Application of aqueous biphasic systems as strategy to purify tannase from Aspergillus tamarii URM 7115

The aims of the current study are to assess the influence of polyethylene glycol (PEG) concentration, molar mass, pH, and citrate concentrations on aqueous biphasic systems based on 2⁴ factorial designs, as well as to check their capacity to purify tannase secreted by Aspergillus tamarii URM 7115. Tannase was produced through submerged fermentation at 26°C for 67?h in Czapeck-Dox modified broth and added with yeast extract and tannic acid. The factorial design was followed to assess the influence of PEG molar mass (M_PEG 600; 4,000 and 8,000?g/?mol), and PEG (C_PEG 20.0; 22.0 and 24.0% w/w) and citrate concentrations (C_CIT 15.0, 17.5, and 20.0%, w/w), as well as of pH (6.0, 7.0, and 8.0) on the response variables; moreover, partition coefficient (K), yield (Y), and purification factor (PF) were analyzed. The most suitable parameters to purify tannase secreted by A. tamarii URM 7115 through a biphasic system were 600 (g/mol) M_PEG, 24% (w/w) C_PEG, 15% (w/w) C_CIT at pH 6.0 and they resulted in 6.33 enzyme partition, 131.25% yield, 19.80 purification factor and 195.08 selectivity. Tannase secreted by A. tamarii URM 7115 purified through aqueous biphasic systems composed of PEG/citrate can be used for industrial purposes, since it presents suitable purification factor and yield.     

Purification and characterization of crystallins by aqueous two-phase extraction

Crystallins are a family of water-soluble proteins that constitute up to 90% of the water-soluble proteins in mammalian eye lenses. We present in this paper an alternative purification method for these proteins using polyethylene glycol/dextran aqueous two-phase extraction. Under the appropriate conditions, we were able to recover the γ-crystallin fraction essentially free of the remaining proteins. High concentrations of salt at a neutral pH maximize the recovery of γ-crystallins in the top phase and minimize the contamination by the other proteins present in the lenses. The proposed protocol decreases the separation time by about 50% The complex partition behavior observed for these proteins reflects a delicate balance between protein/phase-forming species (various polymers and salts) and protein/protein interactions. This is evidenced in part, by the role played by the largest proteins in this group as a “pseudo” phase-forming species.     

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.     

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.     

The scanning electron microscopic study of the infection and conidial development of Aspergillus tamarii Kita on its host, the silkworm, Bombyx mori Linn.

Development of Aspergillosis on the integument of the silkworm, Bombyx mori Linn., was examined by scanning electron microscopy. Aspergillosis is a fungal disease caused by an insect mycopathogen Aspergillus tamarii Kita, which infects the silkworms in countries where sericulture (the rearing of silkworms)is prevalent. The present study showed the course of infection and the conidial development of A. tamarii on the integument of B. mori. Five different strains (KA, NB₁₈, NB₄ D₂, NB₇ and PM) of B. mori were inoculated on their body surface with ca. 1 × 10⁶ conidia/ml. Among the five breeds tested, the conidial germination was greatest on the larval surface of KA breed, and least on PM. Most of the conidia germinated on the cuticle approximately 8–12 hours after inoculation, forming a suctorial appressorium within 24 hours. The hyphae reached the hemocoel, where they grew and multiplied extensively, forming a mycelial complex and causing death of the host larva in about 5–6 days. The death of the host was followed by growth of the fungus through mesodermal and epidermal tissues, leading to larval mummification about 6–7 days post-inoculation. Extensive aerial outgrowths of the fungus followed, mostly through the intersegmental regions of larvae. Abundant branched conidiophores developed, forming a confluent yellow brown mat over the entire host body 7 days after inoculation. Each conidiophore had an apical vesicle bearing numerous phialides from which conidia were developed in long chains.     

Properties of Aspergillus tamarii,A. caelatus and related species from acidic tea field soils in Japan

Fungi in Aspergillus section Flavi include both aflatoxin producers and non-producers. Aspergillus caelatus is a recently described non-aflatoxigenic species in this section, which has some common characteristics with A. tamarii, such as yellowish brown color and double walled spores. In contrast to the morphological similarities, all of the A. caelatus isolates tested produced no cyclopiazonic acid whereas most isolates of A. tamarii produce this compound. There are six nucleotide differences that distinguish the DNA sequences of these two species in the regions of ITS1, ITS2, 5.8S rDNA and 28S rDNA and this is a consistent difference. Both species were isolated from acidified field soils, but A. tamarii isolates were more common than A. caelatus in highly acidic soils. This revised version was published online in August 2006 with corrections to the Cover Date.     

