於本研究中，成功插層蛋白質酵素(α-chymotrypsin)於蒙脫土層間中。高分子（D2000）插層之蒙脫土（MMT-D2000）對α-chymotrypsin具有極佳之吸附/插層能力，約為天然蒙脫土（Na-MMT）之2.5倍（MMT-D2000最大之吸附量為4.4 mg/mg clay，Na-MMT為1.7 mg/mg clay）。由X光粉末繞射儀中可得知，MMT-D2000之d-spacing由原本的5.8 nm提高至7.0 nm，亦即α-chymotrypsin與MMT-D2000中之D2000發生置換，而進入其層間。插層之α-chymotrypsin與蒙脫土間具有一定之作用力，不易發生脫落。改變吸附溶液之pH 值，α-chymotrypsin亦能插層進入MMT-D2000層間（除pH 11外），且吸附量隨pH值改變而有差異，於pH 7時最大。α-chymotrypsin吸附於Na-MMT後，其活性大幅下降，且失活速率相當快，酵素活性不因吸附量多寡而改變；插層於MMT-D2000後，其活性並無明顯改變，而與溶液中之α-chymotrypsin相近，酵素活性隨插層量比例增加而增加，且層間的α-chymotrypsin對基質具有一定之選擇性。

In this study, the protein, α-chymotrypsin, was successfully intercalated in the gallery of montmorillonite (MMT). The protein intercalation in MMT preintercalated with polypropylene oxide (D2000) was highly enhanced (4.4 mg/mg clay) as compared to that (1.7 mg/mg clay) adsorbed at the surface of native Na-montmorillonite (Na-MMT). The d-spacing of MMT after α-chymotrypsin intercalation increased from 5.8 to 7.0 nm by XRPD analysis. The result shows that strong interactions occur between α-chymotrypsin and MMT, leading to the pronounced stabilization effect on the enzyme in the gallery of clay. Except for pH 11, the interaction of the enzyme occurred in the whole range of pH used in this study, although the amount intercalation protein varied with pH. The highest extent of intercalation has been observered at pH 7. While α-chymotrypsin adsorbed at the surface of Na-MMT lost most of its enzymatic activity, the catalytic capability of α-chymotrypsin intercalated in MMT toward low molecular weight substrates remained to a significant extent.

