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     

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     

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     

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 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     

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.     

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     

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.     

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%.     

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     

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%.     

Partition of alkaline protease in aqueous two-phase systems of polyethylene glycol 1000 and potassium phosphate

This article presents a study of polyethylene glycol 1000 (PEG1000)/potassium phosphate aqueous two-phase systems (ATPSs) forBacillus subtilis NS99 alkaline protease extraction. The objectives were to evaluate effects of system pH (7.5, 8.5,9.5, and 10.5), and NaCl concentration (0,4,7, and 10% (w/w)) on ATPS binodal curves, effects of system pH, NaCl concentration, and tie-line length (TLL) on alkaline protease partition coefficient (K) and yield (Y%) at room temperature (30±2 ‡C). Casein hydrolysis was used for determination of alkaline protease activity. It was revealed that system pH had the slightest effect on locations of binodal curves (except at pH 10.5). In contrast, addition of NaCl appeared to have a significant effect on phase characteristics since binodal curves of systems with NaCl (4-10% (w/w)) shifted significantly towards the origin in comparison to the ones without NaCl. Increased NaCl concentration from 4 to 10% (w/w), however, showed trivial influence on locations of the binodal curves. Changes of system compositions due to variation in system pH, TLL, and NaCl concentrations obviously resulted in varied obtainable K and Y% of alkaline proteases. Longer TLL and higher pH generally resulted in higher K. In contrast, the lower NaCl concentration, the higher K. Since the same phase volume ration (1:1) was used throughout the experiments, Y% depended solely on K. The most suitable PEG1000/potassium phosphate ATPS was determined at pH 9.5, and comprised PEG1000, potassium phosphate, and NaCl 18.0,13.0, and 0% (w/w), respectively. This system resulted in considerably high K, and Y% of 20.0, and 95.1%, respectively. Information from this study will be important for further development of an ATPS extraction unit for alkaline protease recovery.     

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.     

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.     

Separation and Purification of Aloe Anthraquinones Using PEG/Salt Aqueous Two-Phase System

The anthraquinones were extracted from Curacao aloe leaves. Aqueous two-phase system (ATPS) of polyethylene glycol (PEG)/salt, coupled with spectrophotometry and high performance liquid chromatography (HPLC) were employed for the first time as an attractive alternative for the downstream processing of aloe anthraquinones, mainly for the removal of the impurities without additional steps. The influence factors such as molecular mass and concentration of PEG, type, and concentration of neutral salt, temperature, and pH on the phase partition behavior of ATPS had been studied. Under the optimal condition, the highest extraction yield 90.54% was obtained in PEG phase using PEG-6000/(NH₄)₂SO₄ system to a mass ratio of 2:1 at 40°C, pH 3.0 with 0.6 g sodium chloride added. The reverse extraction of anthraquinones from the PEG phase was achieved with a recovery of 70.15% by adjusting the pH. Meanwhile, the PEG could be recycled. The major components in aloe anthraquinones of aloe-emodin and chrysophanol were analyzed by HPLC before and after ATPS extraction process. Compared with conventional purification methods, this technique can be completed in one operation; besides it is low-cost and environmentally friendly.     

Partitioning of lysozyme in aqueous two-phase systems containing ethylene oxide-propylene oxide copolymer and potassium phosphates

Lysozyme partitioning in EO₅₀PO₅₀/potassium phosphate aqueous two-phase systems (ATPS) was studied. In the work, the influence of EO₅₀PO₅₀, potassium phosphate and sodium chloride concentration in the ATPS on lysozyme partition coefficient and separation yield was examined. In addition, the influence of the pH of potassium phosphate solution was also investigated. A Box–Behnken design was defined, and response surface models for the partition coefficient K and percentage yield of the enzyme in the top phase Y were calculated. Among the examined factors, the NaCl concentration had the highest influence on lysozyme separation parameters. This influence can be explained mainly by the hydrophobic interactions between the protein and the phase-forming components. A maximum partition coefficient K_L1, yield Y_L1 and Y_L2 were predicted for EO₅₀PO₅₀, potassium phosphate and NaCl concentrations of 17.40, 22.67% and 0.85 mol/l, respectively, and for pH 9.0. A good agreement was obtained between the experimental and the predicted results.     

Partitioning of water-soluble vitamins in biodegradable aqueous two-phase systems: Electrolyte perturbed-chain statistical associating fluid theory predictions and experimental validation

Partition coefficients (K) of vitamins (riboflavin, nicotinic acid, nicotinamide, folic acid, cyanocobalamin) in aqueous two-phase systems (ATPS) composed by polyethylene glycol (PEG 4000, PEG 6000) and organic salt (sodium citrate and sodium tartrate) at T = 298.15 K and p = 1 bar have been studied. Data on liquid–liquid equilibria of the ATPS considered in this study have been taken from the literature (PEG-Na₃Citrate) or measured in this work (PEG-Na₂Tartrate) for PEG 4000 and PEG 6000 at T = 298.15 K and p = 1 bar. The experimental K values were validated by electrolyte perturbed-chain-statistical associating fluid theory predictions. The neutral cyanocobalamin has the highest K values among all studied vitamins at any ATPS studied in this work. This finding contrasted with expectations based on literature data which let assume that charged species have typically the highest K values in the considered ATPS. Thus, besides the typically strong charge–charge interactions especially specific forces (e.g., hydrogen bonding) explains the strong PEG-cyanocobalamin interaction resulting in the high K values.     

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.