Screening Of Phthalate Esters Degrading Enzymes And Its Application In Soil Remediation

Phthalate esters（Phthalate esters,PAEs）are a typical class of priority pollutants with endocrine disruption,neurotoxicity,immunotoxicity and other hazards,which can enter the human body through the food chain and pose a serious threat to human health.In the process of agricultural production,due to the large-scale use of agricultural plastic film,farmland soil has become an important gathering place for PAEs.The remediation of farmland soil requires the removal of pollutants while maintaining its ecological function.Therefore,the research on a green remediation technology of farmland soil is of great significance to promote agricultural green production and reduce the risk of human health.In this study,PAEs were taken as the target pollutants.By comparing the degradation of PAEs by five common commercial enzymes,the lipase from Candida sp.was determined as PAEs degrading enzyme.The structural selectivity of lipase to different chain length PAEs was studied in water system,and the degradation products of PAEs were analyzed and the degradation pathway was inferred.The study on the remediation of PAEs-contaminated farmland soil by lipase under different conditions was carried out,and the immobilization of free lipase by biochar and the application of immobilized lipase in farmland soil remediation were explored.The main conclusions of this study are as follows:（1）By comparing the degradation effects of five commercial enzymes on PAEs,it was found that there was no obvious degradation activity in the reaction of laccase（with or without redox mediators）,peroxidase andα-amylase,while carboxylesterase and lipase could effectively degrade 7 different kinds of PAEs,in the system and the degradation efficiency remained between 88.14%and 99.16%.（2）The relative concentrations of PAEs with different chain length showed different degradation trends at different time points in the lipase catalytic reaction system.The catalytic degradation of seven kinds of PAEs by lipase in water system conformed to the first-order degradation kinetics（R²>0.97）.With the increase of PAEs alkyl chain and molecular weight,the degradation rate will gradually slow down.Through the analysis of the correlation between degradation rate constant,half-life and log K_ow,it is known that with the increase of octanol-water partition coefficient of PAEs,its water solubility decreases,the more difficult it is to combine pollutants with enzymes,and the slower the degradation rate is.According to the above analysis,the structural selectivity of lipase to seven kinds of PAEs in aqueous system was in the following order:DMP>DEP>Dn BP>Di BP>DEHP>Dn OP>Dn NP.（3）Under the catalysis of lipase,PAEs was firstly hydrolyzed to its corresponding monoester MPEs,by ester bond,and then the product PA was obtained by one-step hydrolysis.It can be seen that a clearer degradation pathway of pollutants can be obtained by directly acting on the target pollutants by enzymes,which makes the biodegradation mechanism of pollutants clearer.（4）Lipase can obviously degrade PAEs in soil.Under the optimal conditions,the degradation rate of seven kinds of PAEs is kept at 47.5%-73.9%.The control variable method was used to analyze the effects of different factors on the remediation process,and it was found that the lipase concentration at the level of 2 U/g could ensure that the remediation work was effective and economical;the soil p H and temperature directly affected the lipase activity and then affected the degradation effect;when the soil moisture content was kept at 45%,the mobility of lipase in the soil environment was less limited and the degradation rate was faster.Light and soil microorganisms had no significant effect on lipase degradation of PAEs in soil.By fitting the first-order kinetic equation of lipase degradation of PAEs in soil system,it was found that lipase had the same structural selectivity for different PAEs in the same water system,and the degradation efficiency decreased with the increase of PAEs alkyl chain.In addition,the degradation rate constants of different PAEs in soil were lower than those of corresponding PAEs in water system,which was mainly determined by the fluidity of enzyme molecules and target pollutants in the reaction system.（5）The effects of different factors on the enzyme activity recovery rate of immobilized lipase were studied.The results showed that when the immobilization condition was 4 U/m L of lipase concentration,the biochar particle size was 40 meshes and the immobilization reaction time was 5 h,the enzyme activity recovery rate of immobilized lipase could reach 68%and 73%,and its thermal stability and p H stability were better than those of free lipase.By comparing the effects of free lipase and immobilized lipase on PAEs in soil,it was found that when cultured for 168 h at the same enzyme concentration,the degradation efficiency of PAEs in free lipase system was in the range of 42.1%-72.3%,while that in immobilized lipase system was46.8%-76.8%.Immobilized lipase showed a more lasting and effective degradation effect than free lipase.Therefore,the application of immobilized lipase with biochar as carrier in PAEs contaminated farmland soil has strong advantages and practical value.