Purification of lipase produced by a new source of Bacillus in submerged fermentation using an aqueous two-phase system

This work discusses the application of an aqueous two-phase system for the purification of lipases produced by Bacillus sp. ITP-001 using polyethylene glycol (PEG) and potassium phosphate. In the first step, the protein content was precipitated with ammonium sulphate (80% saturation). The enzyme remained in the aqueous solution and was dialyzed against ultra-pure water for 18 h and used to prepare an aqueous two-phase system (PEG/potassium phosphate). The use of different molecular weights of PEG to purify the lipase was investigated; the best purification factor (PF) was obtained using PEG 20,000g/mol, however PEG 8000 was used in the next tests due to lower viscosity. The influence of PEG and potassium phosphate concentrations on the enzyme purification was then studied: the highest FP was obtained with 20% of PEG and 18% of potassium phosphate. NaCl was added to increase the hydrophobicity between the phases, and also increased the purification factor. The pH value and temperature affected the enzyme partitioning, with the best purifying conditions achieved at pH 6.0 and 4°C. The molecular mass of the purified enzyme was determined to be approximately 54 kDa by SDS-PAGE. According to the results the best combination for purifying the enzyme is PEG 8000g/mol and potassium phosphate (20/18%) with 6% of NaCl at pH 6.0 and 4°C (201.53 fold). The partitioning process of lipase is governed by the entropy contribution.     

New, highly efficient formulation of diclofenac for the topical, transdermal administration in ultradeformable drug carriers, Transfersomes

Unmodified and polyethylene glycol (PEG) modified neutral and negatively charged liposomes were prepared by freeze-thaw and extrusion followed by chromatographic purification. The effects of PEG molecular weight (PEG 550, 2000, 5000), PEG loading (0-15 mol%), and liposome surface charge on fibrinogen adsorption were quantified using radiolabeling techniques. All adsorption isotherms increased monotonically over the concentration range 0-3 mg/ml and adsorption levels were low. Negatively charged liposomes adsorbed significantly more fibrinogen than neutral liposomes. PEG modification had no effect on fibrinogen adsorption to neutral liposomes. An inverse relationship was found between PEG loading of negatively charged liposomes and fibrinogen adsorption. PEGs of all three molecular weights at a loading of 5 mol% reduced fibrinogen adsorption to negatively charged liposomes. Protein adsorption from diluted plasma (10% normal strength) to four different liposome types (neutral, PEG-neutral, negatively charged, and PEG-negatively charged) was investigated using gel electrophoresis and immunoblotting. The profiles of adsorbed proteins were similar on all four liposome types, but distinctly different from the profile of plasma itself, indicating a partitioning effect of the lipid surfaces. alpha2-macroglobulin and fibronectin were significantly enriched on the liposomes whereas albumin, transferrin, and fibrinogen were depleted compared to plasma. Apolipoprotein AI was a major component of the adsorbed protein layers. The blot of complement protein C3 adsorbed on the liposomes suggested that the complement system was activated.     

Recovery of endo-polygalacturonase using polyethylene glycol-salt aqueous two-phase extraction with polymer recycling

The partitioning behaviour of endo-polygalacturonase (endo-PG) and total protein from a clarified Kluyveromyces marxianus fermentation broth in polyethylene glycol (PEG)-ammonium sulfate and PEG-potassium phosphate (pH=7) aqueous two-phase systems was experimentally investigated. Both the enzyme and total protein partitioned in the bottom phase for these two kinds of systems. The enzyme partitioning coefficient can be lower than 0.01 in PEG8000-(NH₄)₂SO₄ ATPS with a large phase volume ratio and a moderate tie-line length, which implies the possibility of concentration operation using aqueous two phase partitioning. An ion-exchange separation of high purification efficiency was applied to analyze the clarified and dialyzed fermentation broth. A total purification factor of only 2.3 was obtained, which indicated the high enzyme protein content in the total protein of the fermentation broth. Consequently, the main purpose for separating endo-PG is concentration rather than purification. A separation scheme using an aqueous two-phase extraction process with polymer recycling and a dialysis was proposed to recover endo-PG from the fermentation supernatant of K. marxianus for commercial purpose. A high enzyme recovery up to 95% and a concentration factor of 5 to 8 with a purification factor of about 1.25 were obtained using the single aqueous two-phase extraction process. More than 95% polymer recycled will not affect the enzyme recovery and purification factor. Dialysis was used mainly to remove salts in the bottom phase. The dialysis step has no enzyme loss and can further remove small bulk proteins. The total purification factor for the scheme is about 1.7.     

