α-amylase production in aqueous two-phase systems with Bacillus subtilis

The production of α-amylase (1,4-α-d-glucan glucanohydrolase, EC 3.2.1.1) by Bacillus subtilis has been studied in repeated batch fermentations in aqueous two-phase systems. In a phase system composed of PEG 600, 8% (w/w), PEG 3350, 5% (w/w)/Dextran T 500, 2% (w/w), 82% of the enzyme partitioned to the top phase. The enzyme concentration in the top phase reached 0.85–1.35 U ml^?1 during the fermentations compared with 0.58 U ml^?1 in the reference fermentation. In the phase system composed of PEG 3350, 9% (w/w)/Dextran T 500, 2% (w/w), 73% of the enzyme partitioned to the top phase. However, the enzyme concentration in this phase system reached only 0.35 U ml^?1 in the top phase. The bacterial cells were microscopically observed to partition totally to the bottom phase in the aqueous two-phase system used. The results are discussed in relation to recirculation of cells by immobilizing to a solid matrix. Extraction of the product to the top phase and the effect of the phase polymers, especially PEG, on the production are also discussed.     

Separation of yeast protoplasts from membrane ghosts using an aqueous two-phase system

Protoplasts, prepared from Saccharomyces cerevisiae, were separated from membrane ghosts using an aqueous two-phase system of Dextran T 500, 5% (w/w) and polyethylene glycol (PEG) 8000, 5% (w/w). The protoplast preparation was prewashed in the top phase. After resuspension in fresh top phase an equal volume of bottom phase was added. A protoplast preparation almost free from membrane ghosts was obtained as a precipitate in the bottom phase. The membrane ghosts partitioned to the interphase.     

Correlations for the partition behavior of proteins in aqueous two-phase systems: effect of overall protein concentration

The effect of protein concentration in partitioning in PEG/salt aqueous two-phase systems has been investigated. PEG 4000/phosphate systems in the presence of 0% w/w and 8.8% w/w NaCl have been evaluated using amyloglucosidase, subtilisin, and trypsin inhibitor. Also, a PEG 4000/phosphate system with 3% w/w NaCl was used for alpha-amylase. The concentration of the protein in each of the phases affected its partition behavior. The pattern for the individual proteins was dependent on their physicochemical properties. In the top phase, maximum protein concentration was determined mainly by a steric exclusion effect of PEG, and hydrophobic interaction between PEG and proteins. In the bottom phase, maximum concentration was determined mainly by a salting-out effect of the salts present. As the ionic strength was increased in the systems the concentration in the top phase increased for all proteins. In the bottom phase an increase in ionic strength increased the salting-out effect. Amyloglucosidase had a very low maximum concentration in the PEG-rich top phase which was probably due to its large size (steric exclusion) and low hydrophobicity, and a high concentration in the salt-rich bottom phase due to its high hydrophilicity. In the case of subtilisin and trypsin inhibitor, their high concentrations in the top phase were due to their hydrophobic nature (hydrophobic interaction with PEG) and small size (negligible steric exclusion). The maximum concentration in the bottom phase for trypsin inhibitor was lower than that of subtilisin which was probably due to its higher hydrophobicity and, hence, a stronger salting-out effect. The protein concentration in each of the two phases was correlated with a "saturation"-type equation. The partition coefficient could be satisfactorily predicted, as a function of the overall protein concentration, by the ratio between the "saturation" equations of the two individual phases. Better correlations were obtained when an empirical sigmoidal Boltzmann equation was fitted to the data, since in virtually all cases the partition coefficient is constant at low protein concentration (true partitioning) and changes to a different constant value at a high overall protein concentration. (c) 1996 John Wiley & Sons, Inc.     

Simultaneous production and partition of chitinase during growth of Serratia marcescens in an aqueous two-phase system

Summary Partition and production of the extracellular chitinase from Serratia marcescens were studied in PEG/dextran aqueous two-phase systems. The enzyme partitions into the bottom phase and the cells segregate into the top phase. The best system is 2% (w/v) PEG 20000 and 5% (w/v) dextran T500. The cell growth and enzyme production kinetics are similar in the aqueous two-phase system and in the polymer-free reference system. However, the maximum enzyme concentration in the former system is 1.5 times that in the latter one.     

