Cloning, Mutagenesis and Expression of the Housefly Acetylcholinesterase in Pichia pastoris, and its Potential Application in the Detection of Pesticide Residues

文献类型： 外文期刊

第一作者： Tan, Furong

作者： Tan, Furong;Wang, Ligang;Wang, Jinbin;Wu, Xiao;Zhu, Hong;Zhao, Kai;Jiang, Lingxi;Tao, Shiru;Tang, Xueming;Tan, Furong;Wang, Ligang;Wang, Jinbin;Wu, Xiao;Zhu, Hong;Zhao, Kai;Jiang, Lingxi;Tao, Shiru;Tang, Xueming;Yang, Yan

作者机构：

关键词： Acetylcholinesterase;Mutagenesis;Rapid detection;pesticide residues;Food safety

期刊名称：CONFERENCE ON ENVIRONMENTAL POLLUTION AND PUBLIC HEALTH, VOL 1-2

ISSN：

年卷期： 2010 年

页码：

收录情况： SCI

摘要： The full-length cDNA encoding an acetylcholinesterase (AChE) was cloned and sequenced from the housefly by reverse transcriptase polymerase chain reaction (RT-PCR). The cDNA sequence lacking the signal peptide was inserted into vector pPIC9K and then introduced into the yeast Pichia pastoris. The recombinant protein was expressed in. P. pastoris strain GS115 by methanol induction. Site-directed mutagenesis of the P180V, A262G and Y327T residues, either singly or in combination, was performed by reverse PCR and these mutations increased AChE activity and improved sensitivity to the insecticide, demethoate and chlorpyrifos. The double mutation, GF, and the triple mutation, VGF, showed the highest enzyme activity showing 75.516 U per ml and 75.6E3 U per ml, respectively. Furthermore, the inhibition ratios of the mutants to organophosphate insecticide, dimethoate and chlorpyrifos, increased by about 3.5-4.4 times compared with the wild-type. These results strongly suggested that these residues are the key structural elements controlling AChE activity and sensitivity. The AChE enzyme obtained by this method could be used to detect the organophosphate and carbamate insecticide residues in fruits and vegetables, a characteristic of great potential research and industrial application.

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