Studies On The Immobilization Of Pectinase By Non-magnetic And Magnetic Ion-exchange Resin

Pectinase is a complex group of enzymes that degrade various pectic substances. Pectin, the major component in the middle lamella and primary cell wall of higher plants, has a great impact on the viscosity and turbidity of fruit juices. Pectinase is mainly used for increasing the yield and clarification of fruit juices. Therefore, the use of pectinase in the production of fruit juices is very common.Due to the relatively high cost of pectinase used in fruit juice processing it is often advantageous to have a system in which the enzyme can be recovered or utilized repeatedly. A common method of achieving this aim is to immobilize the enzyme on a solid support. Immobilization technology of pectinase is an effective measure to achieve utilizing repeatedly and improve stability.Many organic and inorganic substances have been used as the support materials. Ion exchange resin is one of the most widely used materials in industry. It has the broad application prospect as the carrier of immobilization for its low price, good mechanical properties, and stable physical performance. Recently, magnetic particles receive considerable attention because of their widely use in biomedicine, cytology, bioengineering and so on. Compared with non-magnetic carriers, enzyme immobilized by magnetic carriers can be more easily recovered and separated from the reaction system.In this article, immobilization of pectinase by non-magnetic and magnetic ion exchange resin was studied. The optimal conditions of immobilization, characteristics of non-magnetic and magnetic immobilized enzyme were mainly investigated in this thesis. Moreover, the external configuration of immobilized enzymes was characterized by scanning electron microscopy (SEM). The main experimental results are as follows:(1) Through primarily research on ten kind of absorption resin and ion exchange resin used widely in industry, we find the best support for the immobilization of pectinase is the anion-exchange resin D380.(2) Pectinase was immobilized on anion-exchange resin D380 using glutaraldehyde as cross-linking reagent. Effect of temperature, pH, time, amount of the enzyme, concentration of glutaraldehyde, cross-linking temperature and time on the activity recovery of immobilized enzyme was mainly investigated in this thesis. The optimumconditions for the immobilization were as follows: 40°C, PH5.5, 6h, 0.75 mL pectinase. The concentration of glutaraldehyde was 0.1%, cross-linking temperature and time were 4°C and 4h respectively. Under the optimal conditions, the activity recovery of immobilized pectinase can reach to more than 81.67%.(3) Compared with free pectinase, the characteristics of immobilized pectinase were as follows: the optimum working temperature was 60"C whereas the free enzyme was 50 °C;the optimum working pH was 4.0 whereas the free enzyme was 3.5. From the data illustrated above, we can draw the conclusion that the thermal stability of the immobilization enzyme has obviously improved. Moreover, the immobilized pectinase had a widely pH stability scope. The operation stability and storage stability are very important targets to evaluate whether the immobilization enzyme has practicability and the commercialization. It was seen that the immobilization enzyme had good operation stability. It can still keep high activity (50%) after ten times operations. Further more, the storage stability of the immobilization enzyme improved obviously than the free enzyme when stored at 4°C.(4) Pectinase was immobilized on magnetic resin using glutaraldehyde as cross-linking reagent. Temperature of the immobilization medium, pH, immobilization time, amount of the enzyme, concentration of glutaraldehyde, cross-linking temperature and time were investigated as immobilization parameters in this thesis. The optimum conditions for the immobilization were as follows: 30'C, PH5.5, 6h, 0.5 mL pectinase. The concentration of glutaraldehyde was 0.01%, cross-linking temperature and time were 10°C and 2h respectively. Under the optimal conditions, the activity recovery of immobilized pectinase can reach to more than 137.5%.(5) Compared with free pectinase, the characteristics of magnetic immobilized pectinase were as follows: the optimum working temperature was 70 °C whereas the free enzyme was 50 °C;the optimum working pH was 4.5 whereas the free enzyme was 3.5. In this research, we can concluded that the thermal stability (between 30~60°C) of the immobilization enzyme has obviously improved. Moreover, the pH stability of the immobilized pectinase improved when the value of pH is between 3.5~4.5. The operation stability is not as good as the pectinase immobilized on non-magnetic resin. It was seen that the immobilization enzyme had a good operation stability. It can still keep high activity (61.11%) after six times operations. Further more, the storage stability of the immobilization enzyme improved obviously than the free enzyme when stored at 4°C.