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Yanmin Lu Wenjuan Lu Wei Wang Qingwei Guo Yanzhao Yang 《Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)》2013,88(3):415-421
BACKGROUND: Aqueous two‐phase extraction is a versatile method for separating biological particles and macromolecules. In the present wok, the feasibility of using PEG 4000/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. RESULTS: Mathematical models concerning the purification of lysozyme from chicken egg white in polyethylene glycol 4000 (PEG 4000)/potassium citrate ATPS are established using response surface methodology. Screening experiments using fractional factorial designs show that the pH of the system significantly affects the recovery and purification of lysozyme. An optimized ATPS was proved to be at pH 5.5 and 30 °C and contained 18% (w/w) PEG, 16% (w/w) potassium citrate, 3.75% (w/w) potassium chloride (KCl). Under those conditions, the specific activity, purification factor and activity yield for lysozyme were 31100 U mg?1, 21.11 and 103%, respectively. CONCLUSION: The PEG 4000/potassium citrate ATPS has the potential to be applied to establish bioprocesses for the primary recovery and partial purification of lysozyme. © 2012 Society of Chemical Industry 相似文献
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Marco Rito‐Palomares Andrew Lyddiatt 《Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)》2000,75(7):632-638
A two‐stage extraction process for the recovery of intracellular proteins from brewers' yeast was selected as a practical model system to study the implementation of polyethylene glycol (PEG)–phosphate aqueous two‐phase systems (ATPS). Disrupted all suspensions generated by homogenisation and bead milling were used to study the impact of cell debris upon the partition behaviour of the intracellular products (bulk protein, fumarase and pyruvate kinase). Regardless of their origin debris particles did not significantly influence the partition behaviour of the intracellular products in selected ATPS distant from the binodal and at volume ratios greater than one. Recycling of used PEG into the initial extraction stage did not significantly influence the protein partition behaviour in batch ATPS. In the polymer recycling studies in continuous ATPS using spray columns, the addition of fresh materials to make up the deficits of phase‐forming chemicals compensate any negative effect of the continuous recycling of the top PEG‐rich phase. The findings of these studies raise the potential application of ATPS processes for protein recovery from complex biological systems. © 2000 Society of Chemical Industry 相似文献
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Karolina Chairez‐Cantu Mirna Gonzlez‐Gonzlez Marco Rito‐Palomares 《Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)》2021,96(1):8-13
Stem cell therapy has emerged as a promising alternative for replacing lost cells involved in neurodegenerative diseases. High efficiency of differentiation and full cell viability are actual challenges to achieve the translation of cell therapies to the clinic. To address this, the construction of aqueous two‐phase systems in three‐dimensional (ATPS‐3D) cultures has been proposed. This technique involves the combination of two polymers in which cells are confined in dextran droplets immersed over a substrate located in a poly(ethylene glycol) phase. The controlled placement of cells in a defined pattern promotes intercellular communication. This review aims to provide insight into the techniques used to enhance neural differentiation and current challenges to achieve the implementation of cell therapies. Cell density, colony size, interconnectivity and an appropriate substrate to modulate paracrine signaling are factors that determine neural differentiation efficiency during the construction of ATPS‐3D cultures. Hence, this contact‐free technique enables the design of neural niches to recapitulate in vivo environments more accurately. © 2020 Society of Chemical Industry (SCI) 相似文献
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Oscar Aguilar Marco Rito‐Palomares 《Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)》2008,83(3):286-293
