Isolation, Characterization Of Carbaryl-Degrading Strains And Cloning Of The Ceha Gene

Carbaryl is a kind of carbamate, it has medium toxicity. It was extensively used in the control of565insect pests in the plants of crops such as fruit trees, vegetables, grain, medicinal materials and tea trees. Carbaryl inhibits the activity of acetylcholinesterase and kills the pests. Because of the extensive and continuous usage of carbaryl, its residual in environment has brought a potential threat to ecological balance and human health. Bioremediation based on the degradation ability of microorganisms is an environment friendly method in the treatment of pollutants.Two bacteria strains FX-1and X9capable of degrdading carbaryl were isolated from the sludge sample collected from the wastewater treating system of a carbaryl pesticide-manufacturing company. Strain FX-1and X9were preliminarily identified as Sphingobium sp. and Rhizobium sp. respectively, according to their physiological&biochemical characteristics and the analysis of their16S rRNA gene sequence.Biological characteristics of strain FX-1and X9were investigated, respectively. The optimal temperature for growth of strain FX-1and X9were both at30℃. The optimal pH for growth were both at7.0. The optimal NaCl concentration were5g·L-1and10g·L-1, respectively. The optimal carbon source for the growth were both glucose, and the optimal nitrogen source for growth were ammomnium sulfate and ammonium nitrate, respectively. They were both resistant to streptpomycin.Strain FX-1and X9could utilize carbaryl as the sole carbon source for growth, and could completely degrade34mg·L-1carbaryl within6h and4h, respectively. The optimal temperature for degradation were30℃and37℃,the optimal pH were8.0and9.0. The aeration level showed obvious influence on the degradation of carbaryl by strain FX-1,while showed almost no influence on that of strain X9. Strain X9could utilize1-naphthol, the hydrolysis product of carbaryl, as the sole carbon source for growth, and could degrade34mg·L-11-naphthol within16h. Large initial inoculum could promote the degradation and shorten the lag phase.The optimal condition for its degradation was30℃and pH7.0. Strain X9could degrade20-200mg·L-11-naphthol.The primers were designed according to the published sequences of the carbaryl degrading related genes. The cehA gene was cloned from strain X9by PCR, its sequence showed99%similarity to the cehA of strain AC100.The cehA gene from X9was ligated to the expression plasmid vector pET32a and expressed in E.coli Rosetta-gami, the protein with the right molecular size (85KDa) was prouduced, it could hydrolyze carbaryl to1-naphthol.