ArticleBiotechnology journal2014
Convenient microtiter plate-based, oxygen-independent activity assays for flavin-dependent oxidoreductases based on different redox dyes.
Article in Biotechnology journal, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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14 citing papers in PubMed, 27 citations in OpenAlex.
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- Deciphering the role of recurrent FAD-dependent enzymes in bacterial phosphonate catabolism.iScience · 2023Article
- Role of extremophiles and their extremozymes in biorefinery process of lignocellulose degradation.Extremophiles : life under extreme conditions · 2021Review
- Developing a cell-bound detection system for the screening of oxidase activity using the fluorescent peroxide sensor roGFP2-Orp1.Protein engineering, design & selection : PEDS · 2020Article
- Recent Development of Extremophilic Bacteria and Their Application in Biorefinery.Frontiers in bioengineering and biotechnology · 2020Review
- Versatile Oxidase and Dehydrogenase Activities of Bacterial Pyranose 2-Oxidase Facilitate Redox Cycling with Manganese PeroxidaseApplied and environmental microbiology · 2019Article
- Assays of D-Amino Acid Oxidase Activity.Frontiers in molecular biosciences · 2017Article
- Electrochemical characterization of the pyranose 2-oxidase variant N593C shows a complete loss of the oxidase function with full preservation of substrate (dehydrogenase) activity.Physical chemistry chemical physics : PCCP · 2016Article
- Oxidation of Phe454 in the Gating Segment Inactivates Trametes multicolor Pyranose Oxidase during Substrate Turnover.PloS one · 2016Article
- Estimating the success of enzyme bioprospecting through metagenomics: current status and future trends.Microbial biotechnology · 2016Review
- Mutation breeding of high 4-androstene-3,17-dione-producing Mycobacterium neoaurum ZADF-4 by atmospheric and room temperature plasma treatment.Journal of Zhejiang University. Science. B · 2015Article
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8 authors at 2 institutions in 1 country.
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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.
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