Crystal Morphology And Activity Of Lysozyme Mediated By Ionic Liquids And Spectroscopic Study On Interaction

Inoic liquids,one room temperature melted salt,have extensive potentials in many areas due to its'good physic-chemical property and designable characteristic.With the development of biotechnology,the advantages of biomacromolecule like protein used in medicine began to be known and valued.What's more,the study on protein crystal morphology and its'secondary structure has a significant meaning to understand protein function,activity control and drug design.However,the obtain of protein crystal which have high activity and also is available for X-ray diffraction is still an unsolved problem in this area.In our previous study,it was found that ionic liquids have a control on the lysozyme.Thus,the study on lysozyme crystallization process,enzyme activity and interactions between ILs and lysozyme has a significant meaning on the obtain of lysozyme crystals with high quility.In this paper,the crystallization process,changes of second structure and enzyme activity were studied by using hen egg-white lysozyme as model protein,hydrophilic ILs[BMIM]BF₄,[BMIM]Cl,[BMIM]Br and[DMIM]I as addictives.The interaction mechanism between ILs and lysozyme has also been discussed.Lysozyme crystals were obtained by cooling crystallization,the mechanism of lysozyme crystallization induced by ILs was preliminary discussed on the basis of results collected by optical microscope,X-ray diffraction and SEM.It is easy to tell that ILs made the crystallization process much easier and controllable.Optical microscope,X-ray diffraction and SEM results revealed that[BMIM]BF₄,[BMIM]Cl,[BMIM]Br only has an impact on the crystal morphology,while[DMIM]I can promote the formation of monoclinic crystal.Besides,the investigation on enzyme activity indicated that the enzyme activity can be enhanced by the crystallization condition control.The changes in lysozyme second structure was studied by UV spectrophotometer and fluorescent spectrometer.UV spectrum revealed that the interaction between ILs and lysozyme is very weak.The fluorescent spectroscopy was analysed by Stern-Volmer equation,it turns out that ILs have a static quenching effect on lysozyme's fluorescence.The information get from the binding site and binding constant illustrated that the interaction between them is weak,which is accord with the UV results.Synchronous fluorescence spectroscopy proved that ILs has a greater impact on Tryptophan?Trp?rather than Tyrosine?Tyr?.By calculating the enzyme activity at different pH,ILs,IL concentration,the interaction between ILs and lysozyme has been studied.The enzyme activity was highest when the IL concentration is 75mM,pH=6.85.The binding site of ILs switched from Trp 62 to the core active site with the increase of IL concentration.