摘要 I
Abstract II
目錄 III
圖目錄 VIII
表目錄 IX
壹、 研究背景與目的 1
一、 研究背景 1
二、 研究目的 2
貳、 文獻回顧 3
一、 稀有醣類 3
1. 稀有醣簡介 3
2. 核糖 3
二、 利用酵素轉換法生產L-核糖 4
1. 簡介 4
2. L-核糖異構酶 4
3. 甘露糖-6-磷酸異構酶 4
4. Actinotalea fermentans ATCC 43279來源之L-RI 5
三、 聚羥基烷酸酯 5
1. 簡介 5
2. 蛋白質純化之應用 6
3. 藥物遞送之應用 7
4. 酵素固定化之應用 8
參、 實驗設計與流程 10
一、 將Ralstonia eutropha H16來源之phaA及phaB基因建構於載體 10
二、 Ralstonia eutropha H16來源之phaC基因與Af-ri基因融合 10
三、 PHA-RI表現及純化 11
肆、 材料與方法 12
一、 實驗材料 12
1. 菌株 12
2. 載體 12
3. 電泳標準品 12
4. 酵素 13
5. 培養基 13
6. 市售套組 13
7. 化學藥品 14
8. 實驗設備 16
二、 藥品配置 18
1. 製備Terrific broth (TB) 培養基 18
2. 製備Nutrient broth (NB) 培養液 18
3. 製備SOC培養基 19
4. Luria-Bertani（LB）平板培養基 19
5. Ampicillin (100 mg/mL) 20
6. Chloramphenicol (34 mg/mL) 20
7. DNA 瓊脂膠體製備 20
8. SDS-PAGE 相關藥劑之製備 21
9. 破菌相關試劑 23
三、 方法 24
1. 基因體 DNA（Total genomic DNA）的萃取 24
2. 設計選殖目標基因之引子對 25
3. 抽取質體DNA 25
4. 將phaA及phaB基因建構在pBBR1MSC-1 plasmid上 26
4.1. 擴增phaAB DNA片段 26
4.2. DNA Clean up 27
4.3. 限制酶剪切 28
4.4. PstI剪切後之pBBR1MSC-1 以鹼性磷酸酶（CIAP）剪切 28
4.5. 剪切或水解後之PCR產物及pBBR1MSC-1之DNA Clean up 28
4.6. 接合作用 28
4.7. 轉形作用 29
4.7.1. 勝任細胞製備 29
4.7.2. 電穿孔轉形 30
4.8. Colony PCR 篩選 30
4.9. 定序 31
4.10. 菌種保存 31
5. 將phaC基因建構於pET-21b-Af-ri質體 31
5.1. 擴phaC基因 31
5.2. 擴增質體pET-21b-Af-ri 之DNA片段 32
5.3. 限制酶剪切 33
5.4. 熱休克（Heat Shock）轉形 33
5.5. Colony PCR 篩選 34
5.6. 定序 35
5.7. 定序菌種保存 35
6. 質體pBBR1MCS-1-phaAB和pET-21b-phaC-Af-ri在E. coli之表現 35
6.1. 抽取質體 DNA 35
6.2. 電穿孔轉形至不同表現宿主 35
6.2.1. 在不同表現宿主之蛋白質表現 36
6.3. 不同IPTG 濃度誘導表現蛋白質 36
7. 聚羥基烷酸酯固定化RI（PHA-RI）的製備及特性探討 36
7.1. PHA-RI的製備 36
7.2. PHA-RI活性測試 37
7.3. 最適作用pH值 38
7.4. 最適作用溫度 38
7.5. 熱穩定性 38
7.6. 貯存試驗 38
8. Af-RI之表現與純化 39
8.1. Af-RI之表現 39
8.2. Af-RI之純化 39
9. Af-RI之特性測試 40
9.1. Af-RI活性測試 40
9.2. 最適作用pH值 40
9.3. 最適作用溫度 40
9.4. 熱穩定性 40
10. 利用PHA-RI生產L-核糖 41
10.1. 反應基質L-核酮糖之製備 41
10.1.1. AI生產L-核酮糖 41
10.1.2. AI菌體培養 41
10.1.3. AI之純化 41
10.1.4. AI活性測定 41
10.1.5. 基質L-核酮糖之製備 42
10.2. L-核糖生產 42
10.3. HPLC-ELSD分析 42
11. PHA-RI重複使用於L-核糖生產 43
伍、 結果與討論 44
一、 建構含phaA及phaB基因之重組質體 44
二、 建構含phaC及Af-ri基因之重組質體 44
三、 PHA-RI表現及純化 45
1. 最佳表現宿主 45
2. PHA-RI奈米顆粒形成於E. coli BL21 Star (DE3)菌體中 45
3. phaC-Af-ri序列分析 46
4. 最適IPTG誘導濃度 46
四、 PHA-RI特性探討 46
1. 最適作用pH值 46
2. 最適作用溫度 47
3. 熱穩定性 47
4. 貯藏試驗 47
五、 利用PHA-RI生產L-核糖 48
六、 PHA-RI重複使用於L-核糖生產 48
陸、 結論 49
柒、 參考文獻 50
捌、 圖表 57

吳泰徵，2016，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提昇其熱穩定性，國立臺灣海洋大學食品科學系碩士學位論文，基隆。
黃幸光，2014，熱穩定性阿拉伯糖異構酶之表現、特性探討、稀有糖類生產及蛋白質工程，國立臺灣海洋大學食品科學系碩士學位論文，基隆。
顏艾，2021，Deinococcus indicus DSM 15307來源重組澱粉蔗糖酶之特性探討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。
魏子庭，2021，利用D-阿洛酮糖表異構酶及L-鼠李糖異構酶的固定化菌體生產稀有醣類，國立臺灣海洋大學食品科學系碩士學位論文，基隆。
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