Isolation of plasmid DNA from cell lysates by aqueous two-phase systems

This work presents a study of the partitioning of a plasmid vector containing the cystic fibrosis gene in polyethylene glycol (PEG)/salt (K2HPO4) aqueous two-phase systems (ATPS). The plasmid was extracted from neutralized alkaline lysates using PEG with molecular weights varying from 200 to 8000. The effects of the lysate mass loaded to the ATPS (20, 40, and 60% w/w) and of the plasmid concentration in the lysate were evaluated. The performance of the process was determined by qualitative and quantitative assays, carefully established to overcome the strong interference of impurities (protein, genomic DNA, RNA), salt, and PEG. Plasmid DNA partitioned to the top phase when PEG molecular weight was lower than 400. The bottom phase was preferred when higher PEG molecular weights were used. Aqueous two-phase systems with PEG 300, 600, and 1000 were chosen for further studies on the basis of plasmid and RNA agarose gel analysis and protein quantitation. The recovery yields were found to be proportional to the plasmid concentration in the lysate. The best yields (>67%) were obtained with PEG 1000. These systems (with 40 and 60% w/w of lysate load) were able to separate the plasmid from proteins and genomic DNA, but copartitioning of RNA with the plasmid was observed. Aqueous two-phase systems with PEG 300 concentrated both plasmid and proteins in the top phase. The best system for plasmid purification used PEG 600 with a 40% (w/w) lysate load. In this system, RNA was found mostly in the interphase, proteins were not detected in the plasmid bottom phase and genomic DNA was reduced 7.5-fold.     

Production of alkaline protease by Bacillus licheniformis in an aqueous two-phase system

Alkaline protease production by Bacillus licheniformis was studied in an aqueous two-phase system composed of 5% (w/w) polyethylene glycol 6000 (PEG 6000) and 5% (w/w) dextran T500. The top phase was continuous and rich in PEG while the bottom phase was dispersed and rich in dextran. The cells were retained in the bottom phase and at the interface. The two-phase system produced less enzyme in total amount than the control in the early phase, but after 50 h the enzyme produced in the control system decreased while the aqueous two-phase system continued its production and finally the total enzyme activity reached 1.3 times that of the control culture. In order to improve the productivity of protease, repeated batch cultivation were successfully carried out four times by optimizing the top phas composition of freshly added media, which resulted in 13.8, 35.9, 27.8 and 34.7 units ml⁻¹ h⁻¹ of protease based on the amounts of replaced top phases, respectively.     

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.     

Application of the response surface methodology for optimization of whey protein partitioning in PEG/phosphate aqueous two-phase system

In order to develop a new strategy for β-lactoglobulin (β-lg) removal from whey protein, partitioning of α-lactalbumin (α-la), β-lg and glycomacropeptide (Gmp) was studied using aqueous two phase systems (ATPS). A system composed of 13% (w/w) polyethylene glycol (PEG, average molar mass 2000 g/mol) and 13% (w/w) potassium phosphate was used at 25°C. A central composite rotatable design (CCRD) associated to the response surface methodology (RSM) was applied to investigate the effects of NaCl concentration and pH on the partition of these proteins. It was found that α-la and Gmp partitioned to the top phase rich in PEG, whereas β-lg partitioned to the bottom phase rich in salt. According to the RSM, optimal conditions for β-lg removal where found where pH was equal to 6.7 and salt concentration was 0.35 mol/L. Under these conditions, the partition coefficient K(α) was 0.48 and K(Gmp) was 0.92. On the other hand, the partition coefficient K(β) was only 0.01. In such conditions β-lg preferentially concentrates in the bottom phase, while the top phase exclusively contains the proteins α-la and Gmp. Fractionation of the proteins from fresh whey was performed in a three stage cross-flow extraction system. The extraction yield for β-lg in the bottom phase was 97.3%, while the yields for α-la and Gmp in the top phase were 81.1% and 97.8%, respectively.     

Enzymatic conversion in aqueous two-phase systems: deacylation of benzylpenicillin to 6-aminopenicillanic acid with penicillin acylase

The conversion of benzylpenicillin (BP) to 6-aminopenicillanic acid (6-APA) using penicillin acylase (penicillin amidohydrolase, EC 3.5.1.11) has been studied in aqueous two-phase systems. In a system composed of 8.9% (w/w) PEG 20000/7.6% (w/w) potassium phosphate the enzyme was almost completely partitioned to the bottom phase (K < 0.01), which allowed repeated batch conversions, recirculating the enzyme several times. The initial specific productivities were 0.31–1.47 μmol 6-APA mg protein^?1 min^?1 in repeated conversions over five steps. The yield obtained from the top phase was 0.47–0.71 mol 6-APA mol BP^?1. The results are discussed in relation to recirculating the enzyme by immobilizing it to a solid matrix. Despite the high phosphate concentration in the bottom phase the system needs to be titrated in order for the reaction to proceed. Titration of the top phase alone protected the enzyme from denaturation by strong alkali used for the titration.     

