聚合物或离子液体/柠檬酸钾双水相分离纯化蛋白质的研究
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摘要
双水相萃取技术(又称水溶液两相分配技术)是近年来出现的、引人注目的、极有前途的分离技术。它作为一种新型的液-液萃取技术已被广泛应用于生物化工、生物化学和细胞生物学等领域,具有广阔的应用前景。本文选取了聚乙二醇、咪唑类溴盐/柠檬酸钾两种类型的双水相体系研究了蛋白质的分离纯化。论文的主要工作归纳如下：
1.聚乙二醇(PEG)、咪唑类溴盐离子液体/柠檬酸钾双水相相平衡的性质研究
用浊点滴定法测定了聚乙二醇、咪唑类溴盐/柠檬酸钾两种类型的双水相体系相图。结果表明,各PEG和离子液体的成相能力分别为PEG6000>PEG4000>PEG2000>PEG1000：[C2mim]Br<[C4mim]Br<[C6min]Br。对于PEG/柠檬酸钾体系,随着温度的升高系线的斜率和长度逐渐增大；外加中性盐对该体系的分相有一定的协助作用。对于咪唑类溴盐离子液体/柠檬酸钾体系,研究发现在浊点附近体系中柠檬酸钾的量对体系的R值和F值影响显著。本章研究为以后实验提供了基础数据和必要的理论指导。
2.聚乙二醇/柠檬酸钾双水相体系萃取牛血清蛋白的研究
首次采用聚乙二醇(PEG)/柠檬酸钾双水相体系实现牛血清蛋白(BSA)的萃取和反萃取。研究了聚乙二醇分子量(PEG1000、PEG2000、PEG4000、PEG6000)、聚乙二醇浓度、柠檬酸钾浓度、牛血清蛋白浓度、体系的pH和体系温度对萃取性能的影响。发现在PEG1000/柠檬酸钾双水相体系中,pH对牛血清蛋白分配的影响显著。BSA在该双水相体系中的分配系数Ka和萃取率Ye随pH增大而增大。当体系的pH和温度分别为7.0和30℃,体系组成为19%(w/w)PEG1000/20%(w/w)柠檬酸钾,BSA浓度为0.75mg/g时,牛血清蛋白在该体系中的萃取率高达99%。通过降低体系的pH值,我们实现了PEG相中牛血清蛋白的反萃取,其反萃取率为92%。反萃取过程不仅实现了牛血清蛋白与聚乙二醇的分离,而且能使聚乙二醇得以重复利用。最终,牛血清蛋白在整个过程中的萃取率(Ye+Ybe)为91%。
3.离子液体双水相体系萃取分离细胞色素c的热力学研究
研究了咪唑类离子液体双水相体系对细胞色素c(Cyt-c)的萃取。研究了离子液体阳离子的烷基链长度、柠檬酸钾浓度、温度、pH等因素对Cyt-c萃取率的影响。得出了萃取Cyt-c过程的相关热力学参数的值(△G°T,△H°T和△S°T)。热力学研究发现疏水作用和静电作用是该体系实现Cyt-c萃取的主导作用力。在最佳条件下,1-已基-3-甲基咪唑溴盐/柠檬酸钾双水相体系将Cyt-c萃取到富含离子液体的上相(萃取率为94%)。从UV-vis和圆二色谱(CD)表征结果可以看出Cyt-c与离子液体之间没有形成化学键并保持了Cyt-c结构特征。与传统液液萃取技术(含有大量的挥发性有机溶剂)相比,离子液体双水相萃取技术占有很大的优势。
4.利用聚乙二醇(PEG)/柠檬酸钾双水相体系从鸡蛋清中分离纯化溶菌酶的研究
双水相萃取技术适用于生物物质的分离,具有良好的发展前景。利用PEG4000/柠檬酸钾双水相体系研究了鸡蛋清中溶菌酶的分离纯化。通过响应曲面法(RSM),确定了从鸡蛋清中提取溶菌酶的最优化条件并建立了溶菌酶回收率、纯化系数和活性产率的数学模型。部分析因实验分析结果显示,pH对溶菌酶的回收和纯化效果均有较大影响。在pH为5.5、温度为30℃的条件下,该体系萃取分离溶菌酶的最优体系组成为：18%(w/w)PEG4000,16%(w/w)柠檬酸钾,3.75%(w/w)KCl。在该条件下,纯化后溶菌酶的活性、纯化系数和活性产率分别为31100U/mg,21.11和103%。实验结果证明PEG4000/柠檬酸钾双水相体系在分离纯化溶菌酶的应用方面具有潜在的应用价值。
5.从木瓜蛋白酶制剂中分离纯化木瓜蛋白酶的研究
利用聚乙二醇(PEG)/柠檬酸钾双水相体系研究了从粗制木瓜蛋白酶制剂中提取木瓜蛋白酶的研究。考察了聚乙二醇分子量(PEG1000、PEG2000、PEG4000、PEG6000)、聚乙二醇的浓度、柠檬酸钾的浓度、外加中性盐(氯化钾)的浓度、pH和温度等因素对木瓜蛋白酶纯化的影响。实验结果显示木瓜蛋白酶倾向于分配到双水相体系中亲水性较强的下相(富含盐的相),且最优化体系为PEG4000/柠檬酸钾体系。在此基础上,我们通过响应面法建立了关于木瓜蛋白酶纯化的数学模型并确定了该体系纯化木瓜蛋白酶的最优化条件。在pH为5.0、温度为30℃的条件下,该体系纯化木瓜蛋白酶的最佳体系组成为：PEG4000浓度15%(w/w),柠檬酸钾浓度17%(w/w),KCl浓度5.0%(w/w)。在该条件下,纯化后木瓜蛋白酶的比活度、纯化系数和活性产率分别为1703.02U/mg,1.89和94%。SDS-PAGE电泳实验显示与酶制剂溶液的蛋白带相比下相溶液的蛋白带明显减少。该体系在木瓜蛋白酶的纯化方面有潜在的应用价值。
Recently, aqueous two-phase extraction (or partition of two aqueous phase system) is a compelling and a very promising technology for separation. As a novel liquid-liquid extraction technology, aqueous two-phase extraction has broad application prospects due to being widely used in bio-chemical industry, biochemistry, cell biology and other fields. The PEG,[Cnmim]Br (n=2,4,6)/potassium citrate aqueous two-phase systems were selected for separation and purification of proteins. The detailed information of the paper is summarized as follows:
1. Liquid-liquid equilibrium of the polyethylene glycol (PEG) or [Cnmim]Br (n=2,4,6)/potassium citrate aqueous two-phase systems
