| 摘要: | 本研究論文主要分為兩個部分：
首先是中空類沸石咪唑骨架材料之合成。此部分是延續 2017 年發表的文章，該篇提出以實驗室發展創新原位合成 (de novo) 進行類沸石咪唑骨架 (Zeolitic imidazolate frameworks) 材料包覆酵素時，材料會對酵素產生侷限性 (Spatial confinement) 之理論。即便有使過氧化氫酶展開的尿素分子存在，被包覆的酵素並不會失去活性。為了更進一步探討侷限性與酵素活性表現之間的關係，本研究將酵素包覆於中空的類沸石咪唑骨架材料 hollow ZIF-8 當中，進而觀察酵素因結構侷限性降低所產生的活性變化。然而由實驗中發現中空材料對於酵素活性的檢測有干擾現象，於是我們將目標轉為研究並開發出新形態中空材料的研發。最後利用了奈米鈀金屬粒子作為標記物，證明已經完成新材料 Pd@8@90 製備，並參考最近文獻發表實驗過程，也成功製作出新型中空複合材料 Pd@8@void-90 以及其他衍生物，此研究成果結果對於後續研究侷限性跟酵素活性之間的關係提供一個嶄新的研究平台。
第二部分為利用機械力化學法固定酵素之探討。本實驗室已經成功的在 2017 年利用機械力化學法合成 UiO-66-F4，今年更藉由此機械力化學法快速的將大小為 240 kDa 的 β-葡糖糖苷酶 (β-glucosidase) 包覆於窗孔為 0.6 奈米的 UiO-66-NH2 中。雖然成功避免了多數的金屬有機骨架材料無法在水相中合成，以及材料窗口過小使得較大的底物 (substrate) 無法進入等問題，然此新穎的酵素包覆方法仍有許多的問題需要解釋。因此本研究對酵素包覆於 UiO-66-NH2 有更進一步的材料剖面影像，證實了酵素是包覆在緊密構築的材料之內而非材料間的空隙。此外，為了證明研磨過程是否會對酵素活性產生影響，我們比較了球磨前後酵素的活性差異，結果說明了酵素在此溫和的合成條件下活性僅些微下降而非喪失活性。最後在活性最佳條件調控的研究中，結果更加地確定兩步法的最佳條件是酵素導入的前後各研磨 8 赫茲 2.5 分鐘。
;There are two parts in this thesis.
Part I: Syntheses of hollow zeolitic imidazolate frameworks:
In 2017, we published a JACS paper regrading an additional strength of the de novo approach by demonstrating that enzymes embedded in met-al-organic frameworks (MOFs) via de novo approach remain functional under a wider range of reaction conditions. The enhanced stability arises from the confinement of the enzyme molecules. We also revealed the mechanism by exposing the embedded enzymes to chemical agents that specifically denature or inhibit free enzymes and examining their resulting catalytic activity for reactions relevant to biological functions etc. Herein, in order to further investigate the influence of spatial confinement on the en-zymatic functionality, we loaded catalase enzymes in crystalline ZIF-8, a subgroup of MOFs, nanoparticles with void to mimic enzymes encapsulated in a less restricted environment. However, those MOF materials, i.e., ZIF-67 and ZIF-8, for preparing hollow MOFs may influence enzymatic function or activity detection. Thus, we explored new materials for avoiding inferences on biological activity. The palladium nanoparticle was utilized as a marker for confirming that a new material of Pd@ZIF-8@ZIF-90 denoted as Pd@8@90 has been successfully prepared. Finally, the new hollow compo-site of Pd@8@void-90 and its derivatives were obtained by using a modified approach based on a recent report. Accordingly, this part work may con-tribute to investigate the influence of spatial confinement on the enzymatic functionality.
Part II: Study on immobilization of enzymes by mechanochemical meth-od:
Recently, we developed a new approach for rapidly encapsulating a glycosidase such as β-glucosidase, disaccharides as substrates, in the robust MOFs of Zr-based UiO-66-NH2 with aperture size about 0.6 nm. This method represents the first report of mechanochemical encapsulation of bi-ocatalysts into MOFs, including the first overall encapsulation method for enzymes into UiO-66 type MOFs, and can be extended to generate robust and recyclable MOF biocomposites, especially MOFs not be given in aqueous, for use in a number of industrial applications. Nonetheless, there were still some questions behind this beautiful story and needed to be further studied. Thus, in this work, we provided FIB-SEM images to show the in-tegrity of internal UiO-66-NH2 where enzymes embedded. We also studied the effect of various milling times during mechanochemical approach toward enzymatic activity. Finally, the optimal condition using 8 Hz and two-step 2.5 min milling for preparing enzyme@UiO-66-NH2 biocomposites show-ing the best biological function. |
