Yeast RNA partition accompanying adsorption in salt/PEG two-phase system

Summary Partitioning of yeast total RNA in a salt/PEG two-phase system, i.e., a potassium phosphate/PEG system and a ammonium sulfate/PEG system, was characterized with regard to the dependence on the molecular weights of PEG and RNA. The shift in RNA partitioning was investigated for a PEG molecular weight range from 300 to 20000. RNA was partitioned mainly to the top phase in the system with PEG of a molecular weight up to 1000, mainly at the interface or almost equally to both phases in the system with PEG of a molecular weight 1000–2000, and mainly to the bottom phase in the system with PEG of more than 2000 in a molecular weight . The effect of PEG molecular weight on partitioning of low molecular weight RNA, less than 5.8S molecule, was qualitatively similar to that of high molecular weight RNA, more than 17S molecule. However, partitioning of high molecular weight RNA was more one-sided than that of low molecular weight RNA. In the system with PEG1000–2000, remarkable adsorption of high molecular weight RNA at the interface was investigated; more than 90% of the high molecular weight RNA added was concentrated. Adsorption of RNA at the interface was quantitatively demonstrated as a novel example of adsorption of a soluble macromolecule in an aqueous two-phase system.     

Role of polymer–protein interaction on partitioning pattern of bovine pancreatic trypsinogen and alpha-chymotrypsinogen in polyethyleneglycol/sodium tartrate aqueous two-phase systems

The partitioning pattern of bovine trypsinogen (TRPz) and alpha-chymotrypsinogen (ChTRPz) was investigated in a low impact aqueous two-phase system formed by polyethyleneglycol (PEG) and sodium tartrate (NaTart) pH 5.00. ChTRPz exhibited higher partition coefficients than TRPz did in all the assayed systems. The decrease in PEG molecular weight and the increase in tie line length were observed to displace the partitioning equilibrium of both proteins to the top phase, while phase volume ratios in the range 0.5–1.5 showed not to affect protein partitioning behaviour. Systems formed by PEG of molecular weight 600 with composition corresponding to a high tie line length (PEG 12.93%, w/w and NaTart 21.20%, w/w) are able to recover most of both zymogens in the polymer-enriched phase. A crucial role of PEG–protein interaction in the partitioning mechanism was evidenced by isothermal calorimetric titrations. The major content of highly exposed tryptophan rests, present in ChTRPz molecule, could be considered to be determinant of its higher partition coefficient due to a selective charge transfer interaction with PEG molecule. A satisfactory correlation between partition coefficient and protein surface hydrophobicity was observed in systems formed with PEGs of molecular weight above 4000, this finding being relevant in the design of an extraction process employing aqueous two-phase systems.     

PEG/NaPA aqueous two-phase systems for the purification of proteases expressed by Penicillium restrictum from Brazilian Savanna

The partitioning of proteases expressed by Penicillium restrictum from Brazilian Savanna in an inexpensive aqueous two-phase system composed of poly (ethylene glycol) (PEG) and sodium polyacrylate (NaPA) was studied. The effects of PEG molecular weight and concentration, as well as NaPA concentration and the concentration of fermented broth on protease partitioning were studied. Partitioning into the top PEG-rich phase was increased in systems with smaller PEG-molecular weight, higher NaPA concentration and lower PEG concentration. For most systems studied, purification has been achieved by directing the biomolecule partition to the opposite phase of the other proteins, providing the enzyme purification. The highest partition coefficient was obtained using 20 wt% NaPA, 4 wt% PEG 2000 g mol⁻¹ and 45 wt% fermented broth, leading to a purification factor of 1.98 and partition coefficient of 37.73. The system showed high mass balances and yield, indicating enzyme stability and applicability for industrial processes. The partitioning results using the PEG/NaPA/NaCl system show that this method could be used to purify or concentrate protease from fermented broth.     

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.     

