Convenient microtiter plate-based, oxygen-independent activity assays for flavin-dependent oxidoreductases based on different redox dyes

Dagmar Brugger, Iris Krondorfer, Kawah Zahma, Thomas Stoisser, Juan M. Bolivar, Bernd Nidetzky, Clemens K. Peterbauer, Dietmar Haltrich

Research output: Contribution to journalArticleResearchpeer-review

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.

LanguageEnglish
Pages474-482
Number of pages9
JournalBiotechnology journal
Volume9
Issue number4
DOIs
StatusPublished - 2014

Fingerprint

Oxidation-Reduction
Oxidoreductases
Coloring Agents
Oxygen
Enzymes
Flavoproteins
lactate 2-monooxygenase
cellobiose-quinone oxidoreductase
Electrons
2,6-Dichloroindophenol
D-Amino-Acid Oxidase
Enzyme Assays
4,6-dinitro-o-cresol
Hydrogen Peroxide

Keywords

  • 2,6-dichlorophenol-indophenol
  • Flavin-dependent oxidoreductases
  • High-throughput screening
  • Methylene green
  • Thionine

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Molecular Medicine
  • Medicine(all)

Cite this

Convenient microtiter plate-based, oxygen-independent activity assays for flavin-dependent oxidoreductases based on different redox dyes. / Brugger, Dagmar; Krondorfer, Iris; Zahma, Kawah; Stoisser, Thomas; Bolivar, Juan M.; Nidetzky, Bernd; Peterbauer, Clemens K.; Haltrich, Dietmar.

In: Biotechnology journal, Vol. 9, No. 4, 2014, p. 474-482.

Research output: Contribution to journalArticleResearchpeer-review

Brugger, Dagmar ; Krondorfer, Iris ; Zahma, Kawah ; Stoisser, Thomas ; Bolivar, Juan M. ; Nidetzky, Bernd ; Peterbauer, Clemens K. ; Haltrich, Dietmar. / Convenient microtiter plate-based, oxygen-independent activity assays for flavin-dependent oxidoreductases based on different redox dyes. In: Biotechnology journal. 2014 ; Vol. 9, No. 4. pp. 474-482
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