BACKGROUND: The potential use of plants as production systems to establish bioprocesses has been established over the past decade. However, the lack of efficient initial concentration and separation procedures affect the generic acceptance of plants as economically viable systems. In this context the use of aqueous two‐phase systems (ATPS) can provide strategies to facilitate the adoption of plants as a base for bioprocesses. Among the crops, soybeans (Glycine max) represent an attractive alternative since potentially they can produce high levels of recombinant protein. In this paper the processing of fractionated soybean extracts using ATPS is evaluated as a first step to recover recombinant proteins expressed in plants, using β‐glucuronidase (GUS; E.C. 3.2.1.31) as a model protein. RESULTS: The evaluation of the effect of system parameters provided the conditions under which the contaminant proteins from fractionated soybean extracts and GUS concentrated in opposite phases. A PEG 600/phosphate system comprising 14.5% (w/w) polyethylene‐glycol (PEG), 17.5% (w/w) phosphate, a volume ratio (Vr) equal to 1.0, and a system pH of 7.0 resulted in the potential 83% recovery of GUS from the complex mixture and an increase in purity of 4.5‐fold after ATPS. CONCLUSIONS: The findings reported here demonstrate the potential of ATPS to process fractionated soybean extract as a first step to isolate and purify a recombinant protein expressed in soybeans. The proposed approach can simplify the way in which recombinant proteins expressed in plants can be recovered. Copyright © 2007 Society of Chemical Industry 相似文献
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Marco Rito‐Palomares Anton P
J Middelberg 《Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)》2002,77(9):1025-1029
In this study the use of an aqueous two‐phase system (ATPS) following the direct chemical extraction of a recombinant viral coat protein, from the cytoplasm of Escherichia coli, is evaluated. The driving force is the need to establish an economically‐viable process for the manufacture of a vaccine against human papilloma infection. The partition behaviour of recombinant L1 protein, the major structural protein of the virus, and DNA was investigated in a polyethylene glycol (PEG)–phosphate system. An evaluation of system parameters including PEG molecular mass and the concentrations of PEG and phosphate was conducted, to estimate conditions under which the L1 protein and DNA partition to opposite phases. ATPS extraction comprising a volume ratio of 1.00, PEG 1000 (18.0%(w/w)) and phosphate (15.0%(w/w)) provided the conditions for accumulation of DNA into the bottom phase and concentration of L1 protein into the opposite phase (ie partition coefficient of DNA; ln KDNA < 0.0 and partition coefficient of L1; ln KL1 > 2.5). The findings reported here demonstrate the potential of ATPS to recover recombinant protein released from E coli by direct chemical extraction. © 2002 Society of Chemical Industry 相似文献
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Marco Rito‐Palomares Lilia Nuez Daniel Amador 《Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)》2001,76(12):1273-1280
A novel process for the recovery of c‐phycocyanin from Spirulina maxima exploiting aqueous two‐phase systems (ATPS), ultrafiltration and precipitation was developed in order to reduce the number of unit operations and benefit from an increased yield of the protein product. The evaluation of system parameters such as PEG molecular mass, concentration of PEG as well as salt, system pH and volume ratio was carried out to determine under which conditions the c‐phycocyanin and contaminants concentrate to opposite phases. PEG1450–phosphate ATPS proved to be suitable for the recovery of c‐phycocyanin because the target protein concentrated in the top phase whilst the cell debris concentrated in the bottom phase. A two‐stage ATPS process with a phase volume ratio (Vr) equal to 0.3, PEG1450 7% (w/w), phosphate 20% (w/w) and system pH of 6.5 allowed c‐phycocyanin recovery with a purity of 2.4 (estimated as the relationship of the 620 nm to 280 nm absorbances). The use of ultrafiltration (with a 30 kDa membrane cut‐off) and precipitation (with ammonium sulfate) resulted in a recovery process that produced a protein purity of 3.8 ± 0.1 and an overall product yield of 29.5% (w/w). The results reported here demonstrated the practical implementation of ATPS for the design of a prototype recovery process as a first step for the commercial purification of c‐phycocyanin produced by Spirulina maxima. © 2001 Society of Chemical Industry 相似文献
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Tanhia Hernandez‐Mireles Marco Rito‐Palomares 《Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)》2006,81(6):989-996
