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%.     

Partitioning behaviour of cephalexin and 7‐aminodeacetoxicephalosporanic acid in PEG/ammonium sulfate aqueous two‐phase systems

In order to develop an aqueous two‐phase system (ATPS) for cephalexin synthesis with extractive bioconversion, the partitioning behaviour of cephalexin and 7‐aminodeacetoxicephalosporanic acid (7‐ADCA) in poly(ethylene glycol) (PEG)/salt ATPS were examined. Parameters such as PEG size, salt type and tie line length were investigated to find a primary extraction system. In PEG400/ammonium sulfate and PEG400/magnesium sulfate systems, the partition coefficient of cephalexin (K_C) was larger than 1 while that of 7‐ADCA (K_A) deviated about 1.5. Addition of neutral salts, surfactants and water‐miscible solvents were also investigated in the primary ATPS in order to improve the separation efficiency. K_C greatly increased when neutral salts and surfactants were added to the PEG400/ammonium sulfate primary systems whereas K_A was only slightly higher than that of the additive‐free ATPS. In an improved ATPS for extractive bioconversion, consisting of PEG400 (20% w/w), ammonium sulfate (17.5% w/w), methanol (5% w/w) and NaCl (3% w/w), a K_C value of up to 15.2 was achieved; K_A was 1.8; K_P (partition coefficient of phenylglycine methyl ester) was 1.2 and the recovery yield of cephalexin was 94.2%. The results obtained from the extractive bioconversion of cephalexin in the improved ATPS showed that it is feasible to perform such an enzymatic process in an ATPS and the system offers the potential as a model for enzymatic synthesis of some water soluble products. © 2001 Society of Chemical Industry     

Purification of Glucose Oxidase and β‐Galactosidase by Partitioning in a PEG‐Salt Aqueous Two‐Phase System in the Presence of PEG‐Derivatives

Abstract

Purification of glucose oxidase from Aspergillus niger and that of β‐galactosidase from Kluyveromyces lactis have been attempted using poly(ethylene glycol) (PEG)‐sodium sulfate aqueous two phase system (ATPS) in the presence of PEG‐derivatives, i.e. PEG‐Coomassie brilliant blue G‐250 and PEG‐benzoate, PEG‐palmitate and PEG‐TMA, respectively. The enzymes showed poor partitioning towards the PEG phase in comparison with other proteins in ATPS containing no ligands. Selective partitioning of other proteins was observed towards the PEG phase in the presence of PEG‐benzoate and PEG‐palmitate enriching β‐galactosidase in the salt phase whereas in the case of glucose oxidase, PEG‐Coomassie brilliant blue G‐250 derivative worked as a better affinity ligand for other proteins. A 19‐fold purification was obtained with the PEG dye derivative after 5 stage cross extractions with 80% recovery of glucose oxidase and an enrichment factor upto ～7 for β‐galactosidase with the PEG‐TMA derivative. The interaction of PEG‐benzoate and PEG‐TMA ligands with the active site of β‐galactosidase has been evaluated by molecular modeling. The effect of the molecular weight of glucose oxidase on its partitioning was confirmed as the molecular simulation shows strong affinity interaction of PEG‐glucoside with the enzyme.     

Impact of polyethylene glycol as additive on the formation and extraction behavior of ionic‐liquid based aqueous two‐phase system

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     

Liquid-liquid extraction of protease from cold-adapted yeast Rhodotorula mucilaginosa L7 using biocompatible and biodegradable aqueous two-phase systems

This work aimed to optimize the extraction of an extracellular protease produced by the cold-adapted yeast Rhodotorula mucilaginosa L7 using aqueous two-phase systems (ATPS) comprising polyethylene glycol (PEG) and sodium citrate or sodium tartrate. First, the biocompatibility of the phase forming agents was assessed. The results obtained with PEG-2000, PEG-4000, and PEG-6000 demonstrated that even at large PEG concentrations (32 wt%) the protease maintains its activity after 3 h of reaction, whereas an increase in salt concentration provokes a gradual decrease in protease stability. Subsequently, the partitioning of the protease in both types of ATPS was assessed, evaluating the effect of temperature, molecular weight, and concentration of PEG on protease purification, using two 2³-full factorial designs. The best partitioning conditions were obtained in PEG-6000/sodium tartrate-based ATPS, at 30ºC (with a yield of 81.09 ± 0.66% and a purification factor of 2.51 ± 0.03). Thus, considering the biodegradable characteristics of the system, the PEG/sodium tartrate ATPS is a viable and economic low-resolution step in protease purification, with a strong potential for future industrial application.     

