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.     

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.     

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.     

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.     

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.     

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 (PEG 1000, 2000, 4000 and 6000), PEG and potassium citrate concentration, BSA concentration (C_BSA) and pH on BSA partition. The pH was found to have significant effects on BSA partition with low molecular weight PEG 1000. The yield of the BSA, 99%, was obtained in the top phase under the following conditions: 19% (w/w) PEG 1000, 20% (w/w) potassium citrate and 0.75 mg/g C_BSA at pH 7.0 and 30 °C. BSA can be re-extracted to a new citrate phase by decreasing the pH of the system with a 92% yield. The back-extraction not only separates the BSA from the polymer, but also allows the polymer to be recycled. The global yield (Y_e + Y_be) is up to 91%.     

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     

Density,refractive index and liquid–liquid equilibrium data of polyethylene glycol 3000 + potassium formate + water at different pH values

New liquid–liquid equilibrium data for polyethylene glycol(PEG) 3000 + CHO_2K + H_2O systems were measured at 298.15 K and pH values of 7.95, 8.40 and 9.98. It was found that an increase in pH caused the binodal curve to be displaced downward and the two-phase region to expand. Accordingly, the binodal curve was adjusted to the Pirdashti equation and the tie-line compositions were correlated using the Othmer–Tobias, Bancroft and Hand equations. The study measured the refractive index and densities of several homogeneous binary and ternary solutions. The solutions were used for calibration within a range of 0% to 30% of the mass of the PEG and potassium formate. The density and refractive index data show a linear variation with the mass fraction of the polymer and the salt. The effect of pH on the binodal, tie-line lengths(TLL) and slope of the tie-line(STL) in the systems was examined. It was found that an increase in pH increased the TLL and decreased the STL. It was observed that the density of the aqueous two-phase system was influenced by the TLL. The difference in density between phases(Δρ) increased as the TLL and pH increased. It was found that the TLL and Δρ showed a linear relationship. The effective excluded volume(EEV) of the PEG was obtained and it was found that EEV also increased as the pH increased.     

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     

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     

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     

Response surface methodology for the evaluation of guanidine hydrochloride partitioning in polymer-salt aqueous two-phase system

The current study employed response surface methodology (RSM) with a face-centered central composite design (CCD) to indicate the essential variables on the partition coefficient of guanidine hydrochloride (GuHCl) in the poly (ethylene glycol) (PEG)-phosphate aqueous two-phase system (ATPS). To evaluate the partition coefficients of GuHCl in the mentioned ATPS, the pH (7.0, 8.5 and 10.0), GuHCl concentration (1.0, 3.5 and 6.0% w/w), PEG molecular weight (2,000, 4,000 and 6,000 gmol^?1) and PEG/potassium phosphate concentrations ratio were selected as independent variables. A quadratic model is suggested to find the impact of these variables. The suggested model has a strong harmony with the experimental data. The results of the model display that the GuHCl concentration and weight percent of the salt in feed have a large and small influence on the GuHCl partitioning.     

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.     

双水相体系萃取精氨酸脱亚胺酶

报道了利用聚乙二醇/硫酸铵双水相体系从自溶NJ402菌粗提酶液中分离纯化精氨酸脱亚氨酶(ADI)的研究结果,为精氨酸脱亚氨酶的分离纯化提供了一种方法。在双水相体系中采用聚乙二醇(PEG)与(NH4)2SO4为组成成分,考察了聚乙二醇(PEG)平均相对分子质量、PEG质量分数、(NH4)2SO4质量分数、pH及NaCl质量分数对精氨酸脱亚氨酶分离纯化效果的影响。最佳双水相体系萃取条件为:聚乙二醇(PEG)平均相对分子质量为1 000,w(PEG1000)=15%,w[(NH4)2SO4]=20%,pH=6.5,室温下从自溶NJ402菌粗提酶液中分离纯化精氨酸脱亚氨酶,纯化倍数达到2.35倍,萃取率达91.1%。     

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     

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     

Separation of Bromelain by Aqueous Two Phase Flotation

Aqueous two phase flotation (ATPF) system of polyethylene glycol (PEG) and potassium phosphate is studied for the separation and partial purification of bromelain from the pineapple fruit (Annanus comosus L. Merryl). The effect of PEG molecular weight (1500–20000), concentration of phase forming components (PEG 12–18% w/w and potassium phosphate 14–20% w/w), system pH, nitrogen flow rate, and flotation time were studied and optimum conditions for ATPF were obtained. At optimum conditions of the system, i.e., 14% w/w PEG 1500, 18% w/w potassium phosphate, 80 mL/min of nitrogen flow rate and pH 7, maximum yield of 91.47% and purification fold of 4.26 were obtained. ATPF was found to be an effective technique for the purification of bromelain from pineapple fruit with higher extraction yield and purification fold as compared to aqueous two phase extraction (ATPE).     

Demixing kinetics of phase systems employed for liquid-liquid extraction and correlation with system properties

The kinetics of phase demixing in liquid-liquid extraction for model systems comprising PEG 4000/potassium phosphate and t-butanol/ammonium sulfate was studied. The kinetics of demixing depicts the entire demixing pattern of phases of the phase system during and after extraction and hence the study is essential prior to design of large scale gravity phase separators. With an increase in composition, both the systems showed increase in demixing rate (decrease in demixing time). At high tie line length (TLL) and phase volume ratio <1, with salt rich phase as continuous phase, both PEG 4000/potassium phosphate and t-butanol/ammonium sulfate systems showed reduction in demixing time by 59% and 50%, respectively as compared to that at low TLL. An empirical equation proposed in the literature for rate of phase demixing was used to correlate the experimental data. The agreement was better for t-butanol/ammonium sulfate system when compared to PEG 4000/potassium phosphate system.     

The phase behavior of n-ethylpyridinium tetrafluoroborate and sodium-based salts ATPS and its application in 2-chlorophenol extraction

In this paper, the aqueous two-phase systems (ATPS) containing n-ethylpyridinium tetrafluoroborate ([EPy]BF4), sodium-based salts, and water were studied and the extraction efficiency of 2-chlorophenol was measured to study the ATPS performance in extracting phenolic compounds. The binodal curves of [EPy]BF4+sodium car-bonate (Na2CO3) ATPS and [EPy]BF4+sodium dihydrogen phosphate (NaH2PO4) ATPS have been determined at 308.15 K, 318.15 K, and 328.15 K and atmospheric pressure. After getting good correlation with Merchuk equa-tion, the binodal curves together with gravimetric method were used to calculate the tie-lines data. Furthermore, the reliability of tie-lines data was verified using Othmer-Tobias and Bancroft equations. Then, the salt influence and temperature influence on the phase behavior were discussed and the results show the salt-outing ability of Na2CO3 is better than NaH2PO4. With the aim of studying the ATPS performance in extracting phenolic com-pounds, extraction efficiency for 2-chlorophenol at different temperatures were studied and the results show that [EPy]BF4+Na2CO3 ATPS is preferred than [EPy]BF4+NaH2PO4 ATPS in applications.     

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.