Production of cyclodextrin homologues using aqueous two-phase system

Cyclodextrin homologues (CDs), produced by cyclodextrin glycosyltransferase (CGTase), were simultaneously partitioned in aqueous two-phase system (ATPS). Partition coefficients of CDs were measured in PEG/salt and PEG/dextran systems. Phosphate, citrate, sulfate were tested as salt. ATPS of PEG/salt and PEG/dextran had the partition coefficients of the CDs, larger than unity. However, PEG/dextran system was observed better than PEG/salt as CGTase activity decreased sharply with salt concentration. Enzymatic reaction occurred mainly in PEG-rich bottom phase because of the low partition coefficient of CGTase. The resulting CDs transferred to the PEG-rich top phase, obeying the diffusional partition. In the ATPS of 7% PEG (M.W. 20,000) and 9% dextran (M.W. 40,000), 7 mg/ml of CDs were obtained in top phase at 4.5 hours.     

Effect of salt additives on protein partition in polyethylene glycol–sodium sulfate aqueous two-phase systems

Partitioning of 15 proteins in polyethylene glycol (PEG)–sodium sulfate aqueous two-phase systems (ATPS) formed by PEG of two different molecular weights, PEG-600 and PEG-8000 in the presence of different buffers at pH 7.4 was studied. The effect of two salt additives (NaCl and NaSCN) on the protein partition behavior was examined. The salt effects on protein partitioning were analyzed by using the Collander solvent regression relationship between the proteins partition coefficients in ATPS with and without salt additives. The results obtained show that the concentration of buffer as well as the presence and concentration of salt additives affects the protein partition behavior. Analysis of ATPS in terms of the differences between the relative hydrophobicity and electrostatic properties of the phases does not explain the protein partition behavior. The differences between protein partitioning in PEG-600–salt and PEG-8000–salt ATPS cannot be explained by the protein size or polymer excluded volume effect. It is suggested that the protein–ion and protein–solvent interactions in the phases of ATPS are primarily important for protein partitioning.     

Affinity separation by protein conjugated IgG in aqueous two-phase systems using horseradish peroxidase as a ligand carrier

A novel affinity separation method in an aqueous two-phase system (ATPS) is suggested, using protein conjugated IgG as a ligand. For verification of the proposed approach, horseradish peroxidase (HRP) and human IgG was used as a ligand carrier and affinity ligand, respectively. The partition of the affinity ligand, human IgG, was controlled by the conjugation of HRP. Two ATPSs, one consisting of potassium phosphate (15%, w/w) and polyethylene glycol (PEG, M.W. 1450, 10%, w/w) and the other of dextran T500 (5%, w/w) and PEG (M.W. 8000, 5%, w/w), were used. The conjugated human IgG-HRP favored a PEG-rich top phase, whereas human IgG, rabbit anti-human IgG and goat anti-mouse IgG preferred a salt or dextran-rich bottom phase. Using the conjugated human IgG-HRP, rabbit anti-human IgG was successfully separated into a PEG-rich top phase from the mixture with goat anti-mouse IgG. The appropriate molar ratio between human IgG-HRP and rabbit anti-human IgG was around 3:1 and 1:1 for the salt and dextran-based ATPS, respectively. The dextran-based ATPS showed a better recovery yield and purity than the salt-based ATPS for the range of test conditions employed in this experiment. The yield and purity of the recovered rabbit anti-human IgG were 90.8 and 87.7%, respectively, in the dextran-based ATPS, while those in the salt-based ATPS were 78.2 and 73.2%.     

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).     

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.     

Response surface methodology for the extraction of wedelolactone from Eclipta alba using aqueous two-phase extraction

Abstract

Aqueous two-phase extraction of wedelolactone from Eclipta alba was studied using the polymer-salt system. The system consisted of polyethylene glycol (PEG) as a top phase (polymer) and sodium citrate as a bottom phase (salt). Process parameters such as PEG concentration, PEG molecular weight, salt concentration, and pH have been optimized using response surface methodology (RSM) with the help of central composite design (CCD). The optimized conditions for aqueous two-phase system (ATPS), in the case of one factor at a time approach, were found as PEG 6000, PEG concentration 18% (w/v), salt concentration 16% (w/v), and pH 7; with maximum extraction yield of 6.52?mg/g. While, RSM studies showed maximum extraction yield of 6.73?mg/g with the optimized parameters as PEG 6000, PEG concentration 18% (w/v), salt concentration 17.96% (w/v), and pH 7. ATPS was found to give a 1.3 fold increase in the extraction yield of wedelolactone as compared to other conventional extraction methods.     

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.     

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.     