Parameters influencing protein extraction for whole broths in detergent based aqueous two-phase systems

The parameters important for an optimisation of cloud point extraction in technical scale were investigated using a genetically engineered fusion protein derived from endoglucanase I expressed in Trichoderma reesei and the nonionic polyoxyethylene Agrimul NRE 1205. The key parameters are temperature, detergent concentration, and additional salts. These parameters are interdependent, thus there is an optimum in the partition coefficient with respect to detergent concentration and a maximum for the partition coefficient and the yield with respect to temperature. These results were confirmed for the detergent C12E5 to demonstrate that these optima are due to the nature of polyoxyethylenes. Cloud point extraction was found to be only slightly affected by pH. In the case studied extraction of whole broth is favourable for a high yield and partition coefficient, since fusion protein adhering to the cells can be solubilized. However some loss of detergent which remains in the fungal biomass was observed. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

The application of aqueous two-phase systems to the purification of pharmaceutical proteins from transgenic sheep milk

Transgenic sheep milk containing the protein human₁-Antitrypsin (AAT) was partitioned in Poly(ethyleneglycol) (PEG)-Sulphate and PEG-Phosphate biphasic systems. Individual partition coefficients for AAT and some of the milk proteins were determined in these systems. The effects of PEG molecular weight, pH and the inclusion of NaCl on the partitioning of the proteins were also studied. It was found that increasing the concentration of NaCl and decreasing the molecular weight of the PEG resulted in an increase of the partition coefficients of the proteins to the upper (PEG) phase. This partitioning effect was greater for the more hydrophobic proteins and particularly in systems having a pH close to the isoelectric point of the protein. Solubilities of the proteins in increasing concentrations of ammonium sulphate were measured in order to investigate the effects of hydrophobic and electrostatic interactions on the partitioning of these proteins in aqueous two-phase systems. Those proteins that precipitated at low levels of ammonium sulphate showed an increase in partition coefficient at low concentrations of NaCl, or they were precipitated at the interface of the phases at low concentrations of NaCl. Proteins that had low salting out constants in ammonium sulphate solutions were relatively unaffected by NaCl in ATPS. It is probable however that conformational changes and the state of aggregation of proteins are also important and should be invoked in describing the partitioning behavior observed for -Lg for example. Comparison of theoretical and experimental values for AAT yield and purity showed clearly that partition coefficients are influenced by the degree of purity and values obtained with purified standards are not necessarily the same as for the same protein present in a complex mixture. Under the most favourable conditions using a 4% w/w loading of transgenic ovine milk, we obtained a 91% yield of AAT in the PEG phase with a purity of 73%.This revised version was published online in October 2005 with corrections to the Cover Date.     

Continuous purification of antibodies from cell culture supernatant with aqueous two-phase systems: From concept to process

An aqueous two-phase extraction (ATPE) process based on a PEG/phosphate system was developed for the capture of human immunoglobulin G and successfully applied to a Chinese hamster ovary and a PER.C6® cell supernatant. A continuous ATPE process incorporating three different steps (extraction, back-extraction, and washing) was set up and validated in a pump mixer-settler battery. Most of the higher molecular weight cell supernatant impurities were removed during the extraction step, while most of the lower molecular weight impurities were removed during the subsequent steps. A global recovery yield of 80% and a final protein purity of more than 99% were obtained for the IgG purification from a CHO cell supernatant, representing a 155-fold reduction in the protein/IgG ratio. For the purification of IgG from a PER.C6® cell supernatant, a global recovery yield of 100%, and a host cell protein purity were attained, representing a 22-fold reduction in the host cell protein/IgG ratio. These results, thus, open promising perspectives for the application of the developed ATPE process as a platform for the capture of antibodies. In fact, this new process has shown the ability to successfully recover and purify different antibodies from distinct cell culture supernatants. This technology can also overcome some of the limitations encountered using the typical chromatographic processes, besides inherent advantages of scalability, process integration, capability of continuous operation, and economic feasibility.     

Production and Partial Purification of Tannase from Aspergillus ficuum Gim 3.6

A novel fungal strain, Aspergillus ficuum Gim 3.6, was evaluated for its tannase-producing capability in a wheat bran-based solid-state fermentation. Thin-layer chromatography (TLC) analysis revealed that the strain was able to degrade tannic acid to gallic acid and pyrogallol during the fermentation process. Quantitation of enzyme activity demonstrated that this strain was capable of producing a relatively high yield of extracellular tannase. Single-factor optimization of process parameters resulted in high yield of tannase after 60 hr of incubation at a pH of 5.0 at 30°C, 1 mL of inoculum size, and 1:1 solid–liquid ratio in the presence of 2.0% (w/v) tannic acid as inducer. The potential of aqueous two-phase extraction (ATPE) for the purification of tannase was investigated. Influence of various parameters such as phase-forming salt, molecular weight of polyethylene glycol (PEG), pH, and stability ratio on tannase partition and purification was studied. In all the systems, the target enzyme was observed to preferentially partition to the PEG-rich top phase, and the best result of purification (2.74-fold) with an enzyme activity recovery of 77.17% was obtained in the system containing 17% (w/w) sodium citrate and 18.18% (w/w) PEG1000, at pH 7.0.     

