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Colorimetric High-Throughput Assay for Alkene Epoxidation Catalyzed by Cytochrome P450 BM-3 Variant 139-3Departamento de Biocatalisis, Instituto de Catalisis y Petroleoquimica (CSIC), Campus Universidad Autonoma de Madrid, Cantoblanco, Madrid, Spain
Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena
Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena frances{at}cheme.caltech.edu
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
Key Words: P450 BM-3 139-3 • alkene epoxidation •
Journal of Biomolecular Screening, Vol. 9, No. 2,
141-146 (2004) This article has been cited by other articles:
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-(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. (Journal of Biomolecular Screening 2004:141-146) 
