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Colorimetric High-Throughput Assay for Alkene Epoxidation Catalyzed by Cytochrome P450 BM-3 Variant 139-3

Alcalde, Miguel and Farinas, Edgardo T. and Arnold, Frances H. (2004) Colorimetric High-Throughput Assay for Alkene Epoxidation Catalyzed by Cytochrome P450 BM-3 Variant 139-3. Journal of Biomolecular Screening, 9 (2). pp. 141-146. ISSN 1087-0571. https://resolver.caltech.edu/CaltechAUTHORS:20180824-100531408

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Abstract

Cytochrome P450 BM-3 variant 139-3 is highly active in the hydroxylation of alkanes and fatty acids (AGlieder, ET Farinas, and FH Arnold, Nature Biotech 2002;20:1135-1139); it also epoxidizes various alkenes, including styrene. Here the authors describe a colorimetric, high-throughput assay suitable for optimizing this latter activity by directed evolution. The product of styrene oxidation by 139-3, styrene oxide, reacts with the nucleophile γ-(4-nitrobenzyl)pyridine (NBP) to form a purple-colored precursor dye, which can be monitored spectrophotometrically in cell lysates. The sensitivity limit of this assay is 50-100 μ Mof product, and the detection limit for P450 BM-3 139-3 is ~0.2 μ Mof enzyme. To validate the assay, activities in a small library of random mutants were compared to those determined using an NADPH depletion assay for initial turnover rates.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1177/1087057103261913DOIArticle
ORCID:
AuthorORCID
Alcalde, Miguel0000-0001-6780-7616
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2004 Published by Sage Publications in association with The Society for Biomolecular Screening. Received Jul 5, 2003 and in revised form Nov 2, 2003. Accepted for publication Nov 17, 2003. This work was supported by the National Science Foundation and by Maxygen, Inc. MA was supported by the Ministerio de Educacion y Cultura, Spain.
Funders:
Funding AgencyGrant Number
NSFUNSPECIFIED
Maxygen, Inc.UNSPECIFIED
Ministerio de Educacion y Cultura (MEC)UNSPECIFIED
Subject Keywords:P450 BM-3 139-3, alkene epoxidation, γ-(4-nitrobenzyl)pyridine, styrene oxide
Issue or Number:2
Record Number:CaltechAUTHORS:20180824-100531408
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180824-100531408
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89146
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:24 Aug 2018 17:13
Last Modified:09 Mar 2020 13:18

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