利用機械力化學法快速合成咪唑骨架材料及其酵素複合材料之探討

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陳靖蕙(Jing-Hui Chen) 查詢紙本館藏

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化學學系

論文名稱

利用機械力化學法快速合成咪唑骨架材料及其酵素複合材料之探討
(Investigation of Rapid Synthesis of Metal-organic Frameworks and Enzyme-MOF Biocomposites via Mechanochemical Approach)

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至系統瀏覽論文 (2029-9-30以後開放)

摘要(中)

酵素具有促進生化反應及專一性等特性，應用範圍廣。但對於環境耐受度低，需要通過固定化提高其穩定性，並實現回收再利用以降低成本。而有機金屬框架材料(Metal-organic Frameworks, MOFs)具備多孔隙以及高穩定性等特質，很適合作為酵素載體。因此本實驗室於2015年開發了類沸石咪唑骨架材料-90 (Zeolitic Imidazolate Framework-90, ZIF-90)封裝過氧化氫酶(Catalase, CAT)的原位創新合成法（de novo approach）。此方法雖為酵素固定化提出了一個新的思維，但耗時長且僅適用在特定的MOFs中。故在2019年，我們改以溶液輔助研磨法（Liquid‐Assisted Grinding），將酵素封裝在UiO-66-NH2等MOFs中。而此方法尚有需要被優化的地方，例如需要使用到有機溶劑、合成時間長達五分鐘、封裝酵素須分為兩步驟而非一鍋法完成以及無法以單晶包覆酵素等，若能改進上述合成的缺陷勢必能使溶液輔助研磨法具備更強的競爭力。
本篇論文成功的透過溶液輔助研磨法在10秒內合成單晶包覆的酵素材料複合物，CAT@ZIF-90，相較於本實驗室之前的研究，酵素活性從2.68 × 10−2提高至1.98 × 10−1。而為了更進一步了解酵素材料複合物的合成機制，利用不同溶液進行溶液輔助研磨，並成功推論出供後續以溶液輔助研磨法合成其他種類MOFs時緩衝溶液選擇的依據。並以上述成果進行酵素材料複合物包覆率提升的研究，得到最高包覆率約為34%，制定出一個新的能有效提高包覆率的策略。

摘要(英)

Enzymes have characteristics such as promoting biochemical reactions and specificity, making them widely applicable. However, their low tolerance to environmental conditions necessitates immobilization to enhance stability and enable recycling to reduce costs. Metal-organic frameworks (MOFs), with their porous nature and high stability, are well-suited as enzyme carriers. Therefore, in 2015, our laboratory developed a de novo approach for encapsulating catalase (CAT) in zeolitic imidazolate framework-90 (ZIF-90). While this method provided a new perspective on enzyme immobilization, it was time-consuming and only applicable to specific MOFs. Consequently, in 2019, we adopted the liquid-assisted grinding (LAG) to encapsulate enzymes in MOFs such as UiO-66-NH2. However, this method still required optimization, such as the use of organic solvents, a synthesis time of up to five minutes, a two-step encapsulation process instead of a one-pot method, and the inability to encapsulate enzymes in single crystals. Improving these synthesis drawbacks would undoubtedly enhance the competitiveness of the LAG method.
This paper successfully synthesized single-crystal encapsulated enzyme-MOF biocomposites, CAT@ZIF-90, in 10 seconds using the LAG. Compared to our previous research, the enzyme activity increased from 2.68 × 10−2 to 1.98 × 10−1. To further understand the synthesis mechanism of enzyme-MOF biocomposites, different solutions were used for LAG, successfully deducing the criteria for selecting buffer solutions for synthesizing other types of MOFs using the LAG. Based on these results, a study to improve the loading of enzyme-MOF biocomposites was conducted, achieving a maximum loading of approximately 34%, and formulating a new strategy that effectively enhances the loading.

關鍵字(中)

★ 酵素固定化
★ 有機金屬框架材料
★ 類沸石咪唑骨架材料
★ 機械力球磨法

關鍵字(英)

★ Enzyme
★ MOFs
★ ZIFs
★ ZIF-90
★ Mechanochemistry

論文目次

摘要..................................................................................................................................i
Abstract ...........................................................................................................................ii
Eligibility........................................................................................................................iv
圖目錄..........................................................................................................................viii
表目錄............................................................................................................................xi
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機械力化學法................................................................................................6
1-4酵素固定化....................................................................................................7
1-5研究動機及目的............................................................................................8
第二章實驗部分......................................................................................................10
2-1實驗藥品......................................................................................................10
2-2實驗儀器......................................................................................................12
2-3實驗儀器之原理..........................................................................................14
2-3-1中量快速球磨機(Mixer Mill) .......................................................14
2-3-2X射線粉末繞射儀(X-ray powder diffractometer).........................17
2-3-3場發掃描式電子顯微鏡(Field-emission Scanning Electron Microscope)...................................................................................................19
2-3-4紫外光可見光分光光譜儀(UV-Visible spectrophotometer) ........21
2-3-5十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)........................................22
2-3-6比表面積及孔隙度分析儀(Surface Area and Pore Size Distribution Analyzer).......................................................................................................24
2-4酵素..............................................................................................................27
2-4-1過氧化氫酶(Catalase, CAT) ............................................................27
2-4-2血紅素(Hemoglobin, Hb) ................................................................27
2-4-3溶菌酶（Lysozyme, LYS) ................................................................28
2-4-4蛋白酶（protease）..........................................................................28
2-5實驗步驟......................................................................................................29
2-5-1機械力化學法合成Enzyme@ZIF-90..............................................29
2-5-2偵測材料中蛋白質濃度...................................................................30
2-5-3酵素金屬有機骨架複合材料之蛋白質凝膠電泳分析...................32
2-5-4偵測過氧化氫酶活性之方法（Ferrous Oxidation in Xylenol orange assay, FOX assay）.......................................................................................34
2-5-5偵測過氧化氫酶在蛋白酶K環境下之活性檢測..........................36
第三章結果與討論—酵素材料複合物的合成與活性探討.................................37
3-1CAT@ZIF-90之合成條件優化與鑑定......................................................37
3-2CAT@ZIF-90之X射線粉末繞射圖譜分析...................................37
3-3掃描式電子顯微鏡影像分析...........................................................44
3-4十二烷基硫酸鈉聚丙烯酰胺凝膠電泳...........................................47
3-5CAT@ZIF-90合成條件與活性探討..........................................................49
3-6緩衝溶液協助MOF生成之合成機制探討...............................................54
第四章結論與未來展望..........................................................................................58
第五章結果與討論—增加酵素材料複合物包覆率之研究.................................60
5-1材料合成條件選擇與增加酵素包覆率策略之研究.................................60
5-2CAT@ZIF-90之合成、鑑定與包覆率探討..............................................62
5-3Hb@ZIF-90之合成、鑑定與包覆率探討................................................64
5-4LYS@ZIF-90之合成、鑑定與包覆率探討..............................................66
第六章結論與未來展望..........................................................................................69
第七章參考文獻......................................................................................................71

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指導教授

謝發坤(Fa-Kuen Shieh)

審核日期

2024-9-25

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