Isolation of alpha-1-antitrypsin from human plasma by partitioning in aqueous biphasic systems of polyethyleneglycol-phosphate

The partitioning of alpha-1-antitrypsin was assayed in biphasic aqueous systems containing potassium phosphate and two polyethyleneglycols of molecular mass 600 and 1000, respectively. In order to isolate the alpha-1-antitrypsin from serum plasma, the partitioning behaviour of human serum albumin, its principal contaminant, was also studied. Several aqueous two-phase systems with different partitioning properties were obtained by varying the PEG1000/PEG600 mass proportion. In systems with PEG1000/PEG600 mass ratio of 8, the optimal difference between the partition coefficients of both proteins was found. Under such conditions, a satisfactory purification was carried out by a three-step extraction procedure. By applying this method the alpha-1-antitrypsin specific activity increased severalfold (nearly 10 times) with a yield of 43%.     

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.     

Purity of spiking agent affects partitioning of prions in plasma protein purification.

Prions are not detectable in the blood or plasma of persons afflicted with classical or variant Creutzfeldt-Jakob disease, and they have never been shown to be transmitted by blood or plasma products. Despite the uncertainty as to the presence and biophysical properties of prions in plasma, prion removal studies have been conducted using brain homogenate or microsomes prepared from prion-infected rodent brains as model prions. In this study, we compare the partitioning of different prion spiking agents, having different biophysical properties, in the processes used for plasma protein purification. We have found that membrane-bound prion spiking agents partition similarly, whereas purified, unbound pathogenic prion proteins can have significantly different partitioning properties depending on the conditions in the production process. We conclude that prion spiking studies for the evaluation of prion reduction in plasma protein purification should employ spiking agents with different biophysical properties to mimic partitioning of the theoretical prion contaminant. This will give greater assurance as to the prion safety margins of the life-saving plasma protein therapeutics and excipients.     

Yeast cell debris and protein partitioning in the poly(ethylene glycol)-poly(vinyl alcohol) biphasic system

Yeast cells, cell debris and protein partitioning have been investigated in the poly(ethylene glycol) (PEG) 8000/poly(vinyl alcohol) (PVA) 10,000 system. Cells and cell debris partition into the lower (PVA) phase over the pH range 4.8-7.5, and with up to 0.37 M KCl at pH 5.9. Protein partitioning is more pH-dependent in the PEG/PVA system than in the PEG/dextran system, and a significant fraction of the total protein is found at the interface at lower pH values. Significant, rapid purification of overproduced pyruvate kinase in a PEG/PVA system containing Blue Sepharose CL-6B particles is demonstrated.     

An aqueous hydroxypropylstarch-poly(ethylene glycol) two-phase system for extraction of alpha-lactalbumin and beta-lactoglobulin from bovine whey.

The partitioning of alpha-lactalbumin and beta-lactoglobulin from bovine whey has been studied in an aqueous poly(ethylene glycol) (PEG)-hydroxypropylstarch two-phase system. The influence of several parameters including concentrations of polymers, sodium phosphate buffer, KSCN, and of PEG palmitate, with and without the presence of Ca2+, on the partitioning of the proteins has been investigated. The separation of the two proteins was demonstrated by counter-current distribution. A purification procedure for both proteins has been developed by using PEG-hydroxypropylstarch two-phase system. This system is compared with the more costly standard system based on PEG and dextran. The possible use of the aqueous two-phase systems for batch extraction for large scale purification of these whey proteins is discussed.     

