Cloning And Functional Analysis Of A Dibutyl Phthalate Hydrolase Gene From Rhodococcus Sp. 2G

With the development of global industrialization,plastic products remains high production.Phthalic acid esters?PAEs?,as important additives in plastic products,can be released into the environment and cause environmental pollution with the extensive use of plastic products.Moreover,PAEs are typical endocrine disruptors and some of them have carcinogenic,teratogenic,and mutagenic effects,which can be transmitted into the human body through the food chain and endanger human health.Dibutyl phthalate?DBP?,as a common PAE compound,is widely detected in environmental media and has been listed as a priority pollutant by many countries or organizations because of its serious toxicity.Therefore,how to effectively control and eliminate PAEs in the environment has become an urgent task.At present,microbial degradation has become one of the important methods for PAEs remediation because of its low cost,efficiency,and no secondary pollution.However,the molecular mechanism of biodegradation of PAEs is still unclear,which greatly limits the application of degrading bacteria in the bioremediation of PAEs-contaminated environment.In the present study,DBP was selected as the target pollutant,and the PAEs degrading bacteria Rhodococcus sp.2G isolated in our previous study was selected as the research object.The DBP hydrolase gene was screened out and cloned by conducting gene library,thus the expression and purification of its exogenous protein were investigated.The characteristics of this hydrolase combining with DBP and its conformation change were further studied by spectroscopy and molecular simulation docking techniques to reveal binding mechanism between the hydrolase and DBP.This study provides not only a theoretical basis for the molecular mechanism of microbial degradation of PAEs,but also genetic resources for the construction and application of highly effective degrading bacteria.The main results are as follows:?1?Construction of the gene library:a gene library of the PAEs degrading bacteria Rhodococcus sp.2G was constructed to obtain the DBP hydrolase gene.About 18,000transformants were generated in the constructed gene library,and a positive transformant named as 2G-I with DBP degrading activity was obtained by a three-step screening method.The result of sequencing analysis showed that the inserted gene fragment was 795 bp with an ORF,which was similar to the ORF of a?/?-hydrolase gene in the whole genome sequence of Rhodococcus sp.2G.The similarity was as high as 100%.The inserted gene was named as Hyd gene.?2?Bioinformatics analysis of the Hyd gene:Hyd gene was used to infer the amino acid sequence of Hyd gene by various bioinformatics softwares.Based on the analysis of protein family and conserved domain,the Hyd protein,belonging to?/?-hydrolase family and a member of PF00561 in?/?-hydrolase?CL0028?,has a typical?/?-hydrolase superfamily structure.In addition,the primary and secondary structure showed that the Hyd protein was composed of 264amino acids with a molecular formula of C₁₂₈₉H₂₀₁₅N₃₆₇O₃₇₂S₆,a molecular weight?M?of 28.79kDa,and an isoelectric point?p I?of 5.27.The contents of?-helix,?-sheet and irregular crimp contributed 42.5%,16.67%and 41.29%,respectively,to protein secondary structures.?3?Prokaryotic expression and purification of the Hyd gene and its enzymatic properties:at20°C,the recombinant Hyd protein was induced by 0.5 mmol/L IPTG overnight.SDS-PAGE analysis indicated that there was a specific expression band at 35 kDa.The expressed protein was purified and validated by Western Blot.The degradation rate of DBP by 0.1 mg Hyd protein was up to 65%within 5 min when the initial concentration of DBP was 27.83 mg/L.DBP can be effectively degraded by this hydrolase within a wide range of temperature?20-50??and pH?6.0-8.0?,while the optimum conditions are 37?and pH 7.5.?4?Binding mechanism between the Hyd protein and DBP:the interaction mode of DBP and Hyd protein was determined by fluorescence spectrometry and molecular docking technique.The results showed that DBP molecule was completely imbedded into the hydrophobic cavity composed of amino acid residues such as Leu29,Ser93,Phe94,Ala97,Pro118,Ile120,Pro123,Ile130,Thr190,Ser191,and Trp192.By binding with Hyd protein to form a ground state complex,the endogenous fluorescent Hyd not only had hydrophobic effect on the hydrophobic amino acid,such as Leu29,Ala97,Ile120,Ile130,and Trp192,but also formed two hydrogen bonds with Thr190 and Ser191.The bond lengths were 2.0?and 2.1?,respectively.In addition,ultraviolet-visible absorption spectra,synchronous fluorescence spectra,and circular dichroism analysis showed that DBP affected the conformation of Hyd protein during the combination with Hyd protein,resulting in a decrease in?-helix content in secondary structure of Hyd protein.The decrease of?-helical content indicated that DBP bound to the amino acids of the Hyd protein polypeptide backbone,and then caused rearrangement and structural changes of the polypeptide chain.Partial stretch of the polypeptide chain resulted in the exposure of certain hydrophobic regions,which in turn provided reaction space for the binding Hyd protein to DBP.