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 μm range, this assay may provide a reliable alternative system for the quantification of haloperoxidase‐activity.

中文翻译：

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使用二维校准方法测定卤过氧化物酶活性的基于荧光的测定系统

筛选有趣的生物催化剂及其随后的动力学表征取决于可靠的活性测定。在这项工作中，建立了一种基于 4-甲基-7-二乙氨基-香豆素卤化的荧光测定法来监测卤代过氧化物酶的活性。由于卤过氧化物酶利用过氧化氢和卤离子通过释放次卤酸来卤化广泛的底物，因此直接量化卤过氧化物酶的活性仍然很困难。使用此处介绍的系统，3-溴-4-甲基-7-二乙氨基香豆素优先形成并通过荧光测量进行监测。由于起始材料和产品具有相似的光谱特性，因此使用二维校准方法允许在单次测量中对每种化合物进行量化。为了验证系统，Curvularia inaequalis被记录下来，产生了这种酶的第一个整体动力学参数。由于检测限和定量限在低μm范围内，该测定法可为卤过氧化物酶活性的定量提供可靠的替代系统。