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研究生: 張紘綸
論文名稱: 建立海藻糖合成酶之高效率純化暨活性篩選系統
指導教授: 李冠群
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2010
畢業學年度: 98
語文別: 中文
論文頁數: 95
中文關鍵詞: 海藻糖海藻糖合成酶
論文種類: 學術論文
相關次數: 點閱:120下載:7
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  • 海藻糖(trehalose)為兩個葡萄糖以α1-1鍵結形成的非還原性雙糖,存在於許多生物體當中,可以作為能量的儲存形式,在脫水等逆境時穩定蛋白質和生物膜的結構,保護細胞免於環境壓力。在食品、化妝、醫藥業上都有廣泛的應用。海藻糖合成酶(trehalose synthase、TreS)為催化麥芽糖分子內鍵結轉化成海藻糖的酵素,並產生副產物葡萄糖,為海藻糖的生合成路徑之一。因為是單一酵素反應,在應用上易於控制流程,且原物料便宜與穩定,在生產海藻糖上有很大的潛力。傳統海藻糖合成酶活性測定的方式有HPLC與海藻糖水解酶(trehalase)呈色法,但這兩種方式有費時、費用昂貴與測定標的物專一性等問題,無法快速大量分析不同活性之TreS。本實驗建立了一套新的微量盤式麥芽糖水解酶(maltase)呈色法來進行大量且快速的活性分析。本法在TreS活性測定上更有效率,準確度高且不會有專一性不足的問題,適用於大量TreS樣品的分析。未來在TreS的蛋白質工程上,如定向演化上可以對大量的突變TreS進行活性篩選,來挑選出高轉化率或是高熱穩定性的突變TreS。

    Trehalose (α-D-glucopyranosyl-1, 1-α-D-glucopyranoside), a non-reducing disaccharide, existing in various organisms can serve as energy storage, and as protectants of protein and lipid from various stress such as cryoprotectants. It has become as an important compound in foods, cosmetics and pharmaceuticals industrials. Trehalose synthase (TreS) is one of the biosynthesis pathways of trehalose, which reversibly catalyzes the intramolecular transglucosylation (isomerization) of maltose to produce trehalose, as well as the minor side-reaction of irreversible hydrolysis of maltose to make glucose. Because maltose is an inexpensive and readily available sugar, it has great potential to produce trehalose from maltose by utilizing the one-step isomerization reaction of TreS. A number of methods have been currently available to measure TreS activity, including HPLC and trehalase-coupled TreS assay. However, these methods are either time-consuming, expensive or with low substrate specificitied, and this makes them not suitable for characterizing TreS in a high-throughput manner. This proposal established a novel 96-well format assay, maltase-coupled TreS assay to colorimetrically determine the isomerization activity of TreS. We also establish a 96-well format protein purified system for high-throughput TreS purification. With these new colorimetric method and purification method, we can perform the biochemical characterization more efficiently, especially, in the directed enzyme evolution of TreS for screening mutant TreS with improved properties, such as high trehalose yield or high thermostability.

