The magnetic chitosan nanoparticles were prepared by reversed-phase suspension method using Span-80 as an emulsifier, glutaraldehyde as cross-linking reagent. And the nanoparticles were characterized by TEM, FT-IR and hysteresis loop. The results show that the nanoparticles are spherical and almost superparamagnetic. The laccase was immobilized on nanoparticles by adsorption and subsequently by cross-linking with glutaraldehyde. The immobilization conditions and characterizations of the immobilized laccase were investigated. The optimal immobilization conditions were as follows: 10 mL of phosphate buffer (0.1 M, pH 7.0) containing 50 mg of magnetic chitosan nanoparticles, 1.0 mg · mL−1 of laccase and 1% (v/v) glutaraldehyde, immobilization temperature of 4°C and immobilization time of 4 h. The immobilized laccase exhibited an appreciable catalytic capability (480 units · g−1 support) and had good storage stability and operation stability. The Km of immobilized and free laccase for ABTS were 140.6 and 31.1 μM in phosphate buffer (0.1 M, pH 3.0) at 37 °C, respectively. The immobilized laccase is a good candidate for the research and development of biosensors based on laccase catalysis.
laccase magnetic chitosan nanoparticles immobilization enzyme activity stability