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Applied and Environmental Microbiology, May 2009, p. 3106-3114, Vol. 75, No. 10
0099-2240/09/$08.00+0     doi:10.1128/AEM.02707-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Cloning, Expression, Characterization, and Biocatalytic Investigation of the 4-Hydroxyacetophenone Monooxygenase from Pseudomonas putida JD1{triangledown} ,{dagger}

Jessica Rehdorf, Christian L. Zimmer, and Uwe T. Bornscheuer*

Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Strasse 4, D-17487 Greifswald, Germany

Received 26 November 2008/ Accepted 20 February 2009

While the number of available recombinant Baeyer-Villiger monooxygenases (BVMOs) has grown significantly over the last few years, there is still the demand for other BVMOs to expand the biocatalytic diversity. Most BVMOs that have been described are dedicated to convert efficiently cyclohexanone and related cyclic aliphatic ketones. To cover a broader range of substrate types and enantio- and/or regioselectivities, new BVMOs have to be discovered. The gene encoding a BVMO identified in Pseudomonas putida JD1 converting aromatic ketones (HAPMO; 4-hydroxyacetophenone monooxygenase) was amplified from genomic DNA using SiteFinding-PCR, cloned, and functionally expressed in Escherichia coli. Furthermore, four other open reading frames could be identified clustered around this HAPMO. It has been suggested that these proteins, including the HAPMO, might be involved in the degradation of 4-hydroxyacetophenone. Substrate specificity studies revealed that a large variety of other arylaliphatic ketones are also converted via Baeyer-Villiger oxidation into the corresponding esters, with preferences for para-substitutions at the aromatic ring. In addition, oxidation of aldehydes and some heteroaromatic compounds was observed. Cycloketones and open-chain ketones were not or poorly accepted, respectively. It was also found that this enzyme oxidizes aromatic ketones such as 3-phenyl-2-butanone with excellent enantioselectivity (E >>100).

* Corresponding author. Mailing address: Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str. 4, D-17487 Greifswald, Germany. Phone: 49-3834-86-4367. Fax: 49-3834-86-80066. E-mail: uwe.bornscheuer{at}uni-greifswald.de

{triangledown} Published ahead of print on 27 February 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, May 2009, p. 3106-3114, Vol. 75, No. 10
0099-2240/09/$08.00+0     doi:10.1128/AEM.02707-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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