Proposed substrate for evaluating alkyl phosphomonoesterase activity in soil

p-Nitrophenyl phosphate is commonly used to assay phosphomonoesterase activity in soil. This substrate has an aromatic chromophore, p-nitrophenol, directly attached to the phosphate portion of the molecule and its rate of hydrolysis may not represent the ability of soil enzymes to degrade alkyl phosphomonesters. The evaluation of a new substrate with which to assay alkyl phosphomonoesterase activity in soil is described. The substrate is a phosphate ester of 4-( p-nitrophenoxy)-1,2-butanediol (PNB). The assay involves a 1 h exposure of the substrate, in maleate buffer (0.1 m, pH 6.5), to l g soil at 37°C. The hydrolysis product, PNB, is chemically oxidized in situ by periodate in the presence of methylamine to produce p-nitrophenol (PNP), which can be quantitatively extracted from soil and measured spectrophotometrically. The initial rate of reaction obeys zero-order kinetics when the substrate concentration is 1 m m. The Michaelis-Menten constant (mean, 0.73 m m) determined in two soils is lower than the K m values for acid phosphatase as reported with PNP-P as substrate. Reproducibility of the results is high (coefficient of variation ranged from 2.1 to 6.2% for four soils).