E. coli. Examination of the substrate spectra using whole-cell biocatalysis revealed a high specificity of the BVMO for aliphatic open-chain ketones. Thus, octyl acetate, heptyl propionate, and hexyl butyrate were quantitatively formed from the corresponding ketone substrates. Several other esters were obtained in conversion >68%. Selected esters were also produced on preparative scale.

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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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Authors and Affiliations

  1. Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Friedrich-Ludwig-Jahn-Str. 18c, 17487, Greifswald, Germany

    Anett Kirschner & Uwe T. Bornscheuer

  2. Institute of Industrial Genetics, Stuttgart University, Allmandring 31, 70569, Stuttgart, Germany

    Josef Altenbuchner

Authors
  1. Anett Kirschner
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  2. Josef Altenbuchner
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  3. Uwe T. Bornscheuer
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Correspondence to Uwe T. Bornscheuer.

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Kirschner, A., Altenbuchner, J. & Bornscheuer, U.T. Cloning, expression, and characterization of a Baeyer–Villiger monooxygenase from Pseudomonas fluorescens DSM 50106 in E. coli . Appl Microbiol Biotechnol 73, 1065–1072 (2007). https://doi.org/10.1007/s00253-006-0556-6

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  • DOI: https://doi.org/10.1007/s00253-006-0556-6

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