Preparation Of Immobilized Laccase On Magnetic Nanoparticles And Its Application In Phenols Wastewater Treatment

In order to improve the stability and reusability of laccase in the application system,in this thesis,we presented a stratgy for laccase immobilization on magnetic nanoparticles through DA self-polymerization process.The immobilized laccase on Fe₃O₄ nanoparticles?Fe₃O₄-PDA-Lac?and the immobilized laccase on silica-coated magnetic nanoparticles?Fe₃O₄@SiO₂-PDA-Lac?were prepared through dopamine?DA?in situ entrapment,and then the immobilized laccase was applied for 4-chlorophenol?4-CP?removal.The main work and innovations were summarized as follows:?1?The Fe₃O₄ nanoparticles were prepared by thermal decomposition method,and then the silica-coated magnetic nanoparticles?Fe₃O₄@SiO₂?were prepared through a sol-gel method.The scanning electron microscope images indicated that Fe₃O₄@SiO₂ have a spherical and uniform size distribution with the average diameter of about 120 nm.The high saturation magnetization of Fe₃O₄@SiO₂?14.68 emu/g?makes it easily to be separated from the reaction system.The results of X-ray diffraction?XRD?and Fourier transform infrared spectroscopy?FTIR?diagrams revealed that the prepared samples were Fe₃O₄ nanoparticles and Fe₃O₄@SiO₂ nanoparticles.?2?The preparing conditions of the Fe₃O₄-PDA-Lac were optimized by response surface methodology.The enzymatic characteristics of the Fe₃O₄-PDA-Lac were investigated.It was found that when laccase concentration at 0.25 mg/mL,DA concentration at 12.74 mg/mL and solution pH at 5.92,the total activity recovery of the Fe₃O₄-PDA-Lac was 88.17%.Compared with the free laccase,the stability and reusability of Fe₃O₄-PDA-Lac were significantly enhanced.After 40 days of storage at 4°C,the residual activity of the Fe₃O₄-PDA-Lac kept about 89%of its initial activity.After resue 10 times,the Fe₃O₄-PDA-Lac still retained 70%of its initial activity.?3?The preparing conditions of the Fe₃O₄@SiO₂-PDA-Lac were optimized by orthogonal experiment.The enzymatic characteristics of the Fe₃O₄@SiO₂-PDA-Lac were investigated.Compare to the laccase immobilized by glutaraldehyde?GA?crosslinking method?Fe₃O₄@SiO₂-GA-Lac?,in the best level,the total activity recovery of the Fe₃O₄@SiO₂-PDA-Lac can reach to 43.28%,but the Fe₃O₄@SiO₂-GA-Lac only exhibited3.33%of the total activity recovery.Compared with the Fe₃O₄@SiO₂-GA-Lac,the Fe₃O₄@SiO₂-PDA-Lac not only exhibited better resistance to a broader pH value,but also enhanced the thermal stability,storage stability and reusability.?4?In the degradation process,the effects of laccase concentration,solution pH and ABTS mediator on the removal efficiency of 4-CP were investigated.The results showed that when solution pH was 6 and laccase concentration was 1.2 U/mL,95%percentage of 4-CP was removed within 8 h enzymatic catalysis.When 50 umol/L of ABTS was added in the reaction system,the removal efficiency of 4-CP kept about 99%within 10 min enzymatic catalysis,which certified that the ABTS as a mediator could enhance the enzymatic reaction rate of the immobilized laccase.After degraded 10 times by the immobilized laccase,the removal efficiency of 4-CP still remained 67%,which exhibited an excellent reusability and operational stability.