牛血清白蛋白在聚乙二醇/葡聚糖双水相体系中分配特性的研究

采用考马斯亮蓝G250染色法测得室温下BSA在PEG/dextran双水相体系中的分配系数。以BSA在PEG/dextran体系的下相富集为目标,研究了PEG的分子量、浓度、dextran浓度以及所加入中性盐的种类与浓度、体系pH诸因素对其分配特性的影响。实验结果表明,在PEG4000/dextran体系中,采用PEG质量分数9%-dextran质量分数9%的浓度组成,同时在pH=7.0,NaC l浓度为0.2 mol.L-1或pH6.0,NaC l浓度为0.34 mol.L-1的工艺条件下萃取BSA均可达最小分配系数,其值为0.014。     

Synthesis and application of a poly(ethylene glycol)-antibody affinity ligand for cell separations in aqueous polymer two-phase systems

The possibility of producing biospecific affinity ligands for separating cells in two polymer aqueous phase systems on the basis of cell surface antigens was investigated. Rabbit anti-human erythrocyte IgG was reacted with cyanuric chloride-activated monomethyl poly(ethylene glycol) (PEG) fractions (molecular weights approximately 200, 1900, and 5000) at various molar ratios of PEG to protein lysine groups. The partition coefficient of the protein in a Dextran/PEG two-phase system increased with increasing degree of modification and increasing PEG molecular weight. There was a concomitant loss in ability to agglutinate human erythrocytes. The ability of the modified IgG to bind to a DEAE-cellulose column was almost eliminated by reaction with the PEG 5000, and was decreased to a lesser extent by PEG 1900. This PEG 1900-modified IgG substantially increased the partition of fresh or fixed human erythrocytes into the PEG-rich phase of a suitable phase system, while having no effect on rabbit cell partition. The partition increase could be inhibited by unmodified anti-human red cell IgG but not by nonspecific unmodified human IgG, demonstrating that the ligand effects were specific for the cell type against which the antibody was raised. A mixture of rabbit and human erythrocytes, which ordinarily have very similar partitions in the phase systems used, could be separated on a countercurrent distribution apparatus using the modified IgG. These results demonstrate the feasibility of producing immunologically specific affinity partition ligands for cell separation.     

Partitioning features of bovine trypsin and alpha-chymotrypsin in polyethyleneglycol-sodium citrate aqueous two-phase systems

The partitioning of bovine trypsin and alpha-chymotrypsin--proteases of similar physico-chemical properties--in different polyethyleneglycol/sodium citrate aqueous two-phase systems was investigated. The effect of different factors such as polyethyleneglycol molecular weight, pH, tie line length, temperature and the presence of an inorganic salt on the protein partition coefficient were analysed. Both a decrease in PEG molecular weight and an increase in pH led to a higher partition coefficient for both enzymes. Aqueous two-phase systems formed by PEG of molecular weight 3350 and citrate pH 5.2 showed the best separation capability which was enhanced in presence of sodium chloride 3%. The transfer of both proteins to the top phase was associated with negative enthalpic and entropic changes.     

Partial purification of penicillin acylase from Escherichia coli in poly(ethylene glycol)–sodium citrate aqueous two-phase systems

Studies on the partition and purification of penicillin acylase from Escherichia coli osmotic shock extract were performed in poly(ethylene glycol)–sodium citrate systems. Partition coefficient behavior of the enzyme and total protein are similar to those described in other reports, increasing with pH and tie line length and decreasing with PEG molecular weight. However, some selectivity could be attained with PEG 1000 systems and long tie line at pH 6.9. Under these conditions 2.6-fold purification with 83% yield were achieved. Influence of pH on partition shows that is the composition of the system and not the net charge of the enzyme that determines the behaviour in these conditions. Addition of NaCl to PEG 3350 systems significantly increases the partition of the enzyme. Although protein partition also increased, purification conditions were possible with 1.5 M NaCl where 5.7-fold purification and 85% yield was obtained. This was possible due to the higher hydrophobicity of the enzyme compared to that of most contaminants proteins.     

