离子液体诱导溶菌酶结晶及机理研究
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摘要
随着生物技术的发展,蛋白质等生物大分子药物不断涌现,其独特的功能特性越来越受到人们的重视。蛋白质结晶过程复杂,影响因素多,且蛋白质晶体体积较小,脱水易碎裂,对X-射线的衍射能力相对较弱,并且对温度特别敏感,长时间暴露在辐射下晶体会损坏。制备适合X-射线衍射的蛋白质晶体是蛋白质结晶的主要技术瓶颈之一,严重制约着蛋白质工程等新型生物技术的发展。
离子液体是指在室温下呈液态的盐类化合物。离子液体在催化领域取得了广泛的应用,在许多的分离过程中被称作“绿色溶剂”。本文就是以溶菌酶为实验对象,研究[C_4mim]BF_4,[C_4mim]Cl,[C_4mim]Br,[bmim]I四种离子液体对溶菌酶结晶过程及晶体性质的影响,并对结晶机理进行初步探讨。
实验采用紫外分光光度法测量了溶菌酶的溶解度,及不同离子液体添加剂对溶解度的影响,并与溶解度经验方程相关联;使用激光动态法测量了离子液体对溶菌酶结晶诱导期的影响,并计算出表面张力和初级成核速率。利用差示扫描量热仪(DSC)及各种热动力学分析方法对溶菌酶晶体热变性及热降解过程进行了热动力学研究,用模型函数法(model-fitting method)和无模型函数法(model-free method)求解动力学方程。
采用冷却结晶获得溶菌酶晶体,通过光学显微镜,偏光显微镜及XRD谱图、傅立叶转换红外谱图、拉曼谱图对离子液体诱导溶菌酶结晶的机理进行了分析和探讨,添加离子液体诱导溶菌酶结晶使溶菌酶晶体形态改变,并且XRD分辨率改善;利用实时在线拉曼光谱仪分析不同离子液体添加剂下过饱和度对溶液中溶菌酶分子结构及分子间作用力的影响;对获得的溶菌酶晶体进行生物活性的测定,研究离子液体作为添加剂诱导溶菌酶结晶对其晶体活性的影响,实验结果表示离子液体作用下,溶菌酶晶体的活性提高。
在实验的基础上使用Material Studio软件Morphology模块中BFDH、Growth Morphology模型对溶菌酶的理论晶习进行预测,预测结果与实验获得两种晶型溶菌酶晶体形状基本一致。
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.
离子液体是指在室温下呈液态的盐类化合物。离子液体在催化领域取得了广泛的应用,在许多的分离过程中被称作“绿色溶剂”。本文就是以溶菌酶为实验对象,研究[C_4mim]BF_4,[C_4mim]Cl,[C_4mim]Br,[bmim]I四种离子液体对溶菌酶结晶过程及晶体性质的影响,并对结晶机理进行初步探讨。
实验采用紫外分光光度法测量了溶菌酶的溶解度,及不同离子液体添加剂对溶解度的影响,并与溶解度经验方程相关联;使用激光动态法测量了离子液体对溶菌酶结晶诱导期的影响,并计算出表面张力和初级成核速率。利用差示扫描量热仪(DSC)及各种热动力学分析方法对溶菌酶晶体热变性及热降解过程进行了热动力学研究,用模型函数法(model-fitting method)和无模型函数法(model-free method)求解动力学方程。
采用冷却结晶获得溶菌酶晶体,通过光学显微镜,偏光显微镜及XRD谱图、傅立叶转换红外谱图、拉曼谱图对离子液体诱导溶菌酶结晶的机理进行了分析和探讨,添加离子液体诱导溶菌酶结晶使溶菌酶晶体形态改变,并且XRD分辨率改善;利用实时在线拉曼光谱仪分析不同离子液体添加剂下过饱和度对溶液中溶菌酶分子结构及分子间作用力的影响;对获得的溶菌酶晶体进行生物活性的测定,研究离子液体作为添加剂诱导溶菌酶结晶对其晶体活性的影响,实验结果表示离子液体作用下,溶菌酶晶体的活性提高。
在实验的基础上使用Material Studio软件Morphology模块中BFDH、Growth Morphology模型对溶菌酶的理论晶习进行预测,预测结果与实验获得两种晶型溶菌酶晶体形状基本一致。
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.
引文
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