利用水相原位創新法合成酵素有機金屬骨架複合材料及酵素失活機制之探討

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题名:	利用水相原位創新法合成酵素有機金屬骨架複合材料及酵素失活機制之探討
作者:	許育慎;Hsu, Yu-Shen
贡献者:	化學學系
关键词:	有機金屬骨架材料;酵素有機金屬骨架材料;水相原位創新法;酵素摺疊效應;MOF;CAT@ZIF-90
日期:	2019-01-21
上传时间:	2019-04-02 14:14:33 (UTC+8)
出版者:	國立中央大學
摘要:	本實驗室於2015年以原位創新合成(de novo approach)的方式將過氧化氫酶(Catalase)包入咪唑骨架材料-90 (ZIF-90)之中。此方法所合成的酵素有機金屬骨架複合材料可藉由ZIF-90的孔洞性質保護酵素不被大分子蛋白質水解酶攻擊的同時，仍能維持酵素活性運作。而本研究則更近一步的利用此材料來探討酵素在蛋白質變性因子－尿素中的表現。　　本研究假設在原位創新法合成中ZIF-90在包覆酵素時，會限制住了酵素周圍的空間，使酵素難以進行構形的變化。為證明酵素在ZIF-90中難以變換構形，本研究將酵素有機金屬骨架複合材料置入變性試劑(Denaturant reagent):尿素之中進行一系探討。預測由於空間限制的作用，即使在尿素環境中，酵素仍保有活性。本研究亦利用中孔矽材(MCF、SBA-15)作為對照組，探討在尿素環境下的活性變化。活性測試(Activity assay)與螢光光譜(Fluorescence spectrum)等實驗結果證明了ZIF-90對酵素產生空間限制的假設，也證明了尿素使酵素失活的機制與酵素構形相關。此外，酵素有機金屬骨架複合材料在其他嚴苛環境如高溫、有機溶劑中能具有活性，同時藉由螢光光譜的結構測定，討論其他嚴苛環境中酵素活性與構形的關聯性。 ;Recently, our group has successfully embedded catalase (CAT) into zeolitic imidazolate frameworks-90 (ZIF-90) via de novo approach. In the de novo approach, the ZIF-90 are grown around the enzyme molecules under a mild synthetic condition. Remarkably, the biological activity of biocomposites is able to be maintained as proteinase K, the enzyme that can digest proteins, is existing with large molecule size due to the size selectivity of ZIF-90 porous. In this work, we hypothesized that the enzyme molecules are confined in the tight mesoporous cavities left in the MOF crystals by growth of the framework around the enzyme molecules which reduces the structural changes of enzymes. In order to test this hypothesis, we exposed the CAT@ZIF-90 to and free CAT to a denature reagent (i.e., urea) and high temperatures (i.e., 80 °C) and examined their resulting catalytic activity, accompanied by fluorescence spectroscopy to monitor the structural conformation changes of the enzymes. The results show that embedded CAT maintains its biological function even when exposed to 6 M urea and 80 °C, respectively, while free CAT shows undetectable activity. A fluorescence spectroscopy study indicates that the structural conformation of the embedded CAT changes less under these denaturing conditions than free CAT. We have not only demonstrated that CAT maintains its biological function under unfolding conditions after being embedded in ZIF microcrystals via a de novo approach but also performed fluorescence spectroscopy to provide an in situ observation that the structural conformation of CAT in ZIFs is mostly maintained.
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