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     

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     

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.     

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     

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     

Recovery and partial purification of thermophilic β-xylosidase derived from recombinant Bacillus megaterium MS941 by aqueous two-phase system

A polymer–salt-based aqueous two-phase system (ATPS) was developed for the effective extraction and purification of extracellular β-xylosidase from the fermentation broth of recombinant Bacillus megaterium MS941. The effect of molecular weight (MW) of polyethylene glycol (PEG), tie-line length (TLL), volume ratio (V_R), crude loading and pH on the recovery performance was evaluated. Under the optimal extraction conditions, β-xylosidase was successfully purified up to 23-fold with a recovery yield of 99% in the bottom salt-rich phase at PEG 4,000/potassium phosphate ATPS comprising TLL of 41.8, V_R of 2.3, crude loading (C_L) of 30% (w/w) at pH 6.     

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     

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     

Aqueous two‐phase systems for the recovery of a recombinant viral coat protein from Escherichia coli

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 K_DNA < 0.0 and partition coefficient of L1; ln K_L1 > 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     

Recovery of Human Interferon Alpha-2b from Recombinant Escherichia coli by Aqueous Two-Phase System

Recovery of periplasmic human recombinant interferon alpha-2b (IFN-α2b) from Escherichia coli rosetta-gami2 (DE3) using a single-step polyethylene glycol (PEG)-potassium phosphate aqueous two-phase system (ATPS) was investigated in this study. The influences of system parameters including PEG molecular weight, tie-line length, volume ratio, crude stock loading, system pH, and sodium chloride (NaCl) concentration (%, w/w) were studied. The results showed that the optimum condition to obtain the high purification factor of IFN-α2b in a single step was achieved by ATPS composed of 4% (w/w) PEG 8000, 13% (w/w) potassium phosphate, 0.5% (w/w) NaCl, 10% (w/w) crude stock, and a system pH of 6.5. A purification factor of 26.3 and recovery yield of 40.7% were obtained from optimized ATPS.     

Aqueous two-phase countercurrent distribution for the separation of c-phycocyanin and allophycocyanin from Spirulina platensis

C-phycocyanin (C-PC) and allophycocyanin (APC) with similar molecular structures were separated, respectively from Spirulina platensis cell homogenate by single extraction and multi-stage countercurrent distribution (CCD) using an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) and potassium phosphate (KPi). The partition coefficients of C-PC and APC were 10.64 and 0.57, respectively, and the extraction selectivity of C-PC was 18.67 from 0.5% (w/w) S. platensis crude extract by single extraction using PEG6000/KPi ATPS (pH 7.0) with 34% (w/w) tie line length (TLL). In ten-stage CCD under the same ATPS extraction condition with 2% (w/w) S. platensis crude extract, the purity of C-PC increased nearly twice and the recovery of APC increased more than nine-fold compared with single extraction. The results displayed that most C-PC (82.1%) followed the mobile phase was enriched in the top phases of the last three tubes, while more APC (41%) remained in the stationary phase was enriched in the bottom phases of the first three tubes in the ten-stage CCD. Hence, aqueous two-phase CCD technology provided an effective and low cost method for C-PC and APC separation from S. platensis cell homogenate directly.     

Separation of catalase from Amsonia orientalis with single step by aqueous two-phase partitioning system (ATPS)

Catalase from Amsonia orientalis was purified by ATPS, and its efficiency was compared against hydrophobic interaction chromatography. Activity recovery and purification fold of purified catalase by ATPS were examined under varying experimental conditions. The effects of various factors such as type of phase-forming salts, (PEG) mass, with their different concentrations, pH and temperature effects on partitioning were investigated. The highest activity recovery (156%) and purification fold (8.67) of catalase were obtained in the ATPS system containing 10% (g/g) PEG4000, 15% (g/g) Na₂SO₄ at pH 6.0 and room temperature. In hydrophobic interaction chromatography, the enzyme has been purified 12.54-fold with 57.5% recovery. The molecular weight of catalase was determined as 75 kDa by SDS-PAGE.     

Extracellular endo-mannanase from Bacillus sp. CFR1601: Economical production using response surface methodology and downstream processing using aqueous two phase system

Bacillus sp. CFR1601, isolated from decaying plant litter, produced an extra-cellular endo-mannanase (198.0 IU/g) under solid state fermentation (SSF) using defatted coconut residue as the prime solid substrate. In order to enhance endo-mannanase production, three component, five level central composite design (CCD) of response surface methodology (RSM) was used. Based on contour plots and variance analysis, optimum conditions for endo-mannanase production from Bacillus sp. CFR1601 were attained when defatted coconut residue was supplemented with sesame oil meal (10.0, w/w), Tween-80 (0.2%, v/v) and inoculated with bacterial cells from log phase (12 h old; OD_600 nm ≈ 3.6). The empirical model developed through RSM brought about 4.04–4.39-fold (800.0–870.0 IU/g) improvement in endo-mannanase yield as compared to un-optimized growth conditions. Downstream processing of endo-mannanase from SSF media was carried out for the first time using polyethylene glycol (PEG)/salt aqueous two phase system (ATPS). ATPS system consisting of a combination of PEG 3350 12.0% (w/w), Na₂SO₄ 12.0% (w/w), protein load 10.0% (w/w) and pH 5.0 resulted in one-sided partitioning of endo-mannanase towards bottom phase with 3.8-fold purification and 95.4% recovery. Second stage ATPS with fresh top phase further improved purification of endo-mannanase to 12.32-fold. Our overall results suggest a cost-effective and integrated process for production and downstream processing of endo-mannanase.     