Simultaneous biosynthesis and purification of two extracellular Bacillus hydrolases in aqueous two-phase systems

Thermostable a-amylase with temperature optimum at 80 °C, molecular mass 58 kDa and pI point 6.9 was purified from a catabolite resistant Bacillus licheniformis strain. The enzyme was sensitive to inhibition by metal ions and N-bromosuccinimide. The partition behaviour of this enzyme in aqueous two-phase systems (ATPS) of the polymer-polymer-water type was investigated and some effects of type, molecular weight and concentration of phase components were studied. Up to 100% retention in the bottom phase of polyethylene glycol 10,000—20,000/dextran 200 system was reached. Best partition conditions were obtained in PEG 10,000—20,000/polyvinyl alcohol 200 systems, where the partition coefficient K increased 750 times to 7.5. Simultaneous production and purification of a-amylase and serine proteinase in PEG-polymer-water ATPS were examined. In the system PEG 6,000/ficoll, up to 90% of the amylase was retained in the bottom phase, whereas about 95% of the total protein (K = 22.8) and 60—75% of the proteinase were in the top phase. Similar separation of the enzymes from laboratory supernatant was obtained in system PEG/Na₂SO₄.     

Characteristics of an aqueous two-phase system for α-amylase production

The characteristics of an aqueous two-phase system for the overproduction of extracellular enzyme through α-amylase fermentation by Bacillus amyloliquefaciens were investigated. With higher molecular weight of polyethylene glycol (PEG) or lower molecular weight of dextran, the partition coefficient of α-amylase was increased. α-Amylase biosynthesis was increased when PEG 6000 was included in the medium compared to the medium without PEG. Phosphate addition to the PEG-dextran system improved the partition coefficient of α-amylase, but deactivated α-amylase severely. By using sodium sulfate instead of phosphate, α-amylase deactivation was negligible, and high partitioning of the enzyme in the top phase was obtained.     

Fusarium solani pisi recombinant cutinase partitioning in peg/potassium phosphate aqueous two-phase systems

Summary An aqueous two-phase system of polyethylene glycol (PEG) and potassium phosphate was developed for extraction of a cutinase from cell debris of a recombinant Escherichia coli strain. Basic studies to identify the primary factors which affect cutinase partition, namely the influence of polymer molecular weight, polymer concentration and pH were carried out using a purified preparation of the cutinase. The enzyme partition coefficient was enhanced with decreasing PEG molecular weight, increasing tie-line length and pH.     

Simultaneous biosynthesis and purification of two extracellular Bacillus hydrolases in aqueous two-phase systems

Thermostable alpha-amylase with temperature optimum at 80 degrees C, molecular mass 58 kDa and pI point 6.9 was purified from a catabolite resistant Bacillus licheniformis strain. The enzyme was sensitive to inhibition by metal ions and N-bromosuccinimide. The partition behaviour of this enzyme in aqueous two-phase systems (ATPS) of the polymer-polymer-water type was investigated and some effects of type, molecular weight and concentration of phase components were studied. Up to 100% retention in the bottom phase of polyethylene glycol 10,000-20,000/dextran 200 system was reached. Best partition conditions were obtained in PEG 10,000-20,000/polyvinyl alcohol 200 systems, where the partition coefficient K increased 750 times to 7.5. Simultaneous production and purification of alpha-amylase and serine proteinase in PEG-polymer-water ATPS were examined. In the system PEG 6,000/ficoll, up to 90% of the amylase was retained in the bottom phase, whereas about 95% of the total protein (K = 22.8) and 60-75% of the proteinase were in the top phase. Similar separation of the enzymes from laboratory supernatant was obtained in system PEG/Na2SO4.     

Protein partitioning at the isoelectric point: influence of polymer molecular weight and concentration and protein size

