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Completing the series of BVMOs involved in camphor metabolism of Pseudomonas putida NCIMB 10007 by identification of the two missing genes, their functional expression in E. coli, and biochemical characterization

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Abstract

The camphor-degrading Baeyer–Villiger monooxygenases (BVMOs) from Pseudomonas putida NCIMB 10007 have been of interest for over 40 years. So far the FMN- and NADH-dependent type II BVMO 3,6-diketocamphane 1,6-monooxygenase (3,6-DKCMO) and the FAD- and NADPH-dependent type I BVMO 2-oxo-∆3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) have not been entirely studied, since it was not possible to produce those enzymes in satisfactory amounts and purity. In this study, we were able to clone and recombinantly express both enzymes and subsequently use them as biocatalysts for various mono- and bicyclic ketones. Full conversion could be reached with both enzymes towards (±)-cis-bicyclo[3.2.0]hept-2-en-6-one and with 3,6-DKCMO towards (−)-camphor. Further OTEMO gave full conversion with norcamphor. OTEMO was found to have a pH optimum of 9 and a temperature optimum of 20 °C and converted (±)-cis-bicyclo[3.2.0]hept-2-en-6-one with a k cat/K M value of 49.3 mM−1 s−1.

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Acknowledgments

We are grateful to the Deutsche Bundesstiftung Umwelt (DBU, Osnabrück, Germany, Grant No. AZ13234) for the financial support and to Ina Menyes for the assistance in the laboratory.

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Authors and Affiliations

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

    Maria Kadow, Kathleen Loschinski, Stefan Saß, Marlen Schmidt & Uwe T. Bornscheuer

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  1. Maria Kadow
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  2. Kathleen Loschinski
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  3. Stefan Saß
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  4. Marlen Schmidt
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  5. Uwe T. Bornscheuer
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Correspondence to Uwe T. Bornscheuer.

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Maria Kadow and Kathleen Loschinski equally contributed to this work.

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Kadow, M., Loschinski, K., Saß, S. et al. Completing the series of BVMOs involved in camphor metabolism of Pseudomonas putida NCIMB 10007 by identification of the two missing genes, their functional expression in E. coli, and biochemical characterization. Appl Microbiol Biotechnol 96, 419–429 (2012). https://doi.org/10.1007/s00253-011-3859-1

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