Aqueous two-phase extraction for protein recovery from corn extracts

Corn has been used as an expression host for several recombinant proteins with potential for large-scale production. Cost-effective downstream initial recovery, separation and concentration remain a challenge. Aqueous two-phase (ATP) partitioning has been used to recover and concentrate proteins from fermentation broths and offers advantages for integration of those steps with biomass removal. To examine the applicability of ATP partitioning to recombinant protein purification from corn endosperm and germ, ATP system parameters including poly(ethylene glycol) (PEG) molecular weight (MW), phase-forming salt, tie line length (TLL), and pH were manipulated to control partitioning of extracted native proteins from each fraction. Moderate PEG MW, reduction of phase ratio, and added NaCl effected complete recovery of the hydrophobic model protein lysozyme in the top phase with ca. 5x enrichment and illustrates a favorable match of recombinant protein characteristics, expression host, and separation method. Furthermore, integration of protein extraction with the partitioning reduced the load of contaminating host proteins relative to the more traditional separate steps of extraction followed by partitioning. Performance of the integrated partitioning was hindered by endosperm solids loading, whereas for germ, which has ca. 35x higher aqueous soluble protein, the limit was protein solubility. For more hydrophilic model proteins (the model being cytochrome c), effective separation required further reduction of PEG MW to effect more partitioning of host proteins to the top phase and enrichment of the model protein in the lower phase. The combination of PEG MW of 1450 with 8.5 wt.% NaCl addition (Na(2)SO(4) as the phase-forming salt) provided for complete recovery of cytochrome c in the lower phase with enrichment of 9x (germ) and 5x (endosperm). As a result of lower-phase recovery, the advantage of simultaneous removal of solids is lost. The lower solubility of native endosperm proteins results in higher purity for the same enrichment.     

Purification of rat C6 glioma plasma membranes by affinity partitioning

We have studied the feasibility of purifying rat C6 glioma plasma membranes by a phase partitioning approach. The purification procedure involves cell homogenization and fractionation with an aqueous two-phase polymer system followed by selective affinity purification of plasma membranes by a wheat germ agglutinin-coupled polymer system. We demonstrate that the two-phase affinity partitioning technique is a simple and efficient method of isolating cell plasma membranes with high purity and yield. Furthermore, the isolated plasma membranes retain their functional integrity, as shown by the high-affinity insulin-like growth factor-I (IGF-I) binding capacity of IGF-I receptors.     

Production of immobilized penicillin acylase using aqueous polymer systems for enzyme purification and in situ immobilization

A novel approach for the isolation and purification of penicillin acylase (PA), which couples aqueous two-phase partitioning and enzyme immobilization has been investigated.A PA yield of 90% was achieved by treating E. coli cells with 4% butyl acetate, freeze-thawing step, and pressure homogenization. PA purification (93% recovery) was achieved by (1) removing cell debris via precipitation with polyethylene glycol (PEG 2000); (2) aqueous two-phase partitioning using a PEG 2000 + phosphate system (87% recovery).An in situ enzyme immobilization approach, using oxirane acrylic or aldehyde-agarose beads dispersed in the PEG-rich phase, was explored for the conversion of penicillin G to 6-aminopenicillanic acid. An appropriate immobilization reaction time was found. The catalytic performance of the enzyme, when immobilized, was found not to be affected by recycling of the phase-forming components.     

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.     