We report the partition coefficient, K(p') at the isoelectric point of lysozyme, chymotrypsinogen A, albumin, transferrin, and catalase in 64 different polyethylene(PEG)/ dextran(Dx)/water systems. We study the trends of the partition coefficient with protein type, polymer concentration, and polymer molecular weight. We find that the partition coefficient decreases with increasing tie line length for lysozyme, albumin, transferrin, and catalase for which K(p) is less than 1, but increases for chymotrysinogen for which K(p) is larger than 1. The effect of the tie line length on the partition coefficient is larger for the large proteins than for the small proteins. The partition coefficient decreases with increasing protein molecular weight except for lysozyme suggesting that lysozyme is present as a dimer or a trimer. The partition coefficient decreases with increasing PEG molecular weight, but the magnitude of the increase is larger for the smaller PEG molecular eights and tends to level of at high PEG molecular weight. The partition coefficient increases with increasing dextran (Dx) molecular weight for chymotrypsinogen but decreases for catalase. The partition coefficients of lysozyme, albumin, and transferrin increase with increasing Dx molecular weight from Dx 10(4) to Dx 1.1 x 10(5) and then slightly decrease from Dx 1.1 x 10(5) to Dx 5 x 10(5). The experimental results are analyzed using a statistical thermodynamics model. The experimental results are analyzed using a statistical thermodynamics model. The experiments suggest that protein partitioning at the isoelectric point in aqueous two-phase systems is strongly related to the size of the proteins and polymers. Finally, the impossibility of obtaining data completely independent of polymer concentration is emphasized.     

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.     

Recovery of antibiotics by aqueous two-phase partition —Partitioning behavior of pure acetylspiramycin solution in polyethylene glycol/potassium phosphate aqueous two-phase systems

Summary The effects of average molecular weight of PEG, concentrations of PEG and KH₂PO₄ and pH on the partition equilibrium of acetylspiramycin in PEG/KH₂PO₄ aqueous two-phase systems were studied in detail. The partition coefficients of acetylspiramycin in PEG/ KH₂PO₄ systems were measured at room temperature 25 °C. It was found that acetylspiramycin partitioned unevenly in the aqueous two-phase systems composed of PEG and KH₂PO₄ and could be purified by this technique. A suitable phase-forming system (pH=6.7, 12w/w% PEG2000, 11w/w% KH₂PO₄) was found out after partition coefficient (Kp=42) , extraction ratio (=96%) and recovery ratio(R=98.8%) were investigated comprehensively in this paper.Hua qiang is one of the cooperators of the experimetal.     

Enzymatic hydrolysis of soluble starch in a polyethylene glycol-dextran aqueous two-phase system

Hydrolysis of soluble starch by glucoamylase and β-amylase was investigated as a model reaction in an aqueous two-phase system consisting of polyethylene glycol (PEG) and dextran (DEX). Changes in glucose concentration observed in the batch reaction experiments with glucoamylase were almost identical for the aqueous two-phase and pure water systems, showing that the enzymic reactions investigated were not influenced by the presence of PEG and DEX. The partition of β-amylase into the DEX phase was insufficient compared to that of glucoamylase. Hence, the former enzyme was crosslinked with glutaraldehyde to increase its apparent molecular weight and, as a consequence, the partition coefficient, defined as the concentration ratio of the component partitioned into the PEG phase to that into the DEX phase, was decreased to 17% of that of the original enzyme. In the operation in which the enzyme and substrate are partitioned selectively into the DEX phase and allowed to react there while the product, thus transferring to the PEG phase, is recovered, the aqueous two-phase system with a smaller partition coefficient provided longer operational stability.     

Partitioning of recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) from plant cell suspension culture in PEG/sodium phosphate aqueous two-phase systems

Partitioning of human granulocyte-macrophage colony stimulating factor (hGM-CSF) was achieved in the aqueous two-phase systems (ATPSs) using a crude extract of transgenic tobacco cell suspension culture. This study examined the effects of polyethylene glycol (PEG) molecular weight and concentration and the effects of sodium phosphate concentration in different PEG/sodium phosphate systems on the partition coefficient,K. The best ATPS system was 5% PEG 8,000/1.6 M sodium phosphate after 2 h of incubation at room temperature. In this system, hGM-CSF was partitioned in the PEG-rich phase with a yield of 57.99% andK _hGM-CSF of 8.12. In another system, 3% PEG 10,000/1.6 M sodium phosphate, hGM-CSF was also partitioned primarily in the top phase with a yield of 45.66% andK _hGM-CSF of 7.64 after 2 h of incubation at room temperature.     

