Isolation,Characterization And Gene Function Of Highly Efficient DEHP-degrading Bacteria

di?2-ethylhexy?phthalate?DEHP?is one of the most widely used phthalic acid esters?PAEs?,which have shown increasing environmental concerns worldwide.DEHP as a kind of plasticizers is widely used in the manufacture of plastics and many other industrial and consumer products.Since DEHP is not chemically bound to the hostv polymers,it has the tendency to leach slowly from the host matrix and migrate into the environment?including soil,water and atmosphere?.As a result of the biological enrichment,the mutagenicity,teratogenicity,carcinogenicity and developmental toxicity of DEHP,DEHP have now been listed as one kind of priority pollutants by the US Environmental Protection Agency.Abiotic and biotic degradations were the two degradation methods of PAEs in the environment,but the main mode was microbial degradation.Many microorganisms have been isolated from rivers,soil,and even marine regions for their ability to degrade DEHP aerobically or anaerobically,but the reports about the catabolic pathway and the Related gene function are rarely.This study reports the taerobic degradation of DEHP by a pure culture of Gordonia sp.HS-NH1 which was isolated from activated sludge in south lake?Wuhan?.Gordonia sp.is a kind of important environmental pollutants degradation bacteria,However the study about the degradation of DEHP by Gordonia sp.is rare.In this study,4 bacterial strains were isolated from activated sludge though enrichmnt culture and one of the bacterial strains named HS-NH1 was found to be capable of utilizing DEHP as carbon and energy sources for growth high-efficiencily.16SrRN A and gyrB gene sequence analysis,combined with morphological,biochemical and physiological characterization,revealed that strain HS-NH1 was most closely related to Gordonia sp.The optimum temperature and pH for degradation and growth of strain HS-NH1 were 30? and 7.0 respectively,Gordonia sp.HS-NHl was able to degrade 500 mg/L DEHP to above 90%within 60 hours.A substrate utilization test showed that strain HS-NHlwas also able to degradate many other phthalates,such as dimethyl phthalate?DMP??Diethyl phthalate?DEP??Dibutyl phthalate?DBP??Dioctyl phthalate?DOP??benzyl butyl Phthalate?BBP??Phthalic acid?PA??protocatechuate acid?PCA??elt.DEHP metabolites were detected by HPLC,in the early stage of DEHP degradation,a large number of PA was accumulated,and gradually reduce along with the degradation time.Results showed that the PA is an important metabolic product of DEHP degradation,and degradation of PA is the key step in the DEHP degradation completely,therefore,the separation and identification of related genes are essential for exploring DEHP specific metabolic pathways.Gordonia sp HS-NH1 was whole genome scaned,the gene functions were predicted and annotated.5073 open read frames were abtained,The genes related to DEHP-degradation include phthalate ester hydrolase gene,monoethylhexylphthalate hydrolase gene,phthalate acid catabolic gene cluster,protocatechuate acid catabolic gene cluster,phosphate ABC transporter gene,MFS transporter gene.They were almost accord with the PAEs-degradational genes in other bacterial reported in NCBI.Phthalate acid catabolic gene cluster?Pht gene cluster?which is responsible for the degradation of PA,include phtAa,phtAb,phtAc,phtAd,phtB,phtC,phtR,encoding the 3 '4-phthalate dioxygenase which consists of large subunit,small subunit,ferredoxin subunit and ferredoxin reductase subunit,dihydrodiol dehydrogenase,dihydroxyphthalate decarboxylase and regulatory protein.RT-PCR analysis indicated that phtBAabcdCR constitute a single transcriptional unit,and induced by PA.Gene segments phtAab,phtAcd?phtB?phtC were amplified by PCR,then expressed in Escherichia coli BL21.SDS-PAGE anlysis showed that proteins?53-kDa,22-kDa?were expressed by E.coli BL21 containing pET-phtAab;proteins?7-kDa.43-kDa?were expressed by E.coli BL21 containing pET-phtAcd;protein?30-kDa?was expressed by E.coli BL21 containing pET-phtB;protein?25-kDa?was expressed by E.coli BL21 containing pET-phtC,They were in accordance with the results of DNAMAN7.0 prediction.The activity of crude enzymes were tested by HPLC.The results indicated that PA was oxidated by the products of phtAab and phtcd,subsequently,transformed into PCA by the products of phtB and phtC.Thus DEHP was hydrolyzed to PA,PA was transformed into protocatechuic acid by many kinds of enzymes expressed by pht gene cluster,protocatechuic acid was eventually transformed into CO₂ and H₂O.To sum up,Gordonia sp.HS-NH1 is a high efficient DEHP-degradation bacterium,the genetic level study of Gordonia sp.HS-NH1 layed a foundation for deep understanding of its degradation pathway,improving the degradation efficiency and expanding the scope of its substrate.