合成中空類沸石咪唑骨架材料及空間侷限性對於酵素活性影響之研究

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研究生：	孫庭謙 Ting-Qian Sun
論文名稱：	合成中空類沸石咪唑骨架材料及空間侷限性對於酵素活性影響之研究
指導教授：	謝發坤 Fa-Kuen Shieh
口試委員:
學位類別：	碩士 Master
系所名稱：	理學院 - 化學學系 Department of Chemistry
論文出版年：	2020
畢業學年度：	108
語文別：	中文
論文頁數：	69
中文關鍵詞：	金屬有機骨架材料、類沸石咪唑骨架材料、中空類沸石咪唑骨架材料、酵素、酵素活性
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大多數固定化酵素由於材料和載體可能影響其結構變化、底物的擴散性、阻礙底物活性位點結合等等，其活性常低於未固定化之酵素。因此，具有可調性孔洞又能保護酵素避免受外界失活物質傷害的金屬有機骨架材料 (Metal-organic Frameworks, MOFs) 則替酵素固定化帶來新的發展可能。而本實驗室於2017年發表在JACS 的文章中，提出以原位創新合成方法 (de novo) 進行類沸石咪唑骨架 (Zeolitic imidazolate frameworks) 材料封裝酵素時，材料的空間限制會對酵素產生侷限性 (Spatial confinement) 之理論。同時利用尿素具有讓蛋白質結構展開而失去活性的特性，發現此材料酵素生物複合體因為侷限效應，在高濃度尿素下依然保有生物活性。為了更進一步探討材料的空間限制對於酵素活性表現的影響，如何開發具有保護功能又不會使得酵素自由度受限的材料，便成為一個重要的課題，而中空材料其內部空腔有機會解決空間限制對酵素活性的影響，因此本篇研究選擇中空金屬有機骨架材料作為研究模板。本研究利用不同的合成方法，合成中空(Hollow) 的類沸石咪唑骨架材料 Hollow ZIF-8以及 HZIF-90@ZnTA 並將酵素封裝於其中，探討酵素因空間侷限性減少所造成的活性變化，並比較兩者實驗結果。此外本研究參考最近文獻發表實驗過程，也成功在10分鐘之內利用單寧酸蝕刻，快速的製作出新型中空類沸石咪唑骨架材料 CAT@HZIF-90@ZnTA ，此研究成果結果或許能對於後續研究中空材料以及空間侷限性和酵素活性之間的關係提供一個嶄新的研究平台。

Immobilized enzymes usually showed lower enzymatic activity than free ones after applying for additional materials or supports, which might influence substrates diffusion, active sites accessibility, subunit dissociation, conformational change, especially volume-confined immobilization. It is necessary to find the potential material which overcomes the above-mentioned shortcomings and provides shell protection for embedded biomolecules to against external denaturing agents. Therefore, metal-organic frameworks (MOFs) with tunable porosity, and variable internal surface property for a targeted application, might be a suitable candidate for advanced enzyme immobilization.
Recently, we reported a paper regrading an additional strength of the de novo approach by demonstrating that embedded enzymes in metal-organic frameworks (MOFs) via de novo approach remain biological functions under a wider range of reaction conditions such as urea. The enhanced stability of the of enzyme molecules arises from the confinement effects provided by MOF structure. However, the enzymatic activity is not as well as the activity of free enzymes, which might be caused by the influence of spatial confinement on the enzymatic functionality.
In order to further investigate the influence of spatial confinement on the enzymatic functionality, herein, we synthesized the new hollow structure of Zeolitic imidazolate frameworks (Hollow ZIF-8 and HZIF-90@ZnTA) by using a modified approach based on a recent report , which embedded enzymes inside of void structures that mimic cytoplasmic conditions with less restricted environment. Accordingly, this part work may contribute to investigate the hollow structure material and the influence of spatial confinement on the enzymatic functionality.

