Methodological recommendations for optimizing assays of enzyme activities in soil samples

Assays of enzyme activities in soil samples based on para-nitrophenol (pNP) spectrophotometry are a powerful tool in soil biochemistry. We evaluated potential sources of error and optimization strategies for soil enzyme assays across 12 diverse soils (6 USDA orders, 31-127 mg g−1 soil organic carbon [SOC]), using the activity of soil phosphomonoesterase (PHO) as an example. We hypothesized that dissolved organic matter (DOM) interference, pNP recovery, and substrate concentration would affect calculated enzyme activities, and that this would 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), 0.5 M NaOH + 2.0 M CaCl2 (this study), and 0.1 M Tris (pH 12) + 0.5 M CaCl2 (Klose et al., 2003). Terminations using 0.5 M NaOH increased pNP recovery compared to termination with 0.1 M Tris, but resulted in greater DOM interference (absorbance at 410 nm), which for terminations using NaOH but not Tris was positively correlated with total SOC (R2 = 0.45-0.38). Greatest DOM interference occurred for Andisols for termination with 0.5 M NaOH + 0.5 M CaCl2, which for two Andisols of intermediate SOC (97 and 68 mg g−1) was 1-2 orders of magnitude greater than other soils (346 and 246% overestimation of PHO activity). Increasing CaCl2 concentration (0.5 M-2.0 M) decreased DOM interference, but this effect was less pronounced than the effects of base type or concentration. Enzyme activity tended to be overestimated in assays terminated with NaOH due to DOM interference, and was more greatly underestimated in assays terminated with Tris buffer due to low recovery of pNP, which was soil-specific. Soil PHO Km values, which were not correlated with SOC, varied by soil (4.2-13.3 mM g−1 soil) demonstrating that substrate concentrations routinely employed (typically ≤ 10 mM g−1 soil) are likely insufficient to achieve recommended substrate conditions (5 × Km) for accurate measurement of PHO activity. This study illustrates the importance of a priori determination of soil enzyme Km to achieve conditions nearing substrate saturation, and recommends termination with 0.2 M NaOH + 2.0 M CaCl2, correction for pNP recovery, and correction for DOM absorbance at 410 nm to increase the accuracy of pNP-based enzyme assays in soils. Finally, to improve communication and thus comparison of measured enzyme activities among studies and assay methods (pNP vs 4-methylumbelliferone [MUF]), it is suggested that studies report the concentration of substrate for the final volume used in enzyme assays, report Km values on a soil mass basis, express enzyme activities on a molar pNP basis, and qualify enzyme activities, Km, and Vmax as 'apparent' if corrections for interferences are not performed.