A high-throughput assay method to quantify Baeyer–Villiger monooxygenase activity

An assay for the spectrophotometric determination of Baeyer–Villiger monooxygenase (BVMO) activity is described. Baeyer–Villiger oxidation of p-nitroacetophenone generates the corresponding acetate and subsequent hydrolysis of this ester by an esterase or NaOH results in the formation of p-nitrophenolate. This chromophore can be easily quantified spectrophotometrically at 410 nm. The assay can be performed in a microtiter plate format and is applicable to whole Escherichia coli cells containing recombinant BVMO, crude cell extract as well as using purified enzyme as exemplified for the 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas putida JD1. Furthermore, the assay was used to identify more active HAPMO variants within enzyme mutant libraries generated by error-prone PCR or site-saturation mutagenesis.