Isolation of plasmid DNA from cell lysates by aqueous two-phase systems

This work presents a study of the partitioning of a plasmid vector containing the cystic fibrosis gene in polyethylene glycol (PEG)/salt (K2HPO4) aqueous two-phase systems (ATPS). The plasmid was extracted from neutralized alkaline lysates using PEG with molecular weights varying from 200 to 8000. The effects of the lysate mass loaded to the ATPS (20, 40, and 60% w/w) and of the plasmid concentration in the lysate were evaluated. The performance of the process was determined by qualitative and quantitative assays, carefully established to overcome the strong interference of impurities (protein, genomic DNA, RNA), salt, and PEG. Plasmid DNA partitioned to the top phase when PEG molecular weight was lower than 400. The bottom phase was preferred when higher PEG molecular weights were used. Aqueous two-phase systems with PEG 300, 600, and 1000 were chosen for further studies on the basis of plasmid and RNA agarose gel analysis and protein quantitation. The recovery yields were found to be proportional to the plasmid concentration in the lysate. The best yields (>67%) were obtained with PEG 1000. These systems (with 40 and 60% w/w of lysate load) were able to separate the plasmid from proteins and genomic DNA, but copartitioning of RNA with the plasmid was observed. Aqueous two-phase systems with PEG 300 concentrated both plasmid and proteins in the top phase. The best system for plasmid purification used PEG 600 with a 40% (w/w) lysate load. In this system, RNA was found mostly in the interphase, proteins were not detected in the plasmid bottom phase and genomic DNA was reduced 7.5-fold.     

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.     

Potential of Aqueous Two-Phase Systems constructed on flexible devices: Human serum albumin as proof of concept

In the present research, the potential use of flexible disposable devices, specifically blood bags, for the fractionation of biological products using Aqueous Two-Phase Systems (ATPS) polymer–salt is studied and demonstrated. Purified human serum albumin (HSA) was used as model protein. Experiments were carried out on ATPS polyethylene glycol (PEG)–potassium phosphate constructed on rigid recipients (conical tubes) and flexible devices (blood bags). The device used for ATPS construction had no significant effect on HSA partition behavior. Protein partition towards the top phase was favored on systems constructed using PEG 1000 g/mol and TLL 45% (w/w), achieving up to 85% recovery. On the other hand a recovery of 92% was achieved at the bottom phase when PEG 3350 g/mol and TLL 25% (w/w) were used. Human serum was used as a complex sample on ATPS experiments. Selective fractionation of human serum proteins on ATPS constructed on flexible devices was achieved. ATPS constructed on blood bags required short equilibrium times (< 6 min), meaning it is feasible to use this approach on mass scale. The potential use of flexible disposable devices, for the fractionation of biological products using ATPS polymer–salt was demonstrated.     

Optimization of conditions for bacteriocin extraction in PEG/salt aqueous two-phase systems using statistical experimental designs

The bacteriocin nisin was extracted in PEG/salt aqueous two-phase systems (ATPS) using the property that the systems can extract hydrophobic proteins. The concentrations of the phase-forming components, PEG 4000 and Na(2)SO(4), were optimized for nisin recovery by means of statistical experimental designs, and it was found that they strongly influenced nisin recovery. The optimal composition of ATPS was found to be 15.99% (w/w) PEG 4000 and 15.85% (w/w) Na(2)SO(4) (pH 2), and the optimal ATPS allowed an 11.60% increase of nisin recovery compared to the standard method of nisin assay.     

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     

Distribution of cells between solid/liquid and liquid/liquid interfaces

The use of aqueous two-phase systems (ATPSs) and each system's individual phase-forming species to prevent Streptococcus sanguis attachment onto hydroxyapatite discs was explored. The strategy that we followed was to attach the cells to a solid surface in the presence of an additional interface. Conditions under which, simultaneously, the phase-forming species form two phases and the cells proliferate were identified. Growth curves were constructed in the presence of various polymers and salts commonly used to prepare ATPSs. Several aqueous two-phase systems were selected such that bacterial growth was comparable to that observed in pure medium. Cells were allowed to attach to hydroxyapatite discs for 7 days in the presence of varying concentrations of media, media with polymer, media with salt, and media with ATPS. Streptococcus sanguis attachment to the disks was evaluated by scanning electron microscopy. The addition of a PEG/Na(2)SO(4) ATPS to high concentrations of yeast-tryptone (YT) media (>65%) and of a PEG/MgSO(4) ATPS to nutrient-limited media reduces surface coverage of S. sanguis to less than 10%. Comparison of the attachment levels for the systems containing PEG/Na(2)SO(4) to media containing the individual phase-forming species and to the YT reference systems indicated that nutrient availability did not affect attachment.     

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.     

