Amidase Activity in Soils: I. Method of Assay

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Abstract
Amidase [acylamide amidohydrolase, EC (Enzyme Commission) 3.5.1.4] is the enzyme that catalyzes the hydrolysis of amides and produces the corresponding carboxylic acid and ammonia. The detection of this enzyme in soils is reported, and a simple, sensitive, and precise method to assay amidase is described. The method involves determination by steam distillation of the NH4+ produced by amidase activity when soil is incubated with buffered (0.1M THAM, pH 8.5) amide solution and toluene at 37°C. The amide compounds studied included formamide, acetamide, and propionamide. The procedure developed gives quantitative recovery of NH4‐N added to soils and does not cause chemical hydrolysis of the substrates. Results showed that this soil enzyme has its optimum activity at buffer pH 8.5 and is inactivated at temperatures above 60°C. By varying the substrate concentration it was found that the initial velocity of the amidase reaction is optimum at 0.05M substrate. The initial rates of NH4‐N released obeyed zero‐order kinetics. Steam sterilization destroyed, and formaldehyde, sodium fluoride, and sodium arsenite inhibited, amidase activity in soils. Assay of amidase activity in the absence of toluene indicated that acetamide and propionamide may induce the synthesis of this enzyme by soil microorganisms.

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