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An isofenphos-methyl hydrolase (Imh) capable of hydrolyzing the P-O-Z moiety of organophosphorus pesticides containing an aryl or heterocyclic group.

Research paper by Rong R Li, Yuan Y Liu, Jian J Zhang, Kai K Chen, Shunpeng S Li, Jiandong J Jiang

Indexed on: 29 Nov '11Published on: 29 Nov '11Published in: Applied Microbiology and Biotechnology



Abstract

Organophosphorus pesticide (OP) hydrolases play key roles in the degradation and decontamination of agricultural and household OPs and in the detoxification of chemical warfare agents. In this study, an isofenphos-methyl hydrolase gene (imh) was cloned from the isocarbophos-degrading strain of Arthrobacter sp. scl-2 using the polymerase chain reaction method. Isofenphos-methyl hydrolase (Imh) showed 98% sequence identity with the isofenphos hydrolase from Arthrobacter sp. strain B-5. Imh was highly expressed in Escherichia coli BL21 (DE3), and the His(6)-tagged Imh was purified (1.7 mg/ml) with a specific activity of 14.35 U/mg for the substrate isofenphos-methyl. The molecular mass of the denatured Imh is about 44 kDa, and the isoelectric point (pI) value was estimated to be 3.4. The optimal pH and temperature for hydrolysis of isofenphos-methyl were pH 8.0 and 35 °C, respectively. The secondary structure of Imh shows that Imh is a metallo-dependent hydrolase, and it was found that Imh was completely inhibited by the metalloprotease inhibitor 1,10-phenanthroline (0.5 mM), and the catalytic activity was restored by the subsequent addition of Zn(2+). Interestingly, Imh had a relatively broader substrate specificity and was capable of hydrolyzing 12 of the tested oxon and thion OPs with the P-O-Z moiety instead of the P-S(C)-Z moiety. Furthermore, it was found that the existence of an aryl or heterocyclic group in the leaving group (Z) is also important in determining the substrate specificity. Among all the substrates hydrolyzed by Imh, it was assumed that Imh preferred P-O-Z substrates still with a phosphamide bond (P-N), such as isofenphos-methyl, isofenphos, isocarbophos, and butamifos. The newly characterized Imh has a great potential for use in the decontamination and detoxification of agricultural and household OPs and is a good candidate for the study of the catalytic mechanism and substrate specificity of OP hydrolases.