| Chitosan and cellulase are similar in structure, so cellulase can hydrolyze chitosan. A magnetic field three-phrase fluidized bed can be introduced in high viscosity reaction system, so that the disadvatage that low mass transfer coefficient and poor reaction efficiency in traditional liquid-solid fluidized bed in batch operation can be improved. The reactor not only can realize continuous production, but also is convenient for the recovery of enzyme. In this study, starch with high biocompatibility is chosen as the shell material of magnetic polymer microspheres, and Fe3O4 with high magnetic responsiveness as core material. Immobilized magnetic microspheres are prepared by immobilizing cellulase with magnetic starch microspheres through physical adsorbtion, covalent bonding and cross linking, and they are easily seperated and recovered in external magnetic field.The optimum reaction condition of free-cellulase hydrolysis was determined. The concentration of reducing sugar was measured by DNS, and the influences of substrate concentration, enzyme dosage and reaction time on the enzymatic reaction were studied. The optimum reaction conditions were as follow: reaction temperature at 55℃with pH 5.8, substrate concentration of 20 mg/mL, cellulase dosage of 100 U/g and reaction time of 4 h. TLC analysis confirmed the production of chito-oligosaccharide.Magnetic fluid was prepared by coprecipitation, and magnetic starch microspheres were prepared through oxidation, coprecipitation and reversed suspension crosslinking. The magnetic starch microspheres prepared by reversed suspension crosslinking showed high magnetic responsiveness and wasn't oxidized. The optimum reaction condition were as follow: the emulsifier concentration of 2%, crosslinking temperature at 40℃, 0.375 g Fe3O4 and 0.3 mL epichlorohydrin for one gram of starch, and reaction time of 6 h.Cellulase was immobilized with magnetic starch microspheres through physical adsorbtion-crosslinking, crosslinking with EDC and covalent bonding with dialdehyde starch, respectively. Immobilized cellulase prepared by the last method showed higher enzyme activity yield. The optimum condition of immobilization was investigated, which was 10μl cellulase immobilized with 20 mg magnetic starch microspheres at 10℃with pH 6.5, for 20 h.Study on characterization of magnetically immobilized cellulase showed that the thermal stability and pH stability were better than that of free catlase. The enzyme activity recovery rate was 45% after using for three times, and the storage stability was good.As showed in the IR spectrum, the peak at 1729 cm-1 indicated the aldehyde group and Fe-O peak at 405 cm-1 which showed the existence of Fe3O4. The magnetic microspheres exhibited high magnetic responsiveness in external magnetic field.
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