臺灣博碩士論文加值系統

L-核糖異構酶 (L-ribose isomerase, L-RI) 可催化 L-核糖 (L-ribose) 與 L-核酮糖之間的醛酮異構化反應，此反應為一可逆反應。L-核糖為醛糖，在自然界中極為稀少屬於稀有醣類 (rare sugar)，而其核苷類似物 (nucleoside analogues) 可做為抗病毒藥物及抗癌藥物之前驅物材料。將 Actinotalea fermentans ATCC 43279 來源之基因表現於 Escherichia. coli BL21 Star (DE3)，其重組酵素之最適作用溫度為 45℃，在 50℃ 的半衰期為 58 分鐘。以蛋白質工程增加 Af-RI 之熱穩定性，選擇了酵素四聚體 interface 上的四個殘基 (L69, P78, V139, F141) 做為突變點，並參考 Geodermatophilus. obscurus DSM43160 之研究結果選擇兩個殘基 (D135, A221) 進行突變，另將在酵素的 C 端加上 ATS 序列。分別得到 L69Q、P78K、P78R、P78T、V139T、F141Y、D135G、A221W、R132Q/D134G/D135G 及 P220Q/A221W 之突變型及 Af-RI-ATS，並成功表現於 E. coli BL21 Star (DE3)。原生型、F141Y、R132Q/D134G/D135G 及 P220Q/A221W 之粗酵素液相對活性分別為 100%、77.6%、58.4% 及 26.9%。粗酵素液經 50℃ 下保溫 10 分鐘後之殘餘活性部分，原生型與 F141Y 分別是 27% 及 38.4%。F141Y 酵素經純化後之比活性為 25.1 U/mg；於 50℃ 酵素活性半衰期為 66.6 分鐘，是原生型的 1.14 倍。將含有 Af-RI 之 E. coli BL21 Star (DE3) 以海藻酸鈉製備固定化菌體，使用乳化液相 (emulsification) 方式製備其固定化效率最高可達 67%。而其最適條件為使用 1.2% 海藻酸鈉、25 g/L 之菌體濃度、0.05 M 氯化鈣溶液。以最適條件所製備之固定化菌體最適作用溫度為 45℃，最適作用 pH 值為 7.5 (Tricine-NaOH buffer)，50℃ 活性半衰期為 52.5 分鐘，於 4oC 保存 16 天後活性約餘 50%。

L-Ribose isomerase (L-RI) catalyzes the reversible aldose-ketose isomerization between L-ribulose and L-ribose. L-ribose is an aldose which is not often found in nature. It can be used as a precursor material for antiviral and anticancer drugs. The gene of Actinotalea fermentans ATCC 43279 was overexpressed in Escherichia coli BL21 Star (DE3), the purified recombinant enzyme demonstrated its optimal activity at 45℃ and the half-life at 50℃ was 58 min. To enhance the thermostability of Af-RI by protein engineering, four residues (L69, P78, V139, F141) on the interface of enzyme tetramers and the mutation sites from the study of Geodermatophilus. obscurus DSM43160 have been chosen for mutagenesis (D135, A221). L69Q, P78K, P78R, P78T, V139T, F141Y, D135G, A221W, R132Q/D134G/D135G, P220Q/A221W and Af-RI-ATS were successfully constructed and expresses in E. coli BL21 Star (DE3). The relative activities of the crude extracts containing wild-type, F141Y, R132Q/D134G/D135G and P220Q/A221W was 100%, 77.6%, 58.4%, and 26.9%, respectively. After incubating at 50℃ for 10 min, the residual activities of wild-type and F141Y were 27% and 38.4%, respectively. After purification, the specific activity of F141Y Af-RI was 25.1 U/mg; the half-life at 50℃ was 66.6 min, which was 1.14 times of wild-type. The cells containing Af-RI were immobilized by entrapment in the beads of alginate and cured by treatment with CaCl2 solution, the immobilization efficiency was up to 67%. The optimum conditions are 1.2% alginate, 25 g/L cells, and 0.05 M CaCl2 solution. The immobilized cells showed its optimal activity at 45℃ and pH 8.0 (Tricine-NaOH buffer). The half-life of immobilized cells at 50℃ was 52.5 min, and the activity was about 50% after 16 days of storage at 4℃.

摘要…………………………………………………………………………………i
Abstract……………………………………………………………………………ii
目錄………………………………………………………………………………..iii
圖目錄……………………………………………………………………………..vi
表目錄…………………………………………………………………………….vii
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. 稀有醣類簡介 3
2. 稀有醣類之用途及功效 3
3. L-核糖 (ribose) 3
4. L-核糖之藥物應用 3
二、 核糖的生產 4
1. L-核糖異構酶 4
2. L-阿拉伯糖異構酶 5
3. 甘露糖-6-磷酸異構酶 5
4. L-甘露醇-1-脫氫酶 5
5. 核糖醇脫氫酶 6
三、 不同來源 L-RI 之特性 6
1. 來自 A. fermentans ATCC 43279之 L-RI 6
2. 來自 Acinetobacter sp. DL-28 之 L-RI 6
3. 來自 C. parahominis MB426 之 L-RI 7
4. 來自 G. obscurus DSM43160之 L-RI 7
四、 L-RI 之晶體結構及序列分析 7
1. L-RI 結構 7
2. L-RI 序列分析 8
五、 ATS 序列 (acidic tails of synulein peptide) 8
六、 蛋白質聚體之次單位間的交互作用 9
七、 E. coli BL21 Star (DE3) 大腸桿菌表現宿主 10
八、 固定化菌體 10
1. 固定化簡介 10
2. 固定化方法 10
3. 固定化材料 11
參、 實驗設計與流程 13
一、 Af-RI 之突變菌株 13
二、 利用 ATS 序列增加酵素穩定性 13
三、 固定化菌體之方法與特性探討 13
肆、 實驗材料與方法 14
一、 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 15
6. 培養基 15
7. 化學藥品 15
8. 實驗設備 17
9. 軟體 18
二、 實驗步驟及方法 19
1. 藥品配置 19
2. 選擇適當之突變點 22
3. 置備小量 plasmid DNA 22
4. 定位點突變 24
5. 將 A. fermentans ATCC 43279 之 C 端加入 ATS 序列 27
6. 電穿孔轉形 27
7. 篩選突變型菌株 29
8. SDS-PAGE 31
9. 蛋白質表現純化 32
10. 蛋白質特性探討 33
11. 固定化菌體 35
伍、 結果與討論 39
一、 突變點之選擇 39
二、 篩選突變型酵素活性及熱穩定性 39
1. Af-RI 之定位突變 39
2. Af-RI 粗酵素液活性與熱穩定性之篩選 39
三、 純化 F141Y Af-RI 特性及熱穩定性 40
1. 大量表現與純化 40
2. 最適作用溫度 41
3. 最適作用 pH 值 41
4. 熱穩定性 41
5. 酵素動力學參數 42
四、 固定化菌體 42
1. 固定化菌體材料與條件 42
2. 固定化菌體之特性 43
陸、 結論 45
柒、 參考文獻 46
捌、 圖表 52
玖、 附錄 78

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