Study On Lysozyme Crystallization Induced By Ionic Liquids And Its Mechanism

With the development of biotechnology, biological macromolecular drugs such as protein quikely increase, whose unique functional properties have received more and more attention. The process of protein crystallization is a complicate process, and there are many factors to affect the crystallization process. Besides, protein crystals are small and easy to fragment when dehydrated, and its X-ray distraction ability is poor. Protein crystals are particular sensitive to temperature and are easy to damage when exposed to radiation in long time. So the main technical bottleneck of protein crystallization, which seriously restricts the development of new biotechnology such as protein engineering, is to prepare crystals proper for X-ray distraction.Ionic liquids, called as liquid salts at room temperature, have been extensively studied, as catalysts and catalytic support, which are also considered as"green solvents"for various separation processes. In this thesis, the crystallization process,crystal properties and crystallizing mechanism of lysozyme with ionic liquids were systematic studied as the following:The solubility of lysozyme in the buffer solution with different ionic liquids was measured by US spectrophotometry method, and the solubility curves were consistent with the experiential equations of solubility. The induce time of lysozyme crystallization with different ionic liquids was measured by dynamic-temperature- changing-laser-method, and the interfacial tension and primary nucleation rate were calculated.The thermodynamics of denaturation and decomposition of lysozyme were investigated using DSC. The model-fitting and model-free methods were used to analyze the thermodynamics.The crystals were obtained by cooling crystallization from saturated solution with different ionic liquids. The crystallization mechanism of lysozyme with ionic liquids was analyzed and discussed through optical microscope, polarizing microscope, X-ray diffraction, FT-IR spectra and Raman spectra. The ionic liquids produced changes in crystal morphology. Crystals grown using ionic liquids as precipitating agents or as additives provided X-ray diffraction resolution better than that obtained without ionic liquids. The effecting of super-saturation of lysozyme solution with ionic liquids on molecular structure and molecular intermolecular forces was analyzed by real-time online Raman spectra. Beside, the biological activity of lysozyme crystal induced by different ionic liquids was determined, which indicated that the ionic liquids have better effect on the lysozyme activity.The Material Studio software was used to predict the morphologies of lysozyme. The morphologies, which were predicted by two models, BFDH and Growth Morphology, were ultimately consistent with the morphologies of lysozyme obtained in the laboratory.