臺灣博碩士論文加值系統

革蘭氏陰性大腸桿菌與革蘭氏陽性枯草桿菌 (Bacillus subtilis) 最常被用於異源重組蛋白質的生產。相較於大腸桿菌，枯草桿菌表現系統所產生之重組蛋白不具脂多醣 (lipopolysaccharide) 。脂多醣是著名的內毒素，會對人體與其他哺乳類動物造成系統性發炎反應。為了可在高溫下生產安全性較高之食品或醫療用途海藻糖，本研究藉由聚合酶鏈反應及配合其他基因重組技術，利用實驗室已選殖至大腸桿菌表現系統，分別含有 Sulfolobus solfataticus ATCC35092 之 treX、treY、treZ 及 S. acidocaldarius ATCC33909 之 treY 基因的重組質體作為序列來源，建構出能在枯草桿菌表現的 pHT254 質體，再將重組質體轉形至枯草桿菌中表現，透過細胞破碎及熱處理較簡化之純化方式獲得酵素。最後以澱粉為基質，利用枯草桿菌表現的異澱粉酶 (isoamylase)、麥芽寡糖苷海藻糖生成酶 (MTSase) 及麥芽糖苷海藻糖水解酶 (MTHase) 生產海藻糖。結果顯示，將 isoamylose 及不同菌種來源之 MTSase (SsMTSase / SaMTSase) 搭配 MTHase，以不同澱粉為基質，其中不同菌種來源之 MTSase 皆以馬鈴薯生澱粉為基質，最終海藻糖產量較佳，分別為79.0% / 74.4%。接著以馬鈴薯生澱粉為基質於不同溫度反應，得知在 80oC 下反應，不同菌種來源之 MTSase 海藻糖最終產量較高，分別為 82.2% / 79.0%。後續將以每克馬鈴薯生澱粉添加不同之酵素量，於 80oC 下反應，在不同酵素添加量之最終海藻糖產量，200 U / 10 U / 10 U (isoamylase / MTSase / MTHase) 為 82.3% / 77.7%、400 U / 20 U / 20 U為 82.1% / 78.8%、800 U / 40U / 40U 為 82.2% / 79.0%。由結果顯示不同添加量之酵素海藻糖最終產量並無顯著差異，綜合以上結果，海藻糖量產實驗將以馬鈴薯生澱粉為基質，每克澱粉添加 isoamylase / MTSase / MTHase = 200 U / 10 U / 10 U 之酵素量並於 80oC下進行。由結果得知，以 80 mL 含 8 克馬鈴薯生澱粉總反應體積試驗，以isoamylose 及不同菌種來源之 MTSase (SsMTSase / SaMTSase) 搭配 MTHase，最終海藻糖產量分別為 80.0% 及 73.5%，經分析發現與小量反應體積最終海藻糖產量無顯著差異。

The gram-negative bacterium Escherichia coli and gram-positive Bacillus subtilis are the most commonly used organisms for the productions of heterologous recombinant proteins. B. subtilis has a general advantage over E. coli that it has no lipopolysaccharides. These lipopolysaccharides are well-known as endotoxins, which can induce a systemic inflammatory response in humans or other mammals. In order to produce trehalose with higher safety for food and medical purposes at high temperatures, this study has used B. subtilis to express thermostable trehalose-producing related enzymes. The trehalose-producing related genes, including treX、treY、treZ of Sulfolobus solfataticus and treY of S. acidocaldarius, were subcloned respectively into pHT254 vector by using PCR and other gene recombination techniques. Then the recombinant plasmids were respectively transferred into B. subtilis by using electroporation, and the expressed enzymes were obtained simply by cell disruption and heat treatment. The trehalose-producing related enzymes, including isoamylase, MTSase (including SsMTSase and SaMTSase) and MTHase, were used to produce trehalose from different sources of starch. The results show that both MTSases can use raw potato starch as substrate to obtain better trehalose yields, which are 79.0% and 74.4% for SsMTSase and SaMTSase, respectively. The trehalose productions from raw potato starch at different temperatures have shown that the better yields of 82.2% and 79.0% for SsMTSase and SaMTSase, respectively, can be obtained at 80oC. When three different amounts of enzymes (isoamylase / MTSase / MTHase) were added to produce trehalose, the trehalose yields were 82.3% / 77.7% (SsMTSase /SaMTSase), 82.1% / 78.8% and 82.2% / 79.0%, for 200 U / 10 U / 10 U, 400 U / 20 U / 20 U and 800 U / 40U / 40U, respectively. The yields were not significant affected by the amounts of enzymes added. Finally, trehalose productions from raw potato starch were carried out at 80oC and the amounts of enzymes isoamylase / MTSase / MTHase = 200 U / 10 U / 10 U were added. In the reaction volume of 80 mL, trehalose yields of 80.0% and 73.5% were obtained for SsMTSase and SaMTSase, respectively, and there were no significant difference in trehalose yields between reaction volumes of 1.5 and 80 mL.

