臺灣博碩士論文加值系統

摘要 I
Abstract II
目錄 III
圖目錄 VI
表目錄 VIII
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 3
1. 簡介 3
2. 生產 3
3. D-阿洛酮糖 3
4. D-阿洛糖 4
二、 D-阿洛酮糖表異構酶 (D-psicose 3-epimerase, DPE) 5
三、 L-鼠李糖異構酶 (L-rhamnose isomerase, L-RhI) 6
四、 大腸桿菌表現宿主 6
1. ClearColiTM BL21 (DE3) 6
2. BL21 (DE3)-CodonPlus-RIL 7
五、 固定化 7
1. 簡介 7
2. 方法 7
3. D-阿洛酮糖表異構酶之菌體固定化 8
六、 共表現 8
參、實驗設計與流程 10
一、 LCATS 與 TsRhI 共同建構在 pET-21b 載體上 10
二、 LCATS 與 TsRhI 共同建構在 pETDuet-1 載體上 10
三、 利用固定化菌體生產 D-阿洛酮糖及 D-阿洛糖 11
肆、實驗材料與方法 12
一、 材料 12
1. 菌株 12
2. 質體 12
3. 電泳標準品 12
4. 酵素 12
5. 培養基 13
6. 市售套組 13
7. 化學藥品 13
8. 軟體 14
二、 儀器設備 14
三、 藥品配製 16
1. Luria-Bertani (LB) 培養液 16
2. Terrific Broth (TB) 培養液 16
3. SOC Broth 16
4. Ampicillin (100 mg/mL) 16
5. Chloramphenicol (34 mg/mL) 16
6. DNA 瓊脂膠體製備 17
7. SDS-PAGE 相關藥劑之製備 17
8. 蛋白質定量相關試劑之製備 18
9. 細胞裂解相關試劑 18
四、 方法 19
1. 製備質體DNA 19
2. LCATS及TsRhI基因建構在 pET-21b 上 20
3. 轉形作用 22
4. Colony PCR篩選 22
5. 將 LCATS 及 TsRhI 基因建構在 pETDuet-1 上 23
6. 蛋白質表現 26
7. 製備粗酵素液 27
8. 蛋白質純化 28
9. 活性測定 29
10. 蛋白質定量 30
11. 菌體固定化 30
12. 以固定化菌體生產 D-阿洛酮糖及 D-阿洛糖 31
13. 樣品分析 32
伍、結果與討論 33
一、 建構含 LCTAS 及 TsRhI 基因之重組質體並探討表現 33
1. 將 LCTAS 及 TsRhI 建構在 pET-21b 上 33
2. 將 LCTAS 及 TsRhI 建構在 pETDuet-1 上 33
3. pETDuet-TsRhI-LCATS 表現及酵素純化 34
二、 固定化菌體生產 D-阿洛酮糖及 D-阿洛糖 34
1. 固定化條件及生產條件 34
2. 重複使用性 36
3. 保存穩定性 36
陸、結論 38
柒、參考文獻 39
捌、圖表 46
玖、附錄 79

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