Methodological Recommendations for Optimizing Assays of Enzyme Activities in Soil Samples.

Assays of enzyme activities in soil samples using para-nitrophenol (pNP)-linked substrates are a powerful tool in soil biochemistry. We evaluated potential error sources and optimization strategies for soil enzyme assays across 12 diverse soils (6 orders, 31-127 mg g^-1 soil organic carbon [SOC]) using the phosphomonoesterase (PHO) as an example. We hypothesized that dissolved organic matter (DOM) interference, pNP recovery, and substrate concentration would affect calculated enzyme activity, and that this would further reflect the method of assay termination: 0.5 M NaOH + 0.5 M CaCl₂ (Tabatabai, 1994), 0.2 M NaOH + 2.0 M CaCl₂ (Schneider et al., 2000), 0.1 M Tris (pH 12) + 0.5 M CaCl₂ (Klose et al., 2003), and 0.5 M NaOH + 2.0 M CaCl₂. Terminations using 0.5 M NaOH increased pNP recovery compared to 0.1 M Tris, but yielded greater DOM absorbance at 410 nm. Increasing CaCl₂ concentration (0.5 to 2.0 M) decreased DOM interference, but this effect was less pronounced than that of base type or concentration. Enzyme activity was overestimated in assays terminated with NaOH due to DOM interference, and underestimated in assays terminated with Tris buffer due to low recovery of pNP. Soil PHO K_m values, which were not correlated with SOC, varied by soil type (1.0 – 6.8 mM per g soil) demonstrating that substrate concentrations routinely employed (typically ≤ 10 mM per g soil) are likely insufficient to achieve substrate concentrations (5 × K_m) considered necessary for accurately determing enzyme activity. This study (1) illustrates the importance of a priori determination of soil enzyme K_m to achieve conditions nearing substrate saturation, and recommends (2) termination with 0.2 M NaOH + 2.0 M CaCl₂, (3) correction for pNP recovery, and (4) correction for DOM absorbance at 410 nm to increase the accuracy of pNP-based enzyme assays in soils.