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研究生:曾彥綸
研究生(外文):Yan-Lun Tseng
論文名稱:Simiduia agarivorans SA1T所產蛋白酶之純化與特性探討
論文名稱(外文):Purification and Characterization of a Protease from Simiduia agarivorans SA1T
指導教授:李宗徽李宗徽引用關係
口試委員:謝文陽林俊茂
口試日期:2013-07-05
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:生藥學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:74
中文關鍵詞:Simiduia agarivorans SA1T;蛋白酶;pH;蛋白酶抑制劑;界面活性劑;金屬離子
外文關鍵詞:Simiduia agarivorans SA1T ;serine protease;pH value;protease inhibitor;surfactant;metal ion
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Simiduia agarivorans SA1T 具良好蛋白酶活性,本研究對此菌株所產生的蛋白酶進行分離、純化並探討該酵素的一般性質。探討最佳化培養條件顯示,使用250 mL具凹槽錐形瓶,添加 0.75% tryptone peptone、pH 7.8、培養液 50 mL、25 °C 下,震盪培養 72 小時後,蛋白酶的活性最佳。粗酵素液經由硫酸銨沉澱、透析、並以SDS-PAGE單離出分子量約 120 kDa 的蛋白酶,其最適反應溫度和 pH 分別為 60 °C,和 pH 9;在溫度 4-37 °C 及 pH 8下,酵素有良好穩定性;當超過 37 °C, 則蛋白酶無法穩定維持活性。此蛋白酶在 5 mM 酵素抑制劑 phenylmethanesulfonylfluride (PMSF) 的作用下活性明顯受到抑制,由此推測此酵素為絲胺酸型蛋白酶 (serine type protease) 。在界面活性劑影響方面,此蛋白酶在 SDS 含量超過 0.5% (g/mL) 時活性開始降低,SDS 濃度增至 2% 時活性完全受到抑制。在金屬離子影響方面, 5 mM 的 Mg2+ 和 Ca2+ 會提高蛋白酶的活性,而 Hg2+、Ni2+ 和 Cu2+ 則會降低蛋白酶活性。蛋白酶活性表現在 1.25% (w/v) NaCl下活性最佳,在 2.5% NaCl 下則不具耐受性;蛋白酶與基質液 (酪蛋白) 反應的最佳濃度為12.5 mg/mL。
A protease of Simiduia agarivorans SA1T was purified and characterized in this study. Optimal protease yielding conditions for the culture were determined. For the best protease production, the culture was incubated at 25 °C with 50 mL PY broth (pH 7.8) in 250 mL flask and shaken for 72 hours (75 rpm). Tryptone peptone (0.75%) was added as an additional nutrition in PY broth. The protease was purified by SDS-PAGE after ammonium sulfate precipitation, dialysis, and desalting. The molecular mass of the protease as determined by SDS-PAGE was approximately 120 kDa. The protease showed optimum activity at alkaline pH 9 and temperature 60 °C, and was stable at pH 8 and 4-37 °C, respectively. The protease was characterized as a serine protease, since it was inactivated by 5 mM phenylmethanesulfonylfluride (PMSF). When the protease was treated with various surfactants, the activity was completely inhibited by 2% (g/mL) SDS. Among the metal ions tested, the enzyme activity was found to be enhanced by 5 mM Mg2+ and Ca2+, and was inhibited when treated with 5 mM Hg2+, Ni2+ and Cu2+. The protease showed optimum activity at salt (NaCl) concentration 1.25% (g/mL). However, when salinity was increased to 2.5%, the protease lost 80% of its activity. The activity of this protease was increased with increases of substrate concentrations up to 12.5 mg/mL and was inhibited as the concentrations of substrates were more than 50 mg/mL.
目錄

