Laccase as a natural green catalyst has high catalytic efficiency,broad substrate specificity,fewer requirements for cofactors and specific environmental conditions,nontoxicity,etc.,and thus plays a powerful role in a vast of industrial areas:biopulping and papermaking,biosynthesis,enhancement of fiber properties,food processing,biosensor manufacturing,and bioconversion and refinement of agricultural and forestry wastes,especially biodegradation and bioremediation of environmental contaminants. However,the use of free laccase for practical applications is still limited due to its low stability and high production costs. Immobilization can overcome some of the aforementioned limitations by improving the thermostability of the enzyme and its resistance to extreme conditions and chemical agents. Additionally,the immobilized laccase may be easily separated from its reaction products,allowing the enzymes to be employed in continuous bioreactor operations. In this study,a laccase Tolacc-T secreted by a white rot fungal strain Trametes orientalis with high laccase-producing capacity screened previously,was purified and immobilized onto chitosan carrier with crosslinker glutaraldehyde,named Tolacc-T@Chit@GA. The optimal conditions for acquiring Tolacc-T@Chit@GA were 0.7%（V/V）glutaraldehyde concentration,4 h crosslinking time,6.0 mL enzyme amount,and 6 h immobilized time. The pH adaptability,resistance to thermal denaturation,and storage stability of Tolacc-T@Chit@GA were apparently enhanced compared with Tolacc-T. The operational stability and durability during multiple reuses also demonstrated superiority;the relative activity of Tolacc-T@Chit@GA still remained over 80% after 7 cycles of continuous use. Additionally,Tolacc-T@Chit@GA was capable of decolorizing multifarious dyes,in particularly metal-complex dye naphthol green B,whose partially metabolic by-products were identified as 1-naphthylamine,2-naphthol,1-amino-2-naphthol,1-nitroso-2-naphthol,and 1-nitroso-2-naphthol-6-sulfonate through GC-MS detection. These findings suggest that the immobilized laccase Tolacc-T@Chit@GA from T. orientalis presents better stability and reusability and therefore could be used in many applications.
