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

Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/91811

Title:	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
Authors:	陳冠佑;Chen, Kuan-Yu
Contributors:	化學學系
Keywords:	金屬有機框架;中空材料;蛋白質折疊;Zeta 電位;酵素固定化;metal-organic frameworks;hollow MOF;protein folding;Zeta potential;enzyme immobilization
Date:	2023-08-14
Issue Date:	2023-10-04 14:43:10 (UTC+8)
Publisher:	國立中央大學
Abstract:	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@HZIF 90，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 5.8710-2。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%。;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 semi permeable 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 5.8710-2, but also demonstrated promising performance in semi permeable membrane applications, achieving a maximum activity recovery of 34.9%.
Appears in Collections:	[Graduate Institute of Chemistry] Electronic Thesis & Dissertation

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