Institute for Bio- and Geosciences I: Bioorganic Chemistry, Forschungszentrum Jülich GmbH, D-52426, Jülich, Germany.
Institut für Bioorganische Chemie, Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich, D-52426, Jülich, Germany.
ChemistryOpen. 2020 Sep 18;9(9):959-966. doi: 10.1002/open.202000184. eCollection 2020 Sep.
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 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.
筛选具有有趣生物催化活性的酶及其后续的动力学特征分析依赖于可靠的活性检测方法。在这项工作中，建立了一种基于 4-甲基-7-二乙氨基香豆素卤化作用的荧光检测方法来监测过氧化物酶活性。由于过氧化物酶利用过氧化氢和卤化物离子通过释放次卤酸来卤化广泛的底物，因此直接定量过氧化物酶活性仍然很困难。在本系统中，优先形成 3-溴-4-甲基-7-二乙氨基香豆素，并通过荧光测量进行监测。由于起始材料和产物具有相似的光谱特性，因此采用二维校准方法来允许在单个测量中定量每种化合物。为了验证该系统，记录了来自 的钒依赖氯化过氧化物酶的二维米氏动力学，为该酶提供了第一个整体动力学参数。该检测方法的检测限和定量限在低 μm 范围内，可能为过氧化物酶活性的定量提供可靠的替代系统。
