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-nitrop henol, directly attached to the phosphate portion of the molecule and its rate of hydrolysis may not represent the ability of soil enzymes t o degrade alkyl phosphomonesters. The evaluation of a new substrate wi th which to assay alkyl phosphomonoesterase activity in soil is descri bed. The substrate is a phosphate ester of 4-(p-nitrophenoxy)- 1,2-but anediol (PNB). The assay involves a 1 h exposure of the substrate, in maleate buffer (0.1 M, pH 6.5), to 1 g soil at 37-degrees-C. The hydro lysis 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 subs trate concentration is 1 mm. The Michaelis-Menten constant (mean, 0.73 mM) determined in two soils is lower than the K(m) values for acid ph osphatase 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).