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.

Full text not available from this repository.
Link to published version:: https://doi.org/10.1177/1087057106287930
Related URLs:

    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 - Does NOT include content added after October 2018: Biomolecular Sciences Research Centre
    Identification Number: https://doi.org/10.1177/1087057106287930
    Page Range: 553-556
    Depositing User: Ann Betterton
    Date Deposited: 19 Feb 2008
    Last Modified: 18 Mar 2021 21:45
    URI: http://shura.shu.ac.uk/id/eprint/418

    Actions (login required)

    View Item View Item

    Downloads

    Downloads per month over past year

    View more statistics