Phase diagrams were determined by the turbidimetric titration method as the one used previously. The results showed that the ability of PEG and ionic liquid for two-phase formation followed the order:PEG6000> PEG4000> PEG2000> PEG1000;[C2mim]Br<[C4mim]Br<[C6mim]Br. To PEG/potassium citrate system, the slope of the equilibrium line and the tie line length increased as the increasing of temperature; the additional salt plays a supportive role in this system. To [Cnmim]Br (n=2,4,6)/potassium citrate system, the phase volume ratio (R) and enrichment factor (F) were significantly affected by the amount of potassium citrate near the cloud point of the system. The objects of this work are to provide the basic data and the necessary theoretical guidance.
2. Bovine serum albumin partitioning in polyethylene glycol (PEG)/potassium citrate aqueous two-phase systems
The extraction and back-extraction of bovine serum albumin (BSA) have been studied by liquid-liquid extraction with poly(ethylene glycol)(PEG)/potassium citrate aqueous two-phase system (ATPS). In this work, the ATPS was examined with regard to the effects of PEG molecular weight (PEG1000,2000,4000and6000), PEG and potassium citrate concentration, BSA concentration (CBSA), pH and temperature on BSA partition. The pH was found to have significant effects on BSA partition with low molecular weight PEG1000. The yield of the BSA,99%, was obtained in the top phase under the following conditions:19%(w/w) PEG1000,20%(w/w) potassium citrate and0.75mg/g CBSA at pH7.0and30℃. BSA can be re-extracted to a new citrate phase by decreasing the pH of the system with a92%yield. The back-extraction not only separates the BSA from the polymer, but also allows the polymer to be recycled. The global yield (Ye+Ybe) is up to91%.
3. Thermodynamic studies of partitioning behavior of cytochrome c in ionic liquid-based aqueous two-phase system
The ionic liquid/aqueous two-phase extraction systems (ATPSs) based on imidazolium ionic liquids were used to extract cytochrome c. Effects of the alkyl chain length of cation of the ionic liquids, concentration of potassium citrate, as well as the extraction temperature and pH value on the extraction efficiency have been investigated. The thermodynamic parameters (△GTo,△HTo and△STo) associated with Cyt-c partitioning in aqueous two phase systems were determined. Thermodynamic studies indicated that the partitioning of Cyt-c was driven by both hydrophobic interactions and electrostatic interactions in the extraction process. Under the optimum conditions, experiment results show that94%of the cytchrome c could be extracted into the ionic liquid-rich phase in a one-step extraction. The structural characterization of Cyt-c in the IL ATPS was investigated by UV-vis and Circular Dichroism (CD) spectra. The results demonstrated that no direct bonding interaction observed between ionic liquid and cytochrome c, while the native properties of the cytochrome c were not altered. Compared with traditional liquid-liquid extractions, which use toxic organic solvents, ionic liquid/aqueous two phase extraction offers clear advantages due to no use of volatile organic solvent and low consumption of imidazolium ionic liquids.