    目錄 I 表目錄 VII 圖目錄 VIII 附錄目錄 X 摘要 XI Abstract XII 壹、緒論 1 貳、研究目的 8 参、研究材料與方法 9 一、重組質體建構 9 1. 建構Deinococcus radiodurance trehalose synthase gene重組質體 9 2. 建構Rice trehalase gene重組質體 9 3. 建構Human trehalase gene重組質體 9 4. 建構隨機突變trehalose synthase gene質體 11 a. Error-prone Polymerase Chain Reaction 11 b. 建構隨機突變Deinococcus radiodurance trehalose synthase gene質體 12 5. 質體取得 16 6. DNA定序分析 16 二、重組蛋白的表達與純化 16 1. 質體轉型 16 a. 勝任細胞的製備 17 b. Escherichia coli轉型作用 17 2. 蛋白質的表達 18 a. Deinococcus radiodurance trehalose synthase的表達 18 b. Rice trehalase的表達 18 c. Human trehalase的表達 19 d. 96孔盤型式蛋白質的表達 19 3. 蛋白質的純化 20 a. Fast Protein Liquid chromatography(FPLC) 20 b. 蛋白質透析 20 4. 高效能96孔盤型式蛋白質的純化與透析 21 a. 高效能96孔盤型式蛋白質的純化 21 b. 高效能96孔盤型式蛋白質去鹽 22 c. PD MultiTrap G-25回收再利用 22 5. 不可溶蛋白質重新摺疊 23 三、蛋白質分析 23 1. 電泳 23 a.蛋白質電泳試劑 23 b. SDS-聚丙醯胺膠體溶液調配法 24 c. 膠體的製備 25 d. 5X SDS sample buffer製備與電泳條件 25 e. 電泳膠體的染色 26 2. 蛋白質的定量 26 3. 酵素活性測定 27 a. Trehalase受質專一性測定 27 b. Maltase受質專一性測定 29 c. Trehalose synthase活性測定-Maltase-coupled trehalose synthase assay 29 d. EDTA與Imidazole對Maltase-coupled trehalose synthase assay的影響 30 e. 固定化Deinococcus radiodurance trehalose synthase活性測定 31 f. High-performance liquid chromatography(HPLC) 31 肆、結果 33 一、高效能96孔盤型式蛋白質純化系統的建立 33 1. 蛋白質粗萃取液對trehalose synthase活性測定的影響 33 2. 高效能96孔盤式蛋白質純化效果的最適化 33 3. Imidazole與EDTA對maltase-coupled trehalose synthase assay的影響 34 4. 高效能96孔盤型式蛋白質去鹽 34 二、Trehalose synthase高效能96孔盤型式活性測定系統的建立 35 1. Trehalase專一性對trehalose synthase活性測定的影響 35 2. Maltase專一性對trehalose synthase活性測定的影響 35 3. Maltase使用量與反應時間 36 4. Maltase-coupled trehalose synthase assay準確性的驗證 36 5. 高專一性 trehalase的取得與trehalase-coupled trehalose synthase assay的可行性分析 37 三、結合高效能96孔盤型式蛋白質純化與活性測定進行trehalose synthase活性分析 38 1. 96孔盤誘導與純化的產量評估 38 2. Trehalose synthase反應時間的確立 39 3. Trehalose synthase固定化對活性的影響 39 四、建構隨機突變之trehalose synthase gene質體 39 1. Error-prone PCR 40 a. Megaprimer PCR 40 b. 限制酵素切割與DNA接合 41 c. Escherichia coli in vivo recombination 41 2. Error-prone rolling circle amplification 41 伍、討論 43 一、高效能96孔盤型式蛋白質純化系統的建立 43 1. 蛋白質粗萃取液對trehalose synthase活性測定的影響 43 2. 高效能96孔盤型式蛋白質純化的最適化 43 3. EDTA與Imidazole對Maltase-couple trehalose synthase assay的影響 44 4. 高效能96孔盤型式的蛋白質去鹽 45 5. PD MultiTrap G-25回收再利用 46 二、Deinococcus radiodurance trehalose synthase高效能96孔盤型式活性測定系統的建立 47 1. Trehalase-coupled trehalose synthase assay 47 2. Maltase-coupled trehalose synthase assay 48 三、高效能96孔盤型式蛋白質純化與活性測定進行 TRES活性分析 50 1. Trehalose synthase反應時間的確立 50 2. Trehalose synthase固定化對活性的影響 50 四、建構隨機突變Trehalose synthase gene之質體 51 1. Error-prone PCR 51 a. Megaprimer PCR 52 b. 限制酵素切割與DNA接合 53 c. E. coli in vivo recombination 54 2. Error-prone rolling circle amplification 55 陸、參考文獻 57

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