Protocol for mutagenesis of alkene monooxygenase and screening for modified enantiocomposition of the epoxypropane product

PERRY, A. and SMITH, T. J. (2006). Protocol for mutagenesis of alkene monooxygenase and screening for modified enantiocomposition of the epoxypropane product. Journal of biomolecular screening, 11 (5), 553-556.

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Link to published version:: 10.1177/1087057106287930

Abstract

Alkene monooxygenase (AMO) from Rhodococcus rhodochrous B-276 is a 3-component enzyme system encoded by the 4-gene operon amoABCD, which catalyzes the stereoselective epoxidation of aliphatic alkenes yielding primarily the R enantiomer. With propene as the substrate, wild-type AMO yields R-epoxypropane with an enantiomeric excess (e.e.) of 83%. The presumed site of alkene oxidation is a dinuclear iron center situated within the large subunit of the epoxygenase component, AmoC. Substantial problems with the expression of recombinant AMO were previously overcome. In this study, the authors have further developed this expression system to allow amoC to be subjected to mutagenesis by means of error-prone PCR, with the aim of developing a system that could be used to manipulate the enantioselectivity of the enzyme. The mutants were screened for altered stereoselectivity in the propene/epoxypropane reaction by a whole-cell assay, solvent extraction, and chiral gas chromatography analysis protocol that is suitable for scale up to several thousand mutants and that is estimated to detect differences in e.e. of as little as 5%.

Item Type: Article
Uncontrolled Keywords: alkene monooxygenase, epoxygenase, directed evolution, propene, epoxypropene
Research Institute, Centre or Group: Biomedical Research Centre
Identification Number: 10.1177/1087057106287930
Depositing User: Ann Betterton
Date Deposited: 19 Feb 2008
Last Modified: 09 Dec 2009 18:23
URI: http://shura.shu.ac.uk/id/eprint/418

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