Aqueous two-phase extraction for the purification of alkaline agarases from culture extracts of Pseudomonas aeruginosa AG LSL-11

The agarases were purified for the first time an using aqueous two-phase system (ATPS) consisting of polyethylene glycol (PEG) and phosphate salt. The three extracellular, alkaline agarases produced by Pseudomonas aeruginosa AG LSL-11 were efficiently extracted into the top PEG-rich layer. The influencing factors on the partition of agarases--molecular weight of the PEG, system pH, system temperature, and NaCl concentration--were investigated. All the factors were found to have a significant effect on the partition of agarases except NaCl. The optimal ATPS parameters for the partitioning and purification of agarases were found to be 12% PEG 600 and 11.9% (w/w) phosphate salt at pH 8.0 and 4°C. All three agarases were concentrated in the top PEG phase with 6.19-fold purity and 71.21% recovery. The ATPS was found to be more convenient and economical than the conventional ion-exchange chromatography (IEC) method for extraction of three agarases and could be significantly employed for the purification of agarases from fermentation broth.     

Purification and Characterization of Polyphenol Oxidase From Waste Potato Peel by Aqueous Two-Phase Extraction

Potato peel from food industrial waste is a good source of polyphenol oxidase (PPO). This work illustrates the application of an aqueous two-phase system (ATPS) for the extraction and purification of PPO from potato peel. ATPS was composed of polyethylene glycol (PEG) and potassium phosphate buffer. Effect of different process parameters, namely, PEG, potassium phosphate buffer, NaCl concentration, and pH of the system, on partition coefficient, purification factor, and yield of PPO enzyme were evaluated. Response surface methodology (RSM) was utilized as a statistical tool for the optimization of ATPS. Optimized experimental conditions were found to be PEG1500 17.62% (w/w), potassium phosphate buffer 15.11% (w/w), and NaCl 2.08 mM at pH 7. At optimized condition, maximum partition coefficient, purification factor, and yield were found to be 3.7, 4.5, and 77.8%, respectively. After partial purification of PPO from ATPS, further purification was done by gel chromatography where its purity was increased up to 12.6-fold. The purified PPO enzyme was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by K_m value 3.3 mM, and V_max value 3333 U/mL, and enzyme stable ranges for temperature and pH of PPO were determined. These results revealed that ATPS would be an attractive option for obtaining purified PPO from waste potato peel.     

Optimisation of aqueous two-phase extraction of human antibodies

The purification of human antibodies in an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) 6000 and phosphate was optimised by surface response methodology. A central composite design was used to evaluate the influence of phosphate, PEG and NaCl concentration and of the pH on the purity and extraction yield of IgG from a simulated serum medium. The conditions that maximise the partition of IgG into the upper phase were determined to be high concentrations of NaCl and PEG, low concentrations of phosphate and low pH values. An ATPS composed of 12% PEG, 10% phosphate, 15% NaCl at pH 6 was further used to purify human monoclonal antibodies from a Chinese Hamster Ovary (CHO) concentrated cell culture supernatant with a recovery yield of 88% in the upper PEG-rich phase and a purification factor of 4.3. This ATPS was also successfully used to purify antibodies from a hybridoma cell culture supernatant with a recovery yield of 90% and a purification factor of 4.1.     

Purification and in situ immobilization of lipase from of a mutant of Trichosporon laibacchii using aqueous two-phase systems

The crude intracellular lipase (cell homogenate) from Trichosporon laibacchii was subjected to partial purification by aqueous two-phase system (ATPS) and then in situ immobilization by directly adding diatomites as carrier to the top PEG-rich phase of ATPS. A partition study of lipase in the ATPS formed by polyethylene glycol–potassium phosphate has been performed. The influence of system parameters such as molecular weight of PEG, system phase composition and system pH on the partitioning behaviour of lipase was evaluated. The ATPS consisting of PEG 4000 (12%) and potassium phosphate (K₂HPO₄, 13%) resulted in partition of lipase to the PEG-rich phase with partition coefficient 7.61, activity recovery 80.4%, and purification factor of 5.84 at pH of 7.0 and 2.0% NaCl. Moreover, the in situ immobilization of lipase in PEG phase resulted in a highest immobilized lipase activity of 1114.6 U g^?1. The above results show that this novel lipase immobilization procedure which couples ATPS extract and enzyme immobilization is cost-effective as well as time-saving. It could be potentially useful technique for the purification and immobilization of lipase.     

Purification of recombinant phenylalanine dehydrogenase by partitioning in aqueous two-phase systems

This study presents the partitioning and purification of recombinant Bacillus badius phenylalanine dehydrogenase (PheDH) in aqueous two-phase systems (ATPS) composed of polyethylene glycol 6000 (PEG-6000) and ammonium sulfate. A single-step operation of ATPS was developed for extraction and purification of recombinant PheDH from E. coli BL21 (DE3). The influence of system parameters including; PEG molecular weight and concentration, pH, (NH(4))(2)SO(4) concentration and NaCl salt addition on enzyme partitioning were investigated. The best optimal system for the partitioning and purification of PheDH was 8.5% (w/w) PEG-6000, 17.5% (w/w) (NH(4))(2)SO(4) and 13% (w/w) NaCl at pH 8.0. The partition coefficient, recovery, yield, purification factor and specific activity values were of 92.57, 141%, 95.85%, 474.3 and 10424.97 U/mg, respectively. Also the K(m) values for L-phenylalanine and NAD(+) in oxidative deamination were 0.020 and 0.13 mM, respectively. Our data suggested that this ATPS could be an economical and attractive technology for large-scale purification of recombinant PheDH.     

Partitioning studies of α-lactalbumin in environmental friendly poly (ethylene glycol)—citrate salt aqueous two phase systems

Aqueous two-phase systems (ATPS) formed by polymer and salt have been utilized to enrich the desired biomolecule into one of the phase with higher yield and purity. The eco-friendly, biodegradable poly ethylene glycol (PEG) and different citrate salts were chosen as ATPS phase components to investigate the partitioning behavior of α-lactalbumin (α-La). System factors and process parameters such as type and concentration of salt, molecular weight and concentration of PEG, pH, temperature and the effect of additives were studied and the results are discussed in detail. PEG 1000–tri-potassium citrate system yields high partition coefficient of 20 with a better yield of 98 % in the top phase. The addition of NaCl as an additive and acidic pH lowers the yield of α-La in the top phase. Influence of phase volume ratio (V _r) on partitioning was studied and found that the partition coefficient remains almost constant along the tie line. High yield was achieved at a V _r of 3.5 at the tie line length of 50.63 (%, w/w).     

Aqueous two-phase systems extraction of alpha-toxin from Clostridium perfringens type A

Two sequential half-fraction designs were applied to studying the alpha-toxin partition produced by Clostridium perfringens type A in aqueous two phase systems (ATPS), as a function of four factors: PEG molar mass and concentration, phosphate concentration and pH. The highest purification factor, yield and partition coefficient results were obtained with PEG 8000 (15%, w/w), phosphate at 20% (w/w) and pH 8.0. This system allows, in a single step, an alpha-toxin purification of 4.6-fold with final activity yield of 230% and partition coefficient of 113.9 in the PEG rich phase.     

Application of the response surface methodology for optimization of whey protein partitioning in PEG/phosphate aqueous two-phase system

In order to develop a new strategy for β-lactoglobulin (β-lg) removal from whey protein, partitioning of α-lactalbumin (α-la), β-lg and glycomacropeptide (Gmp) was studied using aqueous two phase systems (ATPS). A system composed of 13% (w/w) polyethylene glycol (PEG, average molar mass 2000 g/mol) and 13% (w/w) potassium phosphate was used at 25°C. A central composite rotatable design (CCRD) associated to the response surface methodology (RSM) was applied to investigate the effects of NaCl concentration and pH on the partition of these proteins. It was found that α-la and Gmp partitioned to the top phase rich in PEG, whereas β-lg partitioned to the bottom phase rich in salt. According to the RSM, optimal conditions for β-lg removal where found where pH was equal to 6.7 and salt concentration was 0.35 mol/L. Under these conditions, the partition coefficient K(α) was 0.48 and K(Gmp) was 0.92. On the other hand, the partition coefficient K(β) was only 0.01. In such conditions β-lg preferentially concentrates in the bottom phase, while the top phase exclusively contains the proteins α-la and Gmp. Fractionation of the proteins from fresh whey was performed in a three stage cross-flow extraction system. The extraction yield for β-lg in the bottom phase was 97.3%, while the yields for α-la and Gmp in the top phase were 81.1% and 97.8%, respectively.     

Application of a PEG/salt aqueous two-phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract

Aqueous two-phase partition systems (ATPS) have been widely used for the separation of a large variety of biomolecules. In the present report, the application of a polyethylene glycol/phosphate (PEG/phosphate) ATPS for the separation of anti-HIV monoclonal antibodies 2G12 (mAb 2G12) and 4E10 (mAb 4E10) from unclarified transgenic tobacco crude extract was investigated. Optimal conditions that favor opposite phase partitioning of plant debris/mAb as well as high recovery and purification were found to be 13.1% w/w (PEG 1500), 12.5% w/w (phosphate) at pH 5 with a phase ratio of 1.3 and 8.25% w/w unclarified tobacco extract load. Under these conditions, mAb 2G12 and mAb 4E10 were partitioned at the bottom phosphate phase with 85 and 84% yield and 2.4- and 2.1-fold purification, respectively. The proposed ATPS was successfully integrated in an affinity-based purification protocol, using Protein A, yielding antibodies of high purity and yield. In this study, ATPS was shown to be suitable for initial protein recovery and partial purification of mAb from unclarified transgenic tobacco crude extract.     

Recovery of laccase from the residual compost of Agaricus bisporus in aqueous two-phase systems

The potential use of aqueous two-phase systems (ATPS) to establish a viable protocol for the recovery of laccase from the residual compost of Agaricus bisporus was evaluated. The evaluation of system parameters such as poly (ethylene glycol) (PEG) molecular mass, concentration of PEG as well as salt and system pH was carried out to determine under which conditions the laccase concentrates predominantly to the top PEG-rich phase. PEG 1000–phosphate ATPS proved to be suitable for the primary recovery of laccase. An extraction ATPS stage comprising volume ratio equal to 1.0, PEG 1000 18.2% (w/w), phosphate 15.0% (w/w), system pH of 7.0 and loaded with 5% (w/w) of crude extract from residual compost allowed the laccase recovery. The use of ATPS resulted in one-single primary recovery stage process that produced an overall yield of 95%. The results reported here demonstrated the potential application of ATPS for the valorisation of residual material and the potential establishment of a downstream process to obtain value added products with commercial application.     

Direct purification of Burkholderia Pseudomallei lipase from fermentation broth using aqueous two-phase systems

An aqueous two-phase purification process was employed for the recovery of Burkholderia pseudomallei lipase from fermentation broth. The partition behavior of B. pseudomallei lipase was investigated with various parameters such as phase composition, tie-line length (TLL), volume ratio (V_R), sample loading, system pH, and addition of neutral salts. Optimum conditions for the purification of lipase were obtained in polyethylene glycol (PEG) 6000-potassium phosphate system using TLL of 42.2% (w/w), with VR of 2.70, and 1% (w/w) NaCl addition at pH 7 for 20% (w/w) crude load. Based on this system, the purification factor of lipase was enhanced to 12.42 fold, with a high yield of 93%. Hence, the simplicity and effectiveness of aqueous two-phase systems (ATPS) in the purification of lipase were proven in this study.     

Coupling purification and in situ immobilization process of monoclonal antibodies to clenbuterol for immunosensor application

Clenbuterol (CL), which promotes the growth of muscular tissue and the reduction of body fat in pigs and cattle, has been confirmed to be a potential hazard to human health. In this study, a monoclonal antibody to clenbuterol (CL mAb) from a hybridoma culture supernatant was purified by an aqueous two-phase system (ATPS) at different polyethylene glycol (PEG) concentrations, PEG molecular weights, pH values, and NaCl concentrations. Then the CL mAb was immobilized in situ by directly adding polystyrene microspheres (PSMSs) into a PEG phase containing CL mAb. Using the immobilized antibody, an immunosensor was constructed to detect the CL residues in pork samples. The results showed that using an ATPS composed of 15% (w/w) PEG6000, 15% (w/w) phosphate, and 15% (w/w) NaCl at pH 8.0, the partition coefficient was 7.24, the activity recovery was 87.86%, and the purification fold was 2.88. The PEG–CL mAb–PSMS retained approximately 98% of its initial activity after 30-ml phosphate buffer (PBS) washings. After 30 days of storage, the CL mAb–PSMS lost nearly 75% of its activity, whereas the PEG–CL mAb–PSMS retained as much as 95% of its initial activity. Furthermore, the constructed immunosensor obtained recoveries of 90.5 to 102.6% when applied to pork samples spiked with CL.     

Potential Aqueous Two‐Phase Processes for the Primary Recovery of Colored Protein from Microbial Origin

The primary recovery of c‐phycocyanin and b‐phycoerythrin from Spirulina maxima and Porphyridium cruentum, respectively, using an established extraction strategy was selected as a practical model system to study the generic application of polyethylene glycol (PEG)‐phosphate aqueous two‐phase systems (ATPS). The generic practical implementation of ATPS extraction was evaluated for the recovery of colored proteins from microbial origin. A comparison of the influence of system parameters, such as PEG molecular mass, concentration of PEG as well as salt, system pH and volume ratio, on the partition behavior of c‐phycocyanin and b‐phycoerythrin was carried out to determine under which conditions target colored protein and contaminants concentrate to opposite phases. One‐stage processes are proposed for the primary recovery of the colored proteins. PEG1450‐phosphate ATPS extraction (volume ratio (V_R) equal to 0.3, tie‐line length (TLL) of 34 % w/w and system pH 7.0) for the recovery of c‐phycocyanin from Spirulina maxima resulted in a primary recovery process that produced a protein purity of 2.1 ± 0.2 (defined as the relationship of 620 nm to 280 nm absorbance) and a product yield of 98 % [w/w]. PEG1000‐phosphate ATPS extraction (i.e., V_R = 1.0, PEG 1000, TLL 50 % w/w and system pH 7.0) was preferred for the recovery of b‐phycoerythrin from Porphyridium cruentum, which resulted in a protein purity of 2.8 ± 0.2 (defined as the relationship of 545 nm to 280 nm absorbance) and a product yield of 82 % [w/w]. The purity of c‐phycocyanin and b‐phycoerythrin from the crude extract increased 3‐ and 4‐fold, respectively, after ATPS. The results reported herein demonstrated the benefits of the practical generic application of ATPS for the primary recovery of colored proteins from microbial origin as a first step for the development of purification processes.     

Bioprocess intensification: a potential aqueous two-phase process for the primary recovery of B-phycoerythrin from Porphyridium cruentum

A process for the primary recovery of B-phycoerythrin from Porphyridium cruentum exploiting aqueous two-phase systems (ATPS) 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 poly(ethylene glycol) (PEG) molecular mass, concentration of PEG as well as salt, system pH and volume ratio was carried out to determine under which conditions the B-phycoerythrin and contaminants concentrate to opposite phases. PEG 1450-phosphate ATPS proved to be suitable for the recovery of B-phycoerythrin because the target protein concentrated to the top phase whilst the protein contaminants and cell debris concentrated in the bottom phase. An extraction ATPS stage comprising volume ratio (Vr) equal to 1.0, PEG 1450 24.9% (w/w), phosphate 12.6% (w/w) and system pH of 8.0 allowed B-phycoerythrin recovery with a purity of 2.9 (estimated as the relation of the 545-280 nm absorbances). The use of ATPS resulted in a primary recovery process that produced a protein purity of 2.9 +/- 0.2 and an overall product yield of 77.0% (w/w). The results reported demonstrated the practical implementation of ATPS for the design of a primary recovery process as a first step for the commercial purification of B-phycoerythrin produced by P. cruentum.     

Liquid-liquid extraction of a Schistosoma mansoni recombinant protein from an Escherichia coli extract in aqueous two-phase system

This work describes the partition of a Schistosoma mansoni tegumental antigen produced by a recombinant Escherichia coli strain using an aqueous two-phase system composed of polyethylene glycol (PEG) and potassium phosphate. The effects of the polymer molecular weight, tie line length and pH on antigen partitioning were investigated. The detection of the antigen in both phases were determined by ELISA. The system composed of PEG 3550 (19.7% w/w) and potassium phosphate (17.7% w/w) led to a yield of 59% and an antigen purification factor of 3 in the PEG-rich phase. It was observed that the antigen partition in ATPS was strongly affected by the pH value and tie line length. In addition, it was possible in a single step, to remove the cell debris, that precipitated at the interface of the system.     

ATPS applied to extraction of small molecules - polycetides - and simultaneous clarification of culture media with filamentous microorganisms

Aqueous two-phase systems (ATPS) were applied for extraction of small molecules (polycetides) - retamycin, an anthracyclin, and two red pigments, rubropunctamin and monascorubramin - from the whole culture media of Streptomyces olindensis and Monascus purpureus. ATPS allows, in one step, the separation of the small hydrophobic molecules in the PEG rich phase, from the filamentous microorganisms, which remains in the salt phase. Through experimental designs, the main variables and their levels were defined, as follows: for retamycin extraction, PEG 6000 (10%, w/w), phosphate at 20% (w/w) and pH 6.0 led to the higher partition coefficient, K(r) = 8.2, and yield = 91.3%; for red pigments, the statistical analysis indicate PEG 6000 (20%, w/w) and phosphate at 15% (w/w), for a high partition coefficient, (K(pig) = 113 and 150).     

Rotavirus-like particles primary recovery from insect cells in aqueous two-phase systems

Virus-like particles have a wide range of applications, including vaccination, gene therapy, and even as nanomaterials. Their successful utilization depends on the availability of selective and scalable methods of product recovery and purification that integrate effectively with upstream operations. In this work, a strategy based on aqueous two phase system (ATPS) was developed for the recovery of double-layered rotavirus-like particles (dlRLP) produced by the insect cell-baculovirus expression system. Polyethylene glycol (PEG) molecular mass, PEG and salt concentrations, and volume ratio (Vr, volume of top phase/volume of bottom phase) were evaluated in order to determine the conditions where dlRLP and contaminants concentrated to opposite phases. Two-stage ATPS consisting of PEG 400-phosphate with a Vr of 13.0 and a tie-line length (TLL) of 35% (w/w) at pH 7.0 provided the best conditions for processing highly concentrated crude extract from disrupted cells (dlRLP concentration of 5 microg/mL). In such conditions intracellular dlRLP accumulated in the top phase (recovery of 90%), whereas cell debris remained in the interface. Furthermore, dlRLP from culture supernatants accumulated preferentially in the interface (recovery of 82%) using ATPS with a Vr of 1.0, pH of 7.0, PEG 3350 (10.1%, w/w) and phosphate (10.9%, w/w). The purity of dlRLP from culture supernatant increased up to 55 times after ATPS. The use of ATPS resulted in a recovery process that produced dlRLP with a purity between 6 and 11% and an overall product yield of 85% (w/w), considering purification from intracellular and extracellular dlRLP. Overall, the strategy proposed in this study is simpler than traditional methods for recovering dlRLP, and represents a scalable and economically viable alternative for production processes of vaccines against rotavirus infection with significant scope for generic commercial application.     

Correlation for the partition behavior of proteins in aqueous two-phase systems: effect of surface hydrophobicity and charge

Correlations to describe the effect of surface hydrophobicity and charge of proteins with their partition coefficient in aqueous two-phase systems were investigated. Polyethylene glycol (PEG) 4000/phosphate, sulfate, citrate, and dextran systems in the presence of low (0.6% w/w) and high (8.8% w/w) levels of NaCl were selected for a systematic study of 12 proteins. The surface hydrophobicity of the proteins was measured by ammonium sulfate precipitation as the inverse of their solubility. The hydrophobicity values measured correlated well with the partition coefficients, K, obtained in the PEG/salt systems at high concentration of NaCl (r = 0.92-0.93). In PEG/citrate systems the partition coefficient correlated well with protein hydrophobicity at low and high concentrations of NaCl (r = 0.81 and 0.93, respectively). The PEG/citrate system also had a higher hydrophobic resolution than other systems to exploit differences in the protein's hydrophobicity. The surface charge and charge density of the proteins was determined over a range of pH (3-9) by electrophoretic titration curves; PEG/salt systems did not discriminate well between proteins of different charge or charge density. In the absence of NaCl, K decreased slightly with increased positive charge. At high NaCl concentration, K increased as a function of positive charge. This suggested that the PEG-rich top phase became more negative as the concentration of NaCl in the systems increased and, therefore, attracted the positively charged proteins. The effect of charge was more important in PEG/dextran systems at low concentrations of NaCl. In the PEG/dextran systems at lower concentration of NaCl, molecular weight appeared to be the prime determinant of partition, whereas no clear effect of molecular weight could be found in PEG/salt systems.     

Optimization of conditions for acid protease partitioning and purification in aqueous two-phase systems using response surface methodology

Aspergillopepsin I, an acid protease, was purified using an aqueous two-phase system that comprised various combinations of polyethylene glycol (PEG), NaH₂PO₄ and NaCl. Partition of the enzyme depended upon the molecular mass of the PEG and the presence of NaCl. With PEG 1500, 4000 and 6000, the partition coefficients were increased by 1,500-, 1,800- and 560-fold compared to values without NaCl. The presence of NaCl (8.75%, w/w) increased purification by 3.8, 9.5 and 2.8 times into these respective PEGs. The optimal aqueous two-phase system for acid protease purification was developed using response surface methodology. This system contained 17.3% of PEG 4000 (w/w), 15% NaH₂PO₄ (w/w) and 8.75% NaCl (w/w) and provided the best partition coefficient (Ke > 1,100) and yield over 99% in the same phase. The optimal ATPS purification factor of acid protease was over 5.     

Potential application of aqueous two‐phase systems and three‐phase partitioning for the recovery of superoxide dismutase from a clarified homogenate of Kluyveromyces marxianus

Superoxide dismutase (SOD; EC 1.15.1.1) is an antioxidant enzyme that represents the primary cellular defense against superoxide radicals and has interesting applications in the medical and cosmetic industries. In the present work, the partition behavior of SOD in aqueous two‐phase systems (ATPS) (using a standard solution and a complex extract from Kluyveromyces marxianus as sample) was characterized on different types of ATPS (polymer–polymer, polymer–salt, alcohol–salt, and ionic liquid (IL)–salt). The systems composed of PEG 3350‐potassium phosphate, 45% TLL, 0.5 M NaCl (315 U/mg, 87% recovery, and 15.1‐fold purification) and t‐butanol‐20% ammonium sulfate (205.8 U/mg, 80% recovery and 9.8‐fold purification), coupled with a subsequent 100 kDa ultrafiltration stage, allowed the design of a prototype process for the recovery and partial purification of the product of interest. The findings reported herein demonstrate the potential of PEG‐salt ATPS for the potential recovery of SOD. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1326–1334, 2014