臺灣博碩士論文加值系統

麥芽寡糖苷海藻糖生成酶 (MTSase) 能將麥芽寡糖還原端上的 α-1,4 糖苷鍵結轉變為 α-1,1 糖苷鍵結，以產生麥芽寡糖苷海藻糖。由於此酵素對於短鏈基質之活性較差，且有可能是由於對其結合力不足造成，所以本實驗利用電腦模擬 MTSase 與基質的結合方式，期望能找到與基質結合相關的胺基酸殘基。
綜合電腦模擬的結果發現， KX 、 DX 、 EX 、 DX 及 RX 等胺基酸殘基皆位於 subsite -2 到 -4 之間，可與麥芽五糖產生氫鍵，所以將 DX 、 DX 及 RX 於 Sulfolobus solfataricus ATCC 35092 中相對的胺基酸殘基 DX 、 DX 及 RX 突變成 Alanine ，以確認其與基質之氫鍵是否確實存在。
將突變成功之 DNA 片段，於大腸桿菌中表現蛋白質，經過熱處理、離子交換層析及膠體等純化步驟後，這些突變型酵素之比活性分別為 84.26 U/mg (wild-type) 、 1.10 U/mg (DXA) 、 1.21 U/mg (DXA) 及 1.19 U/mg (RXA) ，顯示這些突變確實造成酵素活性大幅下降。
在酵素動力學方面，原生型之 kcat 為 423.64 (s-1) ， KM 為 5.68 (mM) ， kcat/KM 為 74.53 (s-1 mM-1) ； DXA 之 kcat 為 30.19 (s-1) ， KM 為 43.17 (mM) ， kcat/KM 為 0.699 (s-1 mM-1) ； DXA 之 kcat 為 87.16 (s-1) ， KM 為 122.21 (mM) ， kcat/KM 為 0.713 (s-1 mM-1) ； RXA 之 kcat 為 13.41 (s-1) ， KM 為 43.52 (mM) ， kcat/KM 為 0.308 (s-1 mM-1) ，所以其 Δ(ΔG) 分別為 12.93 (kJ/mol) (D411A) 、 12.87 (kJ/mol) (D610A) 及 15.20 (kJ/mol) (R614A) ，表示其活性喪失可能是由於基質與酵素之間的氫鍵消失所造成，所以這也證明了電腦模擬結果的可信度，對於基質結合部位的模擬結果，具有相當高的參考價值。

The maltooligosyltrehalose synthase (MTSase) mainly catalyzes an intramolecular transglycosyl reaction to form maltooligosyltrehalose from maltooligosaccharides by converting the α-1,4-glucosidic linkage at the reducing end to an α-1,1-glucosidic linkage. In addition to transglycosylation reaction, MTSase also catalysis a hydrolysis reaction to release glucose. The hydrolysis activity of MTSase was higher when using maltotriose as substrate than maltooligosaccharides with a higher degree of polymerization (DP), and this phenomenon may relate to the low binding ability of MTSase to low DP substrates. In order to know the residues located in the substrate binding site, computer simulation was used.
The results of computer simulation showed that residues KX, DX, EX, DX and RX are located between subsite -2 and -4, and are hydrogen bonds between enzymes and maltopentaose. In order to confirm the proposed hydrogen bonds between MTSase and maltopentaose, mutants DXA, DXA, and RXA were constructed.
The mutant DNA vectors were obtained by PCR amplification. Then the wild-type and mutant DNA were transformed into E. coli Rosetta (DE3) to express wild-type and mutant MTSases, respectively. The specific activities of purified wild-type, mutant DXA, DXA, and RXA MTSases were 84.26 U/mg, 1.10 U/mg, 1.21 U/mg, and 1.19 U/mg, respectively.
The kcat / KM values of DXA, DXA, and RXA MTSases were lower than that of wild-type MTSase for 69.63 ~ 76.6 folds. This result suggest that the residues X, X and X were the important residues to the activity of MTSase. All mutant MTSases had large changes in Δ(ΔG), suggesting that there are hydrogen bonds between the substrate and residues X, X and X of wild-type MTSase.