A simplified process for the primary recovery and purification of B‐phycoerythrin (BPE) from Porphyridium cruentum exploiting aqueous two‐phase systems (ATPS) and isoelectric precipitation was developed in order to reduce the number of unit operations and benefit from increased purity and yield of the protein product. Evaluation of the partitioning behaviour of BPE in polyethylene glycol (PEG)/sulphate, PEG/dextran and PEG/phosphate ATPS was carried out to determine under what conditions the BPE and contaminants concentrated into opposite phases. An additional stage of isoelectric precipitation at pH 4.0 after cell disruption resulted in an increase in purity of the target protein from the BPE crude extract and enhanced the performance of the subsequent ATPS. PEG1000/phosphate ATPS proved to be suitable after isoelectric precipitation for the recovery of highly purified (defined as absorbance ratio A545 nm/A280 nm > 4.0) BPE with a potential commercial value as high as US$ 50/mg. An ATPS extraction stage comprising 29.5% (w/w) PEG1000, 9.0% (w/w) phosphate, a volume ratio (Vr) equal to 1.0, a system pH of 7.0 and loaded with 40% (w/w) of the BPE extract generated by precipitation allowed BPE recovery with a purity of 4.1±0.2 and an overall product yield of 72% (w/w). The purity of BPE from the crude extract increased 5.9‐fold after isoelectric precipitation and ATPS. The results reported herein demonstrate the benefits of the practical application of isoelectric precipitation together with ATPS for the recovery and purification of BPE produced by P. cruentum as a first step in the development of a commercial purification process. Copyright © 2006 Society of Chemical Industry 相似文献
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引言黄芩是一种疗效确切的常用中药,临床广泛应用于肺炎、肝炎、慢性支气管炎、高血压、化脓性感染及先兆流产等疾病．黄芩甙（baicalin)是黄岑中具有药用价值的主要成分．有抗血小板凝集、抗肿瘤、降血脂和降压利尿等作用,在医学界引起了很大的关注．双水相分配（ATPP）技术自60年代发展以来,已广泛用于生物化学、细胞生物学和生物工程等领域,实现了生物产品的分离和纯化．但由于聚合物回收的困难,在一定程度上阻碍了ATPP技术的应用．最近一个引人注目的聚合物──环氧乙烷（EO）和环氧丙烷（PO)的无规共聚物(EOPO）使这一问题… 相似文献
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Two‐phase polymerization of acrylamide (AM) has been successfully carried out in aqueous poly(ethylene glycol) (PEG) solution with 2,2′‐azobis[2‐(2‐imidazolin‐2‐yl)propane] dihydrochloride (AIBI) as the initiator. A new heterogeneous kinetic model has been developed based on the partitioning of components between the two phases. It was found that polymerization proceeded in both the continuous and dispersed phases, even though the latter was the dominating polymerization locus. Besides the initiator, monomer concentration, and polymerization temperature, the PEG concentration also significantly influences the polymerization rate. With increasing concentration of PEG, gel effects in the aqueous PAM droplets were enhanced and more monomer preferred to polymerize inside the droplets, hence, the polymerization kinetics accelerated. The proposed model can successfully predict the composition of each phase and the polymerization kinetics during the aqueous two‐phase polymerization over a wide range of various reactions conditions. © 2010 American Institute of Chemical Engineers AIChE J, 2011 相似文献
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Tanhia Hernandez‐Mireles Marco Rito‐Palomares 《Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)》2006,81(6):997-1002
A new aqueous two‐phase system (ATPS) based on a degradable polymer called poly(ethylene oxide sulfide) with a molecular weight of 33 000 g mol?1 (identified as PEOS‐12) and potassium phosphate was exploited for the potential recovery of proteins. An initial characterisation of the ATPS was achieved by the construction of a phase diagram for the PEOS‐12/phosphate system. The protein partitioning behaviour of lysozyme and bovine serum albumin (BSA), selected as single model proteins, and B‐phycoerythrin (BPE) produced by Porphyridium cruentum in the new ATPS under increasing tie line length (TLL) conditions at constant phase volume ratio (Vr) and system pH was investigated. Both single proteins partitioned in the new ATPS, initially exhibiting bottom phase preference; however, lysozyme changed phase preference when TLL was increased. Fractionation of a complex model (production of BPE by P. cruentum) using PEOS‐12/phosphate ATPS was performed to evaluate the potential protein recovery from fermentation broth or cell homogenate. The proposed new ATPS proved to be suitable for the potential recovery of BPE from crude extract of P. cruentum. In general, a system comprising Vr = 1.0, 18% (w/w) PEOS‐12, 8% (w/w) phosphate and 30% (w/w) TLL at pH 7.0 provided conditions to concentrate BPE into the bottom phase (i.e. partitioning behaviour of BPE; lnKBPE = ?1.8) with a protein recovery of 84%. The findings reported here demonstrate the potential application of the new ATPS for the recovery of proteins from complex biological suspensions. Copyright © 2006 Society of Chemical Industry 相似文献
