2.5 Enzyme activity measurement
Extracellular enzyme activity was determined using a previously established method (Marx et al., 2001). Fluorogenic methylumbelliferone-based artificial substrates were used to estimate the activity levels of BG, XYL, and NAG, which are involved in soil organic C (BG, XYL) and N (NAG) degradation (Sinsabaugh and Follstad Shah, 2012). The fresh soil (1 g) was prepared by dissolving in 125 mL of 50 mM sodium acetate buffer (pH 5) using low-energy sonication (50 J s−1) for 1 min. A 200-μL aliquot of the slurry was incubated with 50 μL of the corresponding substrate at 20 °C for 4 h in the dark. Ten microliters of 1 M NaOH solution was added to each plate to terminate the reaction before measurement; the fluorescence values were then determined with an automated fluorometric plate reader (Victor3 1420–050 Multi-label Counter; PerkinElmer, Waltham, MA, USA) with an excitation wavelength of 365 nm and an emission wavelength of 450 nm. Enzyme activities were expressed as nM g-1h-1.