目錄.......................................................I
圖目錄.....................................................V
表目錄....................................................VI
中文摘要....................................................1
英文摘要....................................................2
一、前言...................................................3
二、文獻整理...............................................5
1. 海藻糖...................................................5
1.1簡史...................................................5
1.2特性及功能.............................................5
1.3應用...................................................6
1.4 熱穩定酵素轉換法生產海藻糖............................7
2. 麥芽寡糖苷海藻糖生成酶之介紹............................7
2.1 起源及特性............................................8
2.2 反應機制..............................................9
2.2.1 轉糖基機制.........................................9
2.2.2 水解機制...........................................10
3. 麥芽寡糖苷海藻糖生成酶結構之探討.........................10
3.1 麥芽寡糖苷海藻糖生成酶之完整結構......................10
3.2 麥芽寡糖苷海藻糖生成酶活性作用部位結構探討............11
3.3 麥芽寡糖苷海藻糖生成酶和其他 α- 澱粉酶家族酵素結構上
之異同...........................................13
4. 酵素動力學...............................................13
4.1 酵素動力學理論之起源..................................13
4.2 Michaelis – Menten 方程式之意義........................14
5. 電腦自動分子對接 (Automated docking).....................14
5.1 簡介..................................................14
5.2 AutoDock 軟體.........................................15
5.3 AutoDock的應用........................................15
6. 分子動力學模擬 (Molecular Dynamic Simulation)............16
6.1 簡介..................................................16
6.2 NAMD 軟體............................................17
6.3 NAMD的應用..........................................17
三、實驗流程...............................................18
四、實驗材料...............................................19
1. 菌株與質體...............................................19
1.1 treY 基因來源菌株......................................19
1.2 表現 treY 基因之質體...................................19
1.3 保存質體之菌株........................................19
1.4 蛋白質表現系統之生產菌株...............................19
2. 抗生素...................................................20
3. 酵素.....................................................20
3.1聚合酶.................................................20
3.2限制酶.................................................20
4. DNA 標準品 (Marker) ....................................20
5. 蛋白質標準品 (Marker) ...................................20
6. 培養基...................................................21
7. 試液與化學藥品...........................................21
8. 儀器設備.................................................22
9. 電腦軟體.................................................24
五、實驗方法...............................................25
1. AutoDock操作流程........................................25
2. NAMD 操作流程...........................................25
3. 設計點突變 PCR 引子.....................................26
4. 質體 DNA 之製備.........................................27
4.1 DNA 濃度之測定......................................28
5. 點突變 PCR 反應.........................................28
5.1 點突變 PCR 的溶液組成................................28
5.2 點突變 PCR 反應條件..................................29
6. DpnI 剪切 dsDNA template pET-15b-∆H-treY................30
7. 勝任細胞 (competent cell) 的製備.........................30
7.1 超級勝任細胞 (ultra competent cell) (E. coli DH5α) .........30
7.2 電穿孔 (electroporation) 勝任細胞 (E. coli Rosetta DE3).32
8. 突變 DNA 之轉形 (transformation) ......................32
8.1 熱休克................................................33
8.2 電穿孔................................................34
9. 以 colony PCR 篩選轉形株...............................34
9.1 colony PCR 反應的溶液組成.............................35
9.2 colony PCR 反應條件...................................36
9.3 電泳膠片分析..........................................36
10. 序列分析..............................................36
11. 麥芽寡糖苷海藻生成酶的生產與純化......................36
11.1 大腸桿菌的培養與酵素之表現...........................36
11.2 細胞破碎.............................................37
11.3 熱處理...............................................37
11.4 透析.................................................37
11.5 離子交換層析.........................................37
11.6 膠體過濾層析.........................................38
12. 蛋白質膠電泳分.......................................38
12.1 試劑之製備...........................................38
12.2 膠體之製備...........................................40
12.3 膠電泳之操作方法.....................................41
12.4 膠片染色與脫色.......................................41
12.4.1 染劑之製備........................................41
12.4.2 脫色液之製備......................................42
13. 蛋白質的定量...........................................42
13.1製備 Dye-reagent stock solution.........................42
13.2 製備 4× Dye-reagent...................................43
13.3 製備 BSA 標準品.....................................43
14. 麥芽寡糖苷海藻糖生成酶之轉糖苷活性測定..................43
14.1活性定義..............................................44
14.2製備 DNS 試劑........................................44
15. 轉糖苷活性之酵素動力學分析..............................45
六、結果與討論..............................................46
1. 電腦模擬.................................................46
1.1 Automated Docking......................................46
1.2 Molecular Dynamic Simulation............................49
2. 突變基因.................................................51
2.1 突變位置之設計........................................51
2.2 引子設計及點突變 PCR.................................52
3. 酵素純化.................................................53
4. 酵素動力學...............................................53
5. 綜合酵素動力學與電腦模擬的結果...........................55
未來展望....................................................55
七、結論....................................................56
八、參考文獻................................................57
圖一、活性區與基質結合模式之假說。........................63
圖二、Autodock 模擬完成後之示意圖。........................64
圖三、Docking後之麥芽寡糖與酵素活性部位之結合情形。.......65
圖四、麥芽三糖於Docking結果中 mg3-1 與酵素活性部位之結合情形。....................................................66
圖五、麥芽三糖於Docking結果中 mg3-3 與酵素活性部位之結合情形。...................................................67
圖六、能量平衡模擬完成後之示意圖。.........................68
圖七、核苷酸定序之結果。...................................69
圖八、蛋白質含量分析之標準曲線。..........................70
圖九、 DNS 測量麥芽五糖濃度之標準曲線。...................71
圖十、原生型 MTSase 之 (A) 離子交換層析； (B) 膠體過濾層析。.72
圖十一、突變型 DXA MTSase 之 (A) 離子交換層析；
(B) 膠體過濾層析。.....................................73
圖十二、突變型 DXA MTSase 之 (A) 離子交換層析；
(B) 膠體過濾層析。.....................................74
圖十三、突變型 RXA MTSase 之 (A) 離子交換層析；
(B) 膠體過濾層析。.....................................75
圖十四、以 10% SDS-PAGE 分析經加熱、離子交換及膠體過濾層析。.76
圖十五、重組之原生型與突變型 MTSase 經 80℃ 熱處理 2 小時後
之殘留活性。.......................................77
表一、 Docking 後 MTSase 與麥芽五糖可能產生之氫鍵..........78
表二、麥芽三糖、四糖及五糖之 Docking energy 和 free energy
的比較.................................................79
表三、能量平衡後 MTSase 與麥芽五糖可能產生之氫鍵...........80
表四、點突變引子...........................................81
表五、表現於 E.coli Rosetta (DE3) 產生原生型與突變型 MTSase
純化後之酵素比活性....................................82
表六、原生型及突變之MTSase以麥芽五糖為基質之轉糖基動力學
參數...............................................83

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