Phosphodiesterase Activity of Soils

An improved method to assay phosphodiesterase activity in soils is described. It involves extraction and colorimetric determination of the p ‐nitrophenol released when 1 g of soil is incubated with 5 ml of 1m M buffered [0.05 M tris (hydroxymethyl) aminomethane (THAM), pH 8.0] bis‐p ‐nitrophenyl phosphate solution at 37°C for 1 hour. The reagents (0.5 M CaCl 2 and 0.1 M THAM pH 12) used for extraction of the p ‐nitrophenol released give quantitative recovery of p ‐nitrophenol added to soils and do not cause chemical hydrolysis of the substrate, bis‐p ‐nitrophenyl phosphate. Results showed that this soil enzyme has its optimum activity at buffer pH 8.0. The initial rates of p ‐nitrophenol release obeyed zero‐order kinetics. The phosphodiesterase activity of six surface soils studied ranged from 16 to 147 µg p ‐nitrophenol released · g −1 soil · hour −1 . Steam sterilization and formaldehyde destroyed and toluene increased the activity of this enzyme. At 5m M , PO 4 3‐ , EDTA, and citrate inhibited phosphodiesterase activity. The inhibition by orthophosphate showed competitive kinetics. The temperature dependence of the rate constant conformed to the Arrhenius equation up to the point of enzyme inactivation (70°C). The activation energy of phosphodiesterase of the six surface soils studied ranged from 7,860 to 10,390 (avg. = 8,720) calories · mole −1 . By using the Lineweaver‐Burk plot, the K m values ranged from 1.26 to 2.02 (avg. = 1.69)m M and the V max ranged from 52 to 530 (avg. = 303) µg p ‐nitrophenol released · g −1 soil · hour −1 . Phosphodiesterase activity was significantly correlated with organic C in each soil profile and in the surface soils studied.