Reaction of Trigonopsis variabilis d-amino acid oxidase with 2,6-dichloroindophenol: kinetic characterisation and development of an oxygen-independent assay of the enzyme activity
| Autor: | Bernd Nidetzky, Anita Slavica, Waander Riethorst, Christian Trampitsch |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
inorganic chemicals
chemistry.chemical_classification Specificity constant Oxidase test flavoenzyme electron acceptor oxygen biocatalysis enzyme assay Stereochemistry Process Chemistry and Technology D-amino acid oxidase Substrate (chemistry) chemistry.chemical_element Bioengineering Electron acceptor Biochemistry Oxygen Combinatorial chemistry Catalysis Amino acid chemistry.chemical_compound chemistry Hydrogen peroxide |
| Zdroj: | Journal of Molecular Catalysis B: Enzymatic. 32:271-278 |
| ISSN: | 1381-1177 |
| DOI: | 10.1016/j.molcatb.2004.12.011 |
| Popis: | 2,6-Dichloroindophenol (DCIP) is shown to be utilised efficiently as electron acceptor replacing dioxygen in the reaction of Trigonopsis variabilis d -amino acid oxidase (TvDAO) with d -methionine as the substrate. The specificity constant for DCIP reduction at 30 °C is one-twelfth that of oxygen conversion into hydrogen peroxide. Time course analysis of simultaneous consumption of DCIP and dioxygen, recorded on-line by absorption and non-invasive fluorescence quenching, respectively, pinpoints the preferential utilisation of dioxygen; and reveals a maximum DCIP conversion rate that is independent of the initial concentration of dioxygen. A robust direct assay of TvDAO activity has been developed that does not require anaerobic reaction conditions. It was down-scaled to microtitre plate format and overcomes practical limitations of other assays due to the low affinity of TvDAO for dioxygen ( K m ≈ 0.7 mmol L −1 ). |
| Databáze: | OpenAIRE |
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