4. The purification of lysozyme from crude hen egg white using polyethylene glycol (PEG)/citrate aqueous two phase system
Aqueous two-phase extraction is a versatile method suitable for separating biological particles and macromolecules. In the present wok, the feasibility of using PEG4000/potassium citrate aqueous two-phase system (ATPS) for recovering and purifying lysozyme was investigated. Response surface methodology was used to determine an optimized ATPS for purification of lysozyme from crude hen egg white. Mathematical models concerning the purification of lysozyme from chicken egg white in polyethylene glycol4000(PEG4000)/potassium citrate ATPS are established using response surface methodology. Screening experiment by fractional factorial designs shows that the pH of the system significantly affects the recovery and purification of the lysozyme. An optimized aqueous two phase system ATPS was proved to be at pH5.5and30℃and contained18%(w/w) PEG,16%(w/w) potassium citrate,3.75%(w/w) potassium chloride (KC1). Under those conditions, the specific activity, purification factor and activity yield for lysozyme were31100U/mg,21.11and103%, respectively. The PEG4000/potassium citrate ATPS has the potential to be applied to establish bioprocesses for the primary recovery and partial purification of lysozyme.
5. Application of response surface methodology to evaluate the purification papain by aqueous two-phase system
Mathematical models concerning the purification of papain from the crud papain in an aqueous two-phase system (ATPS) which are composed of polyethylene glycol (PEG)/potassium citrate are established with response surface methodology. Effects of PEG molecular weight, the concentration of PEG and potassium citrate, the concentration of additional salt (KC1), the temperature and pH on the papain purification have been investigated. Research results showed that papain tends to partitions to the more hydrophilic salt-rich phase. The steepest ascent method was used to locate the optimal domain. Papain was successfully extracted to the bottom phase of a system composed of15%(w/w) PEG4000,17%(w/w) citrate and5.0%(w/w) KC1at pH5.0and30C. Under those conditions, the specific activity, purification factor and activity yield for papain were1703.02U/mg,1.89and94%, respectively. The purity of extracted papain was confirmed by SDS-PAGE analysis. This ATPS has the potential to be applied to purification of papain.
1.聚乙二醇(PEG)、咪唑类溴盐离子液体/柠檬酸钾双水相相平衡的性质研究
用浊点滴定法测定了聚乙二醇、咪唑类溴盐/柠檬酸钾两种类型的双水相体系相图。结果表明,各PEG和离子液体的成相能力分别为PEG6000>PEG4000>PEG2000>PEG1000：[C2mim]Br<[C4mim]Br<[C6min]Br。对于PEG/柠檬酸钾体系,随着温度的升高系线的斜率和长度逐渐增大；外加中性盐对该体系的分相有一定的协助作用。对于咪唑类溴盐离子液体/柠檬酸钾体系,研究发现在浊点附近体系中柠檬酸钾的量对体系的R值和F值影响显著。本章研究为以后实验提供了基础数据和必要的理论指导。
2.聚乙二醇/柠檬酸钾双水相体系萃取牛血清蛋白的研究
