利用溫和水相法快速包封酵素並合成鋁基底之金屬有機骨架材料

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魏名妤(Ming-Yu Wei) 查詢紙本館藏

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化學學系

論文名稱

利用溫和水相法快速包封酵素並合成鋁基底之金屬有機骨架材料

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至系統瀏覽論文 (2027-7-1以後開放)

摘要(中)

由於酵素具有高效催化與專一性特定反應的展現，所以在工業上被廣泛應用—造紙、紡織、清潔劑等。但也因為酵素在嚴苛環境下的不穩定性，如有機溶劑，並且催化完後難以與產物在反應溶液中分離，因此在應用上存在許多限制。對於如何能夠增加酵素穩定性與酵素固定化實用性上的課題，顯得十分重要且有潛力。本實驗室於 2015 年
首次開發出以原位創新合成法(de novo)，在水相室溫下以類沸石咪唑
骨架材料 ZIF-90 包覆過氧化氫酶(Catalase)，利用金屬有機骨架材料
的孔洞性質，允許受質(substrate)進入材料催化，同時酵素受到孔洞材
料保護防止被大分子蛋白質水解酶分解，提供紡織工業漂白水處理議
題的一項有效方法。2017 年更進一步發表此生物性複合材料 enzyme@ZIF-90 研究，發現指出金屬有機骨架材料對酵素提供空間侷限性的效果，此作用降低酵素在展開劑尿素的環境下所造成結構的展開與失活程度。雖然此溫和水相合成系統可順利製備酵素類沸石咪唑骨架材料生物複合體且具有良好催化效果，且可以抵抗較嚴苛的環境，但受限於類沸石咪唑骨架材料小孔洞性質，在受質和酵素的選擇上造成了限制。為了擴展更大孔洞 MOF 材料的水相綠色合成法，本實驗室緊接著於 2019、2021 年成功使用 UiO-66 及 Zn-MOF-74 等大孔洞金屬有機骨架材料於包封酵素，更加延伸了此類生物性複合材料的應用範疇。
於醫療方面，使用鋁基在 20 世紀已有建樹，1926 年免疫學家
Alexander T. Glenny 首次使用鋁鹽作為疫苗之佐劑，經過實驗證實後持續沿用之今。再來著眼於生物相容性，以鋁為基底的金屬有機骨架材料，其被認為是最有前途的 MOFs 之一，由於其優異的骨架穩定性已被廣泛研究。目前分類的大宗有 MIL(拉瓦錫材料研究所)、CAU (基爾大學)等，MIL 代表之一的材料 MIL-53 於 2003 年被發現、被證實具有呼吸效應。
本篇論文成功在溫和水相下於一小時內快速合成鋁基底之金屬有機骨架材料，並藉由提供不同幅度的加熱環境能使其更迅速被合成。過程減少了有機溶劑的使用，更加符合現今對於綠色化學之環保提倡。此材料並能夠成功包覆過氧化氫酶及脂肪酶，有鑒於該材料以金屬鋁為基底，如此具有生物安全性及相容性，在動物測試等能夠相對減少生物毒性，生物性複合材料領域中也擁有更廣的應用。疫苗方面，佐劑對於疫苗上的助益亦相當顯著—能夠減少疫苗所需劑量、擴大疫苗免疫、增加抗體反應的幅度等。因此利用鋁基 MOF 作為佐劑或是其他生物製劑例如單株抗體(Monoclonal Antibody, Mab)，將更具有應用潛力。

摘要(英)

Enzymes have been widely used in industrial applications such as pulp and paper, texile, and detergents, due to their high efficiency and specificity in catalyzing reactions. However, there are lots of restrictions owing to their instability in harsh environments, i.e., organic solvent, and their difficulty in separating from productions in the reaction solution. Therefore, it is particularly important and potential for enzyme immobilization that can increase the stability and practicality of the enzyme. In our previous report in 2015, we developed an innovative one- pot de novo mild water-based approach to encapsulate an enzyme, catalase, into a zeolitic imidazolate framework-90(ZIF-90), a sub group of Metal- organic Frameworks (MOFs), in which MOFs is capable of protecting embedding enzyme from protease and maintaining its biological activity. Furtherly in 2017, our follow-up study on biocomposites was showing the MOF material is able to provide spatial confinement for enzymes, reducing the structural expansion under the environment of denature reagent such as urea. However, the small pores of the zeolitic imidazolate framework limit the selectivity of enzymes and substrates in the system of enzyme@ZIF-90. Afterward, our laboratory also reported about encapsulating enzymes with large-pore metal-organic frameworks, UiO-66 and Zn-MOF-74, via water-based approach in order to expand the applications of biocomposites.
As we mention about medical treatment, the usage of aluminum-based materials has been done in 20th centuries. Aluminum salt was first used by the immunologist, Alexander T. Glenny, in 1926. And it continues been used until now after it was successfully verified through experiments. Recently, we are focusing on aluminum-based MOFs with biocompatibilites as one of the most promising MOFs which have been widely investigated because of their excellent framework stability. Nowadays the main categories are MIL (for Materials Institute Lavoisier), CAU (for Christian- Albrechts-University) etc. The representative material, MIL-53, was discovered in 2003 and it was verified that it has breathing effect.
In this study, we found that aluminum-based MOFs can be successfully synthesized in one hour via the mild water-based method. Additionally, the synthetic time can be adjusted in a shorten period as temperature raised. As the decrease of amount of organic solvent used among the synthetic process, it can be more close to the meaning of so-called ”green chemistry”. This material can also successfully encapsulate catalase and lipase. Because of its aluminum-based characteristic, this material with biocompatibility can lower the biotoxicity through animal testing, and it will pave the way for further applications owing to its extraordinary potential and performance. As it goes to vaccines, adjuvant has tremendous effect on vaccines, such as lower the dose of vaccines, enlarge the vaccines immune, and widen the extent of antibody reactions. Therefore, using aluminum-based MOFs as adjuvant or other biological products, like monoclonal antibody, will be more potential toward applications.

