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Characterization of different biocatalyst formats for BVMO-catalyzed cyclohexanone oxidation
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  • Lisa Bretschneider,
  • Ingeborg Heuschkel,
  • Afaq Ahmed,
  • Katja Bühler,
  • Rohan Karande,
  • Bruno Bühler
Lisa Bretschneider
Helmholtz-Centre for Environmental Research - UFZ
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Ingeborg Heuschkel
Helmholtz-Centre for Environmental Research - UFZ
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Afaq Ahmed
Helmholtz-Centre for Environmental Research - UFZ
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Katja Bühler
Helmholtz Center for Environmental Research
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Rohan Karande
Helmholtz-Zentrum für Umweltforschung GmbH - UFZ
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Bruno Bühler
UFZ, Leipzig, Germany
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Abstract

Cyclohexanone monooxygenase (CHMO), a member of the Baeyer-Villiger monooxygenase family, is a versatile biocatalyst that efficiently catalyzes the conversion of cyclic ketones to lactones. In this study, an Acidovorax-derived CHMO gene was expressed in Pseudomonas taiwanensis VLB120. Upon purification, the enzyme was characterized in vitro and shown to feature a broad substrate spectrum and up to 100% conversion in 6 h. Further, we determined and compared the cyclohexanone conversion kinetics for different CHMO-biocatalyst formats, i.e., isolated enzyme, suspended whole cells, and biofilms, the latter two based on recombinant CHMO-containing P. taiwanensis VLB120. Biofilms showed less favorable values for KS (9.3-fold higher) and kcat (4.8-fold lower) compared to corresponding KM and kcat values of isolated CHMO, but a favorable KI for cyclohexanone (5.3-fold higher). The unfavorable KS and kcat values are related to mass transfer- and possibly heterogeneity issues and deserve further investigation and engineering, in order to exploit the high potential of biofilms regarding process stability. Suspended cells showed an only 1.8-fold higher KS, but 1.3- and 4.2-fold higher kcat and KI values than isolated CHMO. This together with the efficient NADPH regeneration via glucose metabolism makes this format highly promising from a kinetics perspective.

Peer review status:ACCEPTED

29 Jan 2021Submitted to Biotechnology and Bioengineering
29 Jan 2021Submission Checks Completed
29 Jan 2021Assigned to Editor
31 Jan 2021Reviewer(s) Assigned
13 Mar 2021Review(s) Completed, Editorial Evaluation Pending
13 Mar 2021Editorial Decision: Revise Minor
03 Apr 20211st Revision Received
03 Apr 2021Submission Checks Completed
03 Apr 2021Assigned to Editor
04 Apr 2021Review(s) Completed, Editorial Evaluation Pending
04 Apr 2021Editorial Decision: Accept