Modeling the protein partitioning in aqueous polymer two-phase systems: influence of polymer concentration and molecular weight

Aqueous polymer two-phase system provides a powerful method for separation and purification of biomaterials. Among various factors, polymer concentration and polymer molecular weight are essential and have strong impact on the protein partitioning in these systems. Based on the modified Pitzer's model, a simple expression has been obtained for correlating protein partitioning in aqueous polymer two-phase systems with varying polymer concentration and different polymer molecular weights. Using only one group of parameters for each target protein, the partition coefficients of five proteins (lysozyme, chymotrypsinogen-A, bovine serum albumin, transferrin and catalase) in 16 sets of polyethylene glycol (PEG)/dextran systems were correlated. In addition, partition behavior of lactate dehydrogenase in PEG/hydroxypropyl starch systems were measured, correlated and partially predicted. A comparison of calculated and experimental data indicated that the model provides good correlation and prediction abilities on the protein partitioning in aqueous polymer two-phase systems with a wide range of polymer concentration and molecular weight.     

Separation selectivity of aqueous polyethylene glycol-based separation systems: DSC, LC and aqueous two-phase extraction studies

The distribution behavior of n-alcohols, ketones and nitroalkanes in aqueous liquid chromatography with a column packed with polyethylene glycol (PEG) gel, TSKgel Ether-250, was compared with that in aqueous two-phase systems (ATPSs) formed from PEG and Na₂SO₄ or (NH₄)₂SO₄. The plots of the distribution data obtained for the PEG gel system against those for the ATPS reveal that the separation selectivities exerted by the PEG gel system and the PEG-based ATPS are approximately the same. Differential scanning calorimetry studies on aqueous PEG solutions suggest that PEG polymer forms a hydration structure of which the composition is 50% (w/w) PEG or the hydration number per ethylene oxide is 2.4 and the separation selectivity of the PEG-water systems can be attributed to partition of solute compounds into the hydrated PEG polymer structure.     

Experimental studies on phase equilibria of PEG-water-dextran/ammonium sulfate systems and partitioning of albumin into two water-continuous phases

The clouding points and equilibrium concentrations of PEG/dextran/water and PEG /ammonium sulfate/water systems were experimentally determined for different molecular weights of PEG by titration method and direct determination of concentrations. In phase diagrams of PEG-water with ammonium sulfate or dextran, the addition of salt or dextran induced the phase separation of PEG-rich phases near the PEG-lean phases having the different partitioning of PEG. The concentrations of PEG in PEG-rich phases increase as the amounts of PEG or salt and dextran increase, while the concentrations of PEG in aqueous media decrease in any cases. The higher the molecular weight of PEG has, the wider the two-phase regions are. In dextran DT10 systems, the partition coefficients of egg albumin into PEG-lean phases increase with concentrations of dextran and the molecular weight of PEG. In ammonium sulfate systems, the partitioning coefficients showed a maximum, having lower partitioning at the very high and low concentration of salts. It is also observed that as the amounts of albumin increase, the partitioning of albumin into PEG-lean phase increases.     

金属螯合双水相亲和分配技术分离纳豆激酶的研究

利用金属螯合亲和双水相分配技术对纳豆激酶的分离纯化进行了研究。考察了双水相系统、聚合物的分子量和浓度、亲和配基加入量、pH值、相比以及生物质加入量等因素对亲和分配的影响。结果表明,双聚合物系统比聚合物／无机盐系统更有利于纳豆激酶亲和分配;pH值和亲和配基加入量是影响分配的关键因素。优化的分配条件为：2．6％聚乙二醇,20．2％羟丙基淀粉,5％亲和配基PEG-IDA—Cu(Ⅱ),相比12,pH8．2,发酵液加入量15％。分配系统放大到100g,仍保持一致的酶活收率(90％)和纯化因子(2.0)。设计了两次分配分离流程,纯化因子达到3．52,总收率为81％。     

Combined bioreaction and separation in centrifugal fields using zonal rotors

Combined biosynthesis and product separation has been successfully performed for the first time using a zonal centrifugal bioreactor–separator. The biosynthesis of dextran polymer from sucrose using the dextransucrase enzyme was investigated in order to evaluate bioreactor performance. The bioreactor consisted of a bowl, fitted with a Reorienting gradient rotor which facilitated substrate and enzyme loading and the reproducible unloading of solutions from the system. This allowed the distribution of substrate, enzyme and product materials in the bowl at the end of each trial to be accurately determined. Studies have indicated that combined bioreaction–separation is possible using this system. However, viscosity build-up in the bioreactor must be minimised in order to increase the yield of polymer product per unit time and improve product separation.     

