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\begin{document}

\title{Characterization of different biocatalyst formats for BVMO-catalyzed
cyclohexanone oxidation}



\author[1]{Lisa Bretschneider}%
\author[1]{Ingeborg Heuschkel}%
\author[1]{Afaq Ahmed}%
\author[2]{Katja Bühler}%
\author[3]{Rohan Karande}%
\author[4]{Bruno Bühler}%
\affil[1]{Helmholtz-Centre for Environmental Research - UFZ}%
\affil[2]{Helmholtz Center for Environmental Research}%
\affil[3]{Helmholtz-Zentrum für Umweltforschung GmbH - UFZ}%
\affil[4]{UFZ, Leipzig, Germany}%


\vspace{-1em}



  
  \date{May 22, 2024}


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\begin{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 \emph{Acidovorax}-derived CHMO gene was expressed in
\emph{Pseudomonas taiwanensis} VLB120. Upon purification, the enzyme was
characterized \emph{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
\emph{P. taiwanensis} VLB120. Biofilms showed less favorable values for
K\textsubscript{S} (9.3-fold higher) and k\textsubscript{cat} (4.8-fold
lower) compared to corresponding K\textsubscript{M} and
k\textsubscript{cat} values of isolated CHMO, but a favorable
K\textsubscript{I} for cyclohexanone (5.3-fold higher). The unfavorable
K\textsubscript{S} and k\textsubscript{cat} 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\textsubscript{S}, but 1.3- and 4.2-fold higher
k\textsubscript{cat} and K\textsubscript{I} values than isolated CHMO.
This together with the efficient NADPH regeneration via glucose
metabolism makes this format highly promising from a kinetics
perspective.%
\end{abstract}%



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