Soil Enzyme Activity Quantification

Catalase activity was determined using a titrimetric method [37 ]. The residual H₂O₂ was determined by titrated with KMnO₄ in the presence of H₂SO₄ after 30 min of reaction. Catalase activity was expressed as mL 0.1 mol·L⁻¹ KMnO₄ consumed g⁻¹·soil day⁻¹. Urease activities were determined according to a method described by Liu et al. [38 (link)] and Fang et al. [39 (link)]. Urease activity was expressed as mg NH₃–N released g⁻¹·soil day⁻¹. Sucrase activity was determined by a method described by Guan [37 ] with minor modifications. Briefly, a 5 g soil sample was treated by the addition 1 mL of toluene, followed by 15 mL sucrose and 5 mL phosphate buffer [mix 94.6 mL of Solution A (dissolve 13.61 g of KH₂PO₄ in water and dilute to 1 L) and 3.6 mL of Solution B (dissolve 35.81 g of NaH₂PO₄ in water and dilute to 1 L, pH 5.5)]. After incubation at 37 °C for 24 h, the suspension was filtered through Whatman 1001-090 filter paper (Whatman International, Maidstone, UK) and 0.5 mL of filtrate was treated with 1.5 mL salicylic acid and held for 5 min at 100 °C. Once the solution had cooled, sufficient deionized water was added to make the volume up to 25 mL. The intensity of the pink color that developed after 60 min was measured spectrophotometrically at 508 nm, using a UV2300 device (Shanghai Zhongchen instrument Co., Shanghai, China). The quantity of reducing sugar released by sucrase activity was determined by reference to a calibration curve and expressed as mg glucose g⁻¹·soil day⁻¹.