臺灣博碩士論文加值系統

L-核糖及L-核酮糖屬於稀有醣類，其中L-核糖之核苷類似物 (nucleoside analogues) 可抗病毒及抗癌藥物之前驅物材料，其可由 L-阿拉伯糖利用 L-阿拉伯糖異構酶 (L-arabinose isomerase, L-AI) 轉換成 L-核酮糖，再由 L-核糖異構酶 (L-ribose isomerase, L-RI) 轉換成 L-核糖，本研究所使用之 L-AI 來自 T. saccharolyticum NTOU1 (Ts-AI) 之突變型 F270N，而 L-RI 則是使用來自 Actinotalea fermentans ATCC 43279 (Af-RI)，首先以乳糖誘導 Af-RI 表現，發現當接種 5% 隔夜培養菌液於含有 2 g/L TB 培養液在 20oC 下培養 48 小時其可溶性蛋白質表現量最佳，將此條件應用於 4 L 發酵槽進行放大培養，當通氣量為 0.75 vvm 及轉速為 500 rpm 時，可獲得最高菌體收穫量及活性。利用以上所得到的最佳條件培養含有 F270N Ts-AI 菌體，並將兩種酵素利用 HisTrapTM HP 管柱進行純化，而後將純化後酵素固定於 BeaverBeadsTM IDA-Nickel 及 Ni SepharoseTM 6 Fast Flow resin 上，結果顯示將 F270N Ts-AI 酵素固定在 BeaverBeadsTM IDA-Nickel 上其結合率較佳，固定在 Ni SepharoseTM 6 Fast Flow resin 者其固定化效率較好，且最高固定化效率為 10.3%；將 Af-RI 固定在 Ni SepharoseTM 6 Fast Flow resin 上其最高的固定化效率為 17.9%。於固定化菌體的部分，將菌體、海藻酸鈉及界面活性劑混合後，於含有適當界面活性劑之大豆油中進行乳化，最後加入氯化鈣進行固化，並且探討其最適生產條件，得到含有 F270N Ts-AI 及 Af-RI 之固定化菌體顆粒最高固定化效率分別為 64% 及 76%，就熱穩定性來看，所得到含有 F270N Ts-AI 固定化菌體顆粒在 60oC 下半衰期提升了 1.76 倍，而含有 Af-RI 固定化菌體顆粒在 50oC 下半衰期提升了 1.25 倍。於 45oC 利用 F270N Ts-AI 進行 L-核酮糖的生產，於 48 小時後平衡其轉換率約為 8.6%，重複使用 2 次後於相同時間下其轉換率降至 2.5%，接著於相同溫度下進行 Af-RI 進行 L-核糖的生產，發現於 24 小時到達平衡L-核酮糖及L-核糖轉換率為 7.4%，重複使用 1 次後於相同時間下固定化菌體已失活，將兩種固定化菌體填入填充床反應器於 45oC 下進行生產，於 48 小時到達平衡，L-核酮糖及 L-核糖的轉換率分別為 8% 及 20%，而以 HisTrapTM HP 將兩種酵素進行固定化後於 45oC 下進行生產，於 24 小時到達平衡，L-核酮糖及 L-核糖的轉換率分別為 9.3% 及 4.5 %。

L-ribose and L-ribulose are defined as rare sugar. L-Ribose is an important pharmaceutical intermediate for the production of L-form sugar based antivirus drugs. This report firstly used recombinant F270N L-arabinose isomerase from T. saccharolyticum NTOU1 (Ts-AI) to convert L-arabinose into L-ribulose, then used recombinant L-ribose isomerase from Actinotalea fermentans ATCC 43279 (Af-RI) to convert L-ribulose into L-ribose. It was found that Af-RI could be expressed and produced more in soluble form when inoculated with 5% overnight culture in the TB medium containing 2 g/mL lactose and cultured at 20oC for 48 hr. Scale-up from flask to fermenter showed that fermentation under 500 rpm agitation speed and 0.75 vvm airflow rate produced the cells with highest free cell activity and cell wet weight. The same condition was used to scale-up the production of F270N Ts-AI and two enzymes were then purified. The purified enzymes were immobilized on BeaverBeadsTM IDA-Nickel and Ni SepharoseTM 6 Fast Flow resin. It turned out that used BeaverBeadsTM IDA-Nickel as carrier can bind more enzyme, however used Ni SepharoseTM 6 Fast Flow resin can obtain higher immobilization efficiency. The highest immobilized effiencies of F270N Ts-AI and Af-RI are 8.08% and 15.1%, respectively. In term of immobilized cells, it was prepared by emulsification and the optimal condition was assessed. The highest immobilized effiencies of F270N Ts-AI and Af-RI are 64% and 76%, respectivey. The half-lifes and the optimum temperatures of two different immobilized cells have increased. At last, using immobilized cells harboring F270N Ts-AI to produce L-ribulose at 45oC, the reaction reached equilibrium in 48 h with about 8.6% conversion yield of L-ribulose from L-arabinose. The conversion rate of L-ribulose has decreased to 2.5% after three reaction cycles. Then L-ribose was produced by immobilized cells harboring Af-RI at the same temperature. It could reach reaction equilibrium in 24 h with about 7.4% conversion yield of L-ribose from L-ribulose. After two reaction cycles, no L-ribose has been produced. To further exploring the industrial application, production of L-ribulose and L-ribose was performed at 45oC using two kinds of immobilized cells loaded in packed-bed reactor, reaction equilibrium has reached in 48 h with about 20% and 8% conversion yield of L-ribose and L-ribulose, respectively. HisTrapTM HP columns which immobilized two kinds of purified enzymes have been used to produce rare sugars. The reaction equilibrium has reached in 24 h with about 9.3% and 4.5% conversion yield of L-ribose and L-ribulose, respectively.

