Liquid-Liquid Equilibrium of Aqueous Two-Phase Systems Containing Some Synthetic Polyelectrolytes and Polyethylene Glycol

Experimental results are presented for the liquid-liquid equilibrium of aqueous two-phase systems containing a synthetic polyelectrolyte (polysodium acrylate, polysodium methacrylate, and polysodium ethylene sulfonate) and polyethylene glycol at (298.2 and 323.2) K. A total of 40 phase diagrams were obtained, comprising data both of the binodal curve (obtained through cloud-point measurements) and of equilibrium compositions. The influences of temperature, the nature of the polyelectrolyte monomer unit, and the chain length of both types of polymers are analyzed and discussed.

Purification of alpha-toxin from Clostridium perfringens type A in PEG-phosphate aqueous two-phase systems: a factorial study

BACKGROUND: Purification of a-toxin produced by Clostridium perfringens type A in aqueous two-phase systems (ATPS) was studied with a full two-level factorial design on two factors (concentrations of 8000 g mol(-1) PEG and phosphate salt at pH 8.0), to estimate the influence of these factors on the purification results. RESULTS: The partition coefficient (K), purification factor (PF) and activity yield (Y) were strongly influenced by the PEG and phosphate concentrations. Raising the levels of the two factors increased these responses. The highest purification factor (5.7) was obtained with PEG and phosphate concentrations of 17.5% and 15%, respectively. CONCLUSION: These results support the proposal that polymer excluded volume and hydrophobic interactions are the factors that drive the alpha-toxin in PEG/phosphate aqueous two-phase systems. (c) 2008 Society of Chemical Industry

Extraction of Ascorbate Oxidase from Cucurbita maxima by Continuous Process in Perforated Rotating Disc Contactor Using Aqueous Two-Phase Systems

The ascorbate oxidase is the enzyme used to determine the content of ascorbic acid in the pharmaceutical and food industries and clinics analyses. The techniques currently used for the purification of this enzyme raise its production cost. Thus, the development of alternative processes and with the potential to reduce costs is interesting. The application of aqueous two-phase system is proposed as an alternative to purification because it enables good separation of biomolecules. The objective of this study was to determine the conditions to continuously pre-purify the enzyme ascorbate oxidase by an aqueous two-phase system (PEG/citrate) using rotating column provided with perforated discs. Under the best conditions (20,000 g/mol PEG molar mass, 10% PEG concentration, and 25% citrate concentration), the system showed satisfactory results (partition coefficient, 3.35; separation efficiency, 54.98%; and purification factor, 1.46) and proved suitable for the pre-purification of ascorbate oxidase in continuous process.

Separation and partitioning of Green Fluorescent Protein from Escherichia coli homogenate in poly(ethylene glycol)/sodium-poly(acrylate) aqueous two-phase systems

The partitioning of Green Fluorescent Protein (GFP) in poly(ethylene glycol)/Na-poly(acrylate) aqueous two-phase systems (PEG/NaPA-ATPS) has been investigated. The aqueous two-phase systems are formed by mixing the polymers with a salt and a protein solution. The protein partitioning in the two-phase system was investigated at 25 degrees C. The concentration of the GFP was measured by fluorimetry. It was found that the partitioning of GFP depends on the salt type, pH and concentration of PEG. The data indicates that GFP partitions more strongly to the PEG phase in presence of Na2SO4 relative to NaCl. Furthermore, the GFP partitions more to the PEG phase at higher pH. The partition to the PEG phase is strongly favoured in systems with larger tie-line lengths (i.e. systems with higher polymer concentrations). The molecular weight of PEG is important since the partition coefficient (K) of GFP gradually decreases with increasing PEG size, from K ca. 300-400 for PEG 400 to K equal to 1.19 for PEG 8000. A separation process was developed where GFP was separated from a homogenate in two extraction steps: the GFP is first partitioned to the PEG phase in a PEG 3000/NaPA 8000 system containing 3 wt% Na2SO4, where the K value of GFP was 8. The GFP is then re-extracted to a salt phase formed by mixing the previous top-phase with a Na2SO4 solution. The K-value of GFP in this back-extraction was 0.22. The total recovery based on the start material was 74%. (c) 2008 Elsevier B.V. All rights reserved.

