Open AccessProtocol for Mutagenesis of Alkene Monooxygenase and Screening for Modified Enantiocomposition of the Epoxypropane Product
Author(s) -
Ashlee Perry,
Thomas J. Smith
Publication year - 2006
Publication title -
slas discovery
Language(s) - English
Resource type - Journals
eISSN - 2472-5560
pISSN - 2472-5552
DOI - 10.1177/1087057106287930
Subject(s) - rhodococcus rhodochrous , monooxygenase , alkene , mutagenesis , stereoselectivity , propene , chemistry , methane monooxygenase , mutant , stereochemistry , enantiomer , enantiomeric excess , enzyme , biochemistry , gene , rhodococcus , cytochrome p450 , catalysis , enantioselective synthesis
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%.