目錄
摘要 i
Abstract ii
目錄 iii
表目錄 vi
圖目錄 vii
壹、前言 1
貳、文獻整理 3
一、蛋白質表現系統之簡介 3
1. Bacillus 表現系統介紹 3
1.1 B. megaterium 系統 3
1.2 B. subtilis 系統 3
1.3 B. brevis 系統 3
2. 大腸桿菌與枯草桿菌表現系統之比較 4
2.1 大腸桿菌表現系統 4
2.2 枯草桿菌表現系統 4
二、海藻糖之簡介 4
1. 海藻糖之發現與應用 4
2. 海藻糖之特性 5
3. 海藻糖對於疾病之預防與治療 6
3.1 食用安全性 6
3.2 骨質疏鬆症之預防 6
3.3 亨丁頓氏舞蹈症 (Huntington disease) 之減輕 6
3.4 糖尿病之預防 6
4. 海藻糖之生產 7
4.1 酵素轉換法 7
4.2 熱穩定酵素轉換法 8
5. 測定海藻糖產量之方法 9
6. 澱粉特性介紹 9
6.1 澱粉來源與應用 9
6.2 澱粉之化學組成 9
6.3 直鏈澱粉含量 10
6.4 澱粉顆粒型態 10
6.5 澱粉之膨潤率和溶解度 10
7. 以不同澱粉原料生產海藻糖 10
參、實驗設計 12
肆、實驗材料與方法 13
一、實驗材料 13
1. 實驗用質體與菌株 13
1.1 treX、treY 及 treZ 基因來源 13
1.2 質體來源 13
1.3 菌株來源 13
2. 抗生素 13
3. 標準品 13
3.1 DNA 標準品 13
3.2 蛋白質標準品 13
4. 市售套組 14
5. 酵素 14
6. 化學藥品 14
7. 實驗設備 15
8. 藥品配製 16
二、實驗方法 22
1. 擴增目標基因與載體之 DNA 序列 22
1.1 PIPE PCR (Polymerase Incomplete Primer Extension, PIPE) 22
1.2 Restriction Free cloning (RF-cloning) 23
1.3 限制酶剪切 25
2. 利用大腸桿菌將 treX、treY、treZ 及載體 pHT254 進行接合 25
2.1 熱休克 (heat shock) 勝任細胞之製備 25
2.2 熱休克轉形作用 (transformation) 26
2.3 電穿孔 (electroporation) 勝任細胞之製備 26
2.4 電穿孔轉形作用 26
3. 以 colony PCR 篩選轉形株 26
4. 序列分析 27
5. 菌種保存 27
6. 質體 DNA 之製備 27
7. 以電穿孔轉形重組質體至 B. subtilis WB800N 28
7.1枯草桿菌電穿孔勝任細胞之製備 28
7.2枯草桿菌電穿孔轉形作用 28
8. B. subtilis WB800N 之 IPTG 最適添加濃度 29
8.1 蛋白質標準曲線製作 29
8.2 蛋白質濃度之測定 29
8.3 蛋白質電泳分析 29
8.4 膠體製備 29
8.5 膠體電泳之操作方式 30
8.6 膠片之染色與脫色 30
9. Isoamylase、MTSase 及 MTHase 之 大量生產 30
10. Isoamylase、MTSase 及 MTHase 之蛋白質純化與活性測定 30
10.1 Isoamylase、MTSase 及 MTHase 之純化 30
10.2 Isoamylase 之活性測定 31
10.3 以 DNS 法測定 MTSase 與 MTHase 之活性 31
10.4 MTSase 之活性測定 32
10.5 MTHase 之活性測定 32
11. 海藻糖產量試驗 33
11.1利用熱穩定酵素生產海藻糖 33
11.2利用最適條件量產海藻糖 34
11.3 以 HPLC 分析海藻糖之生成 34
伍、結果與討論 36
一、以 DNA 重組技術將海藻糖生成相關基因建構至 pHT254 載體 36
1. PIPE 之 pHT254-SsTreX / SsTreY / SsTreZ 質體建構 36
2. RF-cloning 之 pHT254-SaTreY 質體建構 36
二、B. subtilis WB800N 最適 IPTG 濃度 37
三、Isoamylase、MTSase 及 MTHase 之純化 37
1. Isoamylase 之表現與活性 38
2. MTSase 之表現與活性 38
3. MTHase 之表現與活性 39
四、以熱穩定性海藻糖生成相關酵素生產海藻糖 39
1. 最適澱粉基質測試 39
2. 最適生產溫度測試 40
3. 最適生產酵素添加量測試 41
4. 以最適條件量產海藻糖 41
五、未來期許 42
陸、結論 43
柒、參考文獻 44

