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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题名:	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
作者:	郭蕎菱;Kuo, Chiao-Ling
贡献者:	化學學系
关键词:	有機金屬骨架材料;MOF
日期:	2024-09-19
上传时间:	2024-10-09 15:29:01 (UTC+8)
出版者:	國立中央大學
摘要:	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。 ;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.
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