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研究生:陳尚文
研究生(外文):Chen, Shung-Wen
論文名稱:α-半乳糖苷酶之酵素學探討與應用
論文名稱(外文):Enzymatic Characterization and Application of α- Galactosidase
指導教授:黃贊勳
指導教授(外文):Hwang, Tzann-Shun
口試委員:黃贊勳沈賜川謝建正
口試委員(外文):Hwang, Tzann-ShunShen, Szu-ChuanHsieh, Chien-Cheng
口試日期:2012-12-28
學位類別:碩士
校院名稱:中國文化大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:76
中文關鍵詞:α-半乳糖苷酶
外文關鍵詞:α-Galatosidase
相關次數:
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α-半乳糖苷酶已用於食品工業、造紙工業、醫藥工程等產業上,但在許多工業應用上,α-半乳糖苷酶面臨著不同pH值與產品熱處理過程中的活性問題,進而影響到產品之製成率,因此高熱穩定性、化學穩定性之α-半乳糖苷酶是目前提高產品製成率的解決方法。
本研究利用選殖至大腸桿菌DH5α之嗜熱菌Thermus thermophilus HB8 melA基因,於培養後大量表現耐熱性α-半乳糖苷酶,純化後測試此酵素在不同緩衝液的最佳pH值,並測試不同濃度之金屬離子、Glycerol、BSA、KCl、SDS與儲存溫度對其活性之影響,不同溫度之酵素動力學常數。在對反應液條件的探討結果顯示,純化的 α-半乳糖苷酶在磷酸鹽緩衝溶液中,在pH 7.4 時有最佳之酵素活性;在10 mM 的金屬離子Zn2+與Cu2+存在下,會顯著抑制α-半乳糖苷酶的活性;於反應液中含有0.1%、0.5%、1% 的Glycerol 則有相對較高之活性,但高於 3% 的Glycerol時,α-半乳糖苷酶活性隨著濃度之增加而減少;於反應液中含有濃度 0.5% 至10% 的 BSA可增加酵素之活性,但含有濃度 0.1% 至 1% 的 KCl 對α-半乳糖苷酶活性都無明顯之影響;於反應液中添加 SDS 會使酵素活性劇烈下降;在酵素特性的探討結果顯示,其酵素活性於 25℃、70℃、80℃ 與 90℃ 下半衰期大約分別為607天、71小時、46小時與20小時。Vmax 值、KM 值與 kcat 值於溫度 25℃、70℃ 與 90℃ 分別為2.1、44.8與322.6 μmole/mg∙min;KM值為 0.17、1.71與 2.71 mM;kcat 值為 7.5、161.4與1161.3 sec-1。分析結果顯示出Thermus thermophilus HB8之α-半乳糖苷酶能於高溫下展現高穩定性的特性。

α-Galatosidase (α-GAL) has been applied in various industries, including food industry, paper-making industry and medical application. However, there are some problems concerning the enzyme activity needed to be solved, for example the enzyme need to perform its activity under the broad range of pH and at high temperature, or it may reduce the productivity in industrial applications. Therefore, it is attempted to find a good α-GAL which has the high stability at different temperature and pH to have high productivity.
In this study, the Thermus thermophilus HB8 melA gene was cloned into E. coli DH5α to overexpress T. thermophilus α-GAL. The purified α-GAL was used to examine the enzyme activity with different buffers, different metal ions, and different concentration of glycerol, BSA, KCl, and SDS. According to the results, α-GAL showed the highest activity in sodium phosphate buffer of pH 7.4, and the activity of α-GAL was found to be suppressed by 10 mM Zn2+ and Cu2+ ion in the assay reaction. The activity of α-galatosidase was found to be enhanced when the assay reaction was supplied with glycerol in the final concentration from 0.1% to 1%, but the activity was suppressed when supplied with glycerol in the final concentration of higher than 3%; while the activity was also enhanced when supplied with BSA in the final concentration from 0.5% to 10%. The activity of α-GAL was found to have no influence when the assay reaction supplied with KCl in the final concentration from 0.1% to 1%, but the activity of α-GAL was found to be reduced when the assay reaction containing SDS. The stability of enzyme was also invertigated. Under the optimal assay conditions, the purified enzyme was used to analyze kinetic parameters at different temperature. The half-life of enzyme was found to be 607 days, 71 hours, 46 hours, 20 hours at the storage temperature of 25℃, 70℃, 80℃, 90℃, respectively. The kinetic parameters, Vmax was determined to be 2.1、44.8 and 322.6 μmol/mg.min at 25℃、70℃ and 90℃, respectively; KM was 0.17、1.71 and 2.71 mM at 25℃、70℃ and 90℃, respectively; kcat was 7.5、161.4 and 1161.3 sec-1 at 25℃、70℃ and 90℃, respectively. In this study, the best condition for the activity of T. thermophilus HB8 α-GAL was determined with a good stability in high temperature.