Protein partitioning in poly(ethylene glycol)/sodium polyacrylate aqueous two-phase systems

The partition of hemoglobin, lysozyme and glucose-6-phospate dehydrogenase (G6PDH) in a novel inexpensive aqueous two-phase system (ATPS) composed by poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) has been studied. The effect of NaCl and Na2SO4, pH and PEG molecular size on the partitioning has been studied. At high pH (above 9), hemoglobin partitions strongly to the PEG-phase. Although some precipitation of hemoglobin occurs, high recovery values are obtained particularly for lysozyme and G6PDH. The partitioning forces are dominated by the hydrophobic and electrochemical (salt) effects, since the positively charged lysozyme and negatively charged G6PDH partitions to the non-charged PEG and the strongly negatively charged polyacrylate enriched phase, respectively. (c) 2007 Elsevier B.V. All rights reserved.

Aqueous two-phase extraction using thermoseparating copolymer: a new system for phenolic compounds removal from hemicelullosic hydrolysate

BACKGROUND: The hydrolysis of hemicellulosic material can provide liquor with high xylose concentration (which can be used as a fermentation medium) and phenolic compounds (Phs), potentially immunostimulating compounds. However, these hydrolysates must be detoxified in order to remove the Phs that can act as inhibitors in bioconversions. RESULTS: Aqueous two-phase systems composed of thermoseparating copolymers were used for rice straw hydrolysate detoxification. The hydrolysis process was able to promote chemical breakdown of 85% of the total hemicellulose content, 14% of the cellulose, and 2% of the lignin. The hydrolysate obtained contained 19.7 g L-1 of xylose and several phenolic compounds, such as vanillin, vanillic acid, ferullic acid, etc. The phenolics extraction was studied as a function of copolymer molar mass (1100 g mol(-1), 2000 g mol(-1) and 2800 g mol(-1)), their percentages (from 5% to 50%) and Phs initial concentration. Phenolic compounds extraction of around 80% was obtained under the following conditions: 20% (w/w) and 35% (w/w) copolymer 1100 g mol-1, 35% (w/w) copolymer 2000 g mol(-1) and 35% (w/w) copolymer 2800 g mol(-1) at 25 degrees C. CONCLUSIONS: The results demonstrated the viability of this method for the removal of Phs from rice straw hydrolysate, which has potential uses in bioconversion processes. (c) 2007 Society of Chemical Industry.

Liquid-liquid extraction of proteases from fermented broth by PEG/citrate aqueous two-phase system

This work deals with the use of an aqueous two-phase system (ATPS) of PEG/citrate to remove proteases from a Clostridium perfringens fermentation broth. To plan the experimental tests and evaluate the corresponding results, three successive experimental designs were employed, for which the PEG molar mass (M-PEG) and concentration (C-PEG), the citrate concentration (C-C) and the pH were selected as independent variables, while the purification factor (PF), the partition coefficient (K), the activity yield (Y) and the selectivity (S) were selected as responses. PF of proteases in the top phase was shown to increase with increasing MPEG and decreasing Cc, whereas a completely opposite trend was observed for K. On the other hand, Y was favored by simultaneous decreases in both these variables, while S decreased with increasing Cc. Therefore, selecting a simultaneous increase in PF and Y as the most desirable result, the best performance of the system was obtained using M-PEG = 10-000 g/mol C-PEG = 22% (w/w) and C-c = 8.0% (w/w) at pH 8.5. Under these conditions, the activity yield was very high (131 %) but the purification factor (4.2) and the selectivity (4.3) were lower than those ensured by more selective purification methods. According to these results, the ATPS seems to be an interesting alternative primary concentration/decontamination step for vaccine preparation from C. perfringens fermented broth. (C) 2007 Elsevier B.V. All rights reserved.

Phase equilibria of lysozyme precipitation with the volatile salt ammonium carbamate

Lysozyme precipitation induced by the addition of the volatile salt ammonium carbamate was studied through cloud-point measurements and precipitation assays. Phase equilibrium experiments were carried out at 5.0, 15.0 and 25.0 degrees C and the compositions of the coexisting phases were determined. A complete separation of the coexisting liquid and solid phases could not be achieved. Nevertheless it was possible to determine the composition of the solid precipitate through the extensions of experimental tie lines. The same precipitate was found at all temperatures. Lysozyme enzymatic activities of the supernatant and precipitate phases were also determined. The activity balance suggests that ammonium carbamate preserves lysozyme activity after the salting-out precipitation. (C) 2010 Elsevier B.V. All rights reserved.

Comparison of oxygen mass transfer coefficient in simple and extractive fermentation systems

Aeration and agitation are important variables to ensure effective oxygen transfer rate during aerobic bioprocesses: therefore, the knowledge of the volumetric mass transfer coefficient (k(L)a) is required. In view of selecting the optimum oxygen requirements for extractive fermentation in aqueous two-phase system (ATPS), the k(L)a values in a typical ATPS medium were compared in this work with those in distilled water and in a simple fermentation medium. in the absence of biomass. Aeration and agitation were selected as the independent variables using a 2(2) full factorial design. Both variables showed statistically significant effects on k(L)a, and the highest values of this parameter in both media for simple fermentation (241 s(-1)) and extractive fermentation with ATPS (70.3 s(-1)) were observed at the highest levels of aeration (5 vvm) and agitation (1200 rpm). The k(L)a values were then used to establish mathematical correlations of this response as a function of the process variables. The exponents of the power number (N(3)D(2)) and superficial gas velocity (V(s)) determined in distilled water (alpha = 0.39 and beta = 0.47, respectively) were in reasonable agreement with the ones reported in the literature for several aqueous systems and close to those determined for a simple fermentation medium (alpha=0.38 and beta=0.41). On the other hand, as expected by the increased viscosity in the presence of polyethylene glycol, their values were remarkably higher in a typical medium for extractive fermentation (alpha=0.50 and beta=1.0). A reasonable agreement was found between the experimental data of k(L)a for the three selected systems and the values predicted by the theoretical models, under a wide range of operational conditions. (C) 2009 Elsevier B.V. All rights reserved.

Evaluation of the effect of ammonium carbamate on the stability of proteins

BACKGROUND: The use of the volatile salt ammonium carbamate in protein downstream processing has recently been proposed. The main advantage of using volatile salts is that they can be removed from precipitates and liquid effluents through pressure reduction or temperature increase. Although previous studies showed that ammonium carbamate is efficient as a precipitant agent, there was evidence of denaturation in some enzymes. In this work, the effect of ammonium carbamate on the stability of five enzymes was evaluated. RESULTS: Activity assays showed that alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1), lysozyme (1,4-beta-N-acetylmuramoylhydrolase, EC 3.2.1.17) and lipase (triacyl glycerol acyl hydrolase, EC 3.1.1.3) did not undergo activity loss in ammonium carbamate solutions with concentrations from 1.0 to 5.0 mol kg(-1), whereas cellulase complex (1,4-(1,3 : 14)-beta-D-glucan 4-glucano-hydrolase, EC 3.2.1.4) and peroxidase (hydrogen peroxide oxidoreductase, EC 1.11.1.7) showed an average activity loss of 55% and 44%, respectively. Precipitation assays did not show enzyme denaturation or phase separation for alpha-amylase and lipase, while celullase and peroxidase precipitated with some activity reduction. Analysis of similar experiments with ammonium and sodium sulfate did not affect the activity of enzymes. CONCLUSION: Celullase and peroxidase were denatured by ammonium carbamate. While more systematic studies are not available, care must be taken in designing a protein precipitation with this salt. The results suggest that the generally accepted idea that salts that denature proteins tend to solubilize them does not hold for ammonium carbamate. (C) 2010 Society of Chemical Industry

Stability of clavulanic acid under variable pH, ionic strength and temperature conditions. A new kinetic approach

Clavulanic acid (CA) is a beta-lactam antibiotic that alone exhibits only weak antibacterial activity, but is a potent inhibitor of beta-lactamases enzymes. For this reason it is used as a therapeutic in conjunction with penicillins and cephalosporins. However, it is a well-known fact that it is unstable not only during its production phase, but also during downstream processing. Therefore, the main objective of this study was the evaluation of CA long-term stability under different conditions of pH and temperature, in the presence of variable levels of different salts, so as to suggest the best conditions to perform its simultaneous production and recovery by two-phase polymer/salt liquid-liquid extractive fermentation. To this purpose, the CA stability was investigated at different values of pH (4.0-8.0) and temperature (20-45 degrees C), and the best conditions were met at a pH 6.0-7.2 and 20 degrees C. Its stability was also investigated at 30 degrees C in the presence of NaCl, Na(2)SO(4), CaCl(2) and MgSO(4) at concentrations of 0.1 and 0.5 M in Mcllvaine buffer (pH 6.5). All salts led to increased CA instability with respect to the buffer alone, and this effect decreased in following sequence: Na(2)SO(4) > MgSO(4) > CaCl(2) > NaCl. Kinetic and thermodynamic parameters of CA degradation were calculated adopting a new model that took into consideration the equilibrium between the active and a reversibly inactivated form of CA after long-time degradation. (C) 2009 Elsevier B.V. All rights reserved.

