PhD ceremony Ms. H.M. Dudek: Redesign of Baeyer-Villiger monooxygenases for synthetic applications

PhD ceremony: Ms. H.M. Dudek, 12.45 uur, Academiegebouw, Broerstraat 5, Groningen

Dissertation: Redesign of Baeyer-Villiger monooxygenases for synthetic applications

Promotor(s): prof. M.W. Fraaije

Faculty: Mathematics and Natural Sciences

Biocatalysis employs enzymes for performing chemical reactions which may lead to more efficient and environmentally friendly processes. Nowadays, it is possible to tailor properties of enzymes using protein engineering methods. The main goal of the research of Hanna Dudek was engineering phenylacetone monooxygenase (PAMO), a member of the Baeyer–Villiger family, into an efficient catalyst. First, she attempted changing coenzyme and substrate specificity of PAMO by site-specific mutagenesis. This resulted in moderate improvements of the enzyme properties, but nevertheless increased the knowledge on this class of enzymes. On the other hand, the limited success of the rational-engineering experiments underscored the need for alternative approaches. Therefore, she put efforts in developing an activity assay for screening libraries of PAMO mutants. The new assay was based on using a secondary enzyme for detecting activity of PAMO, and it was found to be a reliable way of testing activity of large number of enzyme variants. Equipped with the knowledge on the active site of PAMO and the efficient screening method, we proceeded with preparation and screening of a library of mutants which yielded a variant of PAMO with extended substrate scope. This research comprises an example of a protein engineering cycle which includes enzyme characterisation, development of suitable screening methods, design and screening of libraries, ultimately leading to the isolation of variants with improved properties. Most importantly, the presented approach, which was shown successful for PAMO, can be applied in engineering of other monooxygenases.