Enzyme Immobilization Using Magnetic Nanoparticles And Its Applications

1,2,3-Trichloropropane(TCP)is a kind of industrial pollutant that threatens human beings,and it has the risk of canceration after long-term exposure.After long-term research,it is found that degradation of this kind of compound by biological enzyme not only conforms to the green concept,but also reduces the cost,so it has become a new research hotspot.Three key enzymes,haloalkane dehalogenase,halohydrin dehalogenase and epoxide hydrolase,need to be used in biodegradation.However,the common free enzyme is easy to inactivate during use,and the stability of free enzyme is lower than that of immobilized enzyme.The more critical disadvantage is that the free enzyme is difficult to be separated from the system and reused,which limits the industrial application of biological enzyme and leads to an increase in the use cost.Immobilized enzyme can not only improve the stability and operability of enzyme,but also be reused,which solves the disadvantages of free enzyme.In order to facilitate recycling,the immobilization strategy of cross-linked enzyme aggregates(CLEAs)connected to surface-modified magnetic nanoparticles was adopted in this paper,and this strategy was applied to three key enzymes in the biological pathway of TCP degradation,which was optimized and applied to TCP degradation.Firstly,the surface of magnetic nanoparticles was modified with amino groups.The modification principle mainly depended on hydrolysis of silylation reagent APTES in acidic environment and reaction with hydroxyl groups on the surface of magnetic nanoparticles.The modification was confirmed to be successful by Fourier transform infrared spectroscopy and transmission electron microscopy.Then,combined with the CLEAs immobilization technology in literature,through the optimization of magnetic nanoparticle size,dosage,bovine serum albumin dosage,ammonium sulfate concentration,glutaraldehyde concentration,cross-linking time and other conditions,the immobilized epoxide hydrolase activity retained more than 55%,the immobilized haloalkane dehalogenase retained more than 80%,and the immobilized halohydrin dehalogenase retained more than 90%.After performance test,it was found that the immobilized enzyme with magnetic nanoparticles combined with CLEAs technology has higher thermal stability,storage stability and organic tolerance than free enzyme,which proves that this method has broad application prospects,and then the immobilized enzyme will be applied to TCP degradation pathway.The immobilized three enzymes were used to degrade TCP,and the degradation efficiency of magnetic nano-CLEAs on TCP was verified by detecting the residual concentration of TCP by gas pHase.The efficiency of biodegradation of TCP was optimized by changing the time interval and ratio of enzyme addition.The final optimized reaction system was as follows: adding enzymes at intervals of 10 minutes,the total mass of enzymes was 20 mg,and the mass ratio of enzymes was Dha A: HheC: EchA= 2:2:1.After co-immobilization,TCP was degraded,and finally TCP was degraded by 90% after 5 hours.And 78% of the enzyme activity can still be retained after being used for 6 times,which proves that the system has better efficiency of degrading TCP.