金屬有機骨架材料(Metal-organic frameworks, MOFs)為近幾年研究迅速崛起的孔洞材料，以金屬離子或金屬氧化物及有機配體組成內部具有空腔的結構，其比表面積與吸附性質極高且擁有特定的骨架窗口大小，所以擁有諸多應用，其中一項即為酵素固定化之基板。 近幾年興起一種原位創新合成(de novo)方法，本實驗室於2015年首先發表CAT@ZIF-90即以MOFs的前驅物與酵素同時於水溶液內合成，縝密包覆酵素特點除了擁有大小篩選性，更在2017年研究發表指出被包覆的酵素有空間限制反摺疊(unfolding)現象，具有傳統吸附型固定化無法達到的優勢。 然而受限於大部分MOFs屬於熱溶劑法合成，使酵素在合成過程會失去活性；能夠水相合成的MOFs也以ZIF系列為主，其窗口(aperture)約為3.5Å使大部分底物無法進入。為了將酵素包覆拓展至其他MOFs，我們提出全新包覆的概念：利用短時間及幾乎無溶劑的機械球磨法能合成金屬有機骨架材料的優點，將酵素一同添加達到包覆效果。本研究以葡萄糖甘酶(BGL)與UiO-66-NH2的組合為範本，突破了傳統酵素固定化的方法，以機械球磨方式成功將BGL包覆到UiO-66-NH2中，並測定催化產物的生成保有活性，也因為UiO-66-NH2的耐酸性及孔洞篩選性質，使得BGL@UiO-66-NH2在蛋白質水解酶protease這樣的大分子環境中，能阻擋BGL被水解而保持催化活性。 此結果提供了Enzyme@MOFs創新合成方式，不再侷限於水相合成系統。期待在未來能同時結合機械球磨法與水相合成法，以原位創新合成概念合成更多類型的酵素金屬有機骨架複合材料，並對酵素做進一步研究。 ;Metal organic frameworks(MOFs) composed by metal ions and organic linkers are porous materials which were emerging and developed rapidly in recent years. Many applications are contributed by high porosity and specific aperture of its structure. One of the MOF applications is used as the platform for enzyme immobilizations. Recently, a de novo approach was used for encapsulating enzymes into MOFs materials, our group successfully preformed that biocomposites have been generated under aqueous and mild condition by encapsulating catalase, enzymes for hydrogen peroxide hydrolysis, into zeolitic imidazolate framework-90 (CAT@ZIF-90). By this approach, enzyme could not only be protected in protease solution by aperture size limitation but also reduce unfolding by MOF structure confinement which difficultly achieved by adsorption immobilization. In addition to ZIFs with 3.5Å aperture which limited penetrability of large substrates, most of MOF materials are synthesized by solvothermal ways under harsh conditions such as organic solvent and high temperature etc. in which the biological activity of enzyme is hardly maintained. To this end, we thought out of the box and proposed another concept that takes the method of mechanochemical approach with advantages of rapidly time and almost non-solvent to immobilize enzymes inside of MOFs. We demonstrated a mechanochemical method for the facile preparation of biocomposites by embedding -glucosidase, enzymes for disaccharide hydrolysis, in a Zr-MOF analog, UiO-66-NH2 and also determined the apparent biological activity. UiO-66-NH2 has a large aperture and high acid resistance, thus demonstrating potential for size-shielding against protease as well as maintaining biological function. Thus, this study provides an alternative route for encapsulating enzymes into MOFs, especially for biocomposites that are difficult to obtain under mild aqueous conditions.
