| Pectinase is enzymes of decompose pectin of the plant. It is mainly applicated in dustry of fruit juice and wine. But because of the liquid enzyme's large dosage, high cost, difficult refine, short shelf life, the difficulty in recovery, influence its application in food industry. Then immobilized enzyme may make up the defects in certain degree. At the same time, it can also reduce the pollution of exogenous substance on fruit products. But pectin in the plant cell wall doesn't exist solely. It generally exist combinateing with cellulose. Pectinase can't decode compact protective structure of cellulose without the synergism of cellulase, so can't hydrolyze well pectin.At present, immobilization of pectinase has been researched. But study on immobilization of pectinase by magnetic carrier is less. Studies on the Co-immobilization of pectinase and cellulase have rarely been reported. In this article, the pectinase was immobilized on the magnetic chitosan microspheres, and studied kinetics of reaction on immobilized pectinase. It provided a theoretical basis for the design of immobilized enzyme reactor and determination of the operating conditions. Meanwhile, according to the synergistic effect of pectinase and cellulose, preliminary exploration on immobilized bi-enzyme by applying technology of co-immobilization, opened up new fields of application for the two kinds of enzymes.The main results were as follows:1. First nanometer Fe3O4 particle was synthesized by coprecipitation, then magnetic chitosan microspheres were prepared by the suspension cross-linking technique. Finally, the size, morphology, structure, size distribution and magnetic response properties were studied by means of TEM, SEM, FT-IR, MASTERSIZE. The results indicate: the size of magnetic chitosan microspheres were about 50 nano-sizes, distribution was narrow, and the morphology was all sphericity. FT-IR results show the structure of magnetic microspheres and the Fe3O4 particle was coated completely by the chitosan. The spectrophotometry indicate:magnetic microspheres have strong magnetic response properties. It is a kind of superior carrier of immobilization of enzyme. 2. Through the experiment of kinetics of enzyme-catalyzed reaction, results showed that pH, temperature, substrate concentration, product concentration had strong effects to the rate of the enzyme catalysis reaction, and the product inhibition and high concentration substrate inhibition were produced. Km of immobilized pectinase was 2.67, and liquid pectinase was 2.25. So the affinity immobilized pectinase to substrate was reduced. This may be the result of diffusion limitation and steric hindrance of carrier. Inhibition constant of product and substrate were 0.234 and 0.386 respectively, accordingly gained reaction rate equation.3. By the study on allocation effect, outer diffusional limitation, inner diffusional limitation, allocation effect on immobilized enzyme reaction system, kinetics model and were given respectively.4. By the test of co-immobilization of pectinase and cellulase with adsorption method, the optimum conditions of the reaction were as follows: glutaraldehyde concentration 5%, the volume of two enzymes ratio 3:2, adsorption time 6h. Through the comparison of free pectinase and cellulose with co-immobilized two enzymes, the result of experiment on the property of enzyme were as follows: their optimal temperature is rise, optimal pH is low, but range wide, thermal stability were all enhanced, half life time of pectinase and cellulose were 45d and 32d respectively. The results had industrial application value.
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