Growth and Secondary Metabolite Production by Hairy Roots of Tagetes Patula in Aqueous Two-Phase Systems

Hairy roots of Tagetes patula have been grown in aqueous two-phase systems. After selecting suitable polymers from single-phase experiments (in which salt phases were unable to support growth in the desired concentrations) several two-phase systems were tested for their influence on cell growth and thiophene production. Cell growth occurred in all aqueous two-phase systems, but the highest growth rate was achieved in normal medium. There was no difference in thiophene production between medium and aqueous two-phase systems. The partition of thiophenes favoured slightly the more hydrophobic top phase in most cases, while the cells were confined to the bottom phase. One aqueous two-phase system (15% polyethyleneglycol 10,000 and 15% Reppal PES 200) was tested in a stirred tank reactor with normal medium as a control. The growth rate in medium was higher than in the aqueous two-phase system, while the thiophene production per unit cell weight was in the same range for both systems. The excretion of thiophenes in the reactor with the aqueous two-phase system was about ten times as high as in the control reactor. The amount excreted was however still not more than 3% of the total production.     

Optimization of bovine serum albumin partition coefficient in aqueous two-phase systems

Partitioning of proteins in aqueous two-phase systems has been shown to provide a powerful method for separating and purifying mixtures of biomolecules by extraction. These systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. There are many factors which influence the partition coefficient K, the ratio of biomolecule concentration in the top phase to that in the bottom phase, in aqueous two-phase systems. The value of the partition coefficient relies on the physico-chemical properties of the target biomolecule and other molecules and their interactions with those of the chosen system. In this work, the partition behavior of pure bovine serum albumin in aqueous two-phase systems was investigated in order to see the effects of changes in phase properties on the partition coefficient K. The concentration of NaCl and pH were considered to be the factors having influence on K. Optimal conditions of these factors were obtained using the Box-Wilson experimental design. The optimum value of K was found as 0.0126 when NaCl concentration and pH were 0.14 M and 9.8, respectively, for a phase system composed of 8% (w/w) polyethylene glycol 3,350 - 9 (% w/w) dextran 37,500 - 0.05 M phosphate at 20 °C.     

Relative hydrophobicity of organic compounds measured by partitioning in aqueous two-phase systems

Partitioning of a variety of organic compounds, the majority of which represent therapeutic drugs, was examined in an aqueous dextran–polyethylene glycol (Dex–PEG) two-phase system containing 0.15 M NaCl in 0.01 M sodium phosphate buffer at pH 7.3 and in an octanol–buffer (0.15 M NaCl in 0.01 M sodium phosphate buffer, pH 7.3) system. The possibility of introducing compounds to be partitioned in an aqueous two-phase system with dimethyl sulfoxide, and the effect of this solvent on the solute partitioning was explored. Relative hydrophobicity of the compounds was estimated and expressed in equivalent numbers of methylene units. Comparison of the results obtained for several subsets of compounds in the octanol–buffer and in aqueous Dex–PEG two-phase systems clearly demonstrates the advantage of aqueous two-phase partitioning for the hydrophobicity measurements over partitioning in octanol–buffer system.     

The extractive purification of peroxidase from plant raw materials in aqueous two-phase systems

The extractive purification of peroxidase from Armoracia rusticana roots and Glycine max seed coats in temperature-induced and affinity microsphere-containing aqueous two-phase systems was stuied. The extractive purification of peroxidase from Glycine max seed coats was carried out in a temperature-induced aqueous two-phase system formed by Triton X-45, Triton X-100 and sodium acetate at pH 5.5 A 99% yield with a 6-fold purification factor was obtained. When the clear top phase was subjected to concanavalin-A affinity chromatography, the purification factor rose to 41 and the yield dropped to 28%. A two-step purification process for peroxidase from Armoracia rusticana roots was developed by adding concanavalin-A affinity microspheres to a PEG/phosphate aqueous two-phase system. The method allows a 60% recovery of high purity peroxidase (1,860 guaiacol units per mg). A lower recovery rate and degree of purification of this enzyme was achieved after temperature-induced aqueous two-phase partition or acetone precipitation and concanavalin-A affinity column chromatography.     