牛血清白蛋白在聚乙二醇/葡聚糖双水相体系中分配特性的研究

采用考马斯亮蓝G250染色法测得室温下BSA在PEG/dextran双水相体系中的分配系数。以BSA在PEG/dextran体系的下相富集为目标,研究了PEG的分子量、浓度、dextran浓度以及所加入中性盐的种类与浓度、体系pH诸因素对其分配特性的影响。实验结果表明,在PEG4000/dextran体系中,采用PEG质量分数9%-dextran质量分数9%的浓度组成,同时在pH=7.0,NaC l浓度为0.2 mol.L-1或pH6.0,NaC l浓度为0.34 mol.L-1的工艺条件下萃取BSA均可达最小分配系数,其值为0.014。     

Response surface methodology for the extraction of wedelolactone from Eclipta alba using aqueous two-phase extraction

Abstract

Aqueous two-phase extraction of wedelolactone from Eclipta alba was studied using the polymer-salt system. The system consisted of polyethylene glycol (PEG) as a top phase (polymer) and sodium citrate as a bottom phase (salt). Process parameters such as PEG concentration, PEG molecular weight, salt concentration, and pH have been optimized using response surface methodology (RSM) with the help of central composite design (CCD). The optimized conditions for aqueous two-phase system (ATPS), in the case of one factor at a time approach, were found as PEG 6000, PEG concentration 18% (w/v), salt concentration 16% (w/v), and pH 7; with maximum extraction yield of 6.52?mg/g. While, RSM studies showed maximum extraction yield of 6.73?mg/g with the optimized parameters as PEG 6000, PEG concentration 18% (w/v), salt concentration 17.96% (w/v), and pH 7. ATPS was found to give a 1.3 fold increase in the extraction yield of wedelolactone as compared to other conventional extraction methods.     

Two-step process for initial capture of plasmid DNA and partial removal of RNA using aqueous two-phase systems

In this paper, a two-step process for initial capture of plasmid DNA (pDNA) and partial removal of RNA using polyethylene glycol (PEG) and di-potassium hydrogen phosphate aqueous two-phase systems (ATPS) has been investigated. A Kühni-type ATPS extraction column was prepared with 50 ml (12% (w/w) PEG 1450, 12% (w/w) phosphate) of stationary phase and loaded with crude mobile phase (26% (w/w) PEG 1450, 4% (w/w) phosphate and 70% (w/w) lysate) at a flow rate of 6 ml min⁻¹ at an impeller speed of 200 rpm. The experiment was terminated after 100 min, and after complete resettling of the phases, 45 ml of stationary phase was harvested. During a subsequent second extraction step contained 18% (w/w) PEG 300 and 14% (w/w) phosphate, a proportion of RNA, which was also concentrated during the column process, was removed. It was demonstrated that the recovery of pDNA in the second bottom phase was 89.4%, which was similar to the initial recovery after column extraction (92.1%).     

Purification of recombinant cutinase by extraction in an aqueous two-phase system facilitated by a fatty acid substrate

Purification of recombinant wild-type cutinase from the culture supernatant of Saccharomyces cerevisiae by extraction in aqueous two-phase system was investigated. The partition of the enzyme in a polyethylene glycol (PEG)-potassium phosphate system to the top phase was increased with lower molecular weight PEG. Enzyme partition in a 20% PEG/15% phosphate two-phase system was studied in the presence of detergents, fatty acids, and alcohols, respectively. Addition of 0.5% (w/w) butyrate increased the partition coefficient from 17 to 135 and the purification factor from 10 to 23. The effect of butyrate was also confirmed by using the countercurrent mode of extraction. Recovery of cutinase from the top phase was achieved by a secondary extraction into a new salt phase at a lower pH or a lower temperature. A specific interaction of butyrate to the active site of the enzyme was demonstrated by fluorescence spectroscopy. Size exclusion chromatography showed the cutinase-butyrate complex to be over two times the size of the free enzyme.     

Downstream processing of luciferase from fireflies (Photinus pyralis) using aqueous two-phase extraction

The firefly luciferase has been extensively used for sensitive detection of bacteria, gene expression and environmental toxins (biosensors). The aim of the present study was to design a simple and more efficient method for the purification and concentration of luciferase using aqueous two-phase extraction (ATPE). Downstream processing of luciferase from North American Firefly Photinus pyralis was carried out, for the first time, using polymer/salt aqueous two phase system (ATPS) at 4 °C. The enzyme was observed to preferentially partition to the polyethylene glycol (PEG) rich top phase. The best results of purification (13.69 fold) and enzyme activity recovery (118.34%) were observed in the system containing 4.0% (w/w) PEG (1500) and 20.5% (w/w) (NH₄)₂SO₄ with a phase volume ratio of 0.21.     

Separation of recombinant β-glucuronidase from transgenic tobacco by aqueous two-phase extraction

Transgenic plants hold many promises as viable production hosts for therapeutic recombinant proteins. Many efforts have been devoted to increase the expression level of the proteins, but the efforts for developing economic processes to purify those proteins are lacking. In this report, aqueous two-phase extraction (ATPE) was investigated as an alternative for the separation of an acidic recombinant protein, β-glucