Characterization of different biocatalyst formats for BVMO-catalyzed cyclohexanone oxidation

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 K (9.3-fold higher) and k (4.8-fold lower) compared to corresponding K and k values of isolated CHMO, but a favorable K for cyclohexanone (5.3-fold higher). The unfavorable K and k 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 K, but 1.3- and 4.2-fold higher k and K values than isolated CHMO. This together with the efficient NADPH regeneration via glucose metabolism makes this format highly promising from a kinetics perspective.

Baeyer–Villiger Monooxygenases (BVMOs) as Biocatalysts

“…11a 28 Nevertheless, in terms of making this enzyme class even more enticing for potential industrial use in the future, important issues of enzyme stability and enhanced efficiency must yet be satisfactorily addressed and resolved. 29…”