| Alpha amylase widely exists in animals, plants and microorganisms, it can cut off the alpha-1,4glucoside bond in starch, glycogen, oligosaccharide or saccharan at random to produce maltose, oligosaccharide and glucose. Alpha amylase is widely used in food processing, food industry, alcohol industry, fermentation industry, textile industry and other industries, so it is one of the most widely applied enzymes in industrial production. However, the advanced structure of enzyme is very sensitive to the environment, all kinds of physical factors, chemical factors and biological factors can make the loss of enzyme activity. On the other hand, the enzyme used in the reaction can be used only once, and the separation and purification of the products are very difficult. The cost of production is really high. For modern industry, pure enzyme is not an ideal catalyst. Immobilization technology is a research hotpot to overcome the limitations of alpha amylase in enzyme engineering field.In this paper, we selected non-toxic and inexpensive sodium alginate as the carrier and glutaraldehyde as the cross linking agent. Alpha amylase was immobilized by the embedding-crosslinking method. We took the immobilization efficiency and enzyme activity as the index to determine the optimal conditions. Besides, we compared the enzyme properties of the immobilized and free alpha amylase. We prepared the immobilized enzyme with porous structure by using polyethylene glycol as the porogenic agent. The immobilizing method used in this experiment was inexpensive and easy to handle, it could provide feasibility method and theoretical basis for industrial production.Immobilized alpha amylase was prepared using the embedding-crosslinking method. Through the single factor experiment and the orthogonal experiment, the optimum immobilization condition was:the concentration of sodium alginate was1.6%, the concentration of alpha amylase and CMC were found to be0.4%and0.2%respectively, the concentration of CaCl2was2%, the glutaraldehyde concentration was0.4%, the immobilization time was found to be0.5h, and the immobilization efficiency was92.43%. We improved enzyme activity by38%by putting2.5%PEG4000in the optimized system.In the comparison of enzyme properties, we found the optimum reacting temperature and the Km of the immobilized and free alpha amylase were different. The optimum reacting pH was6.0and the optimum reacting temperature was60℃for the free alpha amylase while the optimum reacting temperature of the immobilized alpha amylase was found to be65℃, the optimum reacting pH was the same as the free alpha amylase. The adaptation scope of the immobilized alpha amylase was more wide than the free enzyme. The Km of immobilized enzyme without polyethylene glycol was6.1mg/mL, the Km of immobilized enzyme with polyethylene glycol was3.7mg/mL, and the Km of free enzyme was2.8mg/mL. Thus, after adding a certain amount of polyethylene glycol into the system, the affinity of immobilized enzyme on the substrate has increased, but it was still less than the affinity on the substrate of free enzyme. After being used6times, the relative enzyme activity could remain around71%.We joined different metal ions with the concentration of0.01mol/L into the system to find that common monovalent ions such as potassium ion, ammonium ion and sodium ion had a very little effect on enzyme activity, and in other ions, calcium ion was activated, while iron and manganese ions had inhibitory effect. |
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