摘要 viii
Abstract ix
第一章 前言 1
1.1蛋白酶之分類 1
1.2蛋白酶之應用 1
1.2.1洗滌工業之添加劑 2
1.2.2皮革處理添加劑 2
1.2.3食品工業 2
1.2.4藥物設計 3
1.3蛋白質工程 4
1.3.1蛋白質設計原理 4
1.3.2 蛋白質工程的研究目標 5
1.4 文獻回顧 5
第二章 材料和方法 6
2.2實驗材料與設備 6
2.2.1實驗設備 6
2.2.2實驗藥品 7
2.2.3培養基、液配製 8
2.2.4蛋白質電泳藥品配製 8
2.3蛋白酶活性測試 9
2.4蛋白酶之培養條件探討 10
2.4.1蛋白酶誘導物測試 10
2.4.2不同濃度的蛋白酶誘導物測試 10
2.4.3培養液體積測試 10
2.4.4 SA1T之生長速率及蛋白酶活性 11
2.4.5 SA1T之最佳化培養 11
2.5細菌大量培養及誘導酵素產生 11
2.6粗蛋白酶的製備 11
2.7硫酸銨沉澱和透析 12
2.8蛋白質定量 12
2.9蛋白酶純化 13
2.9.1離子交換層析 13
2.9.2疏水性層析法 13
2.9.3膠體過濾層析 14
2.9.4製備式SDS-PAGE電泳 14
2.10蛋白質之電泳分析 15
2.10.1 Native-PAGE蛋白質染色 15
2.10.2 Native-PAGE活性染色 15
2.10.3 SDS-PAGE蛋白質染色 15
2.10.4 SDS-PAGE活性染色 16
2.11酵素最適反應溫度 16
2.12酵素之熱穩定性 17
2.13酵素最適pH的測定 17
2.14酵素在不同pH下之穩定性 17
2.15金屬離子對酵素活性的影響 17
2.16抑制劑對酵素活性的影響 18
2.17界面活性劑對酵素活性的影響 18
2.18氯化鈉濃度對酵素活性的影響 18
2.19基質液濃度對酵素活性的影響 19
2.20 去汙測試 19
第三章 結果 21
3.1 誘導SA1T蛋白酶產生之培養條件指標 21
3.1.1 不同蛋白酶誘導物測試 21
3.1.2 不同tryptone peptone 濃度測試 21
3.1.3不同培養體積測試 21
3.1.4 SA1T之生長速率及蛋白酶活性比較 21
3.1.5 SA1T之最佳化培養結果 22
3.2 SA1T蛋白酶的純化 22
3.2.1 離心 22
3.2.2 硫酸銨沉澱劃分 22
3.2.3 硫酸銨沉澱及透析 22
3.2.4 管柱層析 23
3.2.5 製備式SDS-PAGE純化法 23
3.3 蛋白酶分子量分析 23
3.3.1 Native-PAGE蛋白質染色 23
3.3.2 Native-PAGE活性染色 24
3.3.3 SDS-PAGE蛋白質染色 24
3.3.4 SDS-PAGE活性染色 24
3.4蛋白酶的特性探討 24
3.4.1 酵素最適反應溫度及熱穩定性 24
3.4.2酵素最適反應 pH 值及耐受性 25
3.4.3金屬離子對酵素活性的影響 25
3.4.4抑制劑對酵素活性的影響 25
3.4.5界面活性劑對酵素活性的影響 25
3.4.6氯化鈉濃度對酵素活性的影響 26
3.4.7酪蛋白濃度對酵素活性的影響 26
3.5 粗酵素液於去汙效果測試 26
第四章 討論 28
第五章 結論 33
參考文獻 34


表目錄

Table 1. Purification of extracellular protease by Simiduia agarivorans SA1T. 39
Table 2. Effects of metal ion on protease activity. 40
Table 3. Effects of inhibitor on protease activity. 41
Table 4. Effects of surfactant on protease activity. 42
Table 5. Optimize culture condition. 43
Table 6. Protease characterizatics compared. 44






















圖目錄
Figure 1. Electron micrographs of strain SA1T 45
Figure 2. Effects of different culture nutritions on SA1T protease activity. 46
Figure 3. Effects of different tryptone peptone concentration on SA1T protease activity. 47
Figure 4. Effects of different medium volume on SA1T protease activity. 48
Figure 5. Time course studies on growth and protease activity. 49
Figure 6. Optimizing culture condition. 50
Figure 10. Ammonium sulfate precipitation. 51
Figure 11. Standard curve for tyrosine 52
Figure 12. Standard curve for BSA 52
Figure 13. SDS-PAGE analysis of protease from Simiduia agarivorans SA1T. 53
Figure 14. Native-PAGE analysis of protease from SA1T. 54
Figure 15. SDS-PAGE analysis of protease from SA1T. 54
Figure 16. Native-PAGE activity staining of protease from SA1T. 55
Figure 17. SDS-PAGE (7 %) activity staining of protease from SA1T. 56
Figure 18. SDS-PAGE (12.5%) activity staining of protease from SA1T. 56
Figure 19. Effects of temperature and thermal stability on purified protease. 57
Figure 20. Effects of different pH on purified protease activity. 58
Figure 21. Effects of different pH on purified protease stability. 58
Figure 22. Effects of different NaCl concentration on purified protease activity. 59
Figure 23. Effects of casein concentration of purified protease activity. 60
Figure 24. Cotton cloths wash analysis. 61
Figure 25. Cotton cloths wash analysis. 62
Figure 26. Cotton cloths wash analysis. 63
Figure 27. Chromotogram of DEAE-FF 64
Figure 28. Chromotogram of DEAE-FF 64
Figure 29. Chromotogram of ANX-FF 65
Figure 30. Chromotogram of Q-FF 65
Figure 31. Chromotogram of Q-FF 66
Figure 32. Chromotogram of Q-FF 66
Figure 33. Chromotogram of CM-FF 67
Figure 34. Chromotogram of CM-FF 67
Figure 35. Chromotogram of SP-FF 68
Figure 36. Chromotogram of SP-FF 68
Figure 37. Chromotogram of Phenyl-FF 69
Figure 38. Chromotogram of Saphacryl S-200 69
Figure 39. Chromotogram of Superdex 75 HR 70
Figure 40. Chromotogram of Fractgel TSK HW 50 70

附錄目錄
Appendix 1. Ammonium sulfate precipitation table. 71
Appendix 2. Standard protocol for casting SDS-PAGE gels. 72
Appendix 3. Buffer solution 73
Appendix 4. PY agar、PY broth 74
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