Aqueous two-phase partition and detergent precipitation of a drug-responsive NADH oxidase from the HeLa cell surface

The partitioning behaviour of a drug (capsaicin)-responsive NADH oxidase (tNOX) activity released from HeLa ceIls by low pH treatment followed by heat and proteinase K was determined. When partitioned in a standard 6.4% PEG 3350/6.4% dextran T-500 two-phase system, the bulk of the tNOX activity was in the dextran-rich lower phase. The activity was inhibited by and bound to the triazine dye, Cibacron blue. Affinity partition, where the Cibacron blue was coupled to amino PEG 5000 and added to the first two-phase separation step, resulted in the partitioning of activity to the upper PEG phase. A second partition with PEG-salts resulted in the release of the tNOX from the Cibacron blue–amino PEG enriched phase into the salt-enriched lower phase. The phase-purified protein exhibited anomalous behavior and tended to multimerize in sodium dodecyl sulphate (SDS) prior to SDS-polyacrylamide gel electrophoresis (PAGE). Multimerization appeared to be enhanced by PEG. The multimerization was enhanced with the reduced protein in the presence of detergent prior to SDS–PAGE. In addition, the activity was precipitated by PEG 8000 at concentrations between 6 and 30% by weight. In the presence of or after exposure to PEG 3350 or PEG 8000, the protein could not be detected by Western blot analysis after SDS–PAGE suggesting that the protein failed to enter the gel even though other HeLa cell surface proteins were unaffected. The anomalous multimerization behavior has thus far precluded the use of phase partition as a practical purification step for the oxidase.     

Thermoseparating water/polymer system: a novel one-polymer aqueous two-phase system for protein purification

In this study we show that proteins can be partitioned and separated in a novel aqueous two-phase system composed of only one polymer in water solution. This system represents an attractive alternative to traditional two-phase systems which uses either two polymers (e.g., PEG/dextran) or one polymer in high-salt concentration (e.g., PEG/salt). The polymer in the new system is a linear random copolymer composed of ethylene oxide and propylene oxide groups which has been hydrophobically modified with myristyl groups (C(14)H(29)) at both ends (HM-EOPO). This polymer thermoseparates in water, with a cloud point at 14 degrees C. The HM-EOPO polymer forms an aqueous two-phase system with a top phase composed of almost 100% water and a bottom phase composed of 5-9% HM-EOPO in water when separated at 17-30 degrees C. The copolymer is self-associating and forms micellar-like structures with a CMC at 12 microM (0.01%). The partitioning behavior of three proteins (lysozyme, bovine serum albumin, and apolipoprotein A-1) in water/HM-EOPO two-phase systems has been studied, as well as the effect of various ions, pH, and temperature on protein partitioning. The amphiphilic protein apolipoprotein A-1 was strongly partitioned to the HM-EOPO-rich phase within a broad-temperature range. The partitioning of hydrophobic proteins can be directed with addition of salt. Below the isoelectric point (pI) BSA was partitioned to the HM-EOPO-rich phase and above the pI to the water phase when NaClO(4)was added to the system. Lysozyme was directed to the HM-EOPO phase with NaClO(4), and to the water phase with Na-phosphate. The possibility to direct protein partitioning between water and copolymer phases shows that this system can be used for protein separations. This was tested on purification of apolipoprotein A-1 from human plasma and Escherichia coli extract. Apolipoprotein A-1 could be recovered in the HM-EOPO-rich phase and the majority of contaminating proteins in the water phase. By adding a new water/buffer phase at higher pH and with 100 mM NaClO(4), and raising the temperature for separation, the apolipoprotein A-1 could be back-extracted from the HM-EOPO phase into the new water phase. This novel system has a strong potential for use in biotechnical extractions as it uses only one polymer and can be operated at moderate temperatures and salt concentrations and furthermore, the copolymer can be recovered.     

Affinity extraction of lactate dehydrogenase by aqueous two-phase systems using free triazine dyes

Affinity partitioning of lactate dehydrogenase (LDH) was studied in polyethylene glycol (PEG) /salt and PEG / hydroxypropyl starch (PES) aqueous two-phase systems, using free triazine dyes as their affinity ligands. The free dyes showed one-sided partition to the top PEG-rich phase and thus enhanced the affinity partitioning effect in the systems. A two-step affinity extraction process has been discussed for large scale purification of LDH from rabbit muscle.Hu Lin is one of the cooperator of the experiment.     

