Isolation Of Paes-degrading Strain?Analysis Of Its Degradation Pathway,Cloning Of Hydrolase Gene And Chara Cterization Of Its Encodinig Enzyme

Phthalate Esters(PAEs),as plasticizers,are widely used in the production of polyvinyl chloride,polyethylene,polypropylene and polystyrene because of their good compatibility,plasticity and processability.Due to their environmental hormone effect,PAEs are easy to cause residues in air,water,soil,food and organisms,which poses a serious threat to the ecological environment and human health.Studies have shown that biodegradation is the main pathway of PAEs's decomposition in the environment.This study started wth the isolation of DBP-degrading bacteria use DBP as study substrate,then the microbial degradation mechanism of DBP was analyzed from the metabolic pathway,gene and enzyme level.The results obtained were as follows.1?Isolation,identification and degradation characteristics of DBP degrading bacteriaA DBP-degrading enrichment was isolated from the sewage discharged from a plastic factory in Nanjing,Jiangsu Province by enrichment culture.A DBP-degrading bacteria and a PA-degrading bacteria were isolated and purified.The DBP-degrading bacteria named PAE-1 were identified as Microbacterium sp.and PA-degrading bacteria designated PAE-2 were identified as Pandoraea sp.by morphology,physiological and biochemical characteristics and phylogenetic analysis of 16S rRNA gene.The results of HPLC/MS analysis showed that strain PAE-1 was responsible for the first two steps of DBP hydrolysis.First,DBP was dealkylated to form MBP(Monobutyl Phthalate),then MBP was further dealkylated to form PA(Phthalic Acid),and strain PAE-2 was responsible for the hydrolysis of PA.When strain PAE-1 and strain PAE-2 were inoculated simultaneously,they could degrade DBP totally.The results of the degradation characteristics of DBP by strain PAE-1 showed the optimum degradation temperature was 30? and the optimum pH was 8.0.Under the optimum conditions,strain PAE-1 could degrade 0.5 mM DBP reached 98%within 12 hours.The strain PAE-1 showed higher conversion rates for dialkyl and monoalkyl phthalate,and the conversion rates for dialkyl phthalate were DPP>DBP>BBP>DHP>DEP>DMP>DEHP,while the conversion rates for monoalkyl phthalate were MBzP>MPP>MMP>MHP>MBP>MEP>MEHP.2?Cloning and functional identification of DBP hydrolase genes dpeH and mpeHDue to the low water solubility of DBP and the ability of degrading bacteria to hydrolyze it into water-soluble products,the degradation function of strain PAE-1 was verified by the transparent circle display.Two different length hydrolase genes dpeH and mpeH were successfully cloned from 15,000 transformants by shot-gun method.The total length of dpeH is 723 bp and DpeH encodes 240 amino acid residues.The amino acid sequence analysis of DpeH shows that the enzyme belongs to esterase.The enzyme with the highest similarity to the amino acid sequence of the enzyme is alpha/beta hydrolase from Microbacterium sp.MED-G48(53%).The full length of mpeH is 915 bp and MpeH encodes 304 amino acid residues.Amino acid sequence analysis shows that MpeH is also a esterase.The enzyme with the highest similarity to MpeH amino acid sequence is a probable secreted lipase from Trichophyton benhamiae CBS 1 12371(25%).The degradation of DBP and its hydrolysates by hydrolases DpeH and MBP was analyzed by HPLC/MS.The results showed that DpeH could hydrolyze DBP to MBP,but could not continue to hydrolyze MBP.MpeH could only hydrolyze MBP to PA,but had no degradation function to DBP.The intermediate fragments mAB of dpeH and mpeH were amplified by reverse transcription PCR and the results showed that dpeH and mpeH belonged to the same transcription unit and their transcription mode was co-transcription.Real-time fluorescence quantitative PCR results showed that both dpeH and mpeH were not induced by substrate DBP,so it was presumed that their expression types were constitutive.3?Enzymatic properties of DBP hydrolase DpeH and MpeHdpeH and mpeH genes were expressed heterologously in E.coli BL21(DE3)and further purified.The identification of DBP hydrolysates by DpeH showed that it could break the C-O bond in DBP and hydrolyze DBP to MBP.Similarly,the identification of MBP hydrolysates by MpeH revealed that MpeH broke the C-O bond in MBP and hydrolyzed MBP to PA.The optimum conditions for DpeH to hydrolyze DBP were 30?and pH 8.0,while the optimum conditions for MpeH to hydrolyze MBP were 30? and pH 9.0.The enzymatic kinetic constants of DpeH and MpeH were determined.It was found that DpeH had higher catalytic efficiency for various dialkyl phthalates.The catalytic efficiency of DpeH was DPP>DBP>BBP>DHP>DEP>DMP>DEHP.The Km and kcat values of DpeH for DBP were 9.60±0.97 ?M and(2.72±0.06)×106 s-1,respectively.MpeH also had higher catalytic efficiency for various monoalkyl phthalates,and the catalytic efficiency was MBzP>MPP>MMP>MHP>MBP>MEP>MEHP,in which the and tcat values of MpeH to MBP were 18.61±2.00 ?M and(5.83±1.00)×105 s-1,respectively.Amino acid sequence alignment revealed that there were esterase conservative sequence GX1SX2G(G)and catalytic active site Ser-Asp-His in DpeH,while another conservative sequence GX1X2L and catalytic active site Ser-His in MpeH.The catalytic sites in DpeH and MpeH were mutated into alanine(Ala)by overlapping extended PCR.After heterologous expression and purification,the hydrolytic activity of the mutant protein DBP and MBP was determined.It was found that all mutants lost their hydrolysis ability.The results showed that Ser-Asp-His and Ser-His were the key amino acid catalytic sites for DpeH and MepeH respectively.