首次采用聚乙二醇(PEG)/柠檬酸钾双水相体系实现牛血清蛋白(BSA)的萃取和反萃取。研究了聚乙二醇分子量(PEG1000、PEG2000、PEG4000、PEG6000)、聚乙二醇浓度、柠檬酸钾浓度、牛血清蛋白浓度、体系的pH和体系温度对萃取性能的影响。发现在PEG1000/柠檬酸钾双水相体系中,pH对牛血清蛋白分配的影响显著。BSA在该双水相体系中的分配系数Ka和萃取率Ye随pH增大而增大。当体系的pH和温度分别为7.0和30℃,体系组成为19%(w/w)PEG1000/20%(w/w)柠檬酸钾,BSA浓度为0.75mg/g时,牛血清蛋白在该体系中的萃取率高达99%。通过降低体系的pH值,我们实现了PEG相中牛血清蛋白的反萃取,其反萃取率为92%。反萃取过程不仅实现了牛血清蛋白与聚乙二醇的分离,而且能使聚乙二醇得以重复利用。最终,牛血清蛋白在整个过程中的萃取率(Ye+Ybe)为91%。
3.离子液体双水相体系萃取分离细胞色素c的热力学研究
研究了咪唑类离子液体双水相体系对细胞色素c(Cyt-c)的萃取。研究了离子液体阳离子的烷基链长度、柠檬酸钾浓度、温度、pH等因素对Cyt-c萃取率的影响。得出了萃取Cyt-c过程的相关热力学参数的值(△G°T,△H°T和△S°T)。热力学研究发现疏水作用和静电作用是该体系实现Cyt-c萃取的主导作用力。在最佳条件下,1-已基-3-甲基咪唑溴盐/柠檬酸钾双水相体系将Cyt-c萃取到富含离子液体的上相(萃取率为94%)。从UV-vis和圆二色谱(CD)表征结果可以看出Cyt-c与离子液体之间没有形成化学键并保持了Cyt-c结构特征。与传统液液萃取技术(含有大量的挥发性有机溶剂)相比,离子液体双水相萃取技术占有很大的优势。
4.利用聚乙二醇(PEG)/柠檬酸钾双水相体系从鸡蛋清中分离纯化溶菌酶的研究
双水相萃取技术适用于生物物质的分离,具有良好的发展前景。利用PEG4000/柠檬酸钾双水相体系研究了鸡蛋清中溶菌酶的分离纯化。通过响应曲面法(RSM),确定了从鸡蛋清中提取溶菌酶的最优化条件并建立了溶菌酶回收率、纯化系数和活性产率的数学模型。部分析因实验分析结果显示,pH对溶菌酶的回收和纯化效果均有较大影响。在pH为5.5、温度为30℃的条件下,该体系萃取分离溶菌酶的最优体系组成为：18%(w/w)PEG4000,16%(w/w)柠檬酸钾,3.75%(w/w)KCl。在该条件下,纯化后溶菌酶的活性、纯化系数和活性产率分别为31100U/mg,21.11和103%。实验结果证明PEG4000/柠檬酸钾双水相体系在分离纯化溶菌酶的应用方面具有潜在的应用价值。
5.从木瓜蛋白酶制剂中分离纯化木瓜蛋白酶的研究
利用聚乙二醇(PEG)/柠檬酸钾双水相体系研究了从粗制木瓜蛋白酶制剂中提取木瓜蛋白酶的研究。考察了聚乙二醇分子量(PEG1000、PEG2000、PEG4000、PEG6000)、聚乙二醇的浓度、柠檬酸钾的浓度、外加中性盐(氯化钾)的浓度、pH和温度等因素对木瓜蛋白酶纯化的影响。实验结果显示木瓜蛋白酶倾向于分配到双水相体系中亲水性较强的下相(富含盐的相),且最优化体系为PEG4000/柠檬酸钾体系。在此基础上,我们通过响应面法建立了关于木瓜蛋白酶纯化的数学模型并确定了该体系纯化木瓜蛋白酶的最优化条件。在pH为5.0、温度为30℃的条件下,该体系纯化木瓜蛋白酶的最佳体系组成为：PEG4000浓度15%(w/w),柠檬酸钾浓度17%(w/w),KCl浓度5.0%(w/w)。在该条件下,纯化后木瓜蛋白酶的比活度、纯化系数和活性产率分别为1703.02U/mg,1.89和94%。SDS-PAGE电泳实验显示与酶制剂溶液的蛋白带相比下相溶液的蛋白带明显减少。该体系在木瓜蛋白酶的纯化方面有潜在的应用价值。
Recently, aqueous two-phase extraction (or partition of two aqueous phase system) is a compelling and a very promising technology for separation. As a novel liquid-liquid extraction technology, aqueous two-phase extraction has broad application prospects due to being widely used in bio-chemical industry, biochemistry, cell biology and other fields. The PEG,[Cnmim]Br (n=2,4,6)/potassium citrate aqueous two-phase systems were selected for separation and purification of proteins. The detailed information of the paper is summarized as follows:
1. Liquid-liquid equilibrium of the polyethylene glycol (PEG) or [Cnmim]Br (n=2,4,6)/potassium citrate aqueous two-phase systems
Phase diagrams were determined by the turbidimetric titration method as the one used previously. The results showed that the ability of PEG and ionic liquid for two-phase formation followed the order:PEG6000> PEG4000> PEG2000> PEG1000;[C2mim]Br<[C4mim]Br<[C6mim]Br. To PEG/potassium citrate system, the slope of the equilibrium line and the tie line length increased as the increasing of temperature; the additional salt plays a supportive role in this system. To [Cnmim]Br (n=2,4,6)/potassium citrate system, the phase volume ratio (R) and enrichment factor (F) were significantly affected by the amount of potassium citrate near the cloud point of the system. The objects of this work are to provide the basic data and the necessary theoretical guidance.
