Assays of enzyme activities using chromogenic substrates that release para-nitrophenol (pNP) and para-nitroaniline (pNA) products are commonly employed in soil science, but these substrates are susceptible to non-enzymatic (i.e., abiotic) hydrolysis. We evaluated abiotic hydrolysis of 10 pNP- and pNA-linked substrates stored over seven days in two matrices of water and modified universal buffer, and with two alkalization methods of 0.5 M NaOH and 0.1 M Tris. We then compared the magnitude of abiotic versus enzymatic hydrolysis of substrates for two soils with high and low enzyme activity. Finally, we quantified substrate abiotic hydrolysis during the incubation (1–2 h at 37 °C). Abiotic hydrolysis of stored substrate solutions remained relatively constant across 7 days, and the base type used in alkalization had a much stronger effect on abiotic hydrolysis than storage time or matrix. Abiotic hydrolysis was generally least for substrates dissolved in water with Tris alkalization and greatest when dissolved in modified universal buffer with NaOH alkalization. The extent of abiotic hydrolysis varied by substrate, and in general was least for ester substrates and greatest for amide substrates. Abiotic hydrolysis was as low as <0.7% for the glycosidic substrate used to assay β-N-acetyl-glucosaminidase, and as high as 52–57% for amide substrates used to assay aminopeptidases. The magnitude of abiotic hydrolysis was more appreciable, and in some cases greater, than total substrate hydrolysis for the soil with overall low enzyme activities. Finally, appreciable abiotic hydrolysis occurred during the incubation, indicating that the commonly employed control for non-enzymatic pNP or pNA products in which substrate solution is added to a soil after the assay incubation is not appropriate. In order to minimize abiotic hydrolysis, we recommend alkalization of assays using 0.1 M Tris instead of 0.5 M NaOH; appreciable decreases in abiotic hydrolysis can also be achieved for water-only assays. To accurately control for abiotic hydrolysis in soil enzyme assays, incubated soil-free substrate-only controls should be used.

使用释放对硝基苯酚（p NP）和对硝基苯胺（p NA）产物的生色底物进行酶活性测定通常用于土壤科学，但这些底物易受非酶促（即非生物）水解作用。我们评估了10 p NP-和p的非生物水解NA连接的底物在两种矩阵的水和改良的通用缓冲液中存储了7天，并使用0.5 M NaOH和0.1 M Tris的两种碱化方法。然后，我们比较了具有高酶活性和低酶活性的两种土壤的底物的非生物水解和酶促水解的幅度。最后，我们定量了孵育过程中底物的非生物水解（在37°C下1-2 h）。储存的底物溶液的非生物水解在7天中保持相对恒定，并且碱化过程中使用的碱类型对非生物水解的影响要比储存时间或基质强得多。对于溶解于底物的Tris碱而言，非生物水解通常最少，而当溶解于NaOH碱化的改良通用缓冲液中时，非生物水解最大。非生物水解的程度因底物而异，通常，对于酯底物而言最少，而对于酰胺底物而言最大。对于用于检测β-N-乙酰基氨基葡萄糖苷酶的糖苷底物，非生物水解低至<0.7％，对于用于分析氨肽酶的酰胺底物，非生物水解低至52–57％。对于酶活性总体较低的土壤，非生物水解的幅度要比总的底物水解幅度更大，在某些情况下甚至更大。最后，在孵育过程中发生了明显的非生物水解，这表明非酶制剂的常用控制方法 对于酶活性总体较低的土壤，非生物水解的幅度要比总的底物水解幅度更大，在某些情况下甚至更大。最后，在孵育过程中发生了明显的非生物水解，这表明非酶制剂的常用控制方法 对于酶活性总体较低的土壤，非生物水解的幅度要比总的底物水解幅度更大，在某些情况下甚至更大。最后，在孵育过程中发生了明显的非生物水解，这表明非酶制剂的常用控制方法在测定温育后将底物溶液添加到土壤中的p NP或p NA产品是不合适的。为了最大程度地减少非生物水解，我们建议使用0.1 M Tris代替0.5 M NaOH碱化测定。仅水测定法也可以实现非生物水解的明显减少。为了在土壤酶测定中准确控制非生物水解，应使用仅不含底物的温育对照。