| Summary: | 碩士 === 元智大學 === 化學工程與材料科學學系 === 102 === Chitin, the abundant polysaccharide and main solid waste in the seafood processing industry, is mainly obtained from crustaceans now. Its oligosaccharides are reported as immunoeffector and tumoricidal agents. Among the oligosaccharides, hexamer is the much potent. ASChi61 with the molecular weight of 61,202, the chitinases producing the hexameric oligochitinosaccharide from the bacteria has been obtained from the soil of mangroves marsh in south Taiwan. The enzyme was identified with activity proteomics techniques and expressed before. It is SDS-resistant chitinase though most of the chitinases are SDS-sensitive. The chitinase activity of ASCHI61 is unaffected in 2-mercaptoethanol additionally. The actual molecular weight was higher greater than that estimated by SDS-PAGE. Those These properties showed that ASCHI61 is stable for resisting against environmental factors and can be applied in industries.
In recent years, numerous recombinant proteins used for industrial, agricultural or biomedical applications have increased dramatically. Mass production of these proteins has a remarkable demand in the market. Escherichia colioffers a means for the rapid and economical production of these proteins. These advantages, coupled with a wealth of biochemical and genetic knowledge, have enabled the production of such economically application proteins such as insulin and bovine growth hormone. These demands have driven the development of a variety of strategies for achieving high level protein expression and low expenditure. Enzymes reaction economics can be improved by enzyme reuse and in enzyme stability afforded by immobilization. Immobilization of proteins to solid supports is advantageous for wide applications. The capacity to retain or recover enzymes also allows biocatalyst separation from product. Therefore continuous processes can be performed. In addition, prevents subsequent process steps also can prevent from carry-through of protein.
In this wok, ASCHI61 was expressed and immobilized with chitosan beads ad CNBr-activated Sepharose. The enzyme performances under optimal process reaction conditions were investigated. In immobilization with chitosan beads, colloidal chitin concentration and pH in the response surface experimental design, optimized conditions came out with 1.3 mg/ml, pH 6.8. And the optimal conditions for ASCHI61 immobilized with CNBr-activated Sepharose werecolloidal chitin concentration of 3.42 mg/ml and reaction duration of 40 min. Both optimization conditions of immobilization were validated. The yields of hexaoligochitin from immobilization were less than that obtained from enzyme suspension. However, the yield can be improved apparently and the cost can be cut down with enzyme immobilization in continuous batch.
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