Abstract
Technical application of cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 in particular, is hindered by limited enzyme stability. In addition, substrate and product inhibition is a well-known challenge of using CHMO. By site-directed mutagenesis two new combinatorial CHMO variants, CHMO M15 L323C-A325C (M15 DS) and CHMO M16 L323C-A325C (M16 DS), were designed to stabilize the enzyme, by incorporating a reported disulfide bridge into the already published parental CHMO variants: CHMO M15 and CHMO M16. Additionally, the newly described BVMO AFL706 from Aspergillus flavus was characterized for epsilon-caprolactone (ECL) synthesis, for which the enzyme showed significantly higher substrate and product tolerance compared to the wild type. The wild type CHMO and the four variants were applied in a convergent cascade coupled with an alcohol dehydrogenase from Thermoanaerobacter ethanolicus for ECL synthesis. M15 DS and M16 DS variants performed better than the wild type and the parental variants under operational conditions. Subsequently, the cascade was further optimized by means of Design of Experiments, doubling the product yield (21 mM, 27%) with a reduced cofactor amount of 0.5 mM NADP+.
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