Genetically engineered charge modifications to enhance protein separation in aqueous two-phase systems: Electrochemical partitioning

We have examined the effect of genetically engineered charge modifications on the partitioning behavior of proteins in dextran/polyethylene glycol two-phase systems containing potassium phosphate. By genetically altering a protein's charge, the role of charge on partitioning can be assessed directly without the need to modify the phase system. The charge modifications used are of two types: Charged tails of polyaspartic acid fused to beta-galactosidase and charge-change point mutations of T4 lysozyme which replace positive lysine residues with negative glutamic acids. The partition coefficient K(p) for these proteins was related to measured interfacial potential differences Deltaphi using the simple thermodynamic model, In K(p) = In K(o) + (F/RT)Z(p) deltaphi. The protein net charge Z(p) was determined using the Henderson-Hasselbalch relationship with modifications based on experimentally determined titration and isoelectric point data. It was found that when the electropartitioning term Z(p) deltaphi was varied by changing the pH, the partitioning of T4 lysozyme was quantitatively described by the thermodynamic model. The beta-galactosidase fusions displayed qualitative agreement, and although less than predicted, the partitioning increased more than two orders of magnitude for the pH range examined. Changes in the partitioning of lysozyme due to the various mutations agreed qualitatively with the thermodynamic model, but with a smaller than expected dependence on the estimated charge differences. The beta-galactosidase fusions, on the other hand, did not display a consistent charge based trend, which is likely due either to the enzyme's large size and complexity or to nonelectrostatic contributions from the tails. The lack of quantitative fit with the model described above suggests that the assumptions made in developing this model are oversimplified. (c) 1994 John Wiley & Sons, Inc.     

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.     

Extraction of protease from Aspergillus tamarii URM 4634 in aqueous two-phase system under continuous and discontinuous process

Abstract

Aqueous two-phase systems have been studied for almost a century to separate biomolecules in harmless conditions. Proteases produced by Aspergillus tamarii URM 4634 were extracted in polyethylene glycol (PEG)/phosphate aqueous two-phase system under discontinuous and continuous (perforated rotative discs column) process. On the discontinuous process, it was evaluated the effect of operational conditions (PEG molar mass and its concentration, phosphate concentration and pH) over the partition coefficient, activity yield and purification factor. Protease partitioned to PEG-phase with partition coefficients up to 55.73. The best process parameters were 17.5% of PEG, with molar mass 8000?g·mol^?1, 15% of phosphate salt at pH 6, with 113.15% of activity yield and purification factor of 2.62. Under continuous extraction, hold up data showed that 57.1% of the discontinuous phase was available for protein extraction. Further, separation achieved 90.0% of efficiency. The yields surpassed 100% in almost all runs, and the best purification factor was 1.84, with both flows of 2?mL·min^?1. Thus, the best operational conditions reached an activity yield of 95.3% and 90.0% of separation efficiency. Hence, aqueous two-phase system PEG/phosphate extraction is an efficient process for separation of proteases produced by Aspergillus tamarii URM 4634, under continuous extraction likewise under discontinuous process.     

Microwave-assisted aqueous two-phase extraction of piceid, resveratrol and emodin from Polygonum cuspidatum by ethanol/ammonium sulphate systems

Microwave-assisted, aqueous two-phase extraction was investigated to obtain effective constituents, including piceid, resveratrol and emodin in Polygonum cuspidatum. An aqueous two-phase system consisting of 25% (w/w) ethanol 21% (w/w) (NH₄)₂SO₄ gave equal yields of piceid, and 1.1- and 1.9-times higher yields of resveratrol and emodin, respectively, than that achieved by microwave-assisted extraction and heat reflux extraction. Three-separate operations, extraction, clarification and concentration, are hereby integrated into a single step to get higher yields at lower cost. This is therefore a potentially useful method for the extraction and purification of target products.     