Investigation of mathematical methods for efficient optimisation of aqueous two-phase extraction

Mathematical strategies were applied to optimise the extraction of recombinant leucine dehydrogenase from E. coli homogenates and endoglucanase 1 from culture filtrates of Trichoderma reesei in polyethylene glycol–phosphate systems. The goal was to test mathematical tools which could facilitate the optimisation procedure in aqueous two-phase systems. A modified simplex approach, the method of steepest ascent and a genetic algorithm were successfully applied and compared. The methods differ in the height of the optimum found, the number of experiments and the time required. The genetic algorithm proved to be an optimisation procedure which can be used well in aqueous two-phase systems. The simplex procedure has to be further improved.     

Enhanced production of lactic acid through the use of a novel aqueous two-phase system as an extractive fermentation system

 In order to enhance the productivity of lactic acid and reduce the end-product inhibition of fermentation, the partitioning and growth of four different strains of lactic acid bacteria in three different aqueous two-phase systems were studied. Polyethyleneglycol/ dextran, polyethyleneglycol/hydroxypropyl starch polymer (HPS), and a random copolymer of ethylene oxide and propylene oxide (EO-PO)/HPS were used as polymer systems. One strain each of Lactococcus lactis subsp. lactis and of Lactobacillus delbrueckii subsp. delbrueckii partitioned completely to the interface and bottom phase in two-phase systems with low polymer concentrations of EO-PO/HPS100 and EO-PO/ HPS200. The growth and production of lactic acid by two of three L. lactis strains in a two-phase system with 5.5% (w/w) EO-PO and 12.0% (w/w) HPS100 were reduced by less than 10% compared with a reference fermentation in a normal growth medium. The viability of L. lactis subsp. lactis ATCC 19435 was maintained for at least 50 h and with four top-phase replacements during extractive fermentation in the EO-PO/HPS100 system. Moreover, when cell density reached the stationary phase in the first extractive fermentation, the lactate production in this aqueous two-phase system was maintained. Received: 2 October 1995/Received revision: 16 January 1996/Accepted: 22 January 1996     

Liquid–liquid extraction of an extracellular alkaline protease from fermentation broth using aqueous two-phase and reversed micelles systems

Summary The study of recovery of an extracellular alkaline protease from fermentation broth produced by Norcadiopsis sp, was carried out with liquid–liquid extraction through sodium di-(2-ethylhexyl) sulphosuccinate/isooctane reversed micelles systems and aqueous two-phase systems (polyethylene glycol/potassium phosphate). The best conditions for extraction and back-extraction with the reversed micelles system was obtained at pH 9.0 and pH 5.0, respectively, showing a yield of protein of 6.16%, a specific activity of 4.10 U/ml and a purification factor of 1.80. The studies using aqueous two-phase systems of polyethylene glycol/potassium phosphate at pH 10.0 showed purification factors of 2 and 5, and protein yield of 11 and 4%, respectively, for polyethylene glycol 550/potassium phosphate and polyethylene glycol 8000/potassium phosphate. The results indicate that the aqueous two-phase systems are more attractive as a first step in the isolation and purification processes.     

Studies on equilibrium of anthranilic acid in aqueous solutions and in two-phase systems: Aromatic solvent–water

The acid–base equilibria of anthranilic acid have been characterized in terms of macro- and microdissociation constants (dissociation constants K_a1, K_a2 and tautomeric constant K_z). On the basis of spectrophotometric investigations the values of the distribution ratio D of anthranilic acid in the two-phase systems: aromatic solvent (benzene, ethylbenzene, toluene, chlorobenzene, bromobenzene)–aqueous solution were obtained. Employing the results of potentiometric titration in the two-phase systems: aromatic solvent–aqueous solution the distribution constant K_D and dimerization constant K_dim values were calculated. The influence of organic solvent polarity and pH of the aqueous phase on the contents of the particular forms of the acid in the two-phase systems were analyzed.     

Bovine serum albumin partitioning in an aqueous two-phase system: Effect of pH and sodium chloride concentration

The partitioning of bovine serum albumin (BSA) in a polyethylene glycol 3350 (8% w/w)–dextran 37 500 (6% w/w)–0.05 M phosphate aqueous two-phase was investigated at different pHs, at varying concentrations of sodium chloride at 20°C. The effect of NaCl concentration on the partition coefficient of BSA was studied for the PEG–dx systems with initial pH values of 4.2, 5.0, 7.0, 9.0, and 9.8. The NaCl concentrations in the phase systems with constant pH value were 0.06, 0.1, 0.2, 0.3, and 0.34 M. It was observed that the BSA partition coefficient decreased at concentrations smaller than 0.2 M NaCl and increased at concentrations greater than 0.2 M NaCl for all systems with initial pHs of 4.2, 5.0, 7.0, 9.0, and 9.8. It was also seen that the partition coefficient of BSA decreased as the pH of the aqueous two-phase systems increased at any NaCl salt concentration studied.     

