title: Convenient microtiter plate-based, oxygen-independent activity assays for flavin-dependent oxidoreductases based on different redox dyes.
abstract: Flavin-dependent oxidoreductases are increasingly recognized as important biocatalysts for various industrial applications. In order to identify novel activities and to improve these enzymes in engineering approaches, suitable screening methods are necessary. We developed novel microtiter-plate-based assays for flavin-dependent oxidases and dehydrogenases using redox dyes as electron acceptors for these enzymes. 2,6-dichlorophenol-indophenol, methylene green, and thionine show absorption changes between their oxidized and reduced forms in the visible range, making it easy to judge visually changes in activity. A sample set of enzymes containing both flavoprotein oxidases and dehydrogenases - pyranose 2-oxidase, pyranose dehydrogenase, cellobiose dehydrogenase, D-amino acid oxidase, and L-lactate oxidase - was selected. Assays for these enzymes are based on a direct enzymatic reduction of the redox dyes and not on the coupled detection of a reaction product as in the frequently used assays based on hydrogen peroxide formation. The different flavoproteins show low Michaelis constants with these electron acceptor substrates, and therefore these dyes need to be added in only low concentrations to assure substrate saturation. In conclusion, these electron acceptors are useful in selective, reliable and cheap MTP-based screening assays for a range of flavin-dependent oxidoreductases, and offer a robust method for library screening, which could find applications in enzyme engineering programs.
authors.affiliation: Food Biotechnology Laboratory, BOKU University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Brugger
authors.forename: Iris
authors.initials: I
authors.lastname: Krondorfer
authors.forename: Kawah
authors.initials: K
authors.lastname: Zahma
authors.forename: Thomas
authors.initials: T
authors.lastname: Stoisser
authors.forename: Juan M
authors.initials: JM
authors.lastname: Bolivar
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
authors.forename: Clemens K
authors.initials: CK
authors.lastname: Peterbauer
authors.forename: Dietmar
authors.initials: D
authors.lastname: Haltrich
mesh_terms: D015086:2,6-Dichloroindophenol / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D004396:Coloring Agents / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D057166:High-Throughput Screening Assays; D008751:Methylene Blue / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010640:Phenothiazines / Q000032:analysis / Q000737:chemistry / Q000378:metabolism
chemical_list: D004396:Coloring Agents; D010640:Phenothiazines; D015086:2,6-Dichloroindophenol; D010088:Oxidoreductases; C028673:methylene green; D008751:Methylene Blue; C009469:thionine
keywords: 2,6-dichlorophenol-indophenol; flavin-dependent oxidoreductases; high-throughput screening; methylene green; thionine