臺灣博碩士論文加值系統

L-核糖 (L-ribose) 為五碳醛糖，可做為抗病毒及抗癌藥物的前驅物，然而其為一種稀有醣類，使得藥物價格貴並侷限了科學及應用上的發展。L-核糖可先藉由 L-阿拉伯糖異構酶將較廉價的 L-阿拉伯糖轉換為 L-核酮糖，再以 L-核糖異構酶 (L-ribose isomerase, L-RI) 轉換成 L-核糖。先前實驗室成員已將 Geodermatophilus obscurus DSM 43160 來源的 L-ri 基因選殖至 Escherichia coli 表現宿主中，並可表現酵素 Go-RI。
本篇研究先使用 SWISS-MODEL 建立 Go-RI 的結構模型後，再以 ENCoM 軟體預測穩定性較佳的突變點，挑選位於酵素表面的十個殘基進行定位突變。得到突變菌株後製備小量粗酵素液進行篩選，原生型、V142Y、A220F 和 A240L 的粗酵素液活性分別為 1.56、49.8、18.2、56.0 U/ml；以 50°C 熱處理粗酵素液 20 min 後，原生型、E139G 及 A225W 的殘餘活性分別為 7.15%、11.6%、28.8%。挑選此五株突變型進行純化，突變型酵素的比活性均較原生型低，然而培養每公升菌液的原生型、E139G、V142Y、A220F、A225W 和 A240L 可得活性分別為 223、347、2610、680、124、1730 U，顯示突變型菌株 V142Y 與 A240L 的酵素表現量明顯高於原生型菌株；熱穩定性方面原生型酵素於 50°C 保溫下的活性半衰期為 10.8 分鐘，突變型 E139G、A225W 分別為 15.9、19.5 分鐘，是原生型的 1.47 及 1.81 倍顯示熱穩定性增加，雖然 V142Y 有較佳的蛋白質表現量但其活性半衰期僅有 1.19 分鐘，而 A240L 則為 11.3 分鐘與原生型酵素無明顯差異。

L-ribose is a pentose monosaccharide, which can be used as a precursor for the synthesis of L-nucleoside analogs to be formulated into antiviral drugs. Because of L-ribose have only rare amount in nature, the scarcity and high price limit the pursuit of applications and research. L-Ribose can be produced from L-arabinose to L-ribose by two steps. The first step is to catalyze the L-arabinose into L-ribulose by L-arabionose isomerase. The second step is to use L-ribose isomerase (L-RI) catalyzing the aldose-ketose isomerization between L-ribose and L-ribulose. The L-ri gene was previously cloned from the genomic DNA of Geodermatophilusobscurus DSM 43160 and expressed Go-RI in Escherichia coli.
This study aims to enhance the activity and thermostability of L-ribose isomerase by protein engineering. First, we utilized the SWISS-MODEL to obtain a modeled structure of Go-RI, and use the ENCoM sever to evaluate the effects of the single mutation of all residues on the thermodynamic stability of Go-RI structure. Then, chose the best ten mutation residues located at the protein surface and constructed these mutations. The crude extracts containing wild-type and mutant Go-RIs have been assayed, and wild-type and mutant V142Y, A220F and A240L Go-RIs have 1.56, 49.8, 18.2 and 56.0 U/ml, respectively. The residual activities of the crude extracts containing wild-type, E139G and A225W Go-RIs after incubating at 50°C for 20 min are 7.15%, 11.6% and 28.8%, respectively. After purifying the wild-type and the above five selected mutant GoRIs, the specific activities of all five mutant Go-RIs are less than that of wild-type Go-RI. Since the total activities of wild-type, E130G, V142Y, A220F, A225W and A240L GoRIs from a liter of culture broth are 223, 347, 2610, 680, 124 and 1730 U, respectively, the expression of mutant V142Y and A240L Go-RIs are greater than that of wild-type Go-RI. The half-lives of mutant E139G (15.9 min) and A225W (19.5 min) Go-RIs at 50°C are 1.47 and 1.81 fold of wild-type (10.8 min), respectively. Although the mutant V142Y Go-RI has the better expression, its half-life at 50°C is only 1.19 min. The half-life of mutant A240L Go-RI is 11.3 min, which is similar to wild-type Go-RI.

目錄 I
圖目錄 III
表目錄 IV
壹、 研究背景與目的 1
一、 研究背景 1
二、 研究目的 1
貳、 文獻整理 2
一、 稀有醣類 2
1. 稀有醣類簡介 2
2. 核糖 2
二、 L-核糖的生產 2
1. L-甘露醇-1-脫氫酶 3
2. L-核糖醇脫氫酶 3
3. L-阿拉伯糖異構酶 4
4. 甘露糖-6-磷酸異構酶 4
5. L-核糖異構酶 4
三、 L-RI之結晶結構及序列分析 5
1. L-RI 之結構 5
2. 與基質及金屬離子結合的相關序列 5
四、 L-RI 的來源 5
1. G. obscurus DSM 43160 來源的L-RI (Go-RI) 6
2. Acinetobacter sp. DL-28來源的L-RI (AcRI) 6
3. C. parahominis MB426來源的L-RI (CeRI) 6
五、 合理化設計決定突變點 7
1. 同源結構模擬 7
2. ENCoM 預測穩定性軟體 7
參、 實驗設計與流程 9
一、 軟體分析及得到突變型菌株 9
1. 結構模擬及突變點分析 9
2. 定位突變 9
二、 篩選及分析突變型酵素之特性及熱穩定性 9
1. 篩選突變型菌株活性及熱穩定性 9
2. 突變型菌株之大量表現與特性探討 10
肆、 實驗材料與方法 11
一、 材料 11
1. 菌株與載體 11
2. 抗生素 11
3. 標準品 11
4. 酵素 11
5. 市售套組 11
6. 培養基 12
7. 化學藥品 12
8. 實驗設備 13
9. 軟體 15
二、 方法 16
1. 電腦模擬 16
2. 製備小量 plasmid DNA 16
3. 定位點突變 17
4. 電穿孔轉形 19
5. 篩選突變型菌株 21
6. SDS-PAGE 23
7. 蛋白質表現及純化 24
8. 蛋白質特性探討 26
伍、 結果與討論 29
一、 軟體模擬與突變點預測 29
1. Go-RI 結構模擬 29
2. ENCoM 預測熱穩定之突變點 29
二、 篩選突變型酵素活性及熱穩定性 30
1. Go-RI 之定位突變 30
2. Go-RI 之粗酵素液活性篩選 30
3. Go-RI 之粗酵素液熱穩定性篩選 30
三、 純化 Go-RI 特性及熱穩定性 31
1. 純化 Go-RI 之大量表現與純化 31
2. 純化 Go-RI 特性與熱穩定性 31
3. 純化 Go-RI 的酵素動力學參數 32
四、 討論 33
陸、 結論 34
柒、 參考文獻 35
捌、 圖 40
玖、 表 57

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