Protein sorption and recovery by hydrogels using principles of aqueous two-phase extraction

Use of the thermodynamic principles of aqueous two-phase extraction (ATPE) to drive protein into a crosslinked gel is developed as a protein isolation and separation technique, and as a protein loading technique for drug delivery applications. A PEG/dextran gel system was chosen as a model system because PEG/dextran systems are widely used in aqueous two-phase extraction and dextran gels (Sephadex(R)) are common chromatographic media. The effects of polymer concentrations and molecular weights, salts, and pH on the partitioning of ovalbumin matched ATPE heuristics and data trends. Gel partition coefficients (Cgel/Csolution) increased with increasing PEG molecular weight and concentration and decreasing dextran concentration (increased gel swelling). The addition of PEG to the buffer solution yielded partition coefficients more than an order of magnitude greater than those obtained in systems with buffer alone, or added salt. A combined salt/PEG system yielded an additional order of magnitude increase. For example, when ovalbumin solution (2.3 mg/mL) was equilibrated with Sephadex(R) G-50 at pH 6.75, the partition coefficients were 0.13 in buffer, 0.11 in buffer with 0.22M KI, 2.3 in 12 wt% PEG-10,000 and 32.0 in 12 wt% PEG-10, 000 with 0.22M KI. The effect of anions and cations as well as ionic strength and pH on the partitioning of ovalbumin also matched ATPE heuristics. Using the heuristics established above, partition coefficients as high as 80 for bovine serum albumin and protein recoveries over 90% were achieved. In addition, the wide range of partition coefficients that were obtained for different proteins suggests the potential of the technique for separating proteins. Also, ovalbumin sorption capacities in dextran were as high as 450 mg/g dry polymer, and the sorption isotherms were linear over a broad protein concentration range.     

Cutinase purification on poly(ethylene glycol)–hydroxypropyl starch aqueous two-phase systems

The partition behaviour of cutinase on poly(ethylene glycol) (PEG)–hydroxypropyl starch aqueous two-phase systems was characterized. The effect of molecular mass of PEG, the pH of the system and tie-line length on cutinase partition coefficient and cutinase yield to the top phase was investigated for systems prepared with a purified hydroxypropyl starch (Reppal PES 100) and a crude one (HPS). The effect of the presence of different salts, such as sodium chloride, sodium sulphate and ammonium sulphate, on cutinase partition was also studied. The results lead to the conclusion that aqueous two-phase systems composed of PEG and hydroxypropyl starch are not efficient in the purification of cutinase. In the majority of cases, the partition coefficients were very close to 1, with pH being the factor which affects most cutinase partition. Partition coefficients were significantly improved when salts were added to the systems. For PEG 4000–Reppal PES 100 [at pH 4.0; 0.5 M (NH₄)₂SO₄], the partition coefficient for cutinase was 3.7, while a value of 12 was obtained for PEG 4000–HPS (at pH 4.0; 1 M NaCl). An isoelectric point (pI) of 7.8 was confirmed for cutinase by constructing a cross partition graphic from the results obtained in the experiments with different salts.     

Enzymatic hydrolysis of cellulose in aqueous two-phase systems. I. Partition of cellulases from Trichoderma reesei

The partitioning of endo-beta-glucanase, exo-beta-glucanase, and beta-glucosidase from Trichoderma reesei QM 9414 in aqueous two-phase systems has been studied with the object of designing a phase system for continuous bioconversion of cellulose. The partitioning of the enzymes in two-phase systems composed of various water soluble polymeric compounds were studied. Systems based on dextran and polyethylene glycol (PEG) were optimal for one-sidedly partitioning the enzymes to the bottom phase. The influence of polymer molecular weights, polymer concentration, ionic composition of the medium, pH, temperature, and adsorption of the enzymes to cellulose on the enzyme partition coefficients (K) were studied. By combining the effects of polymer molecular weight and adsorption to cellulose, K values could be reduced for endo-beta-glucanase to 0.02 and for beta-glucosidase to 0.005 at 20 degrees C in a phase system of Dextran 40-PEG 40000 in the presence of excess cellulose, At 50 degrees C, K values were increased by a factor of two. In a phase system based on inexpensive crude dextran and PEG, the partition coefficient for endo-beta-glucanase was 0.16 and for beta-glucosidase was 0.14 at 20 degrees C with excess cellulose present.