目錄
一、緒論------------------------------------------------------1
二、文獻回顧--------------------------------------------------3
2-1 天然黏土的介紹--------------------------------------------3
2-2 蒙脫土的介紹----------------------------------------------3
2-3 X光繞射原理-----------------------------------------------4
2-4 蛋白質吸附原理--------------------------------------------6
2-5 蛋白質吸附蒙脫土------------------------------------------9
2-6 高分子改質蒙脫土-----------------------------------------14
2-7 蛋白質α-chymotrypsin介紹---------------------------------16
2-8 α-chymotrypsin插層MMT-D2000-----------------------------18
2-9 Bio-Rad蛋白質分析原理-----------------------------------20
2-9 酵素固定化的介紹及應用-----------------------------------20
三、實驗方法與步驟-------------------------------------------22
3-1 實驗藥品及儀器-------------------------------------------22
3-1-1 實驗藥品-----------------------------------------------22
3-1-2 實驗儀器-----------------------------------------------25
3-2 蛋白質溶液分析-------------------------------------------26
3-2-1 測定D2000高分子是否會干擾蛋白質溶液分析----------------26
3-2-2 Bio-Rad蛋白質分析--------------------------------------26
3-2-3 微量Bio-Rad蛋白質分析----------------------------------27
3-3 Chymotrypsin吸附/插層蒙脫土之分析測定--------------------27
3-3-1於不同pH値下，chymotrypsin吸附/插層蒙脫土---------------27
3-3-2 不同chymotrypsin添加量吸附/插層蒙脫土------------------29
3-4 Chymotrypsin在蒙脫土上之固定量---------------------------29
3-4-1 Chymotrypsin吸附/插層MMT-D2000後經不同溶液清洗---------29
3-4-2 Chymotrypsin在蒙脫土上之吸附與固定量比較---------------31
3-5 FPLC測定溶液中D2000量------------------------------------32
3-5-1 D2000檢量線--------------------------------------------32
3-5-2 Chymotrypsin吸附/插層MMT-D2000過程中D2000之置換量------32
3-6 利用XRPD測定蒙脫土之層間距-------------------------------33
3-6-1 測定Na-MMT及MMT-D2000層間距----------------------------33
3-6-2 測定chymotrypsin吸附/插層蒙脫土後之層間距--------------33
3-6-3 測定chymotrypsin吸附/插層MMT-D2000後經清洗過程之層間距-34
3-7 TGA測定--------------------------------------------------34
3-7-1 MMT-D2000、D2000及chymotrypsin之TGA測定----------------34
3-7-2 Chymotrypsin在蒙脫土上之固定量-------------------------35
3-8 Chymotrypsin吸附/插層蒙脫土前後，酵素對小分子基質之活性測定-----------------------------------------------------------35
3-8-1製備測定酵素活性所需的溶劑------------------------------35
3-8-2製備酵素和基質溶液--------------------------------------36
3-8-3 Chymotrypsin吸附/插層蒙脫土前後，酵素活性之測----------37
3-8-4不同chymotrypsin起始添加量對吸附/插層蒙脫土後之酵素活性影響-----------------------------------------------------------37
3-8-5 Chymotrypsin吸附/插層蒙脫土前後，酵素活性之最適溫度----38
3-8-6 Chymotrypsin吸附/插層蒙脫土前後，酵素活性之最適pH值----38
3-8-7 Chymotrypsin吸附/插層蒙脫土前後，於47.5℃下隨時間失活之情形-----------------------------------------------------------39
3-8-8 Chymotrypsin吸附/插層蒙脫土前後的酵素活性對溫度之穩定性40
3-8-9不同酵素濃度下，酵素活性與溫度穩定性之關係--------------40
3-8-10不同chymotrypsin起始添加量的酵素活性對溫度穩定性之影響-41
3-9 Chymotrypsin吸附/插層MMT-D2000前後，酵素對
大分子基質之活性測定-----------------------------------------41
3-9-1 Chymotrypsin分解catalase之活性測定---------------------41
3-9-2 Chymotrypsin吸附/插層MMT-D2000後，酵素分解catalase之活性測定---------------------------------------------------------42
3-10 AFM偵測-------------------------------------------------42
3-10-1 Chymotrypsin吸附/插層MMT-D2000------------------------42
3-10-2 Chymotrypsin吸附/插層MMT-D2000後，酵素分解catalase經16
小時後-------------------------------------------------------43
四、結果與討論-----------------------------------------------44
4-1 蒙脫土基本性質-------------------------------------------44
4-1-1 TGA測定------------------------------------------------44
4-1-2 XRPD測定-----------------------------------------------46
4-1-3 D2000之UV/Vis測定--------------------------------------48
4-2 Chymotrypsin最佳吸附/插層環境----------------------------50
4-2-1在不同pH值下，chymotrypsin吸附/插層蒙脫土之分析---------50
4-2-2 Chymotrypsin吸附/插層MMT-D2000後之AFM圖----------------59
4-3不同chymotrypsin添加量吸附/插層蒙脫土---------------------61
4-3-1比較Na-MMT及MMT-D2000對chymotrypsin之吸附/插層能力------61
4-3-2 Chymotrypsin吸附/插層過程中D2000之置換量---------------71
4-3-3 pH值對不同chymotrypsin添加量之影響---------------------75
4-4 Chymotrypsin於蒙脫土上之固定量---------------------------80
4-4-1不同溶液對插層後之chymotrypsin清洗能力之比較------------80
4-4-2 Chymotrypsin於蒙脫土上之吸附與固定量比較---------------84
4-4-3 利用TGA計算chymotrypsin於蒙脫土上之固定量--------------88
4-5 Chymotrypsin吸附/插層蒙脫土前後，酵素對小分子基質
之活性測定---------------------------------------------------93
4-5-1 Chymotrypsin吸附/插層蒙脫土前後，酵素活性之測定--------94
4-5-2不同chymotrypsin起始添加量對吸附/插層蒙脫土
後之酵素活性影響---------------------------------------------97
4-5-3 Chymotrypsin吸附/插層蒙脫土前後，酵素活性之
最適溫度與pH值----------------------------------------------101
4-5-4 Chymotrypsin吸附/插層蒙脫土前後於47.5℃下隨
時間失活之情形----------------------------------------------104
4-5-5 Chymotrypsin吸附/插層蒙脫土前後的酵素活性對
溫度之穩定性------------------------------------------------107
4-5-6不同酵素濃度下，酵素活性與溫度穩定性之關係-------------110
4-5-7不同chymotrypsin起始添加量的酵素活性對溫度穩定性之影響-113
4-6 Chymotrypsin吸附/插層MMT-D2000前後，酵素對
大分子基質之活性測定----------------------------------------117
4-6-1 Chymotrypsin吸附/插層MMT-D2000前後，
分解catalase之活性測定--------------------------------------117
五、結論----------------------------------------------------122
參考資料----------------------------------------------------124

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