關鍵字(中)

★ 金屬有機骨架材料
★ 水相法
★ 酵素

關鍵字(英)

★ Metal-organic Frameworks
★ MOF

論文目次

摘要 ........................................................................................................... I ABSTRACT ............................................................................................ III
目錄 ......................................................................................................... VI
表目錄 ..................................................................................................... IX
圖目錄 ...................................................................................................... X
第 1 章緒論 ......................................................................................... 1
1-1 金屬有機骨架材料 ..................................................................... 1
1-2 類沸石咪唑骨架材料-90............................................................ 5
1-3 金屬有機骨架材料-74................................................................ 7
1-4 金屬有機骨架材料 NH2-MIL-53(AL)........................................ 9
1-5 酵素固定化於 MOFS 的發展 ................................................... 10
1-6 研究動機以及目的 ................................................................... 13
第2章實驗部分 ............................................................................... 15
2-1 實驗藥品................................................................................... 15
2-2 實驗儀器................................................................................... 18
2-2-1 實驗使用儀器 .................................................................... 18
2-2-2 實驗鑑定儀器 .................................................................... 19 vi
2-3 實驗儀器之原理....................................................................... 20
2-3-1 X 射線粉末繞射圖譜 ........................................................ 20
2-3-2 掃描式電子顯微鏡 ............................................................ 21
2-3-3 紫外光可見光分光光譜儀 ................................................ 22
2-4 酵素........................................................................................... 23
2-4-1 過氧化氫酶 ........................................................................ 23
2-4-2 脂肪酶 ................................................................................ 24
2-5 實驗步驟—MOFS CHEMICAL BIOLOGY ................................... 26
2-5-1 NH2-MIL-53(Al)材料的合成 ............................................ 26
2-5-2 NH2-MIL-53(Al)包覆過氧化氫酶的合成......................... 26
2-5-3 NH2-MIL-53(Al)包覆脂肪酶的合成................................. 27
2-5-4 偵測蛋白質濃度(Bradford Assay) .................................... 27
2-5-5 十二烷基硫酸鈉聚丙醯胺膠體電泳 (SDS-PAGE)......... 29
2-5-6 偵測過氧化氫酶之活性 .................................................... 31
2-5-7 偵測脂肪酶之活性 ............................................................ 33
第 3 章結果與討論 ........................................................................... 35
3-1 優化 NH2-MIL-53(AL)的合成方法.......................................... 35
3-1-1 金屬離子與有機配體比值 ................................................ 35
3-1-2 電子加熱攪拌器 ................................................................ 38
3-1-3 超音波震盪器 .................................................................... 41
3-1-4 Tricine 緩衝溶液 ............................................................... 46
3-2 CAT@NH2-MIL-53(AL)之鑑定與活性實驗........................... 48
3-2-1 X 射線粉末繞射圖譜分析 ................................................ 48
3-2-2 掃描式電子顯微鏡影像分析 ............................................ 49
3-2-3 十二烷基硫酸鈉聚丙醯胺膠體電泳 (SDS-PAGE)......... 50
3-2-4 CA T@NH2-MIL-53(Al)的活性測定 ................................. 51
3-3 LIP@NH2-MIL-53(AL)之鑑定與活性實驗 ............................. 53
3-3-1 X射線粉末繞射圖譜分析 ................................................ 54
3-3-2 掃描式電子顯微鏡影像分析 ............................................ 55
3-3-3 十二烷基硫酸鈉聚丙醯胺膠體電泳 (SDS-PAGE)......... 56
3-3-4 LIP@NH2-MIL-53(Al)的活性測定 ................................... 57
第 4 章結論以及未來展望 ............................................................... 60
參考文獻 ................................................................................................ 61
附錄 ........................................................................................................ 72

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指導教授

謝發坤(Fa-Kuen Shieh)

審核日期

2022-8-18

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