soil enzyme activity and P concerntraion

The activities of AcP, AlP and PD were determined as described by Tabatabai (1994). For AcP and AlP, 1.0 g of fresh soil was mixed with 0.2 mL of toluene, 4 mL of modified universal buffer at pH 6.5 or 11.0, and 1 mL of 0.5 M ρ-nitrophenyl (ρNP) phosphate substrate. The mixture was incubated at 37℃ for 1 h, after which the reaction was stopped by adding 1 mL of 0.5 M CaCl2 and 4 mL of 0.5 M NaOH. A control was conducted for each soil sample, following the same procedure but adding the substrate solution after CaCl2 and NaOH. The mixture was then filtered, and the production of ρNP was measured colorimetrically using a spectrophotometer at 410 nm. PD activity was assayed using a modified universal buffer at pH 8.0, with bis-ρ-nitrophenyl phosphate as the substrate. Activities of phosphatase were expressed as mg ρNP kg soil-1 h-1 using modified pH (6.5) for the determination of albic soil, black soil and brown soil.The enzyme kinetic parameters Vmax (maximum reaction rate) and Km (Michaelis-Menten constant) for APase and PD were determined using a range of substrate concentrations, following the same procedure as described in Section 2.5. The kinetic parameters for AcP and AlP were measured with seven substrate concentrations (i.e. 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM and 70 mM), while PD was measured with six substrate concentrations (10 mM, 20 mM, 30 mM, 40 mM, 50 mM and 60 mM). Each substrate concentration included a negative control. The kinetic parameters Vmax and Km were calculated using the Hanes method (Moscatelli et al., 2012). A straight-line graph can be generated (Fig. S1), where the slope corresponds to 1/Vmax and the intercept is Km/Vmax, facilitating the determination of Vmax and Km.