Towards Practical Baeyer–Villiger‐Monooxygenases: Design of Cyclohexanone Monooxygenase Mutants with Enhanced Oxidative Stability

<jats:title>Abstract</jats:title><jats:p>Baeyer–Villiger monooxygenases (BVMOs) catalyze the conversion of ketones and cyclic ketones into esters and lactones, respectively. Cyclohexanone monooxygenase (CHMO) from <jats:italic>Acinetobacter</jats:italic> sp. NCIMB 9871 is known to show an impressive substrate scope as well as exquisite chemo‐, regio‐, and enantioselectivity in many cases. Large‐scale synthetic applications of CHMO are hampered, however, by the instability of the enzyme. Oxidation of cysteine and methionine residues contributes to this instability. Designed mutations of all the methionine and cysteine residues in the CHMO wild type (WT) showed that the amino acids labile towards oxidation are mostly either surface‐exposed or located within the active site, whereas the two methionine residues identified for thermostabilization are buried within the folded protein. Combinatorial mutations gave rise to two stabilized mutants with either oxidative or thermal stability, without compromising the activity or stereoselectivity of the enzyme. The most oxidatively stabilized mutant retained nearly 40 % of its activity after incubation with H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> (0.2 <jats:sc>M</jats:sc>), whereas the wild‐type enzyme's activity was completely abolished at concentrations as low as 5 m<jats:sc>M</jats:sc> H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>. We propose that oxidation‐stable mutants might well be a “prerequisite” for thermostabilization, because laboratory‐evolved thermostability in CHMO might be masked by a high degree of oxidation instability.</jats:p>