Design Strategies for Integrated β‐Galactosidase Purification Processes

The operational conditions for an aqueous two‐phase system (ATPS) for β‐galactosidase purification were optimized and applied to the design of a purification strategy as an alternative to the primary purification steps. The ATPS proved to be suitable for the recovery and primary enzyme purification. The purification process design developed by ATPS, diafiltration, ion exchange, and diafiltration/ultrafiltration was successful, yielding a more than tenfold purification. The purification strategy design resulted in a powerful integrated purification and recovery process, an evidence of the potential for a scale‐up of the β‐galactosidase purification process.     

Simultaneous recovery of glycyrrhizic acid and liquiritin from Chinese licorice root (Glycyrrhiza uralensis Fisch) by aqueous two-phase system and evaluation biological activities of extracts

Aqueous two-phase system (ATPS) was used for simultaneous purification of glycyrrhizic acid (GA) and liquiritin (LQ) from crude extract of Chinese licorice root. It was revealed that 87% GA and 94% LQ were retrieved in the ATPS top phase, under the optimum conditions of 25% (w/w) ethanol, 30% (w/w) K₂HPO₄ and 4% (w/w) loading sample at 10–40°C. Compared with crude extract, the ATPS top-phase extract exhibited the highest antioxidative activity, but no tyrosinase inhibitory effect. Whereas, the ATPS bottom-phase extract was proved to be effective ABTS radical scavenger and tyrosinase inhibitor, suggesting the potency of the alcohol-salt ATPS purification for the different medicinal purposes.     

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.     

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     

Application of Response Surface Methodology for Optimization of Bromelain Extraction in Aqueous Two-Phase System

Extraction of bromelain from pineapple fruit in an aqueous two phase system (ATPS) composed of polyethylene glycol (PEG) 1500 and potassium phosphate has been studied using response surface methodology. The various process variables such as PEG, potassium phosphate and NaCl concentration, and pH were optimized using a central composite rotatable design (CCRD) of response surface methodology (RSM) based on the partition coefficient, % yield, and purification factor of an enzyme. An optimized ATPS composed of 14% (w/w) PEG 1500, 17.66% (w/w) potassium phosphate and 1 mM sodium chloride at pH 7.5 was used to purify bromelain from a pineapple fruit. With this system, a maximum enzyme partition coefficient of 12.62 and %yield of 90.33 in the top PEG-rich phase with a purification factor of 2.4 was predicted. The enzyme partition coefficient, % yield, and purification factor obtained from experimentation are 12.22, 89.65, and 2.8, respectively, in the top PEG phase. The response model is validated by the closeness between the predicted and experimental results.     

Synthesis and characterization of star‐shaped poly(ethylene glycol)‐block‐poly(L‐lactic acid) copolymers by melt polycondensation

Compared with linear diblock or triblock poly(ethylene glycol)‐block‐poly(L ‐lactic acid) copolymer (PEG‐b‐PLLA), star‐shaped PEG‐b‐PLLA (sPEG‐b‐PLLA) copolymers exhibit smaller hydrodynamic radius and lower viscosity and are expected to display peculiar morphologies, thermal properties, and degradation profiles. Compared with the synthesis routine of PEG‐b‐PLLA form lactide and PEG, the traditional synthesis routine from LA and PEG were suffered by the low reaction efficiency, low purity, lower molecular weight, and wide molecular weight distribution. In this article, multiarm sPEG‐b‐PLLA copolymer was prepared from multiarm sPEG and L ‐lactic acid (LLA using an improved method of melt polycondensation, in which two types of sPEG, that is, sPEG₁ (four arm, Mn = 4300) and sPEG₂ (three arm, Mn = 3200) were chosen as the core. It was found the molecular weight of sPEG‐b‐PLLA could be strongly affected by the purity of LLA and sPEGs, and the purification technology of vacuum dewater and vacuum distillation could help to remove most of the impurities in commercial available LLA. The polymers, including sPEG and sPEG‐b‐PLLA with varied core (sPEG₁ and sPEG₂) and LLA/sPEG feeding ratios, were characterized and confirmed by ¹H‐NMR and ¹³C‐NMR spectroscopy, Fourier transform infrared spectroscopy (FT‐IR) and gel permeation chromatography, which showed that the terminal hydroxyl group in each arm of sPEGs had reacted with LLA to form sPEG‐b‐PLLA copolymers with fairly narrow molecular weight distribution. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Structure–Property–Process Relationship for Blown Films of Bimodal HDPE and Its LLDPE Blend

Blown films of bimodal‐high‐density polyethylene (HDPE) (BPE) and its blend containing 40 wt% of linear low‐density polyethylene (LLDPE) are prepared in various neck‐heights (NHs). The crystal structures of both films are investigated in detail using small‐angle X‐ray scattering and wide‐angle x‐ray diffraction techniques. The results show that the blending of LLDPE notably modifies the crystal structure of BPE, including crystal density (ρ_c), crystallite size of the 110 plane (〈L₁₁₀〉), thickness of the lamellar crystal (L_c), and grain widths of the lamellae. The relationships between NH, crystal structure, and the resistance of dart‐drop impact (DDI) are investigated for both BPE and BPE/LLDPE films. The results indicate that the reorientation of lamellae might be a primary factor responsible for the DDI property. However, large values of ρ_c, L_c, and 〈L₁₁₀〉 are required for the film to achieve high DDI.