目錄
摘要…………………………………………………………………………………I
Abstract……………………………………………………………………………II
目錄………………………………………………………………………………..III
圖目錄……………………………………………………………………………..VI
表目錄…………………………………………………………………………….VII
壹、 前言 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. 簡介 3
2. 應用 3
3. L-核酮糖 (L-ribulose) 3
4. L-核糖 (L-ribose) 3
二、 L-核糖 (L-ribose) 之生產 4
三、 生產L-核糖所需之酵素 5
1. L-阿拉伯糖異構酶 (L-AI) 5
2. L-核糖異構酶 (L-RI) 5
四、 重組蛋白在細菌中之蛋白質表現 6
1. ClearColi BL21 (DE3) 6
2. CondonPlus-RIL 7
3. 乳糖操控組 (Lactose operon) 7
五、 固定化 8
1. 簡介 8
2. 方法 8
3. 固定化材料介紹 9
4. 含 L-AI及L-RI之菌體固定化 10
5. L-核酮糖及 L-核糖之生產 11
參、 實驗設計與流程 12
一、 以乳糖誘導可表現 Af-RI 的大腸桿菌菌體 12
二、 以發酵槽生產可分別表現 F270N Ts-AI 及 Af-RI 的大腸桿菌菌體 12
三、 以 BeaverBeadsTM IDA-Nickel 及 Ni SepharoseTM 6 Fast Flow resin固定化酵素 12
四、 利用乳化法製備固定化菌體 12
五、 L-核糖及 L-核酮糖之生產 13
肆、 實驗材料與方法 14
一、 實驗材料 14
1. 菌株與質體 14
2. 抗生素 14
3. 蛋白質標準品 14
4. 市售套組 14
5. 培養基 14
6. 化學藥品 14
7. 實驗設備 16
二、 藥品配置 17
1. Terrific broth (TB) 培養液 17
2. Super Optimal Broth (SOB) 培養液 18
3. Salt-optimized carbon broth (SOC) 培養液 18
4. 抗生素配置 18
5. Cysteine-carbazole 呈色法試劑 18
6. 定量蛋白質之相關試劑 19
7. SDS-PAGE 相關藥劑製備 19
三、 實驗方法 20
1. 製備小量質體 20
2. 轉形作用 21
3. 蛋白質表現 22
4. 發酵槽生產 24
5. 製備粗酵素液 24
6. 重組型 F270N Ts-AI 及 Af-RI 純化 24
7. 蛋白質定量 25
8. 活性分析與特性探討 25
9. 固定化酵素 27
10. 固定化菌體 27
11. 以固定化菌體及酵素生產 L-核酮糖及 L-核糖 29
12. HPLC-ELSD 分析 30
13. 統計分析 31
伍、 結果與討論 32
一、 Af-RI 表現與純化 32
1. 利用乳糖進行誘導 32
2. 以發酵槽進行大量生產 32
二、 F270N Ts-AI 表現與純化 32
三、 固定化 33
1. 固定化酵素 33
2. 固定化菌體 33
3. 純化酵素與固定化菌體特性比較 35
四、 生產 L-核糖與 L-核酮糖 37
1. 批次生產 37
2. 連續生產 37
陸、 結論 39
柒、 參考文獻 40
捌、 附錄 82

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