Abstract
Objectives
To characterize a novel dimethoate amidohydrolase from Sphingomonas sp. DC-6.
Results
A gene, dmhA, encoding the dimethoate amidohydrolase responsible for transforming dimethoate to dimethoate carboxylic acid and methylamine, was cloned from Sphingomonas sp. DC-6. Sequence analysis and molecular modeling indicate that DmhA shares 31–57 % amino acid sequence identities with other functionally confirmed amidohydrolase. DmhA was expressed in Escherichia coli BL21 (DE3) and purified by Ni–NTA affinity chromatography. The purified DmhA could hydrolyze 4-acetaminophenol, dimethoate and propanil. DmhA activity was optimal at 30 °C and pH 7.5. Hg2+, Zn2+, Cu2+, Cd2+, Tween 80, Triton X-100 or SDS strongly inhibited its activity. The K m and k cat values of DmhA for dimethoate are 0.02 mM and 1.2 s−1, respectively.
Conclusions
DmhA was confirmed to be a novel dimethoate amidohydrolase which could eliminate the toxicity of dimethoate, providing a novel gene resource for the development of pesticide-degrading enzyme preparation and mechanistic study of dimethoate hydrolysis.
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Acknowledgments
This work was supported by the National Natural Science Foundation of China (31570105), the National Natural Science Foundation of China (J1210056 and J1310015) and the Program for New Century Excellent Talents in University (NCET-13-0861).
Supporting information
Supplementary Figure 1-GC-MS profile of the metabolite produced during dimethoate degradation by strain DC-6. A, GC spectra of the sample collected at 0 h; B, GC spectra of the extract obtained from the culture at 4 h; C and D, charged ions mass spectra for the peak at 8.68 and 9.63 min, respectively. Supplementary Figure 2-SDS-PAGE analysis of the purified DmhA. Lane M, protein markers (kDa); lane A, total protein of Escherichia coli BL21(DE3) harbouring pET-29a(+) as control; lane B, total protein of Escherichia coli BL21(DE3) harbouring pET-29a-dmhA, induced by 0.5 Mm IPTG; lane C, purified DmhA.
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Qing Chen and Kai Chen contribute equally to this work.
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Chen, Q., Chen, K., Ni, H. et al. A novel amidohydrolase (DmhA) from Sphingomonas sp. that can hydrolyze the organophosphorus pesticide dimethoate to dimethoate carboxylic acid and methylamine. Biotechnol Lett 38, 703–710 (2016). https://doi.org/10.1007/s10529-015-2027-6
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DOI: https://doi.org/10.1007/s10529-015-2027-6