人類血漿幾丁質酶的分析與鑑別

THE DISTINCTION AND ANALYSIS OF CHITINASES FROM HUMAN PLASMA

徐偉琮(Wei-tsung Hsu)

指導教授 ： 林銘澤 ； 廖純沂

大同大學/工程學院/生物工程學系所/碩士(2006年)

摘要

幾丁質水解酵素廣泛存在於自然界各物種之間，此類酵素可以水解自然界中含量第二豐富的多醣聚合物—幾丁質，屬於醣解酵素的成員之ㄧ。幾丁質水解酵素在特定生物體內如節肢動物及魚類，分別扮演執行組織上的新陳代謝與消化含有幾丁質食物的必要酵素；然而，在人類體內也存在多種的幾丁質水解酵素及類似幾丁質酶的蛋白質，此類酵素及蛋白質的生理功能大多仍然無法有明確的定義。
本研究利用兩種不同的液體層析分離法試圖鑑別人類血漿中之幾丁質水解酵素及相關蛋白質。第一種層析分離方式是以Blue Sepharose 6親和性液體層析管柱與DEAE Sepharose液體層析管柱進行人類血漿中的蛋白質的部份分離，搭配西方墨點法鑑別出一個分子量約為150 kDa且可與Bacillus菌屬chitinase A1的chitin binding domain片段所產生的抗體結合。第二種層析分離方式則是利用幾丁質親和性液體層析管柱與DEAE Sepharose液體層析管柱對人類血漿中的蛋白質進行部份分離，再以SDS-聚丙烯胺電泳及活性染色等方法鑑別出一個分子量約為30 kDa且具有水解glycol chitin能力的幾丁質水解酵素。

關鍵字

幾丁質酶 ； 人類血漿

並列摘要

Chitinolytic enzymes exist in various species in nature. As a glycosyl hydrolases, they can catalyze the hydrolysis of chitin, the second most abundant polysaccharide on earth. Chitinolytic enzymes are necessary in the development of metabolism for arthropoda and fish. In human, there are many kinds of chitinolytic enzymes and chitinase-like proteins. Their physiological functions are yet appreciated.
In this study, chitinase and related proteins from human plasma were identified using liquid chromatography. In one method, Blue Sepharose 6 affinity liquid chromatography and DEAE Sepharose ion exchange liquid chromatography were used to separate proteins from human plasma. A protein with molecular weight of 150 kDa, could be identified by western blot using ChBD antiserum. In other way, chitin affinity liquid chromatography and DEAE Sepharose ion exchange liquid chromatography were used to separate proteins from human plasma. A protein with molecular weight of 30 kDa was identified with ability to catalyze the hydrolysis of glycol chitin by in-situ activity staining on SDS-PAGE.

並列關鍵字

human plasma ； chitinases

參考文獻

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