Practical implementation of aqueous two‐phase processes for protein recovery from yeast

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     

Practical application of aqueous two‐phase systems for the development of a prototype process for c‐phycocyanin recovery from Spirulina maxima

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 (V_r) 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     

Extractive bioconversion of xylan for production of xylobiose and xylotriose using a PEG6000/sodium citrate aqueous two-phase system

Aqueous two-phase system (ATPS) was applied for extraction bioconversion of xylan by xylanase from Trichoderma viride. Phase diagrams for poly (ethylene glycol) (PEG) and sodium citrate were determined at room temperature. The ATPS composed of 12.99% (w/w) PEG6000 and 12.09% (w/w) sodium citrate was favorable for partition of xylanase and used for extraction bioconversion of xylan. Batch hydrolysis demonstrated that higher concentrations of xylobiose and xylotriose were obtained in the PEG6000/sodium citrate ATPS compared to those in the aqueous system. These results present the potential feasibility of production of xylo-oligosaccharides by extraction bioconversion in ATPS.     

Improved recovery of B‐phycoerythrin produced by the red microalga Porphyridium cruentum

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 A_{545 nm}/A_{280 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 (V_r) 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     

Development and Evaluation of PEG-Lithium Citrate Salt Based Aqueous Two Phase System and Its Application in Partitioning of Proteins from Fish Industry Effluent

A aqueous two phase system (ATPS) comprising of PEG (Average mol. Wt: 4000, 6000, 8000) – lithium citrate salt-water systems were studied. The basic studies like binodal curve data generation and equilibrium studies were carried out. Furthermore, the binodal model and Othmer-Tobias and Bancroft models for phase equilibria were used for reproducing the experimental binodal data and phase equilibrium composition data, respectively. Good agreement was obtained with the experimental binodal data and tie line data with the models. The effective excluded volume values were obtained from the binodal model for the present ATPS. The tie line length was determined through the phase equilibrium composition data. This system was used to partition crude proteins of the fish industry effluent. The effects of PEG and salt weight fraction in terms of tie line length and effective excluded volume on partitioning coefficient of crude protein were studied in detail. From the results it was observed that, the crude proteins present in the fish effluent were partitioned in the PEG rich phase and the maximum partition coefficient of 7.82 was obtained. The results are discussed in the context of practical potential of this citrate based ATPS in separating crude proteins from fish industry effluent.     

Extraction of microbial transglutaminase from <Emphasis Type="Italic">Amycolatopsis</Emphasis> sp. fermentation broth using aqueous two-phase system

Partitioning of microbial transglutaminase (MTG) from Amycolatopsis sp. in the polyethylene glycol (PEG)/salt-based ATPS was investigated for the first time. The key parameters such as the molecular weight of PEG (PEG 600-6000), the type and concentration of phase-forming salt (ammonium sulfate or phosphates), the pH of system (pH 5.0-8.5), and the concentration of neutral salt (0-6% NaCl, w/w) were determined. The partition coefficient of the enzyme was not linear with PEG molecular weight; PEG1000 gave better yield than others. The concentration of PEG1000, ammonium sulfate and NaCl, and the system pH showed effects with different extents on specific activity (SA) and yield of the enzyme. In the ATPS of 26% w/w PEG 1000 and 19% w/w ammonium sulfate in the presence of 5% w/w NaCl and at pH 6.0, MTG was partitioned into the PEG-rich phase with a maximum yield of 86.51% and SA was increased to 0.83. The results of SDS-PAGE showed the MTG produced by the test strain differed from the enzymes reported before. Thus, this study proves that ATPS can be used as a preliminary step for partial purification of MTG from Amycolatopsis sp. fermentation broth.     

