Cloning,Expression And Function Of Glutathione S-transferase From DDT-degrading Bacterium Ochrobactrum Sp.DDT-2

Organochlorine pesticides had been widely used in agricultural pests and public health control.Although they have been banned for a long time,the residues of DDT and its main metabolites in the natural environment can still be detected,and there are certain health and ecological risks.Glutathione S-transferase(GSTs)is a conserved family of supergene enzymes with multiple functions,which is ubiquitous in organisms.The main function of GSTs is to catalyze the formation of low toxic and water-soluble compounds from endogenous and exogenous toxic substances and to expel them from the body to reduce or avoid injury to the body.In this paper,the whole genome sequencing of the DDT degrading bacteria Ochrobactrum sp.DDT-2 preserved in the laboratory was carried out,and the main results were as follows:(1)By using Illumina Hiseq and SOAPdenovo(v2.04),gene information of DDT degrading bacteria Ochrobactrum sp.DDT-2 was obtained after sequencing,assembly,optimization and hole filling.By means of assembly sequence,ncRNA information,coding gene,COG,GO and KEGG function annotation,the genome circle map of DDT degrading bacteria Ochrobactrum sp.DDT-2 was drawn.It is predicted that the whole genome annotation sequence numbered U1GM001187 contains dhc gene and encodes glutathione S-transferase,which acts on the transformation of DDT to DDE.(2)Glutathione S-transferase gene dhc was cloned from DDT degrading strain Ochrobactrum sp.DDT-2 by PCR.The recombinant expression strain was constructed.Under the induction of IPTG(Isopropyl beta-D-Thiogalactoside),there were obvious protein bands around 26 kDa.The results showed that DDT,DDE and DDD could be degraded by DDT under the action of dhc gene,and then DDMU was formed by dehydrochlorination.DDE and DDD were degraded to DDMU.(3)The recombinant expressed protein induced by IPTG was purified and its basic enzymatic properties were studied.The purified protease can degrade in a wide range of temperature(20-45?)and pH(6.0-7.0).It can also degrade heptachloride,lindane,chlorothalonil and ?-666.Through the analysis of metabolites,we can conclude that the pathway of action of purified enzymes on metabolites is as follows:?-HCH?pentachlorocyclohexene;Lindan?Pentachlorocyclohexene?Trichlorobenzene;Chlorothalonil?1,3-diamide-tetrachlorobenzene;Heptachlor?Epoxy Heptachlor?The degrading enzymes obtained in this study have good degradability and wide range of substrates,and have good potential application value.