利用機械力學法合成中空酵素金屬有機骨架複合材料之研究

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郭蕎菱(Chiao-Ling Kuo) 查詢紙本館藏

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化學學系

論文名稱

利用機械力學法合成中空酵素金屬有機骨架複合材料之研究
(Encapsulation of Biocatalysts in Hollow Metal-Organic Frameworks via Mechanochemical Approach)

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至系統瀏覽論文 (2029-9-30以後開放)

摘要(中)

本研究旨在解決酵素固定化在金屬有機骨架材料（MOFs）中的相互作用問題。實驗室於 2015 年開發的原位創新水相合成法（de novo mild water-based）雖成功封裝了酵素，但酵素與 MOF 之間的相互作用會影響酵素活性。雖然進一步開發出中空材料合成法（Hollow MOFs, HMOFs）以改善材料與酵素作用問題。然而，製備中空材料蝕刻過程的環境時間與釋放物質仍對酵素產生影響。所以如何為進一步優化蝕刻條件，讓酵素盡量保有原來活性狀態是一個重要課題。
本研究選擇了不同材料製備中空結構，並結合實驗室於 2023 年開發的球磨法，是一種超快速（10 秒）、環境友善且產量高（約 80%）的合成方法。利用 Catalase （CAT）、UiO-66 （Universitetet i Oslo-66）和 ZIF-90 （Zeolitic Imidazole Framework-90）成功合成分級（Hierarchical）材料 CAT-on-UiO-66@ZIF-90。透過材料對蝕刻緩衝液的耐受性差異，選擇性地蝕刻內層材料 UiO-66，並保留了穩定的外層材料 ZIF-90，成功形成中空材料 CAT@HZIF-90。
透過表徵量測結果證實了中空結構的成功合成。同時，活性測試顯示中空材料內酵素的自由度提升，並表現出顯著活性提升，其活性常數從實心材料的 3.5 × 10-2 s-1 提升至空心材料的 7.2 × 10-2 s-1，顯示活性增強了兩倍。與 HZIF-8 相比，其活性為1.1 × 10-2 s-1，顯示 HZIF-90 的活性增強了七倍。
最後，為了未來應用上，能夠結合螢光光譜與中空材料，以研究酵素結構展開與重新摺疊過程，觀察酵素結構的動態變化。探討了中空材料中酵素結構的展開及重新摺疊。用尿素處理並隨後去除後，酵素的結構確實存在重新摺疊的現象。顯示HZIF-90系統在穩定酵素結構方面具有顯著優勢，為未來酵素固定化提供了新的選擇。

摘要(英)

The aim of this study is to address the interaction issues of enzyme immobilization within metal-organic frameworks (MOFs). Although the innovative in-situ synthesis method (de novo mild water-based) developed by the laboratory in 2015 successfully encapsulated enzymes, the interactions between the enzyme and the MOF affected enzyme activity. To improve the interaction between materials and enzymes, hollow material synthesis methods (Hollow MOFs, HMOFs) were developed. However, the etching process used to prepare hollow materials, along with the environmental conditions and released substances like MOF linkers, still impacted enzyme activity. Therefore, optimizing etching conditions to maintain the enzyme’s original activity is a critical issue.
In this study, different MOF materials were selected to prepare hollow structures, combined with the ball milling method developed by our laboratory in 2023. This method is ultra-fast (10 seconds), environmentally friendly, and high-yield (about 80%). Catalase (CAT), UiO-66 (Universitetet i Oslo-66), and ZIF-90 (Zeolitic Imidazole Framework-90) were successfully used to synthesize a hierarchical MOF material, CAT-on-UiO-66@ZIF-90. By utilizing the differential resistance of the materials in buffer to the etching solution, the inner layer material, UiO-66, was selectively etched while the stable outer layer material, ZIF-90, was retained. This process successfully formed the hollow material CAT@HZIF-90.
Characterization measurements confirmed the successful synthesis of the hollow structure. Activity tests showed that the enzyme’s freedom within the hollow material was enhanced, resulting in a significant increase in activity. The activity constant increased from 3.5 × 10-2 s-1 for the solid material to 7.2 × 10-2 s-1 for the hollow material, indicating a twofold increase in activity. Compared to HZIF-8, which had an activity of 1.1 × 10-2 s-1, HZIF-90 demonstrated a sevenfold increase in activity. Subsequent immersion of the material in a proteinase-K solution further confirmed that the enzyme was primarily encapsulated within the MOF.
Finally, for future applications, the combination of fluorescence spectroscopy and hollow materials could be used to study enzyme unfolding and refolding processes, observing the dynamic changes in enzyme structure. The unfolding and refolding of the enzyme structure within the hollow material were explored. After treatment with urea and subsequent removal, the refolding of the enzyme structure was evident. This demonstrates that the HZIF-90 system has significant advantages in stabilizing enzyme structures, providing a new option for future enzyme immobilization.

