Journal
CHEMISTRYOPEN
Volume 9, Issue 9, Pages 959-966Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/open.202000184
Keywords
halogenation; fluorescence spectroscopy; kinetics; enzyme catalysis; hypobromite
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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 fromCurvularia inaequaliswere recorded, yielding the first overall kinetic parameters for this enzyme. With limits of detection and quantification in the low mu mrange, this assay may provide a reliable alternative system for the quantification of haloperoxidase-activity.
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