摘要 vi
英文摘要 viii
壹、 研究背景 1
一、自然界存在之α-半乳糖苷種類 1
二、α-半乳糖苷酶種類與來源 2
三、α-半乳糖苷酶之應用 4
四、α-半乳糖苷酶特性機制之研究 9
五、影響α-半乳糖苷酶活性的因子 11
六、嗜熱菌 13
七、研究目的與預期結果 14
貳、 材料與方法 16
一、試藥及實驗相關酵素 16
二、儀器設備 16
三、實驗方法 16
3.1 Tt-melA 基因於大腸桿菌中之表現 16
3.2 α-半乳糖苷酶之初步純化 16
3.3α-半乳糖苷酶粗萃液之熱處理 17
3.4 鎳親和性管柱層析法 17
3.5膠體過濾層析法 19
3.6蛋白質定量-Bradford法 19
3.7蛋白質聚丙醯胺膠體電泳之分析 20
3.8酵素活性與特性分析 22
3.8.1活性測試 22
3.8.2不同緩衝溶液與pH之測定 23
3.8.3不同金屬離子之測定 24
3.8.4 Glycerol濃度之影響 25
3.8.5不同BSA濃度之影響 26
3.8.6 SDS濃度之影響 27
3.8.7酵素穩定度分析 28
3.8.8不同溫度之動力學常數測試 29
參、 結果與討論 30
一、α-半乳糖苷酶的純化 30
1.1α-半乳糖苷酶之鎳親和性管柱層析 30
1.2α-半乳糖苷酶之離子交換管柱層析 31
1.3重組α-半乳糖苷酶結構的分析 32
二、酵素活性與特性分析 32
2.1酵素最適pH的分析 33
2.2 酵素中雙硫鍵角色的探討 33
2.3金屬離子對酵素活性的影響 34
2.4 Glycerol濃度對酵素活性的影響 34
2.5 BSA對酵素活性的影響 35
2.6 KCl濃度對酵素活性的影響 35
2.7 SDS對酵素活性的影響 36
2.8酵素對熱穩定度的探討 36
2.9不同溫度的動力學常數分析 37
肆、 參考文獻 39
伍、 圖 48

圖目錄

Figure 1. 各種 α-半乳糖苷寡糖之結構 2
Figure 2. Glycosidase 之保留構型機制 10
Figure 3. Glycosidase 之反向構型機制 11
Figure 4. 4-Nitrophenyl-alpha-D-galactopyranoside之結構圖 23
Figure 5. SDS-PAGE蛋白質電泳分析純化的α-半乳糖苷酶 48
Figure 6. SDS-PAGE蛋白質電泳分析鎳親和性管柱層析純化的α-半乳糖苷酶 49
Figure 7. 離子交換管柱層析純化的α-半乳糖苷酶 50
Figure 8. SDS-PAGE 蛋白質電泳分析離子交換管柱層析純化的α-半乳糖苷酶 51
Figure 9. 重組α-半乳糖苷酶的膠體過濾層析法分析 52
Figure 10. 緩衝溶液與pH值對α-半乳糖苷酶活性的影響 53
Figure 11. 雙硫鍵還原劑對α-半乳糖苷酶的影響 54
Figure 12. 金屬離子對α-半乳糖苷酶的影響 55
Figure 13. 甘油 (Glycerol) 對α-半乳糖苷酶的影響 56
Figure 14. 牛血清蛋(BSA)白對α-半乳糖苷酶活性的影響 57
Figure 15. 離子強度對α-半乳糖苷酶活性的影響 58
Figure 16. SDS對α-半乳糖苷酶活性的影響 59
Figure 17. α-半乳糖苷酶於 25℃的穩定度探討 60
Figure 18. α-半乳糖苷酶於 70℃的穩定度探討 61
Figure 19. α-半乳糖苷酶於 80℃的穩定度探討 62
Figure 20. α-半乳糖苷酶於 90℃的穩定度探討 63
Figure 21. α-半乳糖苷酶於 25℃的動力學參數分析 64
Figure 22. α-半乳糖苷酶於 70℃的動力學參數分析 65
Figure 23. α-半乳糖苷酶於 90℃的動力學參數分析 66

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