臺灣博碩士論文加值系統

壹、 研究背景與目的 1
一、 研究背景 1
二、 研究目的 2
貳、 文獻整理 3
一、 稀有醣類 (rare sugars) 3
1. 稀有醣類簡介 3
2. 核糖 3
二、 生產 L-核糖的相關酵素 3
1. L-阿拉伯糖異構酶 3
2. 甘露糖-6-磷酸異構酶 4
3. L-核糖異構酶 4
三、 不同來源之 L-RI 的特性 4
1. Acinetobacter sp. DL-28來源之 L-RI 4
2. Cellulomonas parahominis MB426 來源之 L-RI 4
3. Geodermatophilus obscurus DSM 43160 來源之 L-RI 5
4. Actinotalea fermentans ATCC 43279 來源之 L-RI 5
5. Mycetocola miduiensis 來源之 L-RI 6
6. Cryobacterium sp. N21 來源之 L-RI 6
四、 大腸桿菌宿主 6
1. E. coli DH5α 6
2. E. coli BL21 Star (DE3) 6
五、 定位突變 (Site-directed mutagenesis) 7
1. 定位突變之簡介 7
2. 定位突變之方法 7
六、 提升酵素熱穩定之方法 9
1. 轉譯後修飾 (Post-translational modification，PTM) 9
2. 導入非共價交互作用 9
3. 導入共價交互作用 10
4. 增加脯胺酸 (proline) 或減少甘胺酸 (glycine) 11
5. 蛋白質表面導入精胺酸 (arginine) 11
參、 實驗設計與流程 13
一、 單一定位突變之 Af-RI 13
二、 多重定位突變之 Af-RI 13
肆、 實驗材料與方法 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. 酵素 14
5. 市售套組 14
6. 培養基 14
7. 化學藥品 15
8. 實驗設備 16
9. 軟體 17
二、 藥品配置 19
1. Terrific broth (TB) 培養液 19
2. 抗生素 19
3. Super Optimal Broth (SOB) 培養液 19
4. Super optimal broth with catabolite repression (SOC) 培養液 20
5. 製備 DNA 瓊脂膠體相關試劑 20
6. 破菌 (cell lysis) 相關試劑 20
7. 製備 SDS-PAGE 相關試劑 21
8. Cysteine-carbazole 呈色法試劑 22
9. 蛋白質定量相關試劑 23
三、 實驗方法 24
1. 選擇突變點 24
2. 定位點突變 24
3. 篩選突變形菌株 29
4. 蛋白質表現純化 30
5. 蛋白質特性探討 32
伍、 結果與討論 35
一、 突變點選擇及引子設計並進行定位突變 35
二、 以酵素活性及熱穩定性篩選突變型酵素 35
三、 探討純化突變型 S148R Af-RI 特性 36
1. 大量表現與純化 36
2. 熱穩定性 36
3. 酵素動力學參數 36
4. 最適作用溫度 37
5. 最適作用 pH 值 37
四、 探討純化突變型 N52R/S148R 及 N52R Af-RI 特性 37
1. 選擇雙重定位突變點 37
2. 雙重定位突變 37
3. 大量表現與純化 37
4. 熱穩定性 38
五、 探討純化突變型 S142R Af-RI 之熱穩定性 38
1. 大量表現及純化 38
2. 熱穩定性 38
陸、 結論 40
柒、 參考文獻 41
捌、 圖表 49

吳泰徵，2017，Actinotalea fermentans ATCC 43279 來源重組 L-核糖異構酶之特性與固定化並以蛋白質工程改變其熱穩定性，國立臺灣海洋大學食品科學系碩士學位論文，基隆
林敬瑋，2008，新型單點及多點定位突變方法之開發與利用定向演化進行麥芽寡糖苷海藻糖水解酶的基因重組，國立臺灣海洋大學食品科學系碩士學位論文，基隆
劉俞均，2018，以蛋白質工程及固定化菌體改變 Actinotalea fermentans ATCC 43279 來源重組 L-核糖異構酶之熱穩定性
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