甲基对硫磷高效降解菌的分离鉴定及降解酶基因的克隆表达

从湖北沙隆达农药厂枵水处理池的活性污泥中分离到一株能以甲基对硫磷(MP)和对硝基苯酚(PNP)为唯一碳源生长的细菌HS-D36,经生理生化试验和16S rDNA同源性分析,初步鉴定为施氏假单胞菌(Pseudomonas stuazeri).该菌在3h内对浓度为50mg˙L-1MP的降解率为90%;在12h内能将50mg·L-1PNP完全降解.HS-D36在以MP为唯一碳源的无机盐培养基上可以耐受800mg·L-1的MP,在LB培养基上可耐受浓度为2000mg·L-1的MP.同时,克隆了甲基对硫磷水解酶基因mpd,并在E.coli中获得高效表达.重组甲基对硫磷水解酶粗酶液活性达到52.5 U·mL-1.

Cloning and expression of the mpd gene from a newly isolated methylparathion-degrading strain of bacteria

A bacterial strain,HS-D36,capable of degrading methylparathion was isolated from active sludge of a wastewater treatment pond of a pesticide factory in Hubei province.The strain HS-D36 could use methylparathion (MP) and p-nitrophenol (PNP) as the sole carbon source for growth.Analysis of its physiological and biochemical properties along with a homologous comparison of its 16SrRNA gene sequence suggests that the HS-D36 strain belongs to Pseudomonas stutzeri.Degradation of 50 mg·L^-1 MP and PNP reached 90% completion at 3 h and was fully degraded by 12 h.The strain could tolerate a high concentration of methylparathion 800 mg·L^-1 in a basic medium and up to 2000 mg·L^-1 in LB medium.Meanwhile,the mpd hydrolase gene from the HS-D36 was cloned and expressed in the E.coli BL21(DE3).The partially purified recombinant methylparathion hydrolase showed moderate activity and hydrolysed methylparathion at a rate of 52.5 U·mL^-1.