Data Mining And Catalytic Performance Of Di(2-Ethylhexyl) Phthalate Hydrolase

Phthalates(PAEs),commonly known as plasticizers,are widely used in the production and processing of plastic products.The consumption of di(2-ethylhexyl)phthalate(DEHP)is the largest,exceeding 3 million tons per year.Studies have shown that PAEs can interfere with the endocrine system of humans and animals at low concentrations,it also has carcinogenic,teratogenic and mutagenic effects at high doses.Biodegradation of PAEs has been widely reported as a major and efficient method of degradation over the past few decades.At present,the enzymes that catalyze the hydrolysis of the first ester bond of PAEs mainly act on PAEs with short alkyl side chains.However,the hydrolases of DEHP are rarely reported.To date,the genetic and protein sequences of DEHP-degrading enzymes are still unknown,which hinders our understanding and further application of enzymatic degradation of large side chain PAEs.In this study,the DEHP-degrading strains were screened from the soil by traditional methods.Six strains of catalytic activity against DEHP were isolated from 200 soil samples contaminated with plastics,including four Gordonia sp.,a Rhodococcus sp.,and a Micrococcus sp..Among them,Gordonia polyisoprenivorans 5F showed the highest activity and was selected for further investigation.By analyzing the degradation intermediates,we speculated the degradation pathway of DEHP in strain 5F.Then,17 potential esterase genes were cloned from strain 5F by genomic data mining,and ligated into the pET-28a plasmid vector.The recombinant plasmids were then transformed into E.coli BL21(DE3)for heterologous expression.The target enzyme for degradable DEHP was successfully screened and named as GoEst15,which hydrolyzes DEHP to mono(2-ethylhexyl)phthalate(MEHP).We have purified GoEst15 and confirmed that its molecular weight was 58 kDa.The optimum reaction conditions for GoEst15 were pH 7.0 and 30?.For DEHP,the Km and kcat/Km values for GoEst15 were 0.31 mM and 0.16 s-1mM-1,respectively.The time required for DEHP and DBP to be completely degraded by GoEst15 is 24 hours and 1 hour,respectively.Substrate spectrum test experiments showed that the enzyme could catalyze the degradation of eleven PAEs.GoEst15 is a rare broad-spectrum esterase.In addition,we also discovered the MEHP hydrolase(GoEstMl)in Gordonia polyisoprenivorans 5F,and constructed a co-expression system of GoEst15 and GoEstM1,which can realize one-step conversion of PAEs to PA.In summary,GoEst15 is a newly discovered esterase capable of degrading DEHP.Due to its good degradation and broad substrate spectrum,the enzyme will be a promising biocatalyst that can be used as a starting enzyme for protein engineering to develop green,gentle,efficient technology for future bioremediation,biodegradation of pollutants and water treatment.