Proteases play a crucial role in the soil nitrogen (N) cycle by converting protein to oligopeptides and amino acids. They are often viewed as a bottleneck in terrestrial N cycling; therefore, it is vital that we have robust methods for evaluating protease activity in soil to understand global patterns of protease activity. In response to this, several laboratory-based protease methods have been developed and subsequently modified. However, the validity of these different approaches remains largely unknown. In addition, the lack of standardised protocols makes it difficult to compare protease activity across studies. In this systematic synthesis, we critically evaluate the most common colorimetric and fluorimetric methods used to measure soil protease activity involving 680 independent studies and 1,491 individual assays. To investigate the key regulators of soil protease activity, we collected associated metadata on environmental (mean annual temperature and soil pH) and methodological (assay temperature and pH) factors. Protease activity measured with colorimetric substrates were centred around ca. 1000 nmol product g⁻¹ h⁻¹, whilst rates measured with fluorimetric substrates were lower at ca. 100 nmol product g⁻¹ h⁻¹. Fluorimetric and colorimetric substrates target different proteases which are likely to have different abundances, kinetic parameters, catalytic mechanism or ecological function suggesting why colorimetric substrates have a higher protease activity. We found soil protease activity varied widely around these peaks, likely due to a wide range of environmental or methodological factors that may influence/bias the result. We present the following recommendations for measuring soil protease activity: 1) report assay conditions and soil characteristics, particularly pH and temperature, 2) conduct the assay at either field or optimised pH and temperature conditions, and, 3) check that measurements lie between 0 and 5000 nmol product g⁻¹ h⁻¹. This will help reduce the variation in soil protease activity measurements due to methodological bias and improve reporting of abiotic and biotic associated data. Altogether this will lead to a better understanding of the ecological drivers of protease activity and refine parameterisation of global biogeochemical models.

蛋白酶通过将蛋白质转化为寡肽和氨基酸，在土壤氮（N）循环中起着至关重要的作用。它们通常被视为陆地N循环的瓶颈；因此，至关重要的是，我们必须拥有可靠的方法来评估土壤中的蛋白酶活性，以了解蛋白酶活性的整体模式。响应于此，已经开发并随后修改了几种基于实验室的蛋白酶方法。但是，这些不同方法的有效性在很大程度上仍然未知。另外，由于缺乏标准化的实验方案，因此难以比较研究之间的蛋白酶活性。在此系统的合成中，我们严格评估用于测量土壤蛋白酶活性的最常用的比色法和荧光法，涉及680项独立研究和1,491项单独测定。为了研究土壤蛋白酶活性的关键调控因子，我们收集了有关环境（平均年温度和土壤pH）和方法学（测定温度和pH）因素的相关元数据。用比色底物测得的蛋白酶活性在约1℃附近居中。1000 nmol产品g^-1 h ^-1，而用荧光底物测得的速率则较低。100 nmol产物g ^-1 h ^-1。荧光和比色底物靶向可能具有不同丰度，动力学参数，催化机理或生态功能的不同蛋白酶，这表明为什么比色底物具有更高的蛋白酶活性。我们发现这些峰附近的土壤蛋白酶活性变化很大，这可能是由于可能影响/偏倚结果的各种环境或方法学因素所致。我们提出以下测量土壤蛋白酶活性的建议：1）报告测定条件和土壤特性，尤其是pH和温度，2）在野外或优化的pH和温度条件下进行测定，并且3）检查测量值是否介于0之间和5000 nmol产物g ^-1 h ^-1。这将有助于减少由于方法论偏差引起的土壤蛋白酶活性测量值的变化，并改善非生物和生物相关数据的报告。总而言之，这将有助于人们更好地理解蛋白酶活性的生态驱动因素，并完善全球生物地球化学模型的参数化。