Continuous extraction of β-d-galactosidase from Escherichia coli in an aqueous two-phase system: effects of biomass concentration on partitioning and mass transfer

High concentrations of Escherichia coli disintegrate move the binodial of a poly(ethylene glycol) (PEG) 4000/potassium phosphate aqueous two-phase system towards lower concentrations. It has also been shown that the yield and purification factor of β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) in the PEG phase was gradually improved by moving the experimental system to a composition closer to the binodial. The mass transfer rates of cell debris, total protein, β-d-galactosidase and DNA have been studied and were found to be fast enough to reach equilibrium between the phases after 1.9 s of mixing in a static mixer with 24 mixing elements. A continuous extraction process for β-d-galactosidase from E. coli has been designed on the basis of these studies with a mean residence time of 6.3 min from the disintegrator inlet to the β-d-galactosidase containing PEG-phase outlet of the centrifuge. This PEG phase contained 83.5% of the total β-d-galactosidase with a purification factor of 13.6, and only 2.8% of the total protease activity of the disintegrate. All cell debris and almost all DNA were confined to the potassium phosphate phase.     

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.     

Genetically engineered charge modifications to enhance protein separation in aqueous two-phase systems: Charge directed partitioning

This report continues or examination of the effect of genetically engineered charge modifications on the partitioning behavior of proteins in aqueous two-phase extration. The genetic modifications consisted of the fusion of charged peptide tails to beta-galactosidase and charge-change point mutations to T4 lysozyme. Our previous article examined the influence of these charge modifications on partitioning as a function of interfacial potential difference. In this study, we examined charge directed partitioning behavior in PEG/dextran systems containing small amounts of the charged polymers diethylaminoethyl-dextran (DEAE-dextran) or dextran sulfate. The best results were obtained when attractive forces between the protein and polymer were present. Nearly 100% of the beta-galactosidase, which carries a net negative charge, partitioned to the DEAE-dextran-rich phase regardless of whether the phase was dextran or PEG. In these cases, cloudiness of the protein-rich phases suggest that strong charge interactions resulted in protein/polymer aggregation, which may have contributed to the extreme partitioning. Unlike the potentialdriven partitioning reported previously, consistent partitioning trends were observed as a result of the fusion tails, with observed shifts in partition coefficient (K(p)) of up to 37-fold. However, these changes could not be solely attributed to charge-based interactions. Similarly, T4 lysozyme, carrying a net positive charge, partitioned to the dextran sulfate-containing phase, and displayed four- to sevenfold shifts in K(p) as a result of the point mutations. These shifts were two to four times stronger than those observed for potential driven partitioning. Little effect on partitioning was observed when the protein and polymer had the same charge, with the exception of beta-galactosidase with polyarginine tails. The high positive charge density of these tails provided for a localized interaction with the dextran sulfate, and resulted in 2- to 15-fold shifts in K(p). (c) 1995 John Wiley & Sons, Inc.     

Technical improvement in the purification of enzymes from red algae using an aqueous two-phase partitioning system

The effective elimination of phycobiliproteins from crude enzyme preparation of the red alga Caloglossa continua (Okamura) King et Puttock (Ceramiales, Florideophyceae) was investigated in an aqueous two‐phase partitioning system (ATPS) by changing the concentrations of polyethylene glycol (PEG) and ammonium sulfate (AS). The phycobiliproteins shifted from the AS‐rich lower phase to the PEG‐rich upper phase in high PEG and AS concentrations. The best ATPS condition for the elimination of phycobiliproteins from the lower phase was obtained by the combination of 20% (weight/volume; w/v) PEG and 16% (w/v) AS. However, the recovery of aldolase and mannitol‐1‐phos‐phatase activities was significantly reduced. For purification of the enzymes, a combination of 15% (w/v) PEG and 16% (w/v) AS was the best ATPS condition, because a high specific activity and recovery of the enzymes were obtained. Under these conditions, 98% of the phycobiliproteins were removed from the lower phase. Therefore, the ATPS proved to be a very useful method as a first step in the purification of enzymes from red algae.     

Thermodynamic modeling of the partitioning of biomolecules in aqueous two-phase systems using a modified Flory–Huggins equation

A modified Flory–Huggins equation accounting for the solvation of polymer molecules by water molecules was used to model the phase behavior of aqueous two-phase systems (ATPS) formed by poly(ethylene glycol) (PEG) and dextran. The parameters of the equation were obtained by fitting experimental equilibrium data either accounting for or disregarding dextran polidispersity. The modified equation was subsequently applied to calculate partition coefficients of biomolecules in these systems. It was found that accounting for polidispersity did not affect significantly the calculated phase equilibrium, but increased the agreement of calculated partition coefficients with experimental data. Further improvement was obtained by using a size dependent interaction parameter for dextran pseudo-components.