In:
ChemistryOpen, Wiley, Vol. 9, No. 9 ( 2020-09), p. 959-966
Abstract:
Screening for an interesting biocatalyst and its subsequent kinetic characterization depends on a reliable activity assay. In this work, a fluorometric assay based on the halogenation of 4‐methyl‐7‐diethylamino‐coumarin was established to monitor haloperoxidase‐activity. Since haloperoxidases utilize hydrogen peroxide and halide ions to halogenate a broad range of substrates by releasing hypohalous acids, a direct quantification of haloperoxidase‐activity remains difficult. With the system presented here, 3‐bromo‐4‐methyl‐7‐diethylaminocoumarin is preferentially formed and monitored by fluorescence measurements. As starting material and product share similar spectroscopical properties, a two‐dimensional calibration ap‐proach was utilized to allow for quantification of each compound within a single measurement. To validate the system, the two‐dimensional Michaelis‐Menten kinetics of a vanadium‐dependent chloroperoxidase from Curvularia inaequalis were recorded, yielding the first overall kinetic parameters for this enzyme. With limits of detection and quantification in the low μ m range, this assay may provide a reliable alternative system for the quantification of haloperoxidase‐activity.
Type of Medium:
Online Resource
ISSN:
2191-1363
,
2191-1363
DOI:
10.1002/open.202000184
Language:
English
Publisher:
Wiley
Publication Date:
2020
detail.hit.zdb_id:
2655605-4