Synthesis and functionality of poly(N-vinylalkylamide). X. A novel aqueous two-phase system based on thermosensitive polymers and dextran

The use of thermosensitive polymers in an aqueous two-phase system was studied. Poly(N-isopropylacrylamide) (PNIPAAm) and poly(N-vinylisobutyramide) (PNVIBA) were used as thermosensitive polymers. Both polymers could form aqueous two-phase with dextran, respectively. The phase diagrams of each system were successfully obtained. Using myoglobin as a model protein, a preliminary separation study was performed. The separation ability of both polymers was higher than that from the poly(ethylene glycol)-dextran system. Protein separation ability appeared to be related to the hydrophilic/hydrophobic balance of the polymers. Both PNVIBA and PNIPAAm rich phases maintained their thermosensitivity after two-phase formation. PNVIBA and PNIPAAm are useful as polymers for a functional aqueous two-phase system. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 73: 2545–2548, 1999     

Optimization of Serine Protease Purification from Mango (Mangifera indica cv. Chokanan) Peel in Polyethylene Glycol/Dextran Aqueous Two Phase System

Mango peel is a good source of protease but remains an industrial waste. This study focuses on the optimization of polyethylene glycol (PEG)/dextran-based aqueous two-phase system (ATPS) to purify serine protease from mango peel. The activity of serine protease in different phase systems was studied and then the possible relationship between the purification variables, namely polyethylene glycol molecular weight (PEG, 4000-12,000 g·mol(-1)), tie line length (-3.42-35.27%), NaCl (-2.5-11.5%) and pH (4.5-10.5) on the enzymatic properties of purified enzyme was investigated. The most significant effect of PEG was on the efficiency of serine protease purification. Also, there was a significant increase in the partition coefficient with the addition of 4.5% of NaCl to the system. This could be due to the high hydrophobicity of serine protease compared to protein contaminates. The optimum conditions to achieve high partition coefficient (84.2) purification factor (14.37) and yield (97.3%) of serine protease were obtained in the presence of 8000 g·mol(-1) of PEG, 17.2% of tie line length and 4.5% of NaCl at pH 7.5. The enzymatic properties of purified serine protease using PEG/dextran ATPS showed that the enzyme could be purified at a high purification factor and yield with easy scale-up and fast processing.     

Reactive aqueous two-phase partition of cellulase

Reactive aqueous two-phase partition of cellulase by modification with amphiphilic copolymer was studied. The modifier of cellulase could be directly used as a polymer of two-phase system. The maleic acid anhydride functional group of the synthetic copolymer covalently coupled with the amino groups of cellulase molecule. The degree of modification of the amino groups was controlled by the types of synthetic copolymer or the weight ratio of a synthetic copolymer to cellulase. As the degree of modification of cellulase increased, the modified cellulase moved to the copolymer phase of the copolymer/dextran two-phase system.     

聚乙二醇双水相体系在萃取分离中的应用

聚乙二醇双水相体系具有易分相、价格低廉、萃取高效温和、无毒、对萃取物活性损失小等优点。本文综述了近几年来聚乙二醇双水相体系及其在蛋白质、生物酶、生物活性物质、抗生素、金属离子等萃取分离方面的应用进展,展望了聚乙二醇双水相在分离方面的应用前景和发展方向。     

Cell-based aqueous two-phase systems for therapeutics

Cell-based aqueous two-phase system technologies present an emerging and promising translational tool for the development of next-generation therapeutic applications. Advantages such as biocompatibility, scalability and ease of implementation confirm this possibility. The aim of this perspective article is to present a brief, process-oriented analysis of cell-based aqueous two-phase system strategies for therapeutic purposes, highlighting their potential advantages and limitations. These strategies include regenerative therapies, high-throughput screening platforms and clinical diagnostics. Special attention is given to the implementation of polyethylene glycol and dextran aqueous two-phase systems in the culture, expansion and separation of cells to accomplish novel diagnostic and therapeutic tasks for the healthcare, biotechnology and pharmaceutical industries. © 2019 Society of Chemical Industry     

