Characterization of different biocatalyst formats for BVMO-catalyzed cyclohexanone oxidation

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. Furthermore, we determined and compared the cyclohexanone conversion kinetics for different CHMO-biocatalyst formats, that is, 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_S (9.3-fold higher) and k_cat (4.8-fold lower) compared with corresponding K_M and k_cat values of isolated CHMO, but a favorable K_I for cyclohexanone (5.3-fold higher). The unfavorable K_S and k_cat values are related to mass transfer- and possibly heterogeneity issues and deserve further investigation and engineering, to exploit the high potential of biofilms regarding process stability. Suspended cells showed only 1.8-fold higher K_S , but 1.3- and 4.2-fold higher k_cat and K_I values than isolated CHMO. This together with the efficient NADPH regeneration via glucose metabolism makes this format highly promising from a kinetics perspective.