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
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.