Preparation Of Magnetic Chitosan Microspheres And Application On Laccase Immobilization

Fiber optic biosensing technology has a wide application prospect in medical science, biological engineering, food industries and environment monitoring. The fiber optic biosensor based on oxygen consumption in relation to analyte oxidation of laccase can be applied on clinical medicine examination and disease diagnosis. The biosensing material is the main factor to influence the delicacy, precision and testing scope of the fiber optic biosensor.In this paper, we have done a great deal of experiments on the preparation of immobilized laccase which can be expected to improve the performance of the fiber optic biosensor based on oxygen consumption in relation to analyte oxidation of laccase. Our main works are:1 Magnetic chitosan microspheres were prepared with reversed-phase suspension methodology .Cross-linking and hardening of the spherical particles were achieved by the addition of glutaraldehyde. The testing technologies, such as SEM FTIR, were used to investigate the morphology and structure of the microspheres. The results indicated that the microspheres had well shaped spherical form with smooth surface and Magnetic Fe₃O₄ nanoparticles were packed in the microspheres to form core-shell structure. Its mean particle size was 0.9 U m with a narrow size distribution.2 Laccase was immobilized on magnetic chitosan microspheres by adsorption and cross-linking with glutaraldehyde. The effects of the immobilization conditions on laccase immobilization were studied. The optimal immobilization conditions for laccase were: 10ml of 0.8mg/ml of laccase in phosphate buffer(0.1mol/L,pH = 7.0) reacted with 50 mg of magnetic chitosan microspheres at 25 ℃ for lh and subsequently was kept at 4 ℃ for 2h. Under the immobilization conditions, 24 % of yields of protein coupling was obtained.3 The properties of the immobilized enzyme were investigated. The optimal temperature for immobilized enzyme was 10℃ and 55℃.The immobilized enzyme exhibited the maximal activity at pH 3.0. The Km value of immobilized laccase was 171.1 M, higher than that of free laccase(36.8uM), which means that the immobilized laccase had lower affinity towards the substrate. Nevertheless, the immobilized laccase exhibited remarkably improved stability properties. After the immobilized enzyme was kept at a temperature of 60℃ for 210 min, it still had 74% of initial enzyme activity, while the activity of free enzyme could only remain 19.4% under the same conditions. After 10 cycles of operation, the activity of immobilized enzyme remained over 80% of its initial activity. Therefore, the catalytic efficiency and storage stability of laccase were greatly improved after its immobilization on magnetic chitosan microspheres. In addition, immobilization improved the stability of laccase in the acidic region.