Aim of the project was to improve the enantioselectivity of Cyclohexanone Monooxygenase (CHMO) towards 4 hydroxycyclohexanone in the Baeyer-Villiger reaction using the principle of directed evolution. Mutants were produced by epPCR and saturation
mutagenesis. Reaction conditions were optimized and a medium-throughput gas chromatography-based screening system for the identification of improved mutants was developed, so that 800 mutants per day can be screened. After two rounds of directed evolution
the enantioselectivity of CHMO towards the substrate was increased from 9 to 90%. A mutant of reverse selectivity of 79% was found which was also able to catalyse on other substrates with excellent enantioselectivities (up to 99% ee).