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000884300 1001_ $$0P:(DE-Juel1)176769$$aFejzagić, Alexander V.$$b0
000884300 245__ $$aA Fluorescence‐Based Assay System for the Determination of Haloperoxidase‐Activity Using a Two‐Dimensional Calibration Ap‐proach
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000884300 520__ $$aScreening for an interesting biocatalyst and its subsequent kinetic characterization depends on a reliable activity assay. In this work, a fluorometric assay based on the halogenation of 4‐methyl‐7‐diethylamino‐coumarin was established to monitor haloperoxidase‐activity. Since haloperoxidases utilize hydrogen peroxide and halide ions to halogenate a broad range of substrates by releasing hypohalous acids, a direct quantification of haloperoxidase‐activity remains difficult. With the system presented here, 3‐bromo‐4‐methyl‐7‐diethylaminocoumarin is preferentially formed and monitored by fluorescence measurements. As starting material and product share similar spectroscopical properties, a two‐dimensional calibration ap‐proach was utilized to allow for quantification of each compound within a single measurement. To validate the system, the two‐dimensional Michaelis‐Menten kinetics of a vanadium‐dependent chloroperoxidase from Curvularia inaequalis were recorded, yielding the first overall kinetic parameters for this enzyme. With limits of detection and quantification in the low μm range, this assay may provide a reliable alternative system for the quantification of haloperoxidase‐activity.
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000884300 7001_ $$0P:(DE-Juel1)178077$$aMyllek, Sebastian$$b1
000884300 7001_ $$0P:(DE-Juel1)169329$$aHogenkamp, Fabian$$b2
000884300 7001_ $$0P:(DE-Juel1)171495$$aGreb, Julian$$b3
000884300 7001_ $$0P:(DE-Juel1)128906$$aPietruszka, Jörg$$b4
000884300 7001_ $$0P:(DE-Juel1)128890$$aClassen, Thomas$$b5$$eCorresponding author
000884300 773__ $$0PERI:(DE-600)2655605-4$$a10.1002/open.202000184$$gVol. 9, no. 9, p. 959 - 966$$n9$$p959 - 966$$tChemistryOpen$$v9$$x2191-1363$$y2020
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