利用原位創新法及機械力化學法合成酵素金屬有機骨架複合材料及其活性比較

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姓名

沈玉倢(Yu-Jie Shen) 查詢紙本館藏

畢業系所

化學學系

論文名稱

利用原位創新法及機械力化學法合成酵素金屬有機骨架複合材料及其活性比較

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至系統瀏覽論文 (2026-8-23以後開放)

摘要(中)

金屬有機骨架材料 (Metal-organic Frameworks, MOFs) 近年來成為最熱門的孔洞材料之一，也因具孔洞多樣性、高比表面積以及熱穩定性等多種優勢，使其應用性非常多元化，其中一項應用即為酵素固定化之載體。本實驗室於2015年領導全球，開創出原位創新合成法 (de novo approach)，成功將酵素包覆於MOFs之中，酵素依然保有生物活性，且藉由材料提供的孔洞篩選性得到良好保護，緊接著於2019年再次領先世界，利用機械力化學法 (Mechanochemical method) 將酵素包覆於MOFs之中。上述主要包覆的酵素為過氧化氫酶，其具有受質小且不需要輔酶的特性，然而在鹼性環境下較易失去活性。為了要讓上述系統能夠應用更多的酵素，因此本研究也包覆了脲酶，其具有較慢的反應速率，在實驗進行時可以更容易觀察催化過程，將脲酶包覆在材料當中，將可成為一個觀察酵素催化的全新平台。本研究成功以機械力化學法合成Enzyme@ZIF-90，並透過各種儀器鑑定材料之特性。雖然對於測量脲酶活性並未得到良好的結果，但同時也發現利用機械力化學法合成的CAT@ZIF-90不但與原位水相合成一樣，酵素保留良好的生物活性，但此法具有更高的產率，使其應用在工業上具有極大的潛力。

摘要(英)

Metal-organic frameworks (MOFs) have become one of the most popular porous crystalline materials in recent years. Because of their porous diversity, large specific surface area and thermal stability, they are promising for a wide range of applications, for example enzyme immobilization is one of the attractive areas for our interests. In 2015, our group encapsulated catalase (CAT) enzyme into Zeolitic imidazole framework-90, CAT@ZIF-90, via mild water-based de novo approach, and afterward encapsulated enzymes into MOFs, Enzyme@MOFs, via a mechanochemical method in 2019. One of the mainly above-mentioned enzymes is catalase, which has the characteristics of a small substrate and no need for the coenzyme, but it is easier to lose activity in an alkaline environment. In order to expand the applications of de novo approach, we also encapsulated urease, which has a relatively lower reaction rate that provides a new platform for observing enzyme catalysis. In summary, we successfully synthesized Enzyme@ZIF-90 via a mechanochemical method and identified the characteristics through various instruments. Although urease did not show the expected activity, this method still retains catalase activity and improves the yield. Therefore, it still has a great potential for the further industrial applications.

關鍵字(中)

★ 金屬有機骨架材料
★ 酵素固定化

關鍵字(英)

★ Metal-organic Framworks
★ Enzyme Immobilization

論文目次

目錄
中文摘要 i
Abstract ii
目錄 iii
圖目錄 vii
表目錄 x
第一章緒論 1
1-1金屬有機骨架材料 (Metal-organic Frameworks) 1
1-2類沸石咪唑骨架材料 (Zeolitic Imidazolate Frameworks) 4
1-3機械力化學法 6
1-4酵素固定化 (Enzyme Immobilization) 8
1-5酵素—過氧化氫酶和脲酶 11
1-5-1過氧化氫酶 (Catalase, CAT) 11
1-5-2脲酶 (Urease, URS) 11
1-5-3蛋白酶 (Protease) 12
1-6研究動機與目的 13
第二章實驗部分 14
2-1實驗藥品 14
2-2實驗合成設備與鑑定儀器 17
2-2-1實驗合成設備 17
2-2-2實驗鑑定儀器 18
2-3實驗儀器之原理 19
2-3-1 X光粉末繞射儀 (Powder X-ray Diffractometer, PXRD) 19
2-3-2場發掃描式電子顯微鏡 (Field-emission Scanning Electron Microscope, FE-SEM) 20
2-3-3紫外光可見光分光光譜儀 (Ultraviolet-Visible Spectrophotometer, UV-Vis) 21
2-3-4螢光光譜儀 (Fluorescence Spectrophotometer, FL) 22
2-3-5等溫氮氣吸/脫附測量儀 (Accelerated Surface Area and Porosimetry System, ASAP) 24
2-3-6熱重分析儀 (Thermogravimetric Analyzer, TGA) 27
2-3-7十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE） 28
2-4實驗步驟 30
2-4-1原位創新法 (de Novo approach) 合成Enzyme@ZIF-90 30
2-4-2機械力化學法 (Mechanochemistry) 合成Enzyme@ZIF-90 30
2-4-3原位創新法 (de Novo approach) 合成Enzyme@MOF-74 31
2-4-4偵測材料中蛋白質濃度 32
2-4-5偵測脲酶活性之方法——酚紅 (Phenol red) 以及艾氏試劑 (Ehrlich’s reagent) 34
2-4-6偵測過氧化氫酶活性之方法 37
2-4-7酵素金屬有機骨架複合材料之蛋白質凝膠電泳分析 39
第三章結果與討論 41
3-1類沸石咪唑骨架材料包封脲酶 (URS@ZIF-90) 之材料鑑定 41
3-1-1 X光粉末繞射鑑定 (PXRD) 41
3-1-2掃描式電子顯微鏡影像分析 (SEM) 43
3-1-3 URS@ZIF-90之活性探討 44
3-1-4金屬有機骨架材料-74包封脲酶 (URS@MOF-74) 之相關鑑定 47
3-1-5檢測抑制脲酶之因素 49
3-1-6螢光光譜儀觀察酵素結構變化 50
3-1-7嘗試恢復失活脲酶之活性 51
3-2類沸石咪唑骨架材料包封過氧化氫酶 (CAT@ZIF-90) 之材料鑑定 54
3-2-1 X光粉末繞射鑑定 (PXRD) 54
3-2-2掃描式電子顯微鏡影像分析 (SEM) 56
3-2-3 CAT@ZIF-90之活性探討 57
3-2-4機械力化學法合成CAT@ZIF-90之熱重分析 (TGA) 59
3-2-5機械力化學法合成CAT@ZIF-90之氮氣等溫吸脫附儀分析 61
3-2-6十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (SDS-PAGE) 63
第四章結論與未來展望 65
參考文獻 66

參考文獻

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指導教授

謝發坤(Fa-Kuen Shieh)

審核日期

2021-8-25

推文