Study On Immobilization Of Laccase On Mesoporous Molecular Sieve SBA-15

Enzyme immobilization is an effective approach to enhance the stability of enzymes. Mesoporous materials are ideal carriers to be used for enzyme immobilization due to their nanostructures,and have been actively developed to immobilize enzymes in the recent years. In this work,mesoporous material SBA-15 was prepared as support to immobilize laccase,an oxidase possessing important industrial applications.The mesoporous material SBA-15 was synthesized by a method of hydrothermal crystallization,using a triblockcopolymer EO₂₀PO₇₀EO₂₀(P123) as template and tetraethyl orthosilicate(TEOS) as the silicon source. The obtained SBA-15 was characterized by methods of X-ray diffraction(XRD),and N₂ adsorption-desorption.The result showed that the prepared sample possessed a typical mesoporous structure,with an average pore diameter of 4.835 nm and surface area of 647.2m²/g.Laccase was immobilized to SBA-15 via covalent and glutaraldehyde crosslink method.The optimal immobilization process was conducted as follows:0.03g SBA-15 was mixed with 3.2mL approximately diluted laccase,which concentration was 1.2×10^-4,and 66.56μl of glutaraldehyde,and then the immobilization system was reacted for 7h at pH4.8. The maximal activity recovery of the immobilized laccase was 43.70%.The optimum reaction temperature of the free enzyme and immobilized enzyme were 50℃and 45℃,respectively. The free laccase had maximal activity at pH3.2,while the immobilized enzyme demonstrated an optimum pH of 3.0.Enzyme stability,including thermal,pH and operational stability,were improved after immobilization.There was nearly no activity loss of the immobilized laccase after incubation at pH 4.0～5.0 for 3h,while the free laccase lost more than 15%of its activity. The immobilized enzyme remained 37%of its initial activity after incubation at 65℃for 2h, and there was little activity left for the free laccase under the same conditions.Moreover,the immobilized laccase retained more than 50%of its initial activity after nine batch uses.The immobilized laccase was used to decolorize indigo carmine to assess its potential in dye decolorization.A high decolorization extent was observed for the immobilized laccase in the presence of ABTS.About 94.44%of indigo carmine was decolorized after 1h by the immobilized laccase plus ABTS.However,the decolorization percentage was only 17.07%in the absence of ABTS,indicating that ABTS was an effective redox mediator for the immobilized laccase.