壳聚糖／水溶性高分子共混膜相容性的预测与表征

高分子（聚乙烯醇和聚乙二醇）间的相容性,壳聚糖与极性水溶性高分子在溶液状态时,分子间相互吸引,相容性好,向本体转化而成膜时,CS／PVA体系比CS／PEG体系有更好的本体相容性。原因可归结为PEG不能像PVA一样提供足够多的极性基团与壳聚糖分子形成强烈相互作用,CS与PEG间的氢键强度要弱于CS与PVA间的氢键;另一个方面,PEG结晶能力很强,在由溶液转化为本体而成膜过程中,PEG分子自身之间易于形成较规整的结晶而逐渐使体系发生分相。     

Two-Phase Electrophoresis of Proteins

Abstract

Proteins have been directed into either the top or bottom phase of a polyethylene glycol/dextran aqueous two-phase system by applying an electric field perpendicular to the phase interface. Protein migration across the interface was manipulated by varying polarity, pH, electrophoresis time, field strength, and phase volume ratio. Mixtures of hemoglobin and albumin were separated by operating between isoelectric points and directing oppositely charged proteins into separate phases. Applying 50 V/cm for 2 hours to 58 mL of an equal phase volume two-phase system containing 0.2 g/L of each protein at pH 6 resulted in a bottom phase containing 99% of the hemoglobin and a top phase containing 95% of the albumin. This represents a significant improvement over the separation obtained either by partitioning in the same two-phase system with no applied field or by electrophoresis under the same conditions in homogeneous buffer. The two-phase system divides the electrophoresis device into two distinct regions, providing stability against convective mixing and facilitating product isolation.     

Partition of tannery wastewater proteins in aqueous two-phase poly (ethylene glycol)-magnesium sulfate systems: Effects of molecular weights and pH

The partitioning behavior of soluble proteins from tannery wastewater using aqueous two-phase system (ATPS) was investigated. An ATPS polyethylene glycol (PEG)/MgSO₄ was examined with regard to the effects of PEG molecular weight (MW) and concentration, MgSO₄ concentration, pH and NaCl concentration on protein partition and extraction. The partition coefficients measured for soluble proteins were proportional to the difference in PEG concentration between the phases. The MW and concentration of PEG were found to have significant effects on protein partition and extraction with low MW PEG4000 showing the best conditions for the partitioning of protein in PEG+MgSO₄+water system. Sulfate salt was chosen as the phase-forming salt because of its ability to promote hydrophobic difference between the phases. This system was operated at room temperature . Increase in pH of the system increases the partition coefficient of proteins from tannery wastewater. The addition of sodium chloride showed significant influence on the partition coefficient. ATPS comprising PEG4000-magnesium sulfate provided a means for the recovery of proteins from tannery wastewater. The maximum percentage yield of protein extracted is 82.68%.     

椰油丙基酰胺甜菜碱/聚乙二醇水溶液的分相研究

Phase separation behavior of cocamidopropyl betaine/water/polyethylene glycol (PEG) system was studied. The effects of concentration and molecular weight of PEG on the phase separation behavior were investigated. Clouding occurred when the con-centration of PEG was large enough in the betaine aqueous solution, and the concentration of PEG at cloud point decreased with the increase of PEG molecular weight for a constant betaine concentration. The bottom phase was the PEG-rich phase, and the upper phase was the betaine-rich phase. The volumetric ratio of PEG-rich phase to betaine-rich phase, at the same difference between the PEG concentration and the one at the cloud point, Ccp (0.1 g•ml－1), decreased as the PEG molecular weight increased and approached 1 for higher PEG molecular weight (about 20000), which was similar to the typical aqueous two-phase system. This volumetric ratio depended on the initial PEG concentration, but independent of PEG molecular weight. The concentration ratio of betaine to PEG in both phases depended on the Ccp, independent of PEG molecular weight.     