2. Bovine serum albumin partitioning in polyethylene glycol (PEG)/potassium citrate aqueous two-phase systems
The extraction and back-extraction of bovine serum albumin (BSA) have been studied by liquid-liquid extraction with poly(ethylene glycol)(PEG)/potassium citrate aqueous two-phase system (ATPS). In this work, the ATPS was examined with regard to the effects of PEG molecular weight (PEG1000,2000,4000and6000), PEG and potassium citrate concentration, BSA concentration (CBSA), pH and temperature on BSA partition. The pH was found to have significant effects on BSA partition with low molecular weight PEG1000. The yield of the BSA,99%, was obtained in the top phase under the following conditions:19%(w/w) PEG1000,20%(w/w) potassium citrate and0.75mg/g CBSA at pH7.0and30℃. BSA can be re-extracted to a new citrate phase by decreasing the pH of the system with a92%yield. The back-extraction not only separates the BSA from the polymer, but also allows the polymer to be recycled. The global yield (Ye+Ybe) is up to91%.
3. Thermodynamic studies of partitioning behavior of cytochrome c in ionic liquid-based aqueous two-phase system
The ionic liquid/aqueous two-phase extraction systems (ATPSs) based on imidazolium ionic liquids were used to extract cytochrome c. Effects of the alkyl chain length of cation of the ionic liquids, concentration of potassium citrate, as well as the extraction temperature and pH value on the extraction efficiency have been investigated. The thermodynamic parameters (△GTo,△HTo and△STo) associated with Cyt-c partitioning in aqueous two phase systems were determined. Thermodynamic studies indicated that the partitioning of Cyt-c was driven by both hydrophobic interactions and electrostatic interactions in the extraction process. Under the optimum conditions, experiment results show that94%of the cytchrome c could be extracted into the ionic liquid-rich phase in a one-step extraction. The structural characterization of Cyt-c in the IL ATPS was investigated by UV-vis and Circular Dichroism (CD) spectra. The results demonstrated that no direct bonding interaction observed between ionic liquid and cytochrome c, while the native properties of the cytochrome c were not altered. Compared with traditional liquid-liquid extractions, which use toxic organic solvents, ionic liquid/aqueous two phase extraction offers clear advantages due to no use of volatile organic solvent and low consumption of imidazolium ionic liquids.
4. The purification of lysozyme from crude hen egg white using polyethylene glycol (PEG)/citrate aqueous two phase system
Aqueous two-phase extraction is a versatile method suitable for separating biological particles and macromolecules. In the present wok, the feasibility of using PEG4000/potassium citrate aqueous two-phase system (ATPS) for recovering and purifying lysozyme was investigated. Response surface methodology was used to determine an optimized ATPS for purification of lysozyme from crude hen egg white. Mathematical models concerning the purification of lysozyme from chicken egg white in polyethylene glycol4000(PEG4000)/potassium citrate ATPS are established using response surface methodology. Screening experiment by fractional factorial designs shows that the pH of the system significantly affects the recovery and purification of the lysozyme. An optimized aqueous two phase system ATPS was proved to be at pH5.5and30℃and contained18%(w/w) PEG,16%(w/w) potassium citrate,3.75%(w/w) potassium chloride (KC1). Under those conditions, the specific activity, purification factor and activity yield for lysozyme were31100U/mg,21.11and103%, respectively. The PEG4000/potassium citrate ATPS has the potential to be applied to establish bioprocesses for the primary recovery and partial purification of lysozyme.
5. Application of response surface methodology to evaluate the purification papain by aqueous two-phase system
Mathematical models concerning the purification of papain from the crud papain in an aqueous two-phase system (ATPS) which are composed of polyethylene glycol (PEG)/potassium citrate are established with response surface methodology. Effects of PEG molecular weight, the concentration of PEG and potassium citrate, the concentration of additional salt (KC1), the temperature and pH on the papain purification have been investigated. Research results showed that papain tends to partitions to the more hydrophilic salt-rich phase. The steepest ascent method was used to locate the optimal domain. Papain was successfully extracted to the bottom phase of a system composed of15%(w/w) PEG4000,17%(w/w) citrate and5.0%(w/w) KC1at pH5.0and30C. Under those conditions, the specific activity, purification factor and activity yield for papain were1703.02U/mg,1.89and94%, respectively. The purity of extracted papain was confirmed by SDS-PAGE analysis. This ATPS has the potential to be applied to purification of papain.
引文
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