Abstract

Epoxygenases are attractive enzymes for synthesizing important chemical synthons. Directed evolution of epoxygenase properties to production demands have been limited until recently by a lack of screening systems. The previously reported p-nitrothiophenolate (pNTP) screening system was validated through improving styrene epoxidation activity of P450 BM-3 from Bacillus megaterium. Unlike the catalytically self-sufficient P450 BM-3, most epoxygenases are multi-component systems and often significantly less active. We improved the pNTP screening system for a two-component epoxygenase, styrene monooxygenase StyAB from Pseudomonas species, by enhancing the sensitivity of the pNTP assay from 400 to 140 μM and reducing styrene evaporation from 72 to 52%. These improvements were achieved using methylated β-cyclodextrins (mβ-CD) as inclusion host for styrene. Incorporation of mβ-CD increases styrene availability over the assay period and thus enables screening for improved mutants. The pNTP screening procedure for StyAB was subsequently verified in 96-well microtiter plate screens by gas chromatography analysis of styrene conversions.

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call

Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.