目錄
摘要 I
Abstract II
目錄 III
表目錄 V
圖目錄 VI
壹、 研究背景與目的 1
一、 研究背景 1
二、 研究目的 1
貳、 文獻整理 2
一、 稀有醣類 rare sugars 2
1. 簡介 2
2. 松二糖 2
二、 澱粉蔗糖酶 3
1. 簡介 3
2. 不同來源之AS 4
3. Deinococcus indicus DSM 15307之AS 7
4. GH 13 家族之 AS 晶體結構 8
三、 提升熱穩定性方法 9
1. 氫鍵 hydrogen bonds 9
2. 雙硫鍵 disulfide bond 10
3. 蛋白質表面增加精胺酸 (arginine, Arg, R) 10
4. 減少甘胺酸 (glycine, Gly, G)或增加脯胺酸 (proline, Pro, P) 11
四、 電腦輔助軟體簡介 11
1. SWISS-MODEL 11
2. GETAREA 11
3. PyMOL 12
4. Enzfitter 12
參、 實驗設計與流程 13
一、 以軟體評估突變之可能性 13
二、 重組 Di-AS 之突變與特性分析 13
肆、 實驗材料及方法 14
一、 實驗材料 14
1. 菌株與載體 14
2. 抗生素 14
3. 標準品 14
4. 市售套組 14
5. 酵素 15
6. 化學藥品 15
7. 實驗設備 17
8. 軟體 18
二、 實驗方法 18
1. 選擇突變點 18
2. 定位突變 18
3. 質體轉形至表現宿主 23
4. 突變型 Di-AS之表現與純化 24
5. 突變型 Di-AS之活性分析與特性探討 28
伍、 結果與討論 31
一、 突變點選擇及引子設計並進行定位突變 31
二、 突變型酵素之粗酵素液活性及熱穩定性 31
三、 探討純化突變型酵素特性 32
1. 大量表現與純化 32
2. 熱穩定性 32
3. 酵素動力學參數 33
4. 最適作用溫度 34
5. 最適作用 pH 值 34
陸、 結論 35
柒、 參考文獻 36
捌、 圖表 41

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