Liquid-liquid extraction of commercial and biosynthesized nisin by aqueous two-phase micellar systems

Nisin is a natural additive for conservation of food, and can also be used as a therapeutic agent. Nisin inhibits the outgrowth of spores, the growth of a variety of Gram-positive and Grain-negative bacteria. In this paper we present a potentially scalable and cost-effective way to purify commercial and biosynthesized in bioreactor nisin, including simultaneously removal of impurities and contaminants, increasing nisin activity. Aqueous two-phase micellar systems (ATPMS) are considered promising for bioseparation and purification purposes. Triton X-114 was chosen as the as phase-forming surfactant because it is relatively mild to proteins and it also forms two coexisting phases within a convenient temperature range. Nisin activity was determined by the agar diffusion assay utilizing Lactobacillus sake as a sensitive indicator microorganism. Results indicated that nisin partitions preferentially to the micelle rich-phase, despite the surfactant concentration tested, and its antimicrobial activity increases. The successful implementation of this peptide partitioning, from a suspension containing other compounds, represents an important step towards developing a separation method for nisin, and more generally, for other biomolecules of interest. (C) 2007 Elsevier Inc. All rights reserved.

AN ASSESSMENT ON THE USE OF THE DEBYE-HUCKEL EQUATION FOR THE THERMODYNAMIC MODELING OF AQUEOUS SYSTEMS CONTAINING POLYMERS AND SALTS

In this work, a study on the role of the long-range term of excess Gibbs energy models in the modeling of aqueous systems containing polymers and salts is presented. Four different approaches on how to account for the presence of polymer in the long-range term were considered, and simulations were conducted considering aqueous solutions of three different salts. The analysis of water activity curves showed that, in all cases, a liquid-phase separation may be introduced by the sole presence of the polymer in the long-range term, regardless of how it is taken into account. The results lead to the conclusion that there is no single exact solution for this problem, and that any kind of approach may introduce inconsistencies.

An extension of the Pitzer equation for the excess Gibbs energy of aqueous electrolyte systems to aqueous polyelectrolyte solutions

Pitzer`s equation for the excess Gibbs energy of aqueous solutions of low-molecular electrolytes is extended to aqueous solutions of polyelectrolytes. The model retains the original form of Pitzer`s model (combining a long-range term, based on the Debye-Huckel equation, with a short-range term similar to the virial equation where the second osmotic virial coefficient depends on the ionic strength). The extension consists of two parts: at first, it is assumed that a constant fraction of the monomer units of the polyelectrolyte is dissociated, i.e., that fraction does not depend on the concentration of the polyelectrolyte, and at second, a modified expression for the ionic strength (wherein each charged monomer group is taken into account individually) is introduced. This modification is to account for the presence of charged polyelectrolyte chains, which cannot be regarded as punctual charges. The resulting equation was used to correlate osmotic coefficient data of aqueous solutions of a single polyelectrolyte as well as of binary mixtures of a single polyelectrolyte and a salt with low-molecular weight. It was additionally applied to correlate liquid-liquid equilibrium data of some aqueous two-phase systems that might form when a polyelectrolyte and another hydrophilic but neutral polymer are simultaneously dissolved in water. A good agreement between the experimental data and the correlation result is observed for all investigated systems. (c) 2008 Elsevier B.V. All rights reserved.

Exploitation of a single interface flow system for on-line aqueous biphasic extraction

The exploitation of aqueous biphasic extraction is proposed for the first time in flow analysis This extraction strategy stands out for being environmentally attractive since it is based in the utilization of two immiscible phases that are intrinsically aqueous The organic solvents of the traditional liquid-liquid extractions ale no longer used, being replaced by non-toxic, non-flammable and non-volatile ones. A single interface flow analysis (SIFA) system was implemented to carry out the extraction process due to its favourable operational characteristics that include the high automation level and simplicity of operation, the establishment of a dynamic interface where the mass transfer occurred between the two immiscible aqueous phases, and the versatile control over the extraction process namely the extraction time The application selected to demonstrate the feasibility of SIFA to perform this aqueous biphasic extraction was the pre-concentration of lead. After extraction, lead reacted with 8-hydroxyquinoline-5-sulfonic acid and the resulting product was determined by a fluorimetric detector included in the flow manifold. Therefore, the SIFA single interface was used both as extraction (enrichment) and reaction interface. (C) 2010 Elsevier B.V All rights reserved.