Colorimetric high-throughput assay for alkene epoxidation catalyzed by cytochrome P450 BM-3 variant 139-3.
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 gamma-(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 microM of product, and the detection limit for P450 BM-3 139-3 is ~0.2 microM 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.
Departamento de Biocatalisis, Instituto de Catalisis y Petroleoquimica (CSIC), Campus Universidad Autonoma de Madrid, Cantoblanco, Madrid, Spain.
Miguel
M
Alcalde
Edgardo T
ET
Farinas
Frances H
FH
Arnold
D000475:Alkenes / Q000378:metabolism*; D002384:Catalysis; D003124:Colorimetry / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004925:Escherichia / Q000235:genetics; D005822:Genetic Vectors; D006861:Hydrogen Peroxide / Q000032:analysis; D008958:Models, Molecular; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010957:Plasmids; D020058:Styrene / Q000378:metabolism
D000475:Alkenes; D020058:Styrene; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide