Multi-enzyme Co-immobilization Using Magnetic Biochar

1,2,3-Trichloropropane(TCP)is an environmentally hazardous pollutant mainly found in groundwater.The highly toxic and carcinogenic risk of TCP has attracted widespread attention in recent years,so it is important to study the degradation of such environmental pollutants.For chemical and physical remediation means,biodegradation methods are more efficient and environmentally friendly.In this thesis,haloalkane dehalogenase(Dha A),haloalcohol dehalogenase(Hhe C)and epoxide hydrolase(Ech A)were used to degrade TCP in cascade.In this thesis,the three enzymes were immobilized on suitable carriers to improve their stability and reusability,which is more beneficial for industrial applications.Biochar(BC)is a widely available material with high specific surface area,good stability and environmental friendliness.In this thesis,biochar prepared from rice husk was used for enzyme immobilization in the degradation of TCP by covalent immobilization.Magnetic biochar(MBC)was firstly prepared by modifying magnetic particles with biochar,and the surface of MBC was amino-functionalized by APTES.The biochar was characterized before and after modification by SEM,VSM,FT-IR and thermogravimetric analysis.The results demonstrated the success of biochar modification of magnetic particles and amino-functionalization.Then the conditions of enzyme dosage,glutaraldehyde concentration,ammonium sulfate concentration and immobilization time were optimized for the process of MBC immobilized enzyme.The optimized conditions immobilized Dha A and Hhe C both retained more than 80% of the enzyme activity,and the immobilized Ech A retained more than 60% of the enzyme activity.The enzymatic properties of the immobilized haloalcohol dehalogenases under optimal conditions showed that the MBC-immobilized enzymes were effective in improving the thermal stability,storage stability,and organic tolerance of the enzymes without affecting their enantioselectivity,as well as retaining more than 90% of the initial activity after 10 repeated uses.The degradation efficiency of TCP degradation was 52% after random co-immobilization of the three enzymes.In order to improve the efficiency of the cascade degradation of TCP by the three enzymes,then explored the fusion of Spy Tag(ST)/Spy Catcher(SC)with the above three enzymes to construct three fusion proteins ST-GS-Dha A31,Hhe C-GS-SC and STGS-Ech A-GS-ST.Two immobilization strategies,method 1: First,express SC alone and immobilize it on MBC by covalent immobilization,in the order of ST-GS-Ech A-GS-ST,Hhe C-GS-SC and ST-GS-Dha A31,respectively.Self-assembled and immobilized on SC-modified MBC;Method 2: Hhe C-GS-SC and ST-GS-Dha A31 were self-assembled and co-immobilized with Ech A on MBC.Under optimal conditions,the self-assembled immobilized enzyme degraded TCP by method 1 was 56% efficient.The efficiency of catalytic degradation of TCP after self-assembly and co-immobilization by method 2 is98%,which is 46% higher than that of the three enzymes after random coimmobilization.And the co-immobilized enzyme by method 2 still retained more than80% of the TCP degradation efficiency after 11 uses.