Features of the acid protease partition in aqueous two-phase systems of polyethylene glycol-phosphate: chymosin and pepsin

The partitioning of chymosin (from Aspergilus niger) and pepsin (from bovine stomach) was carried out in aqueous-two phase systems formed by polyethyleneglycol-potassium phosphate. The effects of polymer concentration, molecular mass and temperature were analysed. The partition was assayed at pH 7.0 in systems of polyethyleneglycol of molecular mass: 1450, 3350, 6000 and 8000. Both proteins showed high affinity for the polyethyleneglycol rich phase. The increase of polyethyleneglycol concentration favoured the protein transfer to the top phase, suggesting an important protein-polymer interaction. Polyethyleneglycol proved to have a stabilizing effect on the chymosin and pepsin, increasing its protein secondary structure. This finding agreed with the enhancement of the milk clotting activity by the polyethyleneglycol. The method appears to be suitable as a first step for the purification of these proteins from their natural sources.     

Influence of partition parameters on a recombinant antigen of Schistosoma mansoni expressed on E. coli using poly(ethylene glycol)–hydroxypropyl starch aqueous two-phase system

This work describes the partition of a Schistosoma mansoni tegumental antigen produced by a recombinant Escherichia coli strain using an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) and purified hydroxypropyl-starch (Reppal PES 100). The effects of the polymer molecular weight, tie line length and pH on antigen partitioning were investigated. The detection of the antigen in both phases was determined by ELISA. The system composed of PEG 8000 (5.1% w/w) and Reppal PES 100 (13.0% w/w) led to a yield of 92% and a purification factor of 12 concerning the antigen in the PEG-rich phase. It was observed that antigen partition in ATPSs was strongly affected by the pH and tie line length. In addition, it was possible in a single step, to remove the cell debris, which precipitated at the interface of the system.     

利用聚乙二醇(PEG8000)纯化小球藻病毒FJ-1

为了提取小球藻病毒基因组ＤＮＡ,探索利用３％￣１０％的ＰＥＧ８０００加３％￣７％的ＮａＣｌ沉淀病毒。其中以７％的ＰＥＧ和４％的ＮａＣｌ沉淀效果最好,但其沉淀效率较低。     

Study of the serum albumin-polyethyleneglycol interaction to predict the protein partitioning in aqueous two-phase systems

The theoretical framework based only on the excluded volume forces is not enough to explain the bovine serum albumin partitioning behaviour in aqueous biphasic systems. The goal of this work is to look at the phase separation via the polymer effect on the water structure. Our findings suggest that polyethyleneglycol 600-protein interaction is conducted by van der Waals forces between the hydrophobic surfaces from PEG and protein molecules, which implies the rupture of hydrogen bonds from the structured water in their neighbours. Therefore, the protein will concentrate in the most water-structured phase (polyethyleneglycol) in order to reach the minimal free energy condition. When polyethyleneglycol molecular weight increases, its exclusion from protein surface prevails, thus pushing the bovine serum albumin to the bottom phase.     

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.     

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.     

Effect of salt additives on protein partition in polyethylene glycol–sodium sulfate aqueous two-phase systems

Partitioning of 15 proteins in polyethylene glycol (PEG)–sodium sulfate aqueous two-phase systems (ATPS) formed by PEG of two different molecular weights, PEG-600 and PEG-8000 in the presence of different buffers at pH 7.4 was studied. The effect of two salt additives (NaCl and NaSCN) on the protein partition behavior was examined. The salt effects on protein partitioning were analyzed by using the Collander solvent regression relationship between the proteins partition coefficients in ATPS with and without salt additives. The results obtained show that the concentration of buffer as well as the presence and concentration of salt additives affects the protein partition behavior. Analysis of ATPS in terms of the differences between the relative hydrophobicity and electrostatic properties of the phases does not explain the protein partition behavior. The differences between protein partitioning in PEG-600–salt and PEG-8000–salt ATPS cannot be explained by the protein size or polymer excluded volume effect. It is suggested that the protein–ion and protein–solvent interactions in the phases of ATPS are primarily important for protein partitioning.     

