應用離子液體於蛋白質液相液相萃取

Abstract

室溫離子液體（Room Temperature Ionic Liquids, RTILs）由於擁有高極性、高導電度、低蒸氣壓及高熱穩定性等特殊的化學與物理性質，目前持續吸引廣泛的研究，以探討取代傳統有機溶劑的可行性，包括在液相╱液相萃取、有機合成及無機催化等方面的應用。此外由於離子液體對於實驗環境較為安全且可回收再利用，能減低對環境的汙染，因此被視為綠色溶劑。 蛋白質的純化在生物化學及生物技術上是一項相當重要的工作。傳統上，液相╱液相萃取分離蛋白質的技術，包括雙水相溶劑系統及反微胞萃取兩種。在本研究中發現，藉由調控水溶液的pH值，蛋白質能在水溶液及乙酸乙酯(ethyl acetate)和離子液體 [C4mim][PF6]的混合溶液間進行正向及反向萃取。相似於陰離子型界面活性劑的反微胞萃取，蛋白質在較低pH值時能正向萃取至離子液體相裡，在pH值較高時則反向萃取回水層相。以細胞色素 c (cytochrome c)為例，在pH 2.7時可完全萃取進入離子液體相中，在pH 11時即可全部反向萃取回到水層相;肌紅蛋白(myoglobin)則在pH 5時能完全萃取，但是在反向萃取時即使在pH 13反向萃取率也只有50 %；核糖核酸酶 A (ribonuclease A)在pH 2時萃取率60 %，反向萃取時在pH 11即可全部反向萃取回水層相。除此之外，正、反向萃取達成平衡的速率皆相當快。此液相╱液相萃取技術或許可使用於蛋白質的純化及濃縮，並且開拓離子液體一項新的應用領域。

Due to their unique physicochemical properties, such as high polar, high conductivity, no detectable vapor and relatively high thermal stability, ionic liquids(ILs) continue to attract wide research interests on the capability for replacing traditional volatile organic compounds (VOCs), including in the applications of liquid/liquid extraction, organic synthesis and inorganic catalysis. In addition, ILs are relatively safer than VOCs and recyclable, so they are regarded as green solvents. Protein purification is an important task in biochemistry and biotechnology. Traditionally, two liquid/liquid extraction techniques, i.e. two-phase aqueous polymer and reverse micelle systems, have been used for protein purification and enrichment. In this study, we explore that protein can be transported between aqueous solutions and a mixture of ethyl acetate and ionic liquid [C4mim][PF6] by manipulation of pH. Proteins can be extracted from one aqueous solution at low pH into IL phases and back-extracted to another aqueous solution at high pH. For example, the forward extraction of cytochrome c molecules reaches to an almost quantitative completion at pH 2.7 and back-extracted to an aqueous solution at pH 11. Myoglobin can be completely extracted at pH 5, but the recovery is only about 50 % at pH 13. As for ribonuclease A, the forward extraction reaches ~60 % at pH 2 while the recovery reaches to quantitative completion at pH 11. Both the forward and backward processes are found reaching equilibrium in very short time. Therefore, this technique may be applied in purification and enrichment of proteins and thus extends a new field for the application of ILs.