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- Title
Extending the substrate scope of a Baeyer-Villiger monooxygenase by multiple-site mutagenesis.
- Authors
Dudek, Hanna; Fink, Michael; Shivange, Amol; Dennig, Alexander; Mihovilovic, Marko; Schwaneberg, Ulrich; Fraaije, Marco
- Abstract
Baeyer-Villiger monooxygenase-catalysed reactions are attractive for industrial processes. Here we report on expanding the substrate scope of phenylacetone monooxygenase (PAMO). In order to introduce activity on alicyclic ketones in PAMO, we generated and screened a library of 1,500 mutants. Based on recently published structures of PAMO and its mutants, we selected previously uncharacterised positions as well as known hot-spots to be targeted by focused mutagenesis. We were able to mutate 11 positions in a single step by using the OmniChange method for the mutant library generation. Screening of the library using a phosphate-based activity detection method allowed identification of a quadruple mutant (P253F/G254A/R258M/L443F) active on cyclopentanone. The substrate scope of this mutant is extended to several aliphatic ketones while activity on aromatic compounds typical for PAMO was preserved. Moreover, the mutant is as thermostable as PAMO. Our results demonstrate the power of screening structure-inspired, focused mutant libraries for creating Baeyer-Villiger monooxygenases with new specificities.
- Subjects
BAEYER-Villiger rearrangement; MONOOXYGENASES; MUTAGENESIS; MANUFACTURING processes; ALICYCLIC compounds; PROTEIN engineering
- Publication
Applied Microbiology & Biotechnology, 2014, Vol 98, Issue 9, p4009
- ISSN
0175-7598
- Publication type
Article
- DOI
10.1007/s00253-013-5364-1