摘 要 本研究以酪胺酸酵素改質幾丁聚醣，賦予幾丁聚醣新的官能基，或直接將酪胺酸酵素與幾丁聚醣複合成固定化酪胺酸酵素，探討此諸經酪胺酸酵素修飾之幾丁聚醣在化工程序中的應用。利用酪胺酸酵素之催化能力將3，4二羥基苯甲酸、3，4二羥基苯乙酸及3，4二羥基苯丙酸分別接枝於幾丁聚醣之胺基上，而後探討此三種羧基改質幾丁聚醣對於水中所含鹼性染料crystal violet及bismarck brown Y之吸附移除效率，結果發現以3，4二羥基苯丙酸改質之幾丁聚醣吸附移除效率最佳。在酪胺酸酵素去除水中所含酚之反應中，發現未經改質之幾丁聚醣，亦會吸附蛋白質；經羧基改質之幾丁聚醣可作為酪胺酸酵素之親合吸附劑，其吸附行為遵循Langmuir 等溫吸付及二次動力吸附模式，其中以3，4二羥基苯甲酸改質之幾丁聚醣對酪胺酸酵素溶液中蛋白質之吸附效率最佳。以epichlorohydrin、ethylene glycol diglycidyl ether、及gluteraldehyde為交聯劑，分別將酪胺酸酵素化學鍵結於幾丁聚醣，用於將酪胺酸轉化為多巴，固定化後酪胺酸酵素之溫度穩定性明顯提高，幾丁聚醣上殘留之胺基經遮蔽後，並不會提昇固定化酪胺酸酵素之溫度穩定性，但有助於生產多巴時之操作穩定性。以凝膠包埋、三明治式包埋或化學鍵結之方式，將幾丁聚醣/酪胺酸酵素固著於玻璃石墨電極之表面，製作出可用以偵測出水中酚類化合物濃度之生物電極，凝膠包埋方式可以得到最大應答電流，使用交聯劑增加了偵測穩定性但降低了靈敏度以及延長了應答時間；以epichlorohydrin為交聯劑之凝膠包埋生物電極總體表現最佳。 Abstract In this study chitosan was modified to posses carboxyl group via the enzymatical grafting reaction of tyrosinase, or was used to immobilize tyrosinase. The purpose of this thesis was to find applications of these fabricated chitosans using in chemical process. In chapter three, 3,4-dihydroxybenzoic acid, 3,4- dihydroxyphenylacetic acid, and hydrocaffeic acid were used individually as substrates of tyrosinase to graft onto chitosan. The grafting amounts of these phenol derivatives onto chitosan were examined and the modified chitosan were used in experiments on uptake of the cationic dyes crystal violet and bismarck brown Y by a batch adsorption technique. In chapter four, tyrosinase was used to convert phenol to polyqui then polyquinone was removed by chitosan beads and tyrosinase was adsorbed by those carboxylly modified chitosan beads. The thermodynamic and kinetic models of tyrosinase adsorbed by modified chitosan were investigated. In chapter five, Chitosan was activated with glutaraldehyde, epichlorohydrin and ethylene glycol diglycidyl ether respectively in order to immobilize tyrosinase for the production of L-dopa from L-tyrosine. The effects of coupling agents and amine capping agents on the operation stability of immobilized tyrosinase were studied. A practical route to immobilize tyrosinase on chitosan for producing L-dopa from L-tyrosine was found. Finally in chapter six, chitosan was used to prepared tyrosinase-based biosensor on glassy carbon electrode for detecting the concentration of phenols. The effect of immobilizing methods, including chitosan gel mixed with enzyme, sandwich entrapped enzyme between two pieces of chitosan films, and covalently bonded enzyme on chitosan films, upon the apparent response of biosensor were studied. A highly stable biosensor was fabricated.
