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

  • Saß S
  • Kadow M
  • Geitner K
 et al. 
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Abstract

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. © 2012 Elsevier Ltd. All rights reserved.

Author-supplied keywords

  • Baeyer-Villiger monooxygenase
  • Biocatalysis
  • HAPMO
  • Spectrophotometric assay
  • Whole cell

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