Processing of soybean (Glycine max) extracts in aqueous two‐phase systems as a first step for the potential recovery of recombinant proteins

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     

Encapsulation of diclofenac sodium with acidic copolymer hydrogels based on PEG/poly(N‐isopropylacrylamide‐co‐2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid) semi‐interpenetrating network using in situ loading technique

A pH‐ and temperature‐responsive semi‐interpenetrating copolymer PEG6000/poly(NIPA‐co‐AMPS) (PEG/AMPS‐co‐NIPA SIPN), for short PEG SIPN, was made by ammonium persulfate‐initiated suspension copolymerization of N‐isopropylacrylamide, 2‐acrylamido‐2‐methylpropanesulphonic acid, and N,N′‐methylene‐bis‐acrylamide (MBAA; crosslinker) in the presence of PEG6000. The PEG SIPN copolymer matrices containing nanostructures made in the high‐temperature copolymerization resulted in channels for PEG and facile migration of drugs. In drug encapsulation or drug‐loading process, one can easily ignore or pay less attention to the interaction between a drug and its encapsulation materials; however, the ignored interactions may induce problems in drug properties or the release behavior in use. Sodium diclofenac (DFNa) precipitates as the carboxylic acid form in an acidic environment, and it is challenging to encapsulate sodium diclofenac in such an acidic matrix without precipitation of the sparingly soluble acid form of DFNa on the surface of the polymer substrate. To avoid bulky precipitation in drug loading, an in situ loading technique was developed for producing gel spheres with DFNa uniformly distributed in the polymer matrix. The technique is based on fast polymerization of spherical droplets of a pregel solution in which the drug is dissolved. Diffusion‐loading prodrugs were made in comparison with in situ loading prodrugs in thermal, release kinetics, and release behavior. Drug release profiles (in pH 7.4 phosphate buffer) show that the new drug loading technique gives controlled release during a period of about 7 days at 37°C. By contrast, gel spheres loaded with sodium diclofenac using the conventional diffusion technique produced almost total release of the drug within about 24 h. The thermal stability of sodium diclofenac, the PEG/AMPS‐co‐NIPA SIPN, and the prodrugs made with the SIPN and sodium diclofenac was studied. A near zero‐order release kinetics was found in the in vitro release of sodium diclofenac with in situ loading PEG SIPN prodrug. We have, for the first time, studied sodium diclofenac release behavior from the PEG SIPN hydrogel systems. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Aqueous Two‐Phase Partitioning of Glucose Isomerase from Actinoplanes missouriensis in the Presence of PEG‐Derivatives and its Immobilization on Chitosan Beads

Abstract

Purification of glucose isomerase by its partitioning in a PEG‐salt aqueous two‐phase system (ATPS) in the presence of PEG derivatives has been studied. Selective partitioning of the proteins was observed towards the PEG phase containing PEG‐benzoate and PEG‐palmitate, enriching glucose isomerase in the salt phase. Cross‐current extraction in 4 stages in the presence of PEG‐palmitate gave an enrichment factor of ～5 for the enzyme. After initial purification with ATPS, glucose isomerase was immobilized on cross‐linked chitosan beads. The immobilized enzyme was stable over a wider pH range (5.2–9.0) and showed an optimum pH of 6.5     

New aqueous two‐phase systems based on poly(ethylene oxide sulfide) (PEOS) and potassium phosphate for the potential recovery of proteins

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 (V_r) 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 V_r = 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; lnK_BPE = ?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     

Potential application of aqueous two‐phase systems for the fractionation of RNase A and α‐Lactalbumin from their PEGylated conjugates

BACKGROUND: PEGylation reactions often result in a heterogeneous population of conjugated species and unmodified proteins that presents a protein separations challenge. Aqueous two‐phase systems (ATPS) are an attractive alternative for the potential fractionation of native proteins from their PEGylated conjugates. The present study characterizes the partition behaviors of native RNase A and α‐Lac and their mono and di‐PEGylated conjugates on polyethylene glycol (PEG)—potassium phosphate ATPS. RESULTS: A potential strategy to separate unreacted native protein from its PEGylated species was established based upon the partition behavior of the species. The effect of PEG molecular weight (400–8000 g mol^?1), tie‐line length (15–45% w/w) and volume ratio (V_R; 0.33, 1.00 and 3.00) on native and PEGylated proteins partition behavior was studied. The use of ATPS constructed with high PEG molecular weight (8000 g mol^?1), tie‐line lengths of 25 and 35% w/w, and V_R values of 1.0 and 3.0 allowed the selective fractionation of native RNase A and α‐Lactalbumin, respectively, from their PEGylated conjugates on opposite phases. Such conditions resulted in an RNase A bottom phase recovery of 99%, while 98% and 88% of mono and di‐PEGylated conjugates, respectively were recovered at the top phase. For its part, α‐Lac had a bottom phase recovery of 92% while its mono and di‐PEGylated conjugates were recovered at the top phase with yields of 77% and 76%, respectively. CONCLUSIONS: The results reported here demonstrate the potential application of ATPS for the fractionation of PEGylated conjugates from their unreacted precursors. Copyright © 2010 Society of Chemical Industry     

