| 摘要: | 金屬有機骨架材料 (Metal-organic frameworks, MOFs) 在近幾年來的研究相當的熱門，是由金屬離子或金屬氧化物與有機配體所組成，其具有內部的空腔結構、高比表面積、良好的吸附性質以及擁有特定的窗口大小，因而應用於諸多領域上，其中讓我們感興趣的則是酵素固定化之模板的應用。
然而，多數合成金屬有機骨架材料的方法仍以傳統的加熱法 (Conventional heating) 為主，在合成過程中往往需要大量有機溶劑、費時、高溫的合成環境，對於包覆生物分子於其中的應用性有所限制。本實驗室於2015年利用原位創新合成 (de novo) 法，將ZIFs與酵素同時在水溶液中合成出CAT@ZIF-90，搭配孔洞的篩選性質，進行底物 (substrate) 的催化反應，並抵擋了蛋白質分解酶的作用，達到保護酵素的目的。另外，也在2017年發表以機械力化學法，添加微量的溶劑，在短時間以及室溫的條件下成功的合成出UiO-66-F4。
因此，為了將酵素固定化之應用擴展到其他的MOFs，我們利用了機械球磨法之概念，將酵素一起加入以達包覆之目的。本研究以過氧化氫酶 (CAT) 與Zn-MOF-74的搭配為主體，有別於傳統酵素固定化之方法，機械力化學法以較為溫和的合成環境，成功將CAT包覆於Zn-MOF-74之中，並保有催化活性，為了進一步探討酵素的折疊效應與孔洞的篩選性質，我們分別以尿素 (Urea) 蛋白質展開的變性試劑 (Denature reagent) 與蛋白質分解酶做測試，確認CAT仍具有催化活性。此外，我們也成功將另一種酵素 (如Chymotrypsin) 包覆於Zn-MOF-74，藉由機械力化學法快速合成出來，並做後續的活性鑑定。期望未來能利用此一符合綠色化學的機械力球磨法，合成出更多種類的生物複合材料，以擴展酵素固定化之應用。
;Metal-organic frameworks (MOFs) have been quite popular in the recent years. They are composed of metal ions or metal oxides with organic linkers. Because of their internal cavity structure, high surface area, extraordinary adsorption property and the specific aperture size, they have been used in many fields. Among these fields, we are interested in the application of enzyme immobilized platforms.
However, most of the methods for synthesizing MOF materials are still based on conventional heating, which often requires a large amount of organic solvents, time-consuming, and high temperature synthetic environment. The harsh synthetic conditions place a limitation on applications of encapsulating enzymes into the MOF materials. In our previous report in 2015, we demonstrated a novel approach, so called de novo, for synthesizing the biocomposites of CAT@ZIF-90 by encapsulating enzymes inside MOFs, ZIF-90, under mild aqueous solution. The MOFs could endow enzymes with size selectivity to promote the catalytic reaction of the substrate, prohibiting protease larger than the designed aperture from reaching the catalyst to protect the catalase. In 2017, our group published a report that an eco-friendly and rapid chemical synthesis of UiO-66 analog, UiO-66-F4, was demonstrated at room temperature by the mechanochemistry. Thus, we use this concept of mechanochemical approach to immobilize enzymes inside of MOFs in order to expand the applications of enzyme immobilizations to other MOFs.
Herein, we demonstrated the mechanochemical strategy for the synthesis of biocomposites by embedding catalase, enzymes for hydrogen peroxide hydrolysis, in Zn-MOF-74 via a ball milling process and maintained the enzymatic biological activity. In addition, this synthetic approach is different from the conventional method of enzyme immobilization in which it provides a milder synthetic environment and minimizes the use of organic solvents during synthesis. It is worth noting that the embedded CAT could not only be protected in proteinase K solution by the aperture size selectivity but also reduce unfolding in urea, a denature reagent by spatial confinement of the MOF structure. To demonstrate the generality of the method, chymotrypsin was encapsulating in Zn-MOF-74 via the mechanochemical approach as well as maintaining biological activity. We expect that the developed method can be generally applied to encapsulate enzymes of various sizes into MOFs with varied structures. |