關鍵字(中)

★ 有機金屬骨架材料

關鍵字(英)

★ MOF

論文目次

中文摘要 i
Abstract iii
目錄 vi
圖目錄 ix
表目錄 xii
1 第一章緒論 1
1-1 金屬有機骨架材料 1
1-2 類沸石咪唑骨架材料 4
1-3 UiO-66 7
1-4 機械力化學法 8
1-5 酵素固定化（Immobilized enzyme） 9
1-6 過氧化氫酶 10
1-7 研究動機與目的 12
2 第二章實驗部分 15
2-1 實驗藥品與設備 15
2-1-1 實驗藥品 15
2-1-2 實驗使用儀器 18
2-1-3 實驗鑑定儀器 19
2-2 實驗儀器與方法 20
2-2-1 中量快速球磨機（Mixer Mill） 20
2-2-2 X射線粉末繞射儀（Powder X-ray Diffractometer；PXRD） 23
2-2-3 紫外光/可見光分光光譜儀（UV/VIS Spectrophotometer） 26
2-2-4 場發掃描式電子顯微鏡（Field-emission Scanning Electron Microscope, FE-SEM） 27
2-2-5 穿透式電子顯微鏡（Transmission Electron Microscope； TEM） 29
2-2-6 界面電位分析儀（Zeta Potential Analyzer） 30
2-2-7 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳（SDS-PAGE） 32
2-3 實驗步驟 34
2-3-1 溶劑熱法合成 UiO-66 （Size : ~ 150 nm） 34
2-3-2 機械力球磨法合成中空類沸石咪骨架材料-包覆過氧化氫酶（CAT@HZIF-90） 35
2-4 偵測蛋白質濃度（Bradford Assay） 37
2-5 偵測過氧化氫水溶液之濃度（Ferrous Oxidation-Xylenol orange assay；FOX assay） 39
3 第三章結果與討論 41
3-1 MOF@MOF 材料之選擇 41
3-2 CAT-on-UiO-66@ZIF-90之合成條件探討 43
3-3 CAT@HZIF-90之蝕刻條件探討 47
3-4 CAT@HZIF-90 之中空材料結構鑑定與活性實驗 51
3-4-1 CAT-on-UiO-66@ZIF-90 之 X 射線粉末繞射圖譜鑑定 51
3-4-2 CAT@HZIF-90 之 X 射線粉末繞射圖譜鑑定 52
3-4-3 CAT@HZIF-90之掃描式電子顯微鏡影像分析 53
3-4-4 CAT@HZIF-90之穿透式電子顯微鏡影像分析 54
3-4-5 CAT@HZIF-90之膠體電泳實驗 55
3-4-6 CAT@HZIF-90中空材料之活性測試及比較 56
3-4-7 CAT@HZIF-90浸泡蛋白水解酶-K活性表現之影響 58
3-4-8 HZIF-90與HZIF-8系統活性比較 59
3-4-9 CAT@HZIF-90之酵素結構重新摺疊能力測定 60
4 第四章結論及未來展望 61
參考文獻 63

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指導教授

謝發坤

審核日期

2024-9-19

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