臺灣博碩士論文加值系統

本研究以Escherichia coli BL21(DE3)/pTahl10/pT-groE生成基因重組蛋白Amidohydrolase酵素(AHL)，並以French Press將細胞打碎，冷凍離心收集即粗萃細胞液，再依序經過:硫酸銨沈澱法、弱陰離子交換法(DEAE)，最後再以凝膠過濾法層析純化該酵素並判別其分子量大小。結果顯示經硫酸銨沈澱之後，純化酵素之比活性(Specific activity)為細胞粗萃之7.9倍，再經過弱陰離子交換法之後，酵素活性為細胞粗萃之31.3倍，最後經過Sephacryl S-100 HR凝膠過濾層析法之後，酵素活性可提高至110倍以上。並探討不同處理下之Amidohydrolase酵素之動力學，及評估可能之動力模式及參數，以利往後對該酵素之應用。

In this study, the AHL genes in a recombinant strain E coli. was cultated by fermentation；two plasmids of pTal10 and pT-groE were subsequently transformed into E coli. BL21 host cell for the mass production of Amidohydrolase enzyme. After cultration, the cells were disrupt by Franch pressure and centrifuged to obtained cell-free extract. The purified process were used：ammonium sulfate precipiate 、weak anion exchange chromatography (DEAE) and finally size exclusion chromatography to determine the molecule weight .The experimental results showed that specific Amidohydrolase enzyme activity of the ammonium sulfate was 7.9 fold higher than crude cell. However, after being purified by anion-exchange chromatography packed with DEAE sepharose CL-6B, the enzyme activity was enhanced to 31.3 fold of the whole cell activity. With additional gel filtration treatment packed with S-100 sepharcyl, the specific activity of the purified Amidohydrolase enzyme was 110 fold higher than that of the crude cell extract. The kinetics of Amidohydrolase enzyme was investigated for different forms of the enzyme. The optimal kinetic parameters for the proposed models were estimated numerically.

中文摘要.....................................i
Abstract.....................................ii
目錄.....................................iii
圖目錄.....................................vii
表目錄.....................................viii
第一章 序論............................1
1-1抗生素之定義與分類...................1
1-2研究動機與目的............................2
第二章 研究原理............................4
2-1酵素純化原理............................4
2-1-1 沈澱法 ............................4
2-1-2 凝膠層析過濾 ...................5
2-1-3 離子交換............................6
2-1-4 疏水性界面層析法...................8
2-1-5 親和力層析............................8
2-2 分子量測定法...........................12
2-2-1 凝膠過濾層析法..................12
2-2-2 蛋白質電泳實驗..................13
2-3 酵素反應動力學..................14
2-3-1 基本酵素動力學..................14
2-3-2 其他酵素反應動力學模式.........17
第三章 實驗設備材料與方法..................21
3-1 藥品及其儀器...........................21
3-2 菌種培養與定量測定..................23
3-2-1菌種簡介...........................23
3-2-2培養基的製備...........................23
3-2-3 菌種的培養...........................24
3-2-4 細胞菌液的光學密度測量.........24
3-3 菌體之酵素液之製備..................25
3-3-1 全細胞菌體之製備..................25
3-3-2 粗萃酵素液之製備..................25
3-3-3 純化酵素液之製備..................25
3-3-3-1 沈澱法硫酸銨之製備..................25
3-3-3-2 離子交換分離管柱之製備.........26
3-3-3-3 疏水性分離管柱之製備.........26
3-3-3-4 凝膠過濾層析法分離管柱之製備27
3-3-3-5 除鹽分離管柱之製備..................27
3-4酵素之定量與活性分析..................27
3-4-1 蛋白質總量之測定..................27
3-4-1-1 UV A280之測定法(Absorbance at 280 nm)27
3-4-1-2 Bradford Assay之方法.........27
3-4-1-3 Lowry Assay之方法..................28
3-4-2 酵素之活性分析實驗..................29
3-4-2-1 粗萃酵素之活性分析實驗.........29
3-4-2-2 純化酵素之活性分析實驗.........29
3-4-3 蛋白質電泳實驗..................30
第四章 結果與討論...........................31
4-1 Amidohydrolase酵素純化最適步驟之探討.....31
4-1-1 最佳化之探討..................31
4-1-2 純化程度之測試..................40
4-2 Amidohydrolase酵素之定量分析.........43
4-2-1 分子量測定法..................43
4-2-2 蛋白質總量之測定..................46
4-3環境因素對粗萃和純化酵素之影響.........46
4-3-1粗萃酵素和純化酵素之持久性探討.........46
4-3-2 粗萃酵素和純化酵素之熱穩定性探討.......47
4-3-3 粗萃酵素和純化酵素之酸鹼穩定性探討.....48
4-3-4 粗萃酵素和純化酵素之活化能探討.........49
4-4 Amidohydrolase酵素之動力學研究.........51
4-4-1粗萃及純化酵素之動力學研究.........53
4-4-1-1粗萃酵素之動力學研究.........53
4-4-1-2純化酵素之動力學研究.........53
第五章 結論與建議...........................56
5-1 結論....................................56
5-2展望與建議...........................56
參考文獻....................................58
附錄....................................66

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