Investigations On The Application Of Hydrophilic Ionic Liquid1,3-butyl-imidazole Chloride To Lysozyme Crystallization

Attributting to the designable characteristics and excellent physico-chemical features, ionic liquids have become potential functional materials and exploited widely in various fields. X-ray diffraction analysis is an efficient technique to investigate conformations of proteins, however, it is still a bottleneck for obtaining suitable protein crystals in order to perform X-ray diffraction analysis. Therefore, it is of significance to develop efficient technology to obtain high-quality protein single crystals.In this thesis, hydrophilic ionic liquid1,3-butyl-imidazole chloride (BBimCI) was employed as additive in the crystallization process of lysozyme. We have investigated the effects of the ionic liquid on the crystal morphology, crystal numbers, crystal growth rate and the tolerance to concomitant impurities. In addition, the relevant mechanism for protein crystallization in the presence of ionic liquid was also investigated. The conformational structure of the protein crystal was analysed by X-ray single crystal diffraction.The results indicated that BBimCl could effectively improve the solubility of lysozyme and resulted in a lower supersaturation during the process of crystallization; the negligible vapor pressure of ionic liquid helps to modulate the rate for approaching the supersaturation state and thus contols the rate of crystal growth, which is beneficial to avoid the emergence of polycrystalline crystals and eventually led to the formation of better shaped large single crystals suitable for X-ray diffraction analysis. Moreover, a significantly improved tolerance to the coexistence of impurities for the crystallization of lysozyme was also demonstrated in the presence of BBimCl. The X-ray diffraction analysis results indicated that BBimCI posed no effect on the conformational structure of the lysozyme crystal, while the cation of BBim+located in the interior of lysozyme via interaction with the residues of Trp62, Trp63, Asp101.The direct crystallization of lysozyme from egg white was achieved in the presence of BBimCI after the fresh egg white has been treated with dilution, acidification, heating and lyophilization. The obtaibed single crystals were found to possess high purity and biological activities. This observation indicated the potential of developing crystallization methodology with ILs as additives for the separation/purification of protein species of interest from complex sample matrixes.