Immobilization Of Tyrosinase On Polyacrylonitrile Carrier And Its Application In Degradation Of Phenolic Compounds In Solution

The enzyme tyrosinase is a copper-containing enzyme.Under the influence of oxygen,it can catalyze the oxidation of aromatic compounds.It has a good catalytic activity especially for phenolic substances in aqueous media and is widely used in harmless treatment of phenol-containing wastewater.With glutaraldehyde cross-linking method and polyacrylonitrile as a carrier,this topic uhderwent an immobilization process of tyrosinase.Based on this,the immobilization mechanism,the optimal immobilization conditions and the enzymatic properties were further studied.And the immobilized tyrosinase was used to remove the phenol from the solution and its catalytic oxidation performance was explored.In the second chapter,polyacrylonitrile microspheres were experimentally prepared and activated for modification,and finally tyrosinase was immobilized on it.Subsequently,the important properties of the enzyme before and after immobilization were characterized using infrared spectroscopy,scanning electron microscopy and energy dispersive spectroscopy.The optimized operating conditions for the enzyme immobilization process are as follows:the immobilization time is 20 h;the enzyme solution concentration is 1 mg/mL;glutaraldehyde concentration is 10%;the optimum pH is 7.5;the optimum temperature is 35?.;under the optimum conditions,The maximum protein loading is 18.3 mg/g microspheres and tyrosinase can retain 75%activity after immobilization.In Chapter 3,the experiment explored several important properties of enzymology including the optimum pH of the enzymatic reaction,the optimum temperature,the Michaelis constants(Km).It was found that the optimum pH and temperature of the immobilized tyrosinase reaction were 7.0 and 35?,the Michaelis constant value of 2.41 mM was higher than free enzyme.This result indicated that the affinity of the enzyme to the substrate after immobilization is lower than that of the free one.In Chapter 4,we focused on the catalytic degradation of phenol by immobilized tyrosinase and explored the optimal conditions for the degradation of phenol.The results showed that the immobilized tyrosinase could effectively remove the phenol in solution in the optimal degradation environment(O2 for 10 min,pH 7.0,temperature 40?,time 12h).And its relative activity was still close to 60%after 60 days of storage.Compared with free enzymes,it increased significantly;Besides,it had about 60%relative activity after recycling more than 8 times,which shown a good reusability.In Chapter 5,the biosafety of the catalytic oxidation of immobilized tyrosinase to remove phenol was evaluated using the CCK-8 method.The study found that the survival rate of the cells in the low-concentration phenol solution after the immobilized enzyme treatment was higher than that of the untreated same-component solutions.The results of apoptosis detected by flow cytometry showed that the higher the concentration of phenol,the easier it was to induce apoptosis in L02 cells.And the mechanism of cell cycle analysis revealed that because of the influence of phenol,a large amount of cell proliferation was blocked in the S phase,eventually leading to a decrease in cell viability,which may be due to the phenol-induced Cyclin E denaturation.