Decomplexation of Phosphatase From Extracted Soil Humic Substances With Electron Donating Reagents

A substance with phosphatase activity was isolated from a field soil. The isolation procedure involves two steps. First ammonium humates are formed by intermittent shaking of soil in 1.0 N ammonium acetate (pH 7.0) for 20 h. They are extracted by repeated rinsing with distilled water, after the ammonium acetate has been removed by filtration. The filtrate is dialyzed and freeze-dried. Then the phosphatase active part is isolated from the humic substances by decomplexation with 17.0 mM tetramethyl-p-phenylenediamine (TMePDA). The dialyzed filtrate accounted for 3.2% of the soil's phosphatase activity. During freeze-drying 95.4% of the filtrate's phosphatase activity was lost. A total of 1.77 g of freeze-dried filtrate was obtained per kilogram of soil; it had a phosphatase activity of 9.85 μmol p-nitrophenyl phosphate per gram per hour. Twenty-four percent of this enzyme activity was recovered in the step with TMePDA in a clear supernatant, while 97.3% of the solids was precipitated. The freeze-dried filtrate was found to be clay-free by x-ray analysis. The possibility of microorganisms being the immediate source of the phosphatase extracted is excluded by the initial filtration step, application of toluene, and sterility checks with nutrient agar plates and microscopic examination. The extracted phosphatase activity is heat-sensitive and follows Michaelis-Menten kinetics. It is suggested that the extracted phosphatase activity is enzymatic and resembles a fraction of the free phosphatase enzymes in soil. The bonding mechanism between enzyme and humic substances is discussed. Evidence is presented that free soil phosphatase is bonded to humic substances in the form of electron donor acceptor complexes, the humic substances being the acceptor.