Enzymatic Immobilization Based On Functionalized Mesoporous Silica Carrier And Its Catalytic Aromatics Degradation

Enzymatic degradation of organic pollutants in wastewater has the advantages of high catalytic efficiency, without second pollution, etc. After being immobilized, both thermal stability and structural stability of enzyme can be increased. Immobilized enzyme is easy to be separated from system and can be reused. Amino modified mesoporous silica has great potential in the field of enzyme immobilization, due to its adjustable pore size, large specific surface area and easy separation.In this study, the amino modified mesoporous silica carrier was synthesized directly by hydrothermal synthesis with three block copolymer P123 as template. The immobilization of laccase and cellulase on above synthetic carrier was studied by the method of cross-linking. There are two different cross-linking methods of enzyme immobilization, one is that carrier cross-links with glutaraldehyde at first and then fixed to enzyme(two-step enzyme immobilization for short), the other is that carrier cross-links with glutaraldehyde and fixes to enzyme together(one-step enzyme immobilization for short). Above immobilization methods for laccase and cellulase were studied and compared, and the conditions of enzyme immobilization were optimized. The enzymatic properties of immobilized enzymes were compared with that of free enzymes. The influence factors of 2,4-Dichlorophenol degradation catalyzed by immobilized laccase were investigated. The experimental results show that:(1) The amino modified mesoporous silica has been synthetized directly by the method of hydrothermal synthesis. From the structure characterization of carrier, we can see that amino groups have been successfully connected to the surface of mesoporous silica. The specific surface area and pore size of amino modified mesoporous silica are 33.14m2/g and 4.13 nm, respectively.(2) Comparative study on the methods of immobilization. When the laccase was immobilized, the time and pH of two immobilization methods were almost the same, but the glutaraldehyde and laccase concentration of two-step immobilized laccase is far greater than the one-step. The laccase and glutaraldehyde coexist in reaction system for laccase immobilization of one-step method. The inhibition of glutaraldehyde on laccase is obvious. Therefore, the activity of immobilized laccase by two-step is much greater than the one-step: immobilized laccase activity of one-step was only 239.5 U/g, while that of two-step was 2977.5 U/g. For cellulase immobilization, the immobilization time, pH and the glutaraldehyde concentration of two immobilization methods were roughly the same. And the inhibition of glutaraldehyde on cellulase was higher than that of the laccase, the optimal concentration of cellulase for two-step cellulase immobilization was still far greater than one-step method. Similarly, immobilized cellulase activity of two-step is far greater than the one-step: immobilized cellulase activity of one-step was 384.1 μmol?min-1?g-1, while that of two-step was up to 2123.7 μmol?min-1?g-1. The immobilized enzyme activity shows that the immobilized method of two-step is much better than one-step.(3) Enzymatic properties study of immobilized enzyme. The optimum temperatures of immobilized enzymes by two-step were improved, while that of the one-step immobilized enzymes were not changed compared with free enzymes. At the same time, the adaptability of enzymes to high temperature is greatly improved after being immobilized. The optimum pH of immobilized enzyme was shifted to acidity, the reason is that the amino group on carrier presents alkaline in the solution. After 5 times operation, residual activities of all immobilized enzymes were about 50% of that of free enzyme, the operation stability of immobilized enzyme is still pretty good. The Michaelis constant of free enzyme, two-step and one-step method immobilized enzyme increases in turn, this indicates that the substrate affinity of enzyme is decreased after immobilized, and two-step immobilized enzyme has better substrate catalytic effect than one-step.(4) 2,4-Dichlorophenol degradation catalyzed by immobilized laccase was studied. The alkaline solution is benefit to the degradation of 2,4-Dichlorophenol. The optimum temperature of 2,4-Dichlorophenol removal and degradation by immobilized laccase was 40℃. The removal, adsorption and degradation of 2,4-Dichlorophenol were decrease gradually with the increase of its initial concentration. When the 2,4-dichlorophenol initial concentration increased, its certain reaction products have inhibition to laccase activity, which results in the 2,4-dichlorophenol degradation decreased rapidly. And 2,4-dichlorophenol initial concentration rises, too many molecules in solution can’t be connected with the carrier active site, thus led to the decrease of adsorption rate.