Colorimetric high-throughput assay for alkene epoxidation catalyzed by cytochrome P450 BM-3 variant 139-3

Miguel Alcalde, Edgardo T. Farinas, Frances H. Arnold

Research output: Contribution to journalArticlepeer-review

43 Scopus citations

Abstract

Cytochrome P450 BM-3 variant 139-3 is highly active in the hydroxylation of alkanes and fatty acids (A Glieder, 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 μM of product, and the detection limit for P450 BM-3 139-3 is ∼0.2 μM of 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.

Original languageEnglish (US)
Pages (from-to)141-146
Number of pages6
JournalJournal of Biomolecular Screening
Volume9
Issue number2
DOIs
StatePublished - Mar 2004
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biotechnology
  • Biochemistry
  • Molecular Medicine
  • Pharmacology
  • Drug Discovery

Keywords

  • Alkene epoxidation
  • P450 BM-3 139-3
  • Styrene oxide
  • γ-(4-nitrobenzyl)pyridine

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