臺灣博碩士論文加值系統

於本研究中，利用胰蛋白酶（trypsin）及過氧化氫酶（catalase）與天然蒙脫土（Na+-MMT）及兩種不同之高分子改質之蒙脫土（MMT/POP2000及MMT/POP4000）進行吸附/插層之程序。Trypsin於Na+-MMT及MMT/POP2000皆可成功進行插層，但MMT/POP2000之吸附/插層量相對於Na+-MMT則高出了2.4倍。由粉末式X光繞射儀（XRPD）可知，trypsin進入蒙脫土的層間插層後，其層間為5.7 nm；由POP2000的釋放量得知，trypsin吸附/插層MMT/POP2000時與層間之POP2000發生置換以進入MMT/POP2000的層間。不同的pH吸附環境對trypsin的吸附/插層量具有影響（於pH 4.0時，吸附/插層量最大），且對於trypsin插層與否也具有影響。少量的trypsin吸附/插層於MMT/POP2000時即具有酵素活性；但大量的trypsin吸附/插層於Na+-MMT時才具有酵素活性。
Catalase吸附MMT/POP2000及MMT/POP4000時蒙脫土會發生脫層反應。Catalase對於MMT/POP2000及MMT/POP4000的最大吸附量為Na+-MMT的4倍。不同的pH吸附環境下，對於catalase的吸附量及吸附現象皆有影響（pH環境為4.0時，catalase具有最大之吸附量；於pH 11.0時，不會發生脫層反應）。catalase吸附於Na+-MMT及MMT/POP4000時，蛋白質將不具有酵素活性。

In this study, the interactions of trypsin and catalase with Na+-MMT and POP intercalated MMT were investigated. Intercalation of trypsin into the galleries of Na+-MMT and MMT/POP2000 was successfully achieved. The adsorption/intercalation amount of trypsin in MMT/POP2000 was found 2.4-fold higher than that in Na+-MMT although both of their d-spacings maintained at 5.7 nm. It was observed that the POP molecules originally residing within the galleries of MMT was replaced and released during the trypsin intercalation. Both the surface adsorption and intercalation of trypsin on MMT was significantly influenced by pH, the greatest total adsorption being observed at pH 4.0. The enzymatic activity of trypsin intercalated within galleries of MMT/POP200 was well preserved irrespective of the amount of its total adsorption. However, the enzymatic activity was highly reduced as the low concentration of trypsin was employed in its incubating with Na+-MMT.
Interaction of catalase with MMT/POP2000 and MMT/POP4000, however, led to exfoliation of the clay layered structure. Nevertheless, the enzymes adsorption on MMT/POP2000 and MMT/POP4000 was 4-time higher than that on Na+-MMT. Significant effects of pH on the adsorption of catalase were similarly observed. The layered structure of MMT was only maintained with interacting catalase at pH 11.0. The enzymatic activity of catalase was highly reduced by its adsorption on either Na+-MMT or MMT/POP surfaces probably due to the conformational changes of the enzyme as a result of its extensive interactions with clay.

