Manipulating the stereoselectivity of the thermostable Baeyer-Villiger monooxygenase TmCHMO by directed evolution

Baeyer-Villiger monooxygenases (BVMOs) and evolved mutants have been shown to be excellent biocatalysts in many stereoselective Baeyer-Villiger transformations, but industrial applications are rare which is partly due to the insufficient thermostability of BVMOs under operating conditions. In the pr...

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Published inOrganic & biomolecular chemistry Vol. 15; no. 46; pp. 9824 - 9829
Main Authors Li, Guangyue, Fürst, Maximilian J L J, Mansouri, Hamid Reza, Ressmann, Anna K, Ilie, Adriana, Rudroff, Florian, Mihovilovic, Marko D, Fraaije, Marco W, Reetz, Manfred T
Format Journal Article
LanguageEnglish
Published England Royal Society of Chemistry 2017
Subjects
Biocatalysts
Directed evolution
Enantiomers
Evolution
Industrial applications
Ketones
Methylcyclohexanones
Monooxygenase
Mutants
Oxidation
Saturation mutagenesis
Stereoselectivity
Substrates
Thermal stability
Transformations
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Summary:Baeyer-Villiger monooxygenases (BVMOs) and evolved mutants have been shown to be excellent biocatalysts in many stereoselective Baeyer-Villiger transformations, but industrial applications are rare which is partly due to the insufficient thermostability of BVMOs under operating conditions. In the present study, the substrate scope of the recently discovered thermally stable BVMO, TmCHMO from Thermocrispum municipale, was studied. This revealed that the wild-type (WT) enzyme catalyzes the oxidation of a variety of structurally different ketones with notable activity and enantioselectivity, including the desymmetrization of 4-methylcyclohexanone (99% ee, S). In order to induce the reversal of enantioselectivity of this reaction as well as the transformations of other substrates, directed evolution based on iterative saturation mutagenesis (ISM) was applied, leading to (R)-selectivity (94% ee) without affecting the thermostability of the biocatalyst.
ISSN:1477-0520
1477-0539
DOI:10.1039/c7ob02692g