*2.7. Enzyme Activities*

Two of the major enzymes involved in C and N mineralization—cellulase and protease were assayed to determine how their activities changed over the incubation period. Cellulase activity was determined by a *para*-nitrophenol (*p*NP) method [41] using *p*NP-β-D-cellobioside substrate prepared in modified universal buffer (MUB, pH 6.5). For this purpose, triplicate 1 g soil samples (dry weight equivalent) were treated with 5 mL of 12 mM substrate solution, and the mixture was briefly vortexed and then incubated for 2 h at 37 ◦C. Hydrolysis was terminated immediately following incubation by adding 4 mL of 0.1 M tris buffer (pH 12) and 1 mL of 2 M CaCl2, a 1 mL aliquot of the supernatant was centrifuged to remove sediment, and *p*NP was quantified from absorbance measurements at 405 nm.

Protease assays were performed using the casein-based technique of Ladd and Butler [42] with modifications described by Jesmin et al. [43]. Briefly, 1 g of soil (dry weight equivalent) was treated with 2.5 mL of tris buffer (pH 8) and 2.5 mL of sodium caseinate solution (50 mg casein g−<sup>1</sup> soil) and incubated for 2 h at 50 ◦C on a shaking sand bath. After incubation, trichloroacetic acid was added to terminate hydrolytic activity, the supernatant obtained by centrifugation was treated with Na2CO3 solution and Folin reagent, and the tyrosine released was determined spectrophotometrically at 650 nm.