The effect of phase separation on the structure and permeability of gelatin–dextran films

The effect of polymer–polymer phase separation on the transport properties of gelatin–dextran films has been studied. In the gelatin–dextran–water system, the phase separation and gel formation do not occur due to the presence of a nonsolvent, but depend entirely on the temperature and the total polymer concentration. It has been found that the permeability of polymer blend films to aqueous solutes can be varied not only by changing the volume ratio of the phases, but also by varying the size of the individual domains of the two phases in the films. Under the conditions of restricted demixing (e.g., after gel formation), the course of the phase separation process can be changed by varying the length of time allowed for unrestricted separation. The two most important parameters affecting the result are the total polymer concentration in the casting solution, and the temperature regime. An exponential dependence of the permeation coefficient on the volume fraction of the solvent in the film is shown.     

Optimization of recovery of esterase from Serratia marcescens using combination of the solvent impregnated resin and aqueous two-phase extraction techniques

ABSTRACT

The performance of tunable aqueous polymer phase impregnated resins (TAPPIR) which is the combination of the solvent impregnated resin principle and an aqueous two-phase system for the separation of esterase from Serratia marcescens was evaluated in this study. Different molecular weight of polyethylene glycol (PEG) (2000, 4000 and 6000) at concentration ranging from 5% to 20% (w/w) and potassium citrate were used to construct the aqueous phase in TAPPIR technology. Optimum composition of PEG and salt for esterase partitioning was determined using response surface methodology. The optimum condition for the purification of esterase was impregnation of 25% (w/w) of PEG 2000 into 4 mm porous glass beads and extraction of esterase using 15% (w/w) potassium citrate at pH 8 containing 12% (w/w) crude loading with the addition of 4% (w/w) NaCl. Esterase from S. marcescens was successfully purified by the TAPPIR technology up to 5.32 of purification factor with a yield of 75.98%.     

双水相萃取技术研究新进展

介绍了双水相萃取技术(ATPE)在生物工程下游工程中的应用现状,综述了近年来ATPE相关研究的进展。为了提高ATPE的选择性和分离效率,不但发展了热分离聚合物体系和表面活性剂混合体系等新型双水相系统(ATPS),而且在组成传统ATPS的聚合物上耦联亲和配基的亲和ATPS也得到发展。与物理场作用、其他分离技术和生物过程的集成克服了单一ATPE的某些不足,是ATPE的发展方向。常规萃取设备和连续化操作技术在ATPE中的应用标志着其工业化日趋成熟,但建立溶质在ATPS中分配的热力学模型和相关理论有待进一步完善。     

Tunable Aqueous Polymer Phase Impregnated Resins: A Combination of the Extractant Impregnated Resin Principle and Aqueous Two-Phase Systems

This scientific note introduces a concept supposed to enhance aqueous two-phase extraction by immobilizing one of the two phases of an aqueous two-phase system in porous particles. This approach, developed by the author, is based on solvent impregnated resins. The resulting so-called (tunable) aqueous polymer phase impregnated resins might avoid drawbacks of aqueous two-phase extraction, such as long phase separation times due to persistent emulsification. Due to the novelty of the idea and resulting current lack of fundamental studies, the idea is put up to discussion in order to initiate scientific exchange and hopefully widespread investigation of its true potential.     

PEG/（NH4）2SO4双水相萃取分离茶氨酸的研究

采用聚乙二醇(PEG)/(NH4)2SO4双水相体系萃取分离生产茶多酚所得废液中的茶氨酸,考察了PEG分子量与含量、硫酸铵含量、pH、温度、加盐(KCl、KBr、KI)、茶氨酸含量对双水相及萃取分离茶氨酸的影响。结果表明,PEG/(NH4)2SO4双水相萃取分离茶氨酸的适宜条件是:PEG平均分子量为4 000,质量分数为10%,硫酸铵质量分数为15%,pH约为6,30℃。在此条件下,茶氨酸的分配系数K1=0.16,蛋白质的分配系数K2=0.28,糖类的分配系数K3=9.8,茶氨酸在下相的萃取率为89.5%,可以将茶多酚废液中的茶氨酸与糖类及其他有颜色的杂质分开。