中文摘要    I
ABSTRACT    II
目錄    III
圖目錄    VII
表目錄    IX
第一章  緒論    1
1-1  金屬有機骨架材料    1
1-1-1  簡介    1
1-1-2  類沸石咪唑骨架材料    4
1-1-3  類沸石咪唑骨架材料-8/-67/-90    5
1-2  酵素及酵素固定化    8
1-2-1  簡介    8
1-2-2  過氧化氫酶(Catalase, CAT)    10
1-2-3  胰凝乳蛋白酶(Chymotrypsin, CTRY)    11
1-3  研究動機與目的    12
第二章  實驗部分    13
2-1  實驗藥品    13
2-2  實驗儀器    15
2-3  實驗儀器原理    16
2-3-1  X 射線粉末繞射儀 (Powder X-ray Diffractometer, PXRD)    16
2-3-2  場發掃描式電子顯微鏡 (Field-emission Scanning Electron Microscope, SEM)    17
2-3-3  穿透式電子顯微鏡 (Transition Electron Microscope, TEM)    19
2-3-4  紫外/可見光分光光譜儀 (UV/Vis Spectrophotometer)    19
2-3-5  微孔洞及表面積分析儀 (Micropore Size and Surface Area Analyzer)    20
2-3-6  熱重分析儀 (Thermogravimetric Analyzer, TGA)    22
2-4  實驗步驟    23
2-4-1  ZIF-67 包覆於 ZIF-8 雙層材料之合成    23
2-4-2  中空ZIF-67@ZIF-8 材料之合成    23
2-4-3  胰凝乳蛋白酶包覆於 ZIF-67 與 ZIF-8 雙層結構之合成    24
2-4-4  胰凝乳蛋白酶包覆於中空 ZIF-67@ZIF-8 之合成    24
2-4-5  過氧化氫酶包覆於 ZIF-90 之合成    25
2-4-6  胰凝乳蛋白酶包覆於 ZIF-90 之合成    25
2-4-7  過氧化氫酶/胰凝乳蛋白酶包覆於中空 ZIF-90 之合成    26
2-4-8  材料酵素包覆量之測定    27
2-4-9  過氧化氫酶活性之檢測    28
2-4-10 胰凝乳蛋白酶活性之檢測    29
2-4-11 過氧化氫酶/胰凝乳蛋白酶在尿素環境下之活性檢測    30
第三章  結果與討論    31
3-1  CTRY@ZIF-67@ZIF-8 之雙層結構材料鑑定與活性實驗    31
3-1-1  CTRY@ZIF-67@ZIF-8之X射線粉末繞射儀圖譜分析    31
3-1-2  CTRY@ZIF-67@ZIF-8之穿透式電子顯微鏡影像分析    32
3-1-3  CTRY@ZIF-67@ZIF-8中空材料之活性比較    33
3-2  CAT@HZIF-90@ZnTA 之中空結構材料鑑定與活性實驗    34
3-2-1  CAT@HZIF-90@ZnTA 之X射線粉末繞射儀圖譜分析    34
3-2-2  CAT@HZIF-90@ZnTA 之掃描式電子顯微鏡影像分析    35
3-2-3  CAT@HZIF-90@ZnTA 之穿透式電子顯微鏡影像分析    36
3-2-4  CAT@HZIF-90@ZnTA之氮氣等溫吸脫附實驗分析    37
3-2-5  CAT@HZIF-90@ZnTA之熱重分析實驗    39
3-2-6  CAT@HZIF-90@ZnTA 材料之活性比較    40
3-2-7  CAT@HZIF-90@ZnTA 材料在尿素環境下之活性比較    41
3-2-8  過氧化氫酶在酸性環境下之活性比較    42
3-2-9  材料對於過氧化氫消耗之活性比較    43
3-2-10  CAT@HZIF-90@ZnTA 有無使用針筒過濾器之活性比較    44
3-2-11  CAT@HZIF-90@ZnTA 材料之活性比較(使用針筒過濾器)    45
3-3  CTRY@HZIF-90@ZnTA 之中空結構材料鑑定與活性實驗    46
3-3-1  CTRY@HZIF-90@ZnTA 之X射線粉末繞射儀圖譜分析    46
3-3-2  CTRY@HZIF-90@ZnTA 之穿透式電子顯微鏡影像分析    46
3-3-3  CTRY@HZIF-90@ZnTA 材料之活性比較    47
3-3-4  CTRY@HZIF-90@ZnTA 在尿素環境下之活性比較    47
3-3-5  HZIF-90@ZnTA & ZIF-90材料對於活性的影響    48
3-3-6  胰凝乳蛋白酶在酸性環境下之活性比較    48
第四章  結論與未來展望    49
參考文獻及資料    50


                                

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