Recovery of value‐added globular proteins from tannery wastewaters using PEG – salt aqueous two‐phase systems

Leather processing involves discharge of high‐value soluble globular proteins in the wastewater. The recovery of value‐added products from the wastewaters is gaining more importance in the context of recovery of wealth from waste. The recovery of these globular proteins from tannery wastewater was selected as a practical model system to study the implementation of polyethylene glycol (PEG)‐sulfate aqueous two‐phase systems (ATPS). The partition coefficient of bovine serum albumin is comparable to that of soluble proteins from tannery wastewaters. The influence of concentration of polymer, salt, pH and temperature on the partitioning of soluble proteins from tannery wastewaters has been studied. The PEG6000 + sodium sulfate + water system provide better partitioning of these soluble proteins as compared to PEG6000 + ammonium sulfate system. The maximum protein recovery yield for PEG6000 + sodium sulfate + water system at 20 °C is 92.75%. The influence of temperature indicates the recovery of proteins from tannery wastewater to be better at lower temperature. The findings of these studies raise the potential application of ATPS processes for protein recovery from complex biological systems. Copyright © 2006 Society of Chemical Industry     

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%.     

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.     

The partitioning of protease from Calotropis procera latex by aqueous two-phase systems and its hydrolytic pattern on muscle proteins

The protease from the latex of Calotropis procera was isolated by an aqueous two-phase system (ATPS). Polyethylene glycol (PEG 1000, 2000 and 3000) at a concentration of 12, 15, and 18% (w/w) with salts ((NH₄)₂SO₄, K₂HPO₄ and MgSO₄) at a concentration of 14, 17, and 20% (w/w) were investigated. The highest protease recovery (74.6%) was found in the PEG-rich phase of the system (p < 0.05), comprising of 18% PEG 1000 and 14% MgSO₄. Protein patterns and activity staining showed that the isolated protease had a molecular weight of ∼31 kDa without the oligosaccharide attached to the molecule. Degradation of muscle proteins in beef, farmed giant catfish, and squid was observed by the electrophoresis of sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE). The degradation of myofibrillar proteins (myosin heavy chain: MHC and actin: AC) of farmed giant catfish was higher than that of beef and squid muscles as indicated by the degradation proteins with lower molecular weight.     

The relationship of thermosensitive properties with structure of organophosphazenes

The lower critical solution temperature (LCST) of cyclotriphosphazenes and polyphosphazenes grafted with a hydrophilic poly(ethylene glycol) (PEG) and a hydrophobic amino acid ester (AAE) as side groups could be quantitatively correlated with the overall hydrophobicity given by linear combination of the logarithmic partition coefficients of the corresponding free PEG and AAE measured in the 1-octanol/water system. Thus the LCSTs of phosphazenes were found to be well described as a linear function of a new parameter P_t defined as linear combination of the logarithmic partition coefficients of the free side groups, K_peg and K_aae: where x is the mole fraction of PEG. The LCST of cyclotriphosphazenes has shown to be more sensitive to change in hydrophobicity than polyphosphazenes, indicating that the nature of the molecular structure plays an important role in determining their LCST. The additivity of the logarithms of the two partition coefficients for the parameter may indicate that the contribution of each constituent is independent, providing a facile method for prediction of the LCST of thermosensitive polymers.     

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     

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     

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.     

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.     

金属螯合双水相亲和分配技术分离纳豆激酶的研究

利用金属螯合亲和双水相分配技术对纳豆激酶的分离纯化进行了研究。考察了双水相系统、聚合物的分子量和浓度、亲和配基加入量、pH值、相比以及生物质加入量等因素对亲和分配的影响。结果表明,双聚合物系统比聚合物／无机盐系统更有利于纳豆激酶亲和分配;pH值和亲和配基加入量是影响分配的关键因素。优化的分配条件为：2．6％聚乙二醇,20．2％羟丙基淀粉,5％亲和配基PEG-IDA—Cu(Ⅱ),相比12,pH8．2,发酵液加入量15％。分配系统放大到100g,仍保持一致的酶活收率(90％)和纯化因子(2.0)。设计了两次分配分离流程,纯化因子达到3．52,总收率为81％。     

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.     

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.     

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.     

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     

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     

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.     

The study on the conductivity of styrene-maleic anhydride copolymer electrolytes based on poly(ethylene glycol)400 and LiClO4

Polyelectrolytes, in this study were synthesized from styrene-maleic anhydride (SMA) copolymer, poly(ethylene glycol)400 (PEG400), and lithium perchlorate (LiClO₄). Fourier transform infrared spectroscopy (FTIR), and magic angle spinning (MAS) solid-state NMR were used to monitor the interaction between Li⁺ ions and polymer. The results of FTIR and MAS solid-state NMR indicate the Li⁺ ions are preferentially coordinated to the ether oxygen of PEG. The T_g of the PEG segments in polyelectrolyte increases with LiClO₄ concentration, as determined by differential scanning calorimetry (DSC), indicating that solubility of the Li⁺ ions in the host polymer increases with the PEG content. Impedance spectroscopy (IS) shows that the bulk conductivity of polyelectrolytes and the conductivity behavior obeys the Vogel-Tamman-Fulcher (VTF) equation.     

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.     

Optimization of Rhizopus niveus Lipase Partitioning by an Aqueous Biphasic System

An unconventional aqueous two‐phase system (ATPS) composed of polyethylene glycol (PEG) and sodium carbonate was developed and optimized by employing response surface methodology for separation of Rhizopus niveus lipase. A five‐level central composite design was applied to evaluate the optimal level of three process variables in order to obtain maximum lipase separation. Experimental data were analyzed by regression and a polynomial model was created which was found significant. The maximum partition coefficient was achieved with the system PEG 4000/sodium carbonate. Validation experiments confirmed the high accordance of predicted and experimental results. The optimized ATPS can be applied as a suitable cost‐effective system for lipase extraction.     

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