The Preparation Of Mesoporous Silica And The Immobilized Laccase

Enzyme is widely used in different fields. But free enzyme is easily affected by environment and loses its activity; While the immobilized enzyme is stable, low sensitivity to inhibitors and can be reused.The preparation of mesoporous silica carrier and the surface modification of mesoporous silica carrier and immobilization of laccase were studied in this study. The influence of functional groups, including amino and vinyl, and different immobilization methods on laccase activity was analyzed. The influence of the different functional groups on the mesoporous silica carrier was discussed by investigating the enzyme loading By investigating activity and activity recovery of immobilized laccase, the influence of different methods on immobilization was studied. Finally, the properties of the immobilized laccase and comparison with the free laccase were analyzed.(1) The influential factors of activity of white-rot fungi laccase were researched. The optimal conditions of activity of the laccase were as flows:the wavelength was 600 nm, pH value was 4.0, the reaction temperature was 30℃, the concentration of buffer solution was 0.1 mol/L. Dynamics results:between 1 and 5 minutes, the reaction conformed to the linear equation y= 0.0097 x-0.0016, then the reaction rate decreased, and the reaction stopped afer 16 minutes.(2) The influence of different functional groups was studied. The research showed the amount of loading laccase of SBA-15, SBA-15-NH2 and SBA-15-CH=CH2 were respectively 300.2mg/g,176.8mg/g and 51.6 mg/g. So the functional groups reduce the immobilization amount of the lacase, the vinyl has a specific influence on it.(3) The influential factors of the immobilized laccase by cross-linking and adsorption were analyzed. The conditions of immobilized laccase by adsorption were optimized which could be specified as:pH was 5, laccase solution was 0.5 mg/mL, the immobilized time was 5h. Under the optimal combined conditions, the immobilized laccase showed most promising immobilization where the activity of immobilized laccase was 3005U per gram carrier and the recovery of enzyme activity reached 60.1%. The conditions of immobilized laccase by cross-linking and adsorption were optimized in conditions which could be specified as:4% glutaraldehyde, cross-linked time was 7h, concentration of laccase solution, immobilized time was 6h. Under the optimal combined conditions, the immobilized laccase showed the activity of immobilized laccase was 3615U per gram carrier and the recovery of enzyme activity reached 72.3%. So the approach of cross-linking and adsorption is better than adsorption.(4) T the properties of the immobilized laccase and comparison with the free laccase. The optimum temperature of the immobilized lacease which was fixed on the SBA-15-NH2 was 50 ℃, the free enzyme was 30 "C.The optimum pH of free enzyme was 4, the immobilized laccase was 4.5. After laccase was kept for 20 days, the immobilized laccase by crosslinking adsorption could retain above 79% of initial activity. The immobilized laccase by adsorption could retain above 69% of initial activity. While the free laccase could retain above 29% of initial activity.The experimental results showed that stability of immobilized laccase were improved, compared with free laccase. The Michaelis constant of free laccase was 0.088mmol/L. While immobilized laccase by adsorption was 0.16mmol/L, the immobilized laccase by cross-linking adsorption was 0.53mmol/L. In conclusion, the optimum temperature of immobilized laccase enhances, the optimum pH of immobilized laccase boosts slightly. The immobilized laccase has good stability, but the activity of immobilized laccase declines slightly.