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

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

Enhanced secretion and low temperature stabilization of a hyperthermostable and Ca2+-independent alpha-amylase of Geobacillus thermoleovorans by surfactants

AIMS: Selection of suitable surfactants for enhancing and stabilizing alpha-amylase of Geobacillus thermoleovorans. METHODS AND RESULTS: Geobacillus thermoleovorans was cultivated in shake flasks containing 50 ml of starch-yeast extract-tryptone (SYT) medium with/without surfactants. Titres of the enzyme in media were monitored. The enzyme was also preserved at 4 degrees C with/without surfactants and enzyme activities were determined. Among polyethylene glycol (PEGs) of different molecular weights, PEG 8000 (0.5%, w/v) caused a slight increase in the enzyme titre, while Tween-20, Tween-40 and Tween-60 (0.03%, w/v) exerted a significant stimulatory effect on enzyme secretion. In the presence of SDS, Tween-80 and cholic acid (0.03%, w/v), the enzyme production was nearly twofold higher than that in the control. The anionic (SDS, cholic acid) and non-ionic (Tweens) detergents increased the cell membrane permeability, and thus, enhanced alpha-amylase secretion. Furthermore, anionic surfactants exhibited stabilizing effect on the enzyme during preservation at 4 degrees C. CONCLUSIONS: PEG 8000 and the ionic detergents (SDS, cholic acid and Tween-80) were more effective in the solubilization of cell membrane components, and enhancing enzyme yields than the cationic detergents such as CTAB (N,Cetyl-N,N,N-trimethyl ammonium bromide). Further, these surfactants were found to stabilize the enzyme at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The secretion of Ca2+-independent hyperthermostable alpha-amylase was enhanced in the presence of certain anionic and non-ionic detergents in the medium. Furthermore, the surfactants stabilized the enzyme during preservation at 4 degrees C. The use of this enzyme in starch hydrolysis eliminates the addition of Ca2+ in starch liquefaction and its subsequent removal by ion exchange from sugar syrups.     

Effects of low urea concentrations on protein-water interactions

Solvent properties of aqueous media (dipolarity/polarizability, hydrogen bond donor acidity, and hydrogen bond acceptor basicity) were measured in the coexisting phases of Dextran–PEG aqueous two-phase systems (ATPSs) containing .5 and 2.0 M urea. The differences between the electrostatic and hydrophobic properties of the phases in the ATPSs were quantified by analysis of partitioning of the homologous series of sodium salts of dinitrophenylated amino acids with aliphatic alkyl side chains. Furthermore, partitioning of eleven different proteins in the ATPSs was studied. The analysis of protein partition behavior in a set of ATPSs with protective osmolytes (sorbitol, sucrose, trehalose, and TMAO) at the concentration of .5 M, in osmolyte-free ATPS, and in ATPSs with .5 or 2.0 M urea in terms of the solvent properties of the phases was performed. The results show unambiguously that even at the urea concentration of .5 M, this denaturant affects partitioning of all proteins (except concanavalin A) through direct urea–protein interactions and via its effect on the solvent properties of the media. The direct urea–protein interactions seem to prevail over the urea effects on the solvent properties of water at the concentration of .5 M urea and appear to be completely dominant at 2.0 M urea concentration.     

Extractive fermentation for enhanced gellan-hydrolysing enzyme production by Bacillus thuringiensis H14

