Application of Ionic Liquids to Enzyme Stabilizers for the Heat Stress Tolerance of Enzymes

The heat stress tolerance of hen egg white lysozyme (HEWL) was markedly enhanced by the addition of water-miscible aprotic ionic liquids consisting of 1-ethyl-3-methylimidazolium cation and several kinds of anions to the aqueous solution of HEWL. Inorganic salts and glycerol inhibited the formation of enzyme aggregation to some extent, and enhanced the thermal stability of HEWL, while β-cyclodextrin, Triton-X, and pectin had no effect on the thermal stability of HEWL. On the other hand, ionic liquids could more effectively increase the thermal stability of HEWL, compared to conventional stabilizing agents. Addition of ionic liquids to the aqueous solution of HEWL prevented partially unfolded enzymes from aggregating irreversibly at high temperatures. The thermal denaturation curve of HEWL with ionic liquids was remarkably shifted to higher temperature, compared with that without ionic liquids. Moreover, the remaining activity of HEWL after the heat treatment was strongly dependent upon the kind and concentration of ionic liquids. The remaining activities of HEWL with 1.5 M 1-ethyl-3-methylimidazolium tetrafluoroborate ([emim][BF₄]) and 0.1 M 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([emim][Tf]) exhibited 88 and 68% after the heat treatment at 90°C for 30 min, respectively, although that without ionic liquids was perfectly lost.