快速蝕刻中空金屬有機骨架材料 應用於酵素固定化之研究

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陳冠佑(Kuan-Yu Chen) 查詢紙本館藏

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化學學系

論文名稱

快速蝕刻中空金屬有機骨架材料應用於酵素固定化之研究
(Application of Rapid Etching for Hollow Metal-Organic Frameworks in Enzyme Immobilization Research)

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摘要(中)

本實驗室於 2015 年利用原位創新合成法 (de novo approach)，成功將
酵素在水相環境下封裝在類沸石咪唑骨架材料中 (ZIF-90 和 ZIF-8)。原位創
新合成法具有提供空間侷限性的優點，使得酵素可以在不易受外在因素影
響的情況下繼續進行催化反應。然而，在酵素固定化的過程中，材料和酵素
之間會發生相互作用，進而導致活性下降。基於這一點，本實驗室開發出快
速合成中空金屬有機骨架材料 (Hollow MOFs；HMOFs)，並將其應用於封
裝酵素。中空材料可以降低材料和酵素之間的界面相互作用，此類中空結構
不僅緩解了活性降低的問題，還保留了 MOFs 的多孔選擇性。然而，與酵
素本身相比，酵素在材料中的生物活性並未完全恢復。
在本研究中期望 HMOFs 除了可以提升活性，還期望能結合 MOFs 多
孔選擇性和中空材料內部空間的優勢，期望 HMOFs 展現出半透膜之特性，
並透過本實驗室先前開發之enzyme@HZIF-8材料與尿素作為蛋白質展開劑
進行半透膜應用測試，透過控制殼層厚度和調節變性時間，進而觀察酵素活
性之變化，期望在中空材料中失活的酵素蛋白能在空腔中進行調整並重新
折疊，但酵素表現在 HZIF-8 系統中並未有明顯的改善。
為了近一步提升酵素在中空 MOF 系統中的活性，本研究決定開發新的
生物複合材料 enzyme@HMOF，在實驗中採用不同尺寸之 UiO-66 材料吸附
酵素，同時參考本實驗室 2015 於發表在《JACS》之論文，在外層合成親水
ii
性材料─ZIF-90，從而獲得雙層材料 enzyme-on-UiO-66@ZIF-90。再結合
MOFs對酸鹼值耐受性的不同，崩解內層的 UiO-66材料獲得enzyme@HZIF90，探討內部空腔大小對活性之影響。在本研究中除了保持快速蝕刻步驟，
enzyme@HZIF-90 不僅在活性表現方面相比 nzyme-on-UiO-66@ZIF-90 提高
了三倍，kobs達到 5.87*10-2。在半透膜應用測試中 enzyme@HZIF-90 也有不
錯的表現，活性的回復也可達到 34%。

摘要(英)

The catalase (CAT) was embedded into sodalite (SOD) zeolitic imidazolate
frameworks (ZIF-8 and ZIF-90) via a water-based mild de novo approach
previously reported by our group in 2015. The de novo approach provided
shielding confinement properties, allowing the enzymes to maintain their
biological activity without being affected by external factors like inhibitors.
However, there are challenges for alleviating negative effects attributed by
interactions between the material and the enzyme during enzyme immobilization
process resulting in the decrease in activity. Building upon this, we developed a
new technique for rapid synthesis of hollow Metal-Organic Frameworks (HMOFs)
for encapsulation of enzyme, in which hollow MOFs reduces the interfacial
interactions between the material and enzymes. The hollow structure not only
mitigates the issue of decreased activity, but also retains the porous selectivity of
MOFs. However, the bioactivity of enzyme was not fully recovered comparing it
of free enzyme.
In this study, our aim was to explore the potential of HMOFs to not only
enhance activity but also integrate the porous selectivity of MOFs and the internal
space of hollow materials. Our hypothesis was that HMOFs could exhibit
characteristics resembling those of a semi-permeable membrane. To test this, we
employed the enzyme@HZIF-8 material in conjunction with urea as a protein
denaturing agent, aiming to assess its suitability for application in nanoscale semipermeable membranes. During our experimentation, we observed changes in
enzyme activity by manipulating the thickness of the shell layer and adjusting the
denaturation time. However, despite these efforts, we did not observe any
significant recovery in enzyme activity.
iv
To further enhance the enzyme activity in the hollow MOF system, we have
developed a novel approach to obtain the biocomposite of enzyme@HMOF.
Firstly, we selected UiO-66 particles of different sizes for enzyme adsorption onto
the surface. And then, we synthesized a double-layered material known as
enzyme-on-UiO-66@ZIF-90, following the methodology described in our
previous report from 2015. Moreover, taking advantage of the distinct acid and
alkaline tolerances of MOFs, we dissolved the inner layer of UiO-66, resulting in
the formation of enzyme@HZIF-90. The reason why we use the different sizes of
UiO-66 and it is expected that the activity of enzyme can be enhanced by
increasing the size of the cavity of hollow MOF.
In this study, besides maintaining a rapid etching step, enzyme@HZIF-90 not
only tripled the enzyme activity compared to enzyme-on-UiO-66@ZIF-90, with
a kobs reaching 5.87*10-2, but also demonstrated promising performance in semipermeable membrane applications, achieving a maximum activity recovery of
34.9%.