A new extractive fermentation process using PEG and potassium phosphate aqueous two-phase system (ATPS) was developed for enhanced production of gellan-hydrolysing enzyme by Bacillus thuringiensis H14. Five different Bacillus sp. were tested for their ability to synthesize gellan-hydrolysing enzyme. Bacillus thuringiensis H14 was found to be the best organism for gellan-hydrolysing enzyme production. The enzyme showed maximum activity at pH 7.5 and 40 °C. The partition studies of gellan-hydrolysing enzyme in the system using PEG X (X = 9000, 6000, 4000) and potassium phosphate–water and PEG–sodium citrate–water system indicated at PEG (4000)– potassium phosphate–water is the best system for partitioning of gellan-hydrolysing enzyme into the PEG phase (K = 4.99). Gellan-hydrolysing enzyme production by Bacillus thuringiensis H14 was studied in ATPSs composed of PEG X (X = 9000, 6000, 4000) and potassium phosphate. The top phase is continuous and rich in PEG while the bottom phase is dispersed and is rich in phosphate, microbial cells being mainly retained in the bottom phase. The gellan-hydrolysing enzyme produced during fermentation partitioned into the upper PEG phase and total gellan-hydrolysing enzyme produced was 2.12, 2.29 and 2.40 times higher than that of homogeneous fermentation when the fermentations were carried out using PEG 9000–potassium phosphate–water, PEG 6000–potassium phosphate–water, PEG 4000–potassium phosphate–water systems respectively.     

Polyethylene glycol as a spacer for solid-phase enzyme immobilization

With the aim to improve the performance of enzyme bound to hydrophilic solid phases, their immobilization with polyethylene glycol (PEG) tether have been studied. Sweet potato β-amylase, which hydrolyses the high molecular weight substrate starch and β-galactosidase, which acts on low molecular weight substrates, were used as model enzymes and beaded thiol–agarose as solid phase. Several two step methods for the introduction of the tether using a bis-oxirane homobifunctional PEG as well as a heterobifunctional derivative with a hydroxysuccinimide ester and a maleimide group have been evaluated. Amino groups, native and de novo thiol groups in the enzymes were utilized for immobilization.

The best approach was found to be to first introduce the PEG derivative via one of its reactive groups to the enzyme. Subsequently the formed conjugate was bound to the solid phase by the remaining reactive group.

Attempts to first introduce the PEG tether into the solid phase were not successful.

A high degree of substitution with PEG chains on the enzyme leads to high immobilization yields for both β-amylase and β-galactosidase, but relatively lower gel-bound activity for the former enzyme which is acting on a high molecular weight substrate and thus more sensitive for steric shielding effects. With optimal degree of PEG substitution (which occurred at five times molar excess of the heterobifunctional reagent) the gel-bound activity of β-amylase was increased from 12% (for the derivative without tether) to 31%.     

Porcine pancreatic lipase partition in potassium phosphate-polyethylene glycol aqueous two-phase systems

This research study examined porcine pancreatic lipase partition in aqueous two-phase systems formed by polyethylene glycol-potassium phosphate at pH 6.0, 7.0 and 8.0, the effect of polymer molecular mass, and NaCl concentration. The enzyme was preferentially partitioned into the polyethylene glycol rich phase in systems with molecular mass 4000-8000, while with polyethylene glycol of 10,000 molecular mass it was concentrated in the phosphate rich phase. The enthalpic and entropic changes found due to the protein partition were negative for all the polyethylene glycol molecular mass systems assessed. Both thermodynamic functions were shown to be associated by an entropic-enthalpic compensation effect suggesting that the water structure ordered in the ethylene chain of polyethylene glycol plays a role in the protein partition. The addition of NaCl increased the lipase affinity to the top phase and this effect was most significant in the system polyethylene glycol 2000-NaCl 3%. This system yielded an enzyme recovery more than 90% with a purification factor of approximately 3.4.     

Determinants of liposome partitioning in aqueous two-phase systems: Evaluation by means of a factorial design

This article evaluates the influence of five parameters on liposome partitioning in aqueous two-phase systems (ATPSs), composed of poly(ethyleneglycol) (PEG)/dextran (Dx), using the factorial experimental design together with a multiple regression. Mathematical models to quantify the influence of these parameters, individually and/or jointly, on liposome partitioning in ATPS were developed. The models were statistically tested and verified by experimentation. This approach was then used to define the conditions for the preferential accumulation of liposomes in the top PEG-rich phase. The models predicted a significant effect of liposome surface charge, PEG molecular weight, phase-forming polymer concentration, and phosphate ion concentration on the partition behavior of liposomes. For negatively charged liposomes, it was found that the smaller the molecular weight of PEG and polymer concentration and the larger the phosphate ion concentration, the greater the partition coefficient of the liposomes. No significant effect of pH, at the range of 6-8, on liposome partitioning was noted. This approach has led to the development of an optimal two-phase system where 90% of negatively charged liposomes accumulated in the PEG phase. In addition to the general scientific value of this research, it has a technological importance as ATPSs may be useful for removing the unentrapped drug from liposomes during their preparation for pharmaceutical applications. (c) 1996 John Wiley & Sons, Inc.     

