Recovery of Enzyme Structure and Activity Following Rehydration From Ionic Liquid

Long-term preservation of proteins at room temperature continues to be a major challenge. Towards using ionic liquids (ILs) to address this challenge, here we present a combination of experiments and simulations to investigate changes in lysozyme upon rehydration from IL mixtures using two imidazolium-based ILs. Circular dichroism spectroscopy confirms that lysozyme maintains its secondary structure upon rehydration, even after 295 days. Increasing the IL concentration decreases the activity of lysozyme and is ultimately quenched at sufficiently high IL concentrations, but the rehydration of lysozyme from high IL concentrations completely restores its activity. From simulations we observe kinetic trapping of active site by EMIM⁺, which leads to possible competitive inhibition and results in the diminish of lysozyme activity. Upon rehydration, fast leaving of EMIM⁺ is observed and the availability of active site is restored. In addition, suppression of structure fluctuations is also observed when in high IL concentrations, which also explains the decrease of activity. The return of native protein structure and activity indicates that after rehydration lysozyme returns to its original state.  Our results also suggest a simple route to protein recovery following extended storage.