Technical aspects of separation using aqueous two-phase systems in enzyme isolation processes.

Technical aspects of the separation of aqueous two-phase systems in a commercial separator were studied in detail. For the Gyrotester B, the smallest available separator, a flow rate of 200 ml/min and a length of the regulating screw in the outlet port of 13.5 mm were found as optimal operation parameters for the separation of a poly(ethylene glycol) (PEG)/dextran two-phase system. In the presence of cells and cell debris the characteristics of the carrier two-phase systems are changed, most notably the phase ratio. Nevertheless good separation and high throughput can be maintained up to 30% wet cell material in the complete system. Using this method the enzyme pullulanase was extracted from 6.65 kg Klebsiella pneumoniae in 88% yield in a single step in less than 2 hr. A yield of 90% was predicted for this step based upon laboratory data, indicating that the performance of the extraction and separation can be calculated with the necessary accuracy and the further scale-up of the process should be accomplished quite easily. The hydrophilic polymers Constituting the phase system will often stabilize the enzymes, So that the separation can be carried out at room temperature without extensive cooling. The method of enzyme solubilization or cell disruption is not decisive for the successful extraction of the enzymes, the only limitation being the necessity to find a suitable two-phase system where the desired product and the cells or cell debris will partition in opposite phases. This is shown for α-glucosidase from Saccharomyces carlsbergensis and three aminoacyl-tRNA-synthetases from Escherichia coli. The results obtained demonstrate that aqueous two-phase systems can be separated in commercially available separators with high capacity and efficiency. It can be expected that the advanced separation technology available from chemical engineering studies can also be used for the development of large-scale isolation processes for enzymes involving liquid–liquid partitions.     

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.     

Enhancement of substrate concentration in microbial stereoinversion through one-pot oxidation and reduction by aqueous two-phase system

An extractive biocatalytic method of aqueous two-phase system was employed for stereoinversing (R)-1-phenyl-1,2-ethanediol into (S)-1-phenyl-1,2-ethanediol by Candida parapsilosis CCTCC M203011. It was observed that substrate and product inhibitions in microbial stereoinversion through one-pot oxidation and reduction were removed efficiently by extractive biocatalysis in aqueous two-phase system with PEG 4000/phosphate potassium system, and that the substrate concentration was enhanced from 15 to 30 g/L with product optical purity of 99.02% e.e. and yield of 90% after 60 h. Simultaneously, it was observed that change in cell morphology impedes the further enhancement of substrate concentration in this system but can be reversibly changed after stereoinversion or cultivation in systems without PEG.     

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.     

High concentrations of PEG as a possible uncoupler of the proton motive force: α-Amylase production with Bacillus amyloliquefaciens in aqueous two-phase systems and PEG solutions

Summary -Amylase production with Bacillus amyloliquefaciens was investigated in two different aqueous two-phase systems and in polyethylene glycol (PEG) 600 solutions of different concentrations. The cells did not partition totally to the bottom phases of the aqueous two-phase systems, and the enzyme production was repressed in both systems as well as in PEG 600 solutions. Concomitantly, the cultivation time was prolonged, indicating an increased maintenance metabolism. The surface properties of cells grown in 200 g/kg PEG 600 were investigated by phase partitioning and compared to the surface properties of Bacillus subtilis, which under these conditions showed increased -amylase production. The cells of B. amyloliquefaciens partitioned to the top phase in a PEG-dextran system, whereas the cells of B. subtilis partitioned to the bottom phase. The results are discussed in relation to water activity, oxygen transfer rate and PEG-induced changes of the surface properties of the cells. The possible role of PEG as an uncoupler of the proton motive force at high concentrations is also discussed.     

