Characterization and Malathion Degradability of Carboxylesterase in Wheat Kernels

Abstract

Malathion residue in wheat kernels is enzymatically degraded into malathion monocarboxylic acids during sample preparation for pesticide residue analysis by the Japanese official method. To investigate whether the hydrolyzing enzyme is identical to carboxylesterase (CE), we compared the effects of various inhibitors against CE activity using p-nitrophenyl acetate as substrate with that against malathion-hydrolyzation activity. Although neither CE nor malathion-hydrolyzation activities were affected by EDTA, they were irreversibly suppressed by several serine esterase inhibitors and reversibly inhibited by cholinesterase inhibitor or sulfhydryl compounds. These inhibitions suggested that characteristics of both enzymes in wheat kernels were close to that of cholinesterase, though the enzymes were not able to be exactly classified by a classification method for mammals. When native polyacrylamide gel electrophoresis (PAGE) with esterase-zymography was performed with the addition of eserine sulfate into the sample and deoxycholic acid into the cathode buffer to prevent aggregation of CE isozymes, three clear bands of the isozymes were observed. These isozymes were confirmed by hydrophobic interaction chromatography (HIC). When malathion was reacted with each isozyme partially purified by chromatography techniques and native PAGE, malathion α-monocarboxylic acid as a major metabolite and malathion β-monocarboxylic acid as a minor metabolite were produced. These results suggested that all isozymes in wheat kernels responsible for the degradation of residual malathion in the kernels, though there are differences among malathion degradability by the isozymes.