| As the rapid development of biotechnology, a lot of biological enzyme are consuming every year. Amylase, as an important class of biological catalysts, have a wide range of applications in the pharmaceutical, fine chemical products, sugar-based chemicals, food and feed additives and other fields, its production occupies nearly the entire 50% of total enzyme, and the demand is growing. However, the free amylase are sensitive to the catalytic environment, also easy to inactivation in a variety of physical and chemical factors, and difficult to recovery , the high usage cost , limited its re-use and highly efficient catalytic, so how to make efficient use of amylase become a major research focus. As the emergence of immobilization technologies, re-use and high amylase catalytic of enzyme become the into reality from theory. Immobilized enzyme is preparated through the immobilization technology, compaire to the free enzyme, it shows more advantages: the stability can get greatly improved after the fixed treatment . Based on the above ,this subject study on the amylase immobilization , including the preparation of immobilized good - porous ceramic ball, amylase multi-enzyme system construction, the conditions of immobilization, pHysical and chemical properties, stability of immobilization system , as follows:First, slect fly ash containing calcium slag as raw material, add the glass powder under appropriate proportion, carbon black as pore forming agent, and carboxymethyl cellulose as a binder to prepare porous ceramic ball carrier, and takeα-amylase enzyme as immobilization test object, made a immobilized testing of ceramic ball. The results show that under the ingredients, the fly ash (g): glass powder (g): carbon black (g): carboxymethyl cellulose (g) = 19.7:65:0.3:15, using the ordinary glass powder, sintering temperature under the conditions of 800℃, the porosity of the porous ceramic reach to 57.8%, shows good properties of enzyme immobilization, so it's suitable for enzyme immobilization as new carrier.Secondly, the amylase multi-enzyme system was constructed, including two-enzyme system , three-enzyme system and the four enzyme systems, compare the effect of catalytic hydrolysis of starch, finally determined the following conclusion: The best multi-enzyme system is four-enzyme system ,which was formed by theα-amylase,β-amylase,γ-amylase and isoamylase, and the actixity ratio between them is 1.2:1.2:1.6:1.0. Thirdly, take porous ceramic as carrier, the four-enzyme system as immobilize object,using glutaraldehyde as crosslinking agent, immobilized amylase multi-enzyme system, through the single factor and orthogonal experiments ,research several key factors which affect the recovery rate of immobilized enzyme activity ,taking activity recovery as observed indicators, the results show that a immobilized temperature, immobilized pH, glutaraldehyde concentration, the proportion of carrier and enzyme have a ceatain extent on the activity recovery rate of immobilized enzyme, concentration of glutaraldehyde play the most critical role, followed by the temperature. Study show that the best immobilization conditions : immobilized temperature is 38℃, immobilized pH 4.7, and 2.2% glutaraldehyde concentration, the carrier ratio is 0.8:1 ,under the best conditions of immobilization, activity recovery rate reach 82.67%.Finally, physicochemical properties and stability of immobilized multi-enzyme system were studied, the results showed: immobilized amylase enzymes system optimal temperature is 55℃; optimum catalytic pH 5.4; by double reciprocal mapping method, calculated Vmax = 8.34mmol/l.min, Km= 21.16mg/ml; immobilized amylase enzymes show strong thermal stability under 60℃, while higher than 65℃, the activity loss a lot, showing thermal instability; immobilized amylase enzymes show strong pH stability in the range of 4.5-5.5 ; immobilized enzyme half-life 70d. After 4 times re-use, the activity has been reduced to 73% compaired to the original activity, in the following re-use, the decline trend slow down.
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