應用親和性離子液體於溶菌酶之萃取

Abstract

近年來，室溫離子液體(room temperature ionic liquids, ILs)已廣泛應用於液相/液相萃取、有機合成及催化反應等研究領域。由於離子液體提供較為安全的實驗環境且可回收再利用，能減低對環境的汙染，取代傳統有機溶劑，因此被視為綠色溶劑。 在生物分子液相/液相萃取方面，已有研究發現離子液體可應用於萃取胺基酸和蛋白質，但是萃取並無選擇性。Cibacron Blue F-3GA(CB3GA)是一對蛋白質有親和性的染料配體，本實驗利用CB3GA取代[BMIM][Cl]的陰離子，合成為具有親和性的離子液體[BMIM]3[CB3GA]，將其作為萃取劑，溶於疏水性離子液體[BMIM][PF6]，從水溶液中萃取蛋白質溶菌酶(lysozyme)。 以含有3.8 mM [BMIM]3[CB3GA]之離子液體[BMIM][PF6]，對500 mg/L 溶菌酶水溶液做萃取，在pH 4.0條件下，正向萃取率約為95 %。再以1.0M KCl，pH 8.0水溶液將溶菌酶萃取回水相，其反向萃取率約為93 %，溶菌酶回收率為87 %。隨著pH值降低，正向萃取率越好，反向萃取則相反。此液相/液相萃取系統對於溶菌酶不論是正向萃取或反向萃取，反應很快即能達到平衡。另外，我們也嘗試萃取蛋白質混合溶液，發現對於溶菌酶有相當程度的選擇性。因此，此親和性離子液體具備蛋白質純化和分離的潛力。

In recent years, room temperature ionic liquids (ILs) have been applied to broad research fields, such as liquid /liquid extractions, organic syntheses and catalytic reactions. ILs are recyclable and relatively safer than traditional volatile organic compounds (VOCs), so they are regarded as green solvents. It has been found that ionic liquids can be used in extracting amino acids and proteins from aqueous solutions, however, without selectivity. Cibacron Blue F-3GA (CB3GA) is an affinity dye which has been widely used for protein purification. In this study, IL [BMIM]3[CB3GA] synthesized as an extraction agent, was dissolved in a water-immiscible IL 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) for extraction of lysozyme. The forward extraction efficiency for lysozyme was 95% using a [BMIM][PF6] solution containing 3.8 mM [BMIM]3[CB] while the aqueous phase was pH 4.0. Lysozyme was then extracted back to another aqueous solution of a buffer solution of pH 8.0 with 1.0M KCl. The back extraction efficiency was 93%, thus the lysozyme recovery was 87% for the complete process. As the pH of the aqueous solution decreased, the forward extraction efficiency increased, while the back extraction efficiency decreased. Both the forward and back processes were found reaching equilibrium in very short time. In addition, a protein mixture was tested to examine the selectivity of this extraction system. The preliminary outcome showed good selectivity for lysozyme. This dye–modified IL may provide a potential for protein separation and purification.