一 、緒論--------------------------------------------------------------------------01
二 、文獻回顧--------------------------------------------------------------------03
2.1 天然黏土的簡介------------------------------------------------------03
2.2 蒙脫土的簡介---------------------------------------------------------04
2.3 胰蛋白酶（trypsin）之簡介-------------------------------------------05
2.4 過氧化氫酶（catalase）之簡介--------------------------------------07
2.5 蛋白質吸附原理------------------------------------------------------08
2.6 蛋白質吸附蒙脫土---------------------------------------------------11
2.7 高分子插層改質蒙脫土---------------------------------------------14
2.8 X光繞射原理---------------------------------------------------------17
2.9 Bio-Rad 蛋白質染劑之分析原理---------------------------------20
三 、實驗部分--------------------------------------------------------------------21
3.1 實驗儀器及藥品------------------------------------------------------21
3.1.1 實驗儀器---------------------------------------------------------21
3.1.2 實驗藥品---------------------------------------------------------22
3.2 MMT/POP2000及MMT/POP4000之TGA測定--------------25
3.3 蛋白質溶液之分析---------------------------------------------------25
3.3.1 蛋白質溶液之Zeta-potential 分析--------------------------25
3.3.2 利用Bio-Rad 蛋白質染劑測定蛋白質濃度---------------26
3.3.3 利用Bio-Rad 蛋白質染劑測定微量蛋白質濃度---------26
3.4 蛋白質吸附/插層蒙脫土之分析-----------------------------------27
3.4.1 不同pH值下，蛋白質吸附/插層蒙脫土--------------------27
3.4.2 不同蛋白質的添加量吸附/插層於蒙脫土-----------------28
3.4.3 蛋白質在蒙脫土上之固定量---------------------------------29
3.5 溶液中POP2000之置換量測試------------------------------------30
3.5.1 POP2000檢量線------------------------------------------------30
3.5.2 Trypsin吸附/插層MMT/POP2000過程中，POP2000的置換量-------------------------------------------------------------31
3.6 利用XRPD測定蒙脫土之層間距---------------------------------31
3.6.1 測定Na+-MMT、MMT/POP2000、MMT/POP4000之層間距----------------------------------------------------------------31
3.6.2 測定蛋白質吸附/插層蒙脫土後，蒙脫土之層間距-------32
3.7 製備TEM的切片樣品-----------------------------------------------32
3.7.1 製備Na+-MMT、MMT/POP2000、MMT/POP4000切片樣品----------------------------------------------------------------32
3.7.2 製備catalase吸附於Na+-MMT、MMT/POP2000、MMT/ POP4000的切片樣品--------------------------------------------33
3.8 Trypsin吸附/插層蒙脫土前後，酵素之活性測定---------------33
3.8.1 製備酵素活性樣品---------------------------------------------33
3.8.2 測定在不同trypsin添加量之下吸附/插層蒙脫土後對酵 素活性的影響----------------------------------------------------34
3.9 Catalase吸附蒙脫土前後，酵素之活性測試---------------------35
3.9.1 製備酵素活性樣品---------------------------------------------35
3.9.2 Catalase吸附/插層蒙脫土後對酵素活性的影響----------35
四 、結果與討論-----------------------------------------------------------------37
4.1 蒙脫土基本性質------------------------------------------------------37
4.1.1 蒙脫土之TGA測定--------------------------------------------37
4.1.2 Na+-MMT、MMT/POP2000、MMT/POP4000之XRPD測定----------------------------------------------------------------38
4.2 蛋白質溶液之Zeta-potential測定---------------------------------40
4.3 於不同pH環境下，trypsin吸附/插層蒙脫土之分析-----------41
4.4 不同添加量的trypsin進行吸附/插層蒙脫土之分析-----------50
4.4.1 Trypsin吸附/插層蒙脫土之分析----------------------------50
4.4.2 Trypsin吸附/插層於蒙脫土上之固定量分析-------------62
4.5 Trypsin不同的起始添加量對吸附/插層蒙脫土後之酵素活性影響---------------------------------------------------------------------64
4.6 不同pH環境之下，catalase吸附蒙脫土之分析-----------------70
4.7 Catalase吸附於蒙脫土後，利用TEM觀測蒙脫土的結構變化---------------------------------------------------------------------------78
4.8 不同添加量的catalase吸附蒙脫土之分析-----------------------83
4.8.1 Catalase吸附量之分析----------------------------------------83
4.8.2 添加微量的catalase吸附於MMT/POP4000---------------93
4.8.3 Catalase吸附於蒙脫土上之固定量分析-------------------95
4.9 Catalase吸附蒙脫土前後，酵素活性之測定---------------------97
五 、結論--------------------------------------------------------------------------99
參考文獻------------------------------------------------------------------------101

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