Large scale extraction of alpha-lactalbumin and beta-lactoglobulin from bovine whey by precipitation with polyethylene glycol and partitioning in aqueous two-phase systems.

The milk proteins alpha-lactalbumin and beta-lactoglobulin have been isolated from bovine whey by fractional precipitation with polyethylene glycol (PEG) and hydrophobic partitioning in aqueous PEG-hydroxypropylstarch two-phase systems using PEG-bound palmitate as hydrophobic ligand. The possible use of this combination for large scale purification of these whey proteins is discussed.     

Enhanced metal-affinity partitioning of genetically engineered hirudin variants in polyethylene glycol/dextran two-phase systems

Hirudin variants were constructed to exhibit an increased metal-binding affinity in an attempt to apply a metal-affinity partitioning process in a primary separation step for purification of hirudin. The hirudin variants were genetically engineered to contain additional surface-accessible histidines and produced by recombinant Saccharomyces cerevisiae. The partitioning behavior of these variants was compared with that of the wild type with a single surface-accessible histidine at position 51. Upon the addition of a small amount of Cu(II)IDA-PEG (Cu(II)iminodiacetic acid-polyethylene glycol) ligand to PEG/dextran two-phase systems, the hirudin variants with two or three surface-accessible histidines were more selectively partitioned into the PEG-rich phase than the wild type. Integrating protein engineering to metal-affinity partitioning offers the potential for general application of this technique to facilitate protein isolation, but the genetically engineered protein variants should be carefully constructed in a manner to minimize reduction of native protein activity.     

Rapid purification of fructose-1,6-bisphosphate aldolase from spinach chloroplasts

A rapid procedure for the purification of fructose-1,6-bisphosphate aldolase from spinach chloroplasts is presented which involves two steps; precipitation of bulk protein with polyethylene glycol and partitioning of remaining soluble protein in aqueous two-phase systems. A 94% pure preparation is obtained within 6h with a yield of 19%. A marked difference in the partition behaviour of the aldolase activity from whole leaf tissue suggested that the procedure is less efficient when leaf extract is used as starting material.Abbreviations EDTA Ethylenediamine Tetraacetic Acid - PEG Polyethylene Glycol - SDS Sodium Dodecyl Sulfate     

Isolation of a Aspergillus niger lipase from a solid culture medium with aqueous two-phase systems

The aim of this work is to find the best conditions to isolate lipase from a solid culture medium of Aspergillus niger NRRL3 strains using aqueous two-phase systems formed with polyethylene glycol and potassium phosphate or polyethylene glycol and sodium citrate. We studied the partitioning of a commercial lyophilizate from A. niger. Also, the lipase enzymatic activity was studied in all the phases of the systems and the results indicate that citrate anion increases lipase activity. An analysis by fluorescence spectroscopy of the interaction between lipase and the bottom and top phases of the systems shows that the protein tryptophan-environments are modified by the presence of PEG and salts. Separation of the enzyme from the rest of the proteins that make up the lyophilized was achieved with good yield and separation factor by ATPS formed by PEG 1000/Pi at pH 7, PEG 2000/Ci at pH 5.2 and PEG 4000/Ci at pH 5.2. The above mentioned systems were used in order to isolate extracellular lipase from a strain of A. niger in submerged culture and solid culture. The best system for solid culture, with high purification factor (30.50), is the PEG 4000/Ci at pH 5.2. The enzyme was produced in a solid culture medium whose production is simple and recovered in a phase poor in polymer, bottom phase. An additional advantage is that the citrate produces less pollution than the phosphate. This methodology could be used as a first step for the isolation of the extracellular lipase from A. niger.