Preparation Of Multifunctional Enzyme Immobilization Matrix And Its Application In Aflatoxin B1 Removal

As one of the toxic organic micropollutants,aflatoxin B1(AFB1)is potentially detrimental to human beings and animals,and thus efficient and green removal of AFB1 is imperative.Enzyme,as a highly efficient and specific biocatalyst,has been widely used for the micropollutant removal.However,the industrial applications of enzymes are often hampered by several limitations such as poor stability and difficulty in reuse.Enzyme immobilization can possibly solve these problems,but it also lowers the catalysis efficiency of the enzyme.Aiming at these problems,this thesis focused on the preparation of multifunctional enzyme immobilization matrix,and the catalytic function of the immobilized enzyme was integrated with the adsorption or separation ability of the matrix to enhance the AFB1 removal.The main research contents and results were summarized as follows:Inspired by catecholamine chemistry,tannic acid-3-aminopropyltriethoxysilane(TA-APTES)coating was applied for carier activation and subsequent enzyme immobilization,focusing on its universality on different carriers.It can be found that the TA-APTES coating showed much better performance in enzyme immobilization than glutaraldehyde,genipin,polydopamine activation strategies thanks to its special surface nanostructure and abundant quinone groups.Secondary grafting branched polymer ?-polyglutamic acid on the TA-APTES coating layer further increased enzyme loading(3.5-4.5 times).The TA/APTES ratio and coating time greatly affected enzyme loading and specific activity.The coating with a TA/APTES ratio of 8:1 and coating time of 18 h exhibited the highest enzyme loading and the best enzyme activity.Finally,the universality of TA/APTES coating was demonstrated by applying it on various materials(i.e.polyvinylidene fluoride,polyether sulfone,polyacrylonitrile,nylon membrane,polyester non-woven fabric and stainless steel wire mesh)for immobilizing different enzymes(i.e.horseradish peroxidase(HRP),lipase,pepsin,laccase and invertase)and removing five micropollutants(i.e.bisphenol A,2,4,6-trichlorophenol,AFB1,deoxynivalenol and tetracycline).AFB1 easily adsorbed onto polyamide nanofiltration membrane,then dissolves into the membrane and passes through the membrane via diffusion,resulting in decreasing rejection with time.In order to solve this problem,HRP was covalently immobilized on NF270 nanofiltration membrane modified by TA-APTES coating to prepare multifunctional membrane with both separation and catalytic functions.The results showed that such a multifunctional membrane had a stable AFB1 rejection of more than 90%due to surface repulsion effect(reducing AFB1 adsorption)and pore narrowing effect(increasing diffusion resistance).The presence of H2O2 would induce the enzymatic reaction for AFB1 degradation,further improving the removal efficiency of AFB1 by the synergistic effect of the multifunctional membrane.After the fouled multifunctional membrane was cleaned by the dilute H2O2 solution,the water permeability can be restored to 98.7%of the initial value.In order to explore the possible adsorption-catalysis synergistic mechanism during the removal of AFB1 by the immobilized enzyme,alginate/chitosan(SA/CS)microspheres and alginate/chitosan/montmorillonite(SA/CS/MON)hybrid microspheres were prepared for HRP immobilization,respectively.Compared with the enzyme immobilization via encapsulation or adsorption followed by crosslinking,HRP immobilized on the microsphere surface via covalent bonding showed the highest specific activity and AFB1 removal efficiency.The negatively charged MON could not only adsorb abundant AFB1,but also attract more CS chains on the microsphere surface and bring more active sites for enzyme immobilization and crosslinking,thus the SA/CS/MON microspheres owned stronger anti-swelling ability,higher AFB1 adsorption capacity(0.36 vs.4.5 ?g/g)and enzyme loading(1.07 vs.1.25 mg/g).Although the AFB1 enrichment in the SA/CS/MON-HRP multifunctional microsphere did not promote its degradation by enzymatic catalysis(there are three possible reasons:substrate inhibition,product adsorption and lowing substrate concentration in bulk solution),the reuse stability of the multifunctional microsphere was improved and the enzymatic degradation efficiency of AFB1 increased with reuse cycle,which might be caused by increasing substrate concentration in the bulk solution when the adsorption capacity of the microsphere became saturate.The obtained multifunctional microsphere as a prototype is promising in the AFB1 removal because both the adsorbent and the catalyst can be designed,and their spatial distribution can even be controlled.