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

Autor:	Mark L. Thompson, Kristian Geitner, Marlen Schmidt, Uwe T. Bornscheuer, Lea Talmann, Maria Kadow, Stefan Saß, Dominique Böttcher
Rok vydání:	2012
Předmět:	chemistry.chemical_classification biology Organic Chemistry Monooxygenase biology.organism_classification medicine.disease_cause Biochemistry Esterase Pseudomonas putida Hydrolysis Microtiter plate Enzyme chemistry Biocatalysis Drug Discovery medicine Escherichia coli
Zdroj:	Tetrahedron. 68:7575-7580
ISSN:	0040-4020
Popis:	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.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=doi_________::2c72cda57d9bb984f413b9c8545ff735 https://doi.org/10.1016/j.tet.2012.05.098 Zobrazit plný text záznamu Full Text from ScienceDirect