As microbial enzymatic activities initiate the mineralization of organic matter through the microbial loop, it is important to correctly measure those activities and be able to perform inter‐study comparisons. Enzymatic activity assays are typically carried out using fluorogenic substrate analogs, such as 4‐methylumbelliferone and 7‐amino‐4‐methylcoumarin linked to sugar monomers, phosphate group, or amino acids. However, methodological divergences can be found in aquatic science literature, potentially leading to misestimated activities. To highlight some of those methodological key points, we first addressed the potential occurrence of an inner filter effect (IFE), a fluorometric artifact that affects the relationship between fluorophore concentration and fluorescence intensity, due to absorption of exciting or emitted light. It has never been considered in the context of environmental water studies before, despite significantly affecting measured activities. IFE occurred with two out of three tested spectrofluorometers when assaying proteases, although no IFE was detected for phosphatase assays. We also evaluated how substrate concentration ranges might affect kinetic parameters estimation, revealing that a many existing studies might use insufficient maximum substrate concentration. Finally, for single substrate concentration assays, we argued for the use of saturating substrate concentration, as naturally occurring substrates might compete with the fluorogenic analog at trace level. The amendment of a molecule mimicking natural substrates generated a significant inhibition of natural seawater phosphatases and proteases assayed with trace concentrations of fluorogenic substrate, while almost no inhibition occurred at higher concentrations. Those key points need to be addressed in order to assess enzymatic rates and allow inter‐study comparison.

中文翻译：

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环境水样品中的微生物酶检测：内部过滤效果和底物浓度的影响

由于微生物的酶促活性通过微生物回路启动了有机物的矿化，因此正确测量这些活性并能够进行研究之间的比较非常重要。酶活性测定通常使用荧光底物类似物进行，例如与糖单体，磷酸基团或氨基酸连接的4-甲基伞形酮和7-氨基-4-甲基香豆素。然而，在水生科学文献中发现方法上的分歧，有可能导致活动被错误估计。为了突出这些方法学要点中的一些，我们首先解决了内部滤镜效应（IFE）的潜在发生，该效应是一种荧光制品，它会由于吸收激发光或发射光而影响荧光团浓度与荧光强度之间的关系。尽管它显着影响所测量的活动，但以前从未在环境水研究中考虑过它。在测定蛋白酶时，尽管三分之二的荧光分光光度计均未检测到IFE，但在三分之二的分光光度计中却发生了IFE。我们还评估了底物浓度范围如何影响动力学参数估计，揭示了许多现有研究可能使用的最大底物浓度不足。最后，对于单一底物浓度测定，我们主张使用饱和底物浓度，因为天然存在的底物可能在微量水平上与荧光类似物竞争。模仿天然底物的分子的修饰产生了对天然海水磷酸酶和蛋白酶的显着抑制作用，用痕量浓度的荧光底物测定，而在较高浓度下几乎没有抑制作用。这些关键点需要解决，以便评估酶促速率并进行研究之间的比较。