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.     

Extractive fermentation for enhanced production of alkaline phosphatase from Bacillus licheniformis MTCC 1483 using aqueous two-phase systems

A study was made to find out maximum partitioning of Bacillus licheniformis alkaline phosphatase in different ATPSs composed of different molecular weight of PEG X (X = 2000, 4000, 6000) with salts (magnesium sulphate, sodium sulphate, sodium citrate) and polymers (dextran 40, dextran T500). Physicochemical factors such as effect of system pH, system temperature and production media were evaluated for partitioning of alkaline phosphatase. PEG 4000 [9.0% (w/v)] and dextran T500 [9.6% (w/v)] were selected as most suitable system components for alkaline phosphatase production by B. licheniformis based on greater partition coefficient (k = 5.23). The two-phase system produced fewer enzymes than the homogeneous fermentation (control) in early stage of fermentation, but after 72 h the enzyme produced in the control system was less than that in the ATPS. Total alkaline phosphatase yield in ATPS fermentation was 3907.01 U/ml and in homogeneous fermentation 2856.50 U/ml.     

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

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

Pancreatic serine protease extraction by affinity partition using a free triazine dye

Affinity partitioning combines the partitioning behavior of biological macromolecules in aqueous two-phase systems with the principle of biorecognition. Among the numerous substances that have been evaluated as ligands, the reactive dyes constitute a group of low cost textile dyes which have proved to act as biomimetic ligands for many enzymes. The ability of reactive yellow 2 (RY2) to interact with trypsin (TRP) and chymotrypsin (ChTRP) and its behavior in aqueous two-phase systems formed by polyethylene glycol (PEG) and sodium citrate (NaCit) - were investigated. Different variables such as PEG molecular weight, tie line length and dye concentration were analyzed. RY2 showed to bind specifically to both TRP and ChTRP with affinity constants near to 10(3)M(-1). Its partition equilibrium is practically displaced to the top phase in systems formed by PEG of different molecular weight. Addition of this dye to PEG 8000/NaCit systems until a final concentration of 0.196% (w/w) induced an increase in TRP and ChTRP partition coefficients of at least 2 times over that in the absence of the ligand. These findings demonstrate that RY2 fulfils all the requirements to be considered as an affinity ligand in aqueous two-phase partitioning of TRP and ChTRP.     

Suppression of type III effector secretion by polymers

Bacteria secrete effector proteins required for successful infection and expression of toxicity into host cells. The type III secretion apparatus is involved in these processes. Previously, we showed that the viscous polymer polyethylene glycol (PEG) 8000 suppressed effector secretion by Pseudomonas aeruginosa. We thus considered that other viscous polymers might also suppress secretion. We initially showed that PEG200 (formed from the same monomer (ethylene glycol) as PEG8000, but which forms solutions of lower viscosity than the latter compound) did not decrease effector secretion. By contrast, alginate, a high-viscous polymer formed from mannuronic and guluronic acid, unlike PEG8000, effectively inhibited secretion. The effectiveness of PEG8000 and alginate in this regard was closely associated with polymer viscosity, but the nature of viscosity dependence differed between the two polymers. Moreover, not only the natural polymer alginate, but also mucin, which protects against infection, suppressed secretion. We thus confirmed that polymer viscosity contributes to the suppression of effector secretion, but other factors (e.g. electrostatic interaction) may also be involved. Moreover, the results suggest that regulation of bacterial secretion by polymers may occur naturally via the action of components of biofilm or mucin layer.