關鍵字(中)

★ 金屬有機框架
★ 中空材料
★ 蛋白質折疊
★ Zeta 電位
★ 酵素固定化

關鍵字(英)

★ metal-organic frameworks
★ hollow MOF
★ protein folding
★ Zeta potential
★ enzyme immobilization

論文目次

中文摘要 i
Abstract iii
目錄 v
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 UiO-66 6
1-4 酵素固定化 (Immobilized enzyme) 7
1-5 過氧化氫酶 8
1-6 研究動機與目的 9
第二章實驗部分 12
2-1 實驗藥品與設備 12
2-1-1 實驗藥品 12
2-1-2 實驗使用儀器 15
2-1-3 實驗鑑定儀器 16
2-2 實驗儀器與方法 17
2-2-1 X射線粉末繞射儀 (Powder X-ray Diffractometer；PXRD) 17
2-2-2 紫外光/可見光分光光譜儀 (UV/VIS Spectrophotometer) 19
2-2-3 掃描式電子顯微鏡 (Field-emission Scanning Electron Microscope ; SEM) 20
2-2-4 穿透式電子顯微鏡 (Transmission Electron Microscope； TEM) 21
2-2-5 界面電位分析儀 (Zeta Potential Analyzer) 22
2-2-6 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (SDS-PAGE) 24
2-3 實驗步驟 26
2-3-1 快速蝕刻合成中空類沸石咪骨架材料-包覆過氧化氫酶 (CAT@HZIF-8) 之合成 26
2-3-2 溶劑熱法合成UiO-66 (100 nm) 27
2-3-3 溶劑熱法合成UiO-66 (200 nm) 28
2-3-4 快速蝕刻合成中空類沸石咪骨架材料-包覆過氧化氫酶 (CAT@HZIF-90) 之合成 29
2-4 偵測蛋白質濃度 (Bradford Assay) 30
2-5 偵測過氧化氫水溶液之濃度 (Ferrous Oxidation-Xylenol orange assay；FOX assay) 32
第三章結果與討論 34
3-1 CAT@HZIF-8之半透膜應用測試 34
3-2 CAT-on-UiO-66@ZIF-90之合成條件探討 37
3-3 CAT@HZIF-90 之中空材料結構鑑定與活性實驗 43
3-3-1 CAT@HZIF-90之X射線粉末繞射圖譜鑑定 43
3-3-2 CAT@HZIF-90之掃描式電子顯微鏡影像分析 46
3-3-3 CAT@HZIF-90之穿透式電子顯微鏡影像分析 47
3-3-4 CAT@HZIF-90之膠體電泳實驗 49
3-3-5 CAT@HZIF-90中空材料之活性測試及比較 50
3-3-6 CAT@HZIF-90浸泡蛋白水解酶-K活性表現之影響 53
3-3-7 CAT@HZIF-90之半透膜應用測試 54
第四章結論及未來展望 55
參考文獻 57

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指導教授

謝發坤

審核日期

2023-8-14

推文