Treatment of metal‐containing wastewaters with a novel extractive membrane reactor using sulfate‐reducing bacteria

This work reports a novel system for the treatment of acidic metal‐containing wastewaters, the Extractive Membrane Bioreactor–Sulfate‐Reducing Bacteria (EMB‐SRB) system. In this system, hydrogen sulfide is produced in the bioreactor by the sulfate‐reducing bacteria, transfers through a dense phase membrane, and precipitates metal ions in the wastewater. The non‐porous membrane prevents the SRB from having direct contact with the toxic metals, extremes of pH, or high salinity in the wastewater. Silicone rubber, which is permeable to H₂S but virtually impermeable to ionic species in the system, was used as a membrane. The rate of mass transfer of H₂S across the membrane was studied and found to be well described by a resistances‐in‐series model. k_ov values vary in the range 5 × 10⁻⁶ –10 × 10⁻⁶ ms⁻¹ 1 depending on the membrane thickness. A continuous EMB–SRB system was operated and more than 90% (w/v) of the Zn²⁺ present in a wastewater was removed. A film of metal precipitate was found to build up on the inside (wastewater) side of the membrane, and became the dominant resistance contributing to the overall mass transfer coefficient during operation. © 2001 Society of Chemical Industry     

Application of Aqueous Two‐Phase Systems for the Potential Extractive Fermentation of Cyanobacterial Products

The potential use of aqueous two‐phase systems (ATPS) to establish a viable protocol for the in situ recovery of cyanobacterial products was evaluated. The evaluation of system parameters such as poly (ethylene glycol) (PEG) molecular mass, concentration of PEG and salt was carried out to determine the conditions under which Synechocystis sp. PCC 6803 cell and cyanobacterial products, i.e., β‐carotene and lutein, become concentrated in opposite phases. PEG‐phosphate ATPS proved to be unsuitable for the recovery of cyanobacterial products due to the negative effect of the salt upon the cell growth. The use of ATPS PEG‐dextran (6.6 % w/w PEG 3350, 8.4 % w/w dextran 66900, TLL 17.3 % w/w, V_R 1.0, pH 7) and (4.22 % w/w PEG 8000, 9.77 % w/w dextran 66900, TLL 18 % w/w, V_R 1.0, pH 7) resulted in the growth of cyanobacteria (Synechocystis sp. PCC 6803) and the concentration of lutein in opposite phases. However, β‐carotene was seen to concentrate in the top phase together with the biomass. The results reported here demonstrate the potential application of ATPS to establish the conditions for an extractive fermentation prototype process for the recovery of cyanobacterial products.     

Rheological properties of poly(ethylene glycol)/poly(N‐isopropylacrylamide‐co‐2‐acrylamido‐2‐methylpropanesulphonic acid) semi‐interpenetrating networks

For the increasing demands of multifunctional materials in applications such as drug delivery system, a pH‐ and temperature‐responsive polyelectrolyte copolymer gel system was studied using rheometry. Rheological properties, determined by plate–plate rheometry in oscillatory shear, of hydrogels formed by free radical initiated copolymerization of N‐isopropylacrylamide (NIPA) and 2‐acrylamido‐2‐methylpropanesulphonic acid (AMPS) in the presence of methylene bisacrylamide (MBAA) as crosslinker are compared with the properties of semi‐interpenetrating network (SIPN) polyelectrolyte gels made by incorporation of poly(ethylene glycol) with molar mass 6000 g mol^?1 (PEG6000). Based on our systematic studies for this PEG/SIPN system, the effects of initiator and crosslinker concentration, relative proportions of comonomer units in the main chains, PEG6000 content and temperature on viscoelastic properties, unusual high storage moduli at small strain for the SIPN were discussed. The SIPN gel with characteristics of PEG molecules as well as pH and temperature responsiveness from AMPS and NIPA units has potential application in drug delivery system design. Ice‐like rheological behavior of the PEG/AMPS‐NIPA SIPN gels at low temperature was first time reported and water remains homogeneous without phase separation in PEG/AMPS‐NIPA SIPN hydrogels at low temperature may be considered as an ideal candidate for water storage material. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

The optimization of aqueous two‐phase extraction of lysozyme from crude hen egg white using response surface methodology

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