Study On Immobilization Of Laccase On Mesostructured Cellular Foam

Laccase belonged to a family of muti-copper oxidase has important applied value in treating the wastewater with phenol-containing, paper industry and so on. Immobilization is an effective approach to enhance the stability of enzymes, and mesoporous materials have received great interest as catalyst supports because of their large surface area, highly ordered pore structure, adjustable pore size, good biocompatibility and excellent structure.Siliceous mesocellular foam (MCF) was successfully synthesized by the acid catalyzed solgel reaction of tetraethoxysilane (TEOS) in the presence of triblock copolymer P123 as a structure-directing agent and trimethylbenzene (TMB) as a swelling agent. The obtained MCF was characterized by methods of scanning electron microscopy, FT-IR and N2 adsorption-desorption. The result showed that the specific surface area and the most probable pore size of the obtained MCF were 580.1 m2/g and 18 nm, respectively. Pore volume is 1.16 cm3/g.The optimum conditions for immobilization of laccase on MCF via cross-linking method in the presence of glutaraldehyde were found as follows:the immobilization time of 2 hours and the ratio of enzyme (5.28 U/ml) to MCF of 4 ml/0.02 g with 50μl glutaraldehyde under pH 4 at 30℃. The activity recovery of the immobilized laccase is more than 50%.The characters of the immobilized laccase were also studied and compared with free laccase. The result showed that the optimal pH and reaction temperature of the immobilized laccase were in a wider region than the free laccase, respectively. The pH and thermal stability of enzyme were improved after immobilization. The immobilized laccase remained more than 80% of its initial activity after incubation at 45℃for 2.5 h, and there was only 45% initial activity left for the free laccase under the same conditions. After incubation at pH 4～6 for 3 h, there was still more than 90% of its initial activity left for the immobilized laccase, while the free laccase lost about 40% of its initial activity.The immobilized laccase had a higher Km value than its free counterpart, which indicated a lower affinity for the substrate (ABTS) after immobilization. After 10 cycles of oxidation of a model substrate, immobilized laccase still maintained more than 20% of its initial activity. The operational stability of immobilized laccase proved that it can be recovered and reused. In the dye decolorization test, the maximum decolorization of 97% and 73% were achieved after 7 h in the presence of ABTS for Indigo Blue and Alizarin Red, respectively.