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The concept of aqueous two‐phase polymerization and a new polymerization method for the preparation of water‐soluble polymers are presented. The phase diagram of poly(acrylamide) (PAAm)‐poly (ethylene glycol) (PEG)‐water two‐phase system was measured by the gel permeation chromatography (GPC). The aqueous two‐phase of PAAm‐PEG‐water system can be easily formed. The critical concentration of phase separation was affected by the molecular weight of PEG. The aqueous two‐phase polymerization of acrylamide (AAm) has been successfully carried out in the presence of PEG by using ammonium persulfate (APS) as the initiator. The polymerization behaviors with varying concentration of AAm, initiator and PEG, the polymerization temperature, the molecular weight of PEG, and emulsifier types were investigated. The activation energy of aqueous two‐phase polymerization of AAm was 132.3 kJ/mol. The relationship of initial polymerization rate (Rp0) with APS and AAm concentrations was Rp0 ∝ [APS]0.72 [AAm]1.28. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009 相似文献
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A multiphase chemical equilibrium algorithm is developed which can be used with aqueous systems. The algorithm uses an average chemical potential for each species as a reference chemical potential. Incipient phases can be identified and their proximity to appearance can be determined through their tangent plane distance. Illustrative examples include the calculation of CaSO4 solubility and the calculation of vapour pressures above an SO2‐NaCl‐H2O system, including the prediction of a three‐phase equilibrium. The algorithm proved robust and versatile. The appearance of incipient phases was easily tracked. Future work needs to be done to optimize damping/acceleration coefficients in the calculations. 相似文献
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Impact of polyethylene glycol as additive on the formation and extraction behavior of ionic‐liquid based aqueous two‐phase system 
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下载免费PDF全文 Mohammad Vahidnia Gholamreza Pazuki Shiva Abdolrahimi 《American Institute of Chemical Engineers》2016,62(1):264-274
Developing a novel Ionic‐liquid (IL) based aqueous two‐phase system (ATPS) with polyethylene glycol (PEG) as adjuvant for the separation of biomolecules is studied. This original work involves addition of various concentration of PEG (2000, 4000, and 6000 gr/mol) to 1‐butyl‐3‐methylimidazolium acetate+ potassium hydrogen phosphate ATPS to investigate their subsequent effect on phase diagrams and partitioning coefficient of α‐amylase. In another innovative aspect of this work, response surface methodology (RSM) based on three‐variable central composite design was employed to understand the effect of phase forming components on extraction studies of α‐amylase. The addition of small amount of PEG improved the partitioning coefficient of biomolecule. The effective excluded volume theory was applied to correlate the salting‐out ability. As a result, it can be stated that the proposed system can effectively be used in separation and purification studies instead of task specific ILs. © 2015 American Institute of Chemical Engineers AIChE J, 62: 264–274, 2016 相似文献
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Fang Gao Andrew J. Daugulis 《Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)》2010,85(2):302-306
BACKGROUND: Biphasic systems with immiscible solvents have been studied for in situ product removal, and have shown improvements in bioreactor performance, however, problems associated with solvent biocompatibility, bioavailability and operation have been identified. One alternative is the solid–liquid system in which polymer beads are used, absorbing and removing target compounds from the aqueous phase while maintaining equilibrium conditions. This work aims to identify polymer properties that may be important in polymer selection for selected biotransformation molecules including 2‐phenylethanol, cis‐1,3‐indandiol, iso‐butanol, succinic acid and 3‐hydroxybutyrolactone. RESULTS: Relatively hydrophobic compounds (e.g. 2‐phenylethanol) tend to be absorbed by polymers better than hydrophilic ones (e.g. iso‐butanol) based on partition coefficient tests; values as high as 80 were obtained for the former and < 3 for the latter. Owing to the presence of polar functional groups on these compounds, polar polymers such as Hytrel® performed better than non‐polar ones such as Kraton®. Crystallinity and intermolecular hydrogen‐bonding were also found to be important polymer properties. CONCLUSION: Polymers showed excellent results in absorbing hydrophobic compounds such as aromatic alcohols, and positive results in absorbing hydrophilic compounds but to a lesser extent. Grafting hydrophilic functional groups onto polymers may be a promising approach for extending polymer uptake capabilities and is currently being investigated. Copyright © 2009 Society of Chemical Industry 相似文献