 
表目錄
表一、PIPE 引子組序列 49
表二、RF-cloning 引子組序列 50
表三、表現於枯草桿菌 WB800N 之重組型 isoamylase 純化表 51
表四、表現於枯草桿菌 WB800N 之重組型 SsMTSase 純化表 52
表五、表現於枯草桿菌 WB800N 之重組型 SaMTSase 純化表 53
表六、表現於枯草桿菌 WB800N 之重組型 MTHase 純化表 54
表七、比較不同來源之生澱粉於 70°C 反應 72 小時之海藻糖產量 55
表八、比較於不同溫度下反應 72 小時之海藻糖產量 56
表九、比較於不同酵素添加量反應 72 小時之海藻糖產量 57
表十、比較不同反應總體積於 72 小時之海藻糖產量 58

 
圖目錄
圖一、海藻糖之化學結構 59
圖二、海藻糖生物合成途徑 60
圖三、以 MTSase 及 MTHase 生產海藻糖之流程圖 61
圖四、不同澱粉顆粒之掃描式電子顯微鏡照片 62
圖五、pHT254 載體 63
圖六、利用 PIPE-cloning 與 RF-cloning PCR 擴增產物之電泳分析圖 64
圖七、SsTreX、SaTreY、SsTreY 及 SsTreZ 於枯草桿菌表現之最適 IPTG 誘導 65
圖八、以 SDS-PAGE 電泳分析 isoamylase、SsMTSase、SaMTSase 及 MTHase 純化後之蛋白質表現分佈 66
圖九、不同來源之 10% 澱粉，添加每克含 isoamylase/MTSase/MTHase 為 800 U / 40 U / 40 U 之酵素量，於 70°C 反應 72 小時之海藻糖產量 67
圖十、以 10% 馬鈴薯生澱粉為基質，添加每克含 isoamylase / MTSase / MTHase 為 800 U / 40 U / 40 U 之酵素量，於不同反應溫度作用 72 小時之海藻糖產量 68
圖十一、以 10% 馬鈴薯生澱粉為基質，添加每克含不同濃度之 isoamylase / MTSase / MTHase 酵素量，於 80°C 反應 72 小時之海藻糖產量 69
圖十二、不同反應總體積，以 10% 馬鈴薯生澱粉為基質，添加每克含 isoamylase / MTSase / MTHase 為 200 U / 10 U / 10 U 之酵素量，於 80°C 反應 72 小時之海藻糖產量 70

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