Cultivation of plant cells in aqueous two-phase polymer systems

Summary Suspension cultures ofNicotiana tabacum have been successfully grown in aqueous, two-phase systems comprised of polyethylene glycol (PEG) and dextran in a modified LS medium. Aqueous two-phase systems may be advantageous for plant tissue cultivation since cells can be immobilized in one phase while secondary products are collected and withdrawn in the other phase, thus enhancing productivity. Culture growth rate was compared in a variety of two-phase systems, covering a range of both polymer molecular weight and concentration. Systems exhibiting relatively higher phase miscibility yielded increased growth rates as compared to less miscible phase formulations. The highest observed growth rate occurred in 3% PEG 20000/5% crude dextran and approached growth rates and cell densities of cultures grown in standard LS medium.     

Partition of horseradish peroxidase in aqueous two-phase systems containing polyvinylpyrrolidone

Growing peroxidase utilisation in different industries encourages the search for high benefit/cost ratio purification methods such as aqueous two-phase partition. In this way, the partitioning behaviour of peroxidase from Armoracia rusticanaroots in polyvinylpirrolidone/Reppal and polyvinylpirrolidone/salt aqueous two-phase systems was investigated. Based on these results, a two-step purification process was developed. In the first system (polyvinylpyrrolidone K30/Reppal PES 200, pH 7.0), cell debris and some contaminating proteins were shifted to the bottom phase while peroxidase concentrated in the top phase. After discarding the bottom phase, the second step involved addition of magnesium sulphate thus forming a second aqueous two-phase system. At this step, the enzyme was extracted into the salt-rich bottom phase. The overall yield was 75% and the purification factor 7.3.This revised version was published online in October 2005 with corrections to the Cover Date.     

Separation of proteins and viruses using two-phase aqueous micellar systems

We discuss the utilization of a novel two-phase aqueous nonionic micellar system for the purification and concentration of biomolecules, such as proteins and viruses, by liquid–liquid extraction. The nonionic surfactant n-decyl tetra(ethylene oxide), C₁₀E₄, phase separates in water into two coexisting aqueous micellar phases by increasing temperature. The mild interactions of the C₁₀E₄ nonionic surfactant with biomolecules, combined with the high water content of the two coexisting micellar phases, suggest the potential utility of two-phase aqueous C₁₀E₄ micellar systems for the purification and concentration of biomolecules. In this paper, we review our recent experimental and theoretical studies involving the partitioning of several water-soluble proteins, including cytochrome c, soybean trypsin inhibitor, ovalbumin, bovine serum albumin, and catalase, in the two-phase aqueous C₁₀E₄ micellar system. In addition, we present results of our preliminary experimental investigation on the partitioning of bacteriophages, including φX174, P22, and T4.     

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

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

Stimuli-responsive separation of proteins using immobilized liposome chromatography

The possibility of the stimuli-responsive separation of proteins was investigated using immobilized liposome chromatography (ILC) as novel aqueous two-phase systems. The specific capacity factor (k_s) of β-galactosidase, obtained by analysis of ILC, was varied by changing the pH of the solution and was maximized at the specific pH of 5 (k_s,max=5.57). The k_s values were found to correspond well with their local hydrophobicities, which can be determined by the aqueous two-phase partitioning method. The variation of k_s, therefore, indicates a change in the surface properties of a protein during conformational change under pH stimuli. A similar phenomenon is observed in the case of other proteins (α-glucosidase, k_s,max=11.3 at pH 4; carbonic anhydrase from bovine, k_s,max=6.53 at pH 4). The difference in the height and/or the position of the peaks of the k_s–pH curves of each protein suggests a difference in their pH denaturation in the ILC column. Based on these results, the mutual separation of the above proteins at pH 4 could be successfully performed by selecting their specific capacity factor as a design parameter.     

Quantitation of plasmid DNA in aqueous two-phase systems by fluorescence analysis

The development of aqueous two-phase systems for plasmid purification from Escherichia coli cell lysates requires a reliable DNA quantitation method. Plasmid DNA was quantified by fluorescence using PicoGreen nucleic acid stain. Linearity was obtained up to 40 ng plasmid ml^–1. Two polyethyleneglycol (PEG)/salt systems were studied, PEG 600/K₂HPO₄ and PEG 300/K₂HPO₄. The average plasmid recovery was 41% in the bottom phase of the first system and 35% in the top phase of the second system. This method has proved to be simple and reproducible.