- 學位論文
以幾丁質球為載體進行固定化酵素之開發
Chitin-binding domain based immobilization of γ-PGA depolymerase
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摘要
γ -聚麩胺酸（poly γ-glutamic acid, γ-PGA）為一具極高水溶性，生物可分解性及可食用之生物天然高分子，且該物質本身及其分解物對人體與環境皆無害處。再者γ-PGA之分子量為影響其物性之重要因素，因此如何製備不同分子量之γ-PGA並應用於食品、化妝品、醫藥、農藥等領域將有很大的潛力。為了擴展γ-PGA的運用性及增加聚麩胺酸分解酵素於工業上的使用性，本研究利用基因重組大腸桿菌進行固定化聚麩胺酸分解酵素之開發。運用幾丁質鍵結區域（chitin-binding domain; ChBD）來發展酵素固定化系統，將可產生聚麩胺酸分解酵素（γ-PGA depolymerase）之基因片段ywtD與ChBD做結合，透過幾丁質球（chitin bead）與chitin-binding domain之間專一的親和性質，有效的將目標酵素以共價鍵結的方式固定於幾丁質球的表面。 實驗結果顯示，固定化聚麩胺酸分解酵素具有分解γ-PGA之能力，每克的幾丁質球於4小時內約可吸附12.4 mg 酵素，相較於自由態酵素，固定化酵素對於pH值和溫度有較高耐受度，於pH 7、30oC下有最佳γ-PGA降解能力。此外，於酵素穩定性測試中，固定化酵素於30oC環境下48小時仍保持50%以上的活性；而保存性實驗中，固定化酵素於4oC環境下保存9天仍維持50%的活性；固定化酵素能重覆使用6次且活性仍有50%以上，由此可顯示，固定化聚麩胺酸分解酵素對於未來工業應用推展具有開發潛力。
並列摘要
Poly-?-glutamic acid (?-PGA) is a biopolymer produced by microorganism in which D- and L- glutamate is polymerized via??-amide linkages. A large range of its applications has been suggested due to its characteristics, such as biodegradable, edible, nontoxic toward human or the environment. The molecular weight of ?-PGA plays an important role which effects its application. The objective of this study is to expand the application of??-PGA and increase the utilization of ??-PGA depolymerase in the industry by enhancing the enzyme activity and stability. We used recombinant E. coli to express fusion protein (chitin binding domain and γ-PGA depolymerase) and then direct adsorption on chitin beads as the immobilized enzyme. It was found that one gram of chitin beads could adsorb 12.4 mg of enzyme for 4h. The Immobilized??-PGA depolymerase exhibited higher temperature and pH value tolerance than the free one. The results indicated that the best condition for r-PGA hydrolysis was pH 7 and 30oC. Furthermore, the immobilized enzyme could store under 30 oC for 48h and maintain over 50% of its activity. The storage time of the immobilized ?-PGA depolymerase is 9 day. And the immobilized enzyme could be reused for 6 times. Those results indicated that the immobilized ?-PGA depolymerase could be applied in the industry.
參考文獻
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