Study Of Immobilized Laccase For The Removal Of BPA And DBP Complex Contamination

Two typical estrogenic endocrine disruptors,bisphenol A(BPA)and dibutyl phthalate(DBP),have been widely used in modern agriculture and industry.In addition,the natural coexistence of BPA and DBP is evident in aquatic ecosystems,and humans may be exposed to BPA and DBP coexistence through multiple pathways.It is important to investigate a method that can achieve simultaneous removal of combined BPA and DBP contamination.Enzymes have the ability to degrade pollutants efficiently,and they also exhibit greater biodegradation potential than microorganisms because of their mild operating conditions,sustainability and environmental friendliness.However,the problem of poor stability of enzymes in the environment has become a major concern.Polydopamine(PDA),as a surface modifier,can enhance the stability of enzymes by achieving good immobilization of various enzymes with abundant catechol and amine groups on the surface,and can also participate in the adsorption of organic pollutants.Therefore,this experiment used PDA as a surface modifier and mesoporous silica microspheres(Si O2)as a support for laccase immobilization(PDA-Si O2)through covalent binding,and used response surface design to create an efficient,stable and reusable laccase immobilization method(Lac-PDA-Si O2).The effects of p H,temperature and reaction time on the biodegradation efficiency of BPA and DBP were investigated,and the enzymatic and degradation kinetics of laccase were analyzed and the binding of laccase to BPA and DBP was simulated by molecular docking.In addition,to evaluate the possible catalytic pathways and to determine the toxicity of the final effluent,the degradation products were examined and the toxicity of various intermediates was analyzed.Finally,we also verified the cycling and storage stability of Lac-PDA-Si O2.The main findings of the study are as follows:(1)Laccase was successfully immobilized on the surface of PDA-coated mesoporous Si O2 microspheres,and the optimal conditions for immobilization of laccase by PDA-Si O2 were determined by single-factor tests on immobilization time,temperature for,p H and laccase concentration as well as response surface construction.The BET results showed that the specific surface area,adsorption pore volume and average pore size of Lac-PDA-Si O2 were increased,which This indicates that the modified material has more adsorption sites and exhibits stronger adsorption performance.Meanwhile,the FTIR and XPS spectra of Lac-PDA-Si O2 showed more functional groups,which not only predicted the successful immobilization of laccase,but also the increase of its surface oxygen-containing functional groups(C=O and C-O)could provide moreadsorption sites for the adsorption of BPA or DBP,realizing the simultaneous adsorption and degradation of BPA/DBP by Lac-PDA-Si O2.(2)Under the optimum conditions,the removal efficiency of Lac-PDA-Si O2 for single contamination of BPA or DBP was 2.03 and 1.75 times higher than that of free laccase,respectively.Lac-PDA-Si O2 showed better temperature stability and p H stability in both single and combined contamination treatment of BPA and DBP.Moreover,the removal of BPA and DBP complex contamination by Lac-PDA-Si O2 occurred simultaneously,and both removal efficiencies showed a slight decrease due to adsorption or competition for binding sites.In the enzymatic kinetic analysis,the Km value of Lac-PDA-Si O2 decreased and the Vmax value of the degradation process decreased compared to the free laccase of the same activity,which may be due to the spatial site resistance of the catalytic site.While in the degradation kinetics study,Lac-PDA-Si O2 was no longer in excess relative to BPA and DBP at 50-100 mg/L,and the increment of reaction rate gradually decreased,and the reaction in this concentration range was a primary secondary mixed kinetic reaction.(3)Molecular docking was used to simulate the laccase catalyzed degradation of BPA and DBP,which confirmed the spontaneous proceeding of the reaction,and the degradation process of BPA and DBP was analyzed,and it was inferred that the degradation pathway of Lac-PDA-Si O2 to BPA might be as follows: the break at the isopropyl linking two hydroxybenzenes in BPA,the generation of isopropenyl phenol radical and then oxidized,the generation of 1-methyl-4-The degradation pathway of DBP by Lac-PDA-Si O2 may be the formation of PA from DBP through the successive breakage of ester bonds.The feasibility of degradation of BPA and DBP by Lac-PDA-Si O2 was well demonstrated.(4)The biocompatibility of Lac-PDA-Si O2 was verified by exploring its effect on the early development of zebrafish embryos.After incubation with Lac-PDA-Si O2 for 96 hpf,there was no difference in the survival rate and malformation rate of zebrafish embryos compared to the blank control group,and no developmental differences or malformations were observed under the body view microscope,confirming that the Lac-PDA-Si O2 biocompatibility.It was found that the synthesized Lac-PDA-Si O2 should be considered as a promising alternative for the effective degradation of BPA and DBP under various conditions.