Organic Functional Modification Of Macroporous SiO₂ Block Material And The Application To Laccase Immobilization

Laccase has broad substrate specificity and good stability, its application in the phenolic wastewater treatment has attracted more and more attention. However, free laccase is difficult to recycle and easily inactivated, limiting its application in industry. Enzyme immobilization technology is an effective means to improve laccase stability and make its reuse. Therefore, the practical applications of laccase in industry are widened. In this thesis, macroporous SiO2 block material was organically modified by some methods, and the modified macroporous materials were used as supports for the immobilization of the Novozymes commercial laccase. At last, the degradation of 2,4-DCP by immobilized laccase were researched.1. The macroporous silica block material was epoxy-modified via post-grafting method under solvothermal conditions. The epoxy-functionalized macroporous silica(EMS) was used as support for immobilization of laccase by using covalent binding method. Immobilization conditions were optimized by single factor analysis method. The activity of the immobilized laccase reaches a maximum of 101.7 U/g under the buffer of pH 4.5, initial laccase concentration of 25 mg/ml and immobilization time of 4 h. The immobilized laccase can be used repeatedly, and the remained activity is 43.4% after 10 times of reaction with the ABTS substrate.2. A new macroporous composite silica/chitosan(SiO2/CS) was prepared by adsorbing chitosan on the pore wall of block macroporous silica and used as the support for immobilization of laccase under glutaraldehyde as crosslinking agent condition. The activity recovery of the immobilized laccase was 85.5 % under the buffer of pH 4.5, initial laccase concentration of 40 mg/ml and immobilization time of 4 h. The studied of properties show that the immobilized laccase has good thermal, pH and operational stability.3. The SiO2/CS composite was further modified by glycol diglycidyl ether(GDE) using ethanol as solvent. The epoxy-functionalized SiO2/CS composite(SiO2/CS-GDE) was employed as the support for immobilization of laccase through covalent coupling method. The activity of the immobilized laccase could reaches a maximum of 315.2 U/g under the buffer of pH 4.5, initial laccase concentration of 70 mg/ml and immobilization time of 5 h. The immobilized enzyme can be used repeatedly, and the remained activity is 58.7% after 10 consecutive operations.4. Under alkaline conditions, iminodiacetic acid(IDA), acting as a metal-chelating ligand, was grafted on SiO2/CS-GDE composite, and after absorbing Cu2+ a Cu2+-chelated macroporous composite SiO2/CS-GDE-IDA-Cu2+ was obtained. Laccase was successfully immobilized on SiO2/CS-GDE-IDA-Cu2+ by using ion affinity method. The activity recovery of the immobilized laccase was 110 % under the buffer of pH 4.5, initial laccase concentration of 30 mg/ml and immobilization time of 5 h. The immobilized laccase has very good operational stability, it still retained 83.5% of the original activity after 10 times of reaction with the substrate.5. The immobilized laccase on SiO2/CS was used for the degradation of 2,4-dichlorophenol. The effects of initial concentration of 2,4-DCP, pH, reaction time and temperature on the degradation rate of 2,4-DCP were investigated. Under the optimized conditions, 2,4-DCP(20 mg/L) was degraded up to 65.6%.