2.6 Soil enzyme activity analysis

The activity of seven enzymes (Table 3) involved in C and N cycling were determined using microplate fluorometric assay, according to the procedures of DeForest [35]. Briefly, 1.0 g dry-mass-equivalent of fresh soil was homogenized in 100 mL of 50 mM acetate buffer (pH 8.5). For hydrolase analysis, buffer, sample suspension, 10 mM references and 200 μM substrates (4-methylumbelliferone or 7-amino-4-methylocumarin) were dispensed into the wells of a black 96-well microplate. The microplates were covered and incubated at 25°C for 4 h in the dark, then fluorescence was measured using a microplate fluorometer (Scientific Fluoroskan Ascent FL, Thermo) with 365 nm excitation and 450 nm emission filters. Phenol oxidase and peroxidase were measured in a clear 96-well microplate using the substrate of L-3, 4-dihydroxyphenylalanine (L-DOPA). The dispensed volume and the order of buffer, sample suspension, 25 mM L-DOPA, and 0.3% (w/v) H₂O₂ were the same as the fluorometric enzymes. The microplates were covered and incubated at 20°C for 20 h in the dark, then the activities were determined by measuring the absorbance at 450 nm using the microplate fluorometer. The enzyme activities were expressed in nmol h⁻¹ g⁻¹. For each sample, the geometric mean of the assayed enzyme activities (Gmea), hydrolase (GH) and oxidase (GOR) were calculated as:

Moreover, soil enzyme functional diversity was calculated using the activities of seven enzymes using Shannon’s diversity index (H′_E) as [36]:

where Pi is the ratio of each enzyme activity to the sum of all enzyme activities.