| 摘要: | 酵素/生物固定化的技術於工業應用已有久遠的歷史!然而對此技術應用卻沒有相關的研究報導，關於酵素是如何存活於其中並且保持活性狀態而達到催化的效果。因此，根據本實驗室 2015 年成功利用類沸石咪唑骨架-90 (ZIF-90) 包覆蛋白質的經驗，發展出原位創新合成 (de novo approach) 的方法提供水相溫和的酵素包覆環境。所合成出酵素金屬骨架複合材料，不僅具有孔洞性質可將較小的基質 (Substrate) 送入其中以供過氧化氫酶 (Catalase, CAT) 催化，同時，可以防止大分子蛋白質水解酶的作用，而達到到保護酵素與維持催化的效果。藉由此原位創新合成為研究模組延伸進行更進一步的研究，進行酵素與骨架材料結合的複合材料對於酵素不友善的環境下的探討。
透過一系列的測試與探討，例如在高濃度尿素環境量測酵素活性，探討酵素的摺疊效應以及利用螢光光譜分析酵素結構，證明了藉由原位創新合成法，將酵素局限金屬有機骨架材料 (Metal-organic frameworks, MOFs) 之後，能夠在更嚴苛的條件環境下進行催化反應並且維持其活性。酵素的穩定性增強是來自於 MOFs 圍繞酵素分子而形成的中孔洞腔體將其封裝保護，降低了酵素分子結構的改變。將過氧化氫酶 (Catalase) 嵌入 ZIF-90/-8之中，然後將被包覆與否的過氧化氫酶置入變性試劑 (Denature reagent)，例如尿素和高溫 (如80 ℃) 的環境下，可以發現即使存在於 6 M 尿素與80 ℃高溫環境，被包覆的過氧化氫酶 (即CAT@ZIF-90) 依然能夠保有其分解過氧化氫的活性，反應速率常數 (kobs) 分別為 1.30 × 10−3 和 1.05 × 10−3 sec-1，反觀未被包覆之過氧化氫酶則已失去催化活性。最後，為了更加直接了解酵素分子的結構變化，藉由過氧化氫酶的結構構形所深埋於內部之色胺酸 (Tryptophan) 影響，使用了螢光光譜研究證明，因為過氧化氫酶被 MOFs 所包覆，以至於在某些變性條件 (如尿素) 之下，結構構型變化相較於未被包覆之過氧化氫酶來得少，因此證明酵素在此方式的保護下依然能夠在嚴峻的環境下維持其生物活性。而實驗結果也再次證明了我們假設之理論的正確性，不但能利用生物複合材料保護了酵素，並且探討了材料對於酵素所提供的環境保護機制，創造出新穎、探討酵素生理機制的新模板。
;For last decades, the enzyme/biological immobilization has been widely applied for industry such as textile, beverage, and food etc. However, it is unclear why the embedded enzyme still retain its biological activity. Regarding our previous publication in 2015, a de novo approach was used to encapsulate the enzyme into metal-organic frameworks (MOFs) crystals and able to maintain bioactivity of enzymes. In order to further study of biocomposite by using de novo approach as a model, this work is focusing on the effects of embedded enzyme activities under enzyme-unfriendly environment.
With a series of examinations, we found the enzyme is able to maintain its biological function under a wider range of conditions after being embedded in MOF microcrystals via a de novo approach. We suggest the enhancement of stability arise from confinement of enzyme molecules in the mesoporous cavities in the MOFs, which reduces the structural mobility of enzyme molecules. Additionally, we embedded catalase (CAT) into zeolitic imidazolate frameworks-90/8 (ZIF-90 and ZIF-8), and then exposed both embedded CAT and free CAT to a denature reagent, i.e., urea, and high temperatures, i.e., 80 °C. The embedded CAT still maintains the decomposition ability of hydrogen peroxide with apparent rate constants kobs (s−1) of 1.30 × 10−3 and 1.05 × 10−3 even when exposed to 6 M urea and 80 °C, respectively, in contrast, free CAT shows undetectable activity. A fluorescence spectroscopy study also had shown that the structure conformational change of the embedded CAT is lesser than it of free CAT as incubated in denaturing condition. Therefore, the result of this work indicated that the biological activity of embedded enzyme is still maintained under the harsh environment.
Finally, this work agreed with our hypothesis that not only biocomposites can shield embedded enzyme against structural unfolding, but also explore the mechanism of enzyme inactivation by the use of nanoporous MOFs as well as create a novel model for probing physiological mechanism of biomolecules. |
