臺灣博碩士論文加值系統

本論文嘗試以IRA-900樹脂吸附單成分酵素溶液，實驗所使用的酵素為葡萄糖氧化酶(GOx)。而本研究共分為利用不同樹脂種類及其粒徑大小與不同緩衝液濃度、鹽類濃度和溫度，探討吸附最佳化條件、吸附熱力學，以及脫附條件之探討。
由實驗結果得知，當GOx溶於磷酸緩衝溶液中時，其pH值大於等電點，GOx帶負電。故強陰離子型交換樹脂IRA-900具有最佳之吸附效果；而在相同樹脂克數下，當樹脂粒徑越小時，其吸附量越高；隨著緩衝液濃度升高，GOx之吸附效果降低；隨著pH值、溫度升高，GOx之吸附效果隨之增高，但是活性隨著溫度上升而降低。由實驗結果得知，在使用粒徑為74μm - 250μm樹脂，吸附緩衝液為0.05 M、pH 7.8之磷酸緩衝液，並在25oC恆溫吸附下會有最佳吸附率和活性。
以Semi-reciprocal plot線性迴歸分析，確定樹脂吸附GOx符合Langmuir isothermal model，且酵素在吸附過程中的熱力學變化，均為吸熱反應。
在脫附實驗方面，使用醋酸緩衝溶液當作脫附液會造成酵素的失活，故使用磷酸緩衝溶液當作脫附液；在其條件下，增加脫附緩衝液濃度以及讓其pH值越趨近於等電點，脫附量漸增；當添加KCl濃度增高有助於酵素脫附率之提升，在1.0M KCl的脫附緩衝液濃度下，脫附率趨近100%。

In this study, the application of ion-exchange resin Amberlite IRA-900 as ion-exchange absorbent glucose oxidase (GOx). The effects of adsorption buffers concentration, pH, temperature and salt concentration on single enzyme adsorption and thermodynamics were investigated. Finally, it was discussed with desorption conditions.
For enzyme, the amount of adsorption was higher at small particle size of ion-exchange resin. The results show that the amount of enzyme adsorbed increases as the adsorption buffer concentration, temperature, pH value and salt concentration increase. In addition, the adsorption isotherms well fitted by the Langmuir isothetrmal model from the semi-reciprocal plot analysis.
For desorption condition, the amount of desorption was higher at low pH values. The results show the amount of enzyme desorbed increases as desorption buffer concentration and salt concentration increase.

摘要 I
Abstract II
目錄 III
表目錄 VI
圖目錄 VII
符號說明 IX
第一章緒論 1
1.1 研究動機與目的 1
1.2 葡萄糖氧化酶簡介 2
1.2.1 葡萄糖氧化酶醱酵培養 3
1.2.2葡萄糖氧化酶基本特性與結構 4
1.2.3葡萄糖氧化酶的應用 7
1.3 蛋白質層析純化技術 8
1.3.1 蛋白質層析技術 8
1.3.2 膠體過濾層析 9
1.3.3 親和層析法 10
1.3.4 疏水性層析 11
1.3.5 離子交換層析 12
1.4 生物燃料電池 14
1.4.1 生物燃料電池簡介 14
1.4.2 生物燃料電池優點 14
1.4.3 生物燃料電池分類 15
1.4.3.1 依照使用方法之催化型式分類 15
1.4.3.2 依電子轉移方式分類 16
1.5 葡萄糖氧化酶和生物燃料電池 16
第二章 吸附基礎理論與文獻回顧 17
2.1 吸附基礎理論 17
2.1.1物理吸附 17
2.1.2 化學吸附 17
2.1.3交換吸附 18
2.2等溫吸附模式 19
2.2.1 Langmuir等溫吸附模式 19
2.3 影響吸附因素 20
2.3.1 吸附劑性質 20
2.3.2 蛋白質結構及穩定度 22
2.3.3 pH效應 23
2.3.4 溫度效應 24
2.3.5 鹽類效應 25
2.4 吸附熱力學探討 26
第三章 材料與實驗方法 28
3.1實驗藥品與儀器 28
3.1.1藥品部分 28
3.1.2儀器部分 29
3.2實驗步驟及分析方法 29
3.2.1樹脂前處理 29
3.2.2酵素吸附動力實驗 30
3.2.3酵素吸附實驗 31
3.2.4添加鹽類對酵素恆溫吸附之影響 32
3.2.5酵素脫附動力實驗 33
3.2.6酵素脫附實驗 33
3.2.7添加鹽類對酵素恆溫脫附之影響 34
3.3分析方法 35
3.3.1酵素之濃度分析(enzyme assay) 35
3.3.2濃度分析檢量線 35
3.3.3活性分析 36
第四章結果與討論 38
4.1 不同樹脂種類及粒徑對酵素吸附量之影響 38
4.1.1 不同樹脂種類對酵素吸附量影響 38
4.1.2 不同樹脂粒徑對GOx吸附量影響 42
4.2 酵素等溫吸附探討 45
4.2.1 緩衝液之pH值以及濃度對GOx吸附量影響 45
4.2.2鹽類效應對GOx吸附量影響 47
4.2.3溫度效應對GOx吸附量影響 49
4.2.4 Langmuir吸附參數分析 51
4.2.5 Langmuir吸附參數分析 54
4.3 GOx脫附條件的探討 56
4.3.1緩衝液之pH值以及濃度對GOx脫附量影響 57
4.3.2鹽類效應對GOx脫附量影響 59
第五章 結論 62
參考文獻 64
自述 70

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