The potential application of aqueous two-phase systems for in situ recovery of 6-pentyl-infinity-pyrone produced by Trichoderma harzianum

Commercial production of aroma compounds by de novo microbial biosynthesis has been principally limited by the low productivity so far achieved. Production of 6-pentyl-alpha-pyrone (6PP), a coconut-like aroma compound, by Trichoderma harzianum has been limited by the toxic effect that occurs even at low concentration (<100 ppm). This work evaluated the feasibility of the use of aqueous-two phase systems (ATPS), as in situ extraction systems, in order to overcome the toxic effects of 6PP and to improve culture productivity. The partition behaviour of 6-pentyl-alpha-pyrone and Trichoderma harzianum mycelium in polyethylene glycol (PEG)-salt and PEG-dextran two-phase systems was investigated and it is reported for the first time. The evaluation of system parameters such as PEG molecular mass, concentration of PEG as well as salt, volume ratio (Vr) and dextran molecular mass, was carried out to determine under which conditions the 6PP partitions to the opposite phase that mycelium does. PEG-dextran systems proved to be unsuitable for the in situ recovery of 6PP because either 6PP and biomass partitioned to the same phase or a large extraction phase was required for the process. ATPS extraction comprising Vr = 0.26, PEG 1450 (7.2% w/w) and sulphate (16.6% w/w) provided the best conditions for the maximum accumulation of the biomass into the bottom phase and concentrated the 6PP in the opposite phase (i.e. 86% of biomass and 56% of 6PP of the total amount loaded from the fermentation extract into the ATPS) for ex situ bioseparation. However, this system caused complete inhibition of the growth of the microorganism during the in situ bioseparation, probably as a consequence of the high ionic strength resulting from the salt concentration. Consequently, two ATPS PEG 8000-sulphate (12%/7% and 6%/14%) were evaluated and proved to be more suitable in the potential application for the in situ recovery of 6PP.     

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).     

Partial purification of α-amylase from culture supernatant of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in aqueous two-phase systems

A study was made of the partition and purification of -amylase from a culture supernatant of Bacillus subtilis in the polyethylene glycol (PEG)—citrate aqueous two-phase system (ATPS). Factors that influenced the partition of the protein in this system, including the molecular weight of the PEG, the tie line length of ATPS, the pH value and the sodium chloride concentration, were investigated. Purification of -amylase was attained with a purification factor (PF) of 1.8 and 90% yield at pH 6.0 in a PEG1000-citrate ATPS with short tie line length. By utilizing the salt-out effect of neutral salt, the purification of -amylase was further improved to 2.0 of PF and 80% yield in a PEG3350-citrate ATPS with 4% sodium chloride.     

Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems.

The effectiveness of thermoseparating polymer-based aqueous two-phase systems (ATPS) in the enzymatic hydrolysis of starch was investigated. In this work, the phase diagrams of PEO-PPO-2500/ammonium sulfate and PEO-PPO-2500/magnesium sulfate systems were determined at 25 degrees C. The partition behavior of pure alpha-amylase and amyloglucosidase in four ATPS, namely, PEO-PPO/(NH(4))(2)SO(4), PEO-PPO/MgSO(4), polyethylene glycol (PEG)/(NH(4))(2)SO(4), and PEG/MgSO(4), was evaluated. The effects of phase-forming component concentrations on the enzyme activity and partitioning were assessed. Partitioning of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii was also investigated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. The PEO-PPO-2500/MgSO(4) system was extremely attractive for starch hydrolysis. Polymer-based starch hydrolysis experiments containing PEO-PPO-2500/MgSO(4) indicated that the use of ATPS had a significant effect on soluble starch hydrolysis. Batch starch hydrolysis experiments with PEO-PPO/salt two-phase systems resulted in higher production of maltose or glucose and exhibited remarkably faster hydrolysis. A 22% gain in maltose yield was obtained as a result of the increased productivity. This work is the first reported application of thermoseparating polymer ATPS in the processing of starches. These results reveal the potential for thermoseparating polymer-enhanced extractive bioconversion of starch as a practical technology.     

PEG–salt aqueous two-phase systems: an attractive and versatile liquid–liquid extraction technology for the downstream processing of proteins and enzymes

Nowadays, there is an increasing demand to establish new feasible, efficient downstream processing (DSP) techniques in biotechnology and related fields. Although several conventional DSP technologies have been widely employed, they are usually expensive and time-consuming and often provide only low recovery yields. Hence, the DSP is one major bottleneck for the commercialization of biological products. In this context, polyethylene glycol (PEG)–salt aqueous two-phase systems (ATPS) represent a promising, efficient liquid–liquid extraction technology for the DSP of various biomolecules, such as proteins and enzymes. Furthermore, ATPS can overcome the limitations of traditional DSP techniques and have gained importance for applications in several fields of biotechnology due to versatile advantages over conventional DSP methods, such as biocompatibility, technical simplicity, and easy scale-up potential. In the present review, various practical applications of PEG–salt ATPS are presented to highlight their feasibility to operate as an attractive and versatile liquid–liquid extraction technology for the DSP of proteins and enzymes, thus facilitating the approach of new researchers to this technique. Thereby, single- and multi-stage extraction, several process integration methods, as well as large-scale extraction and purification of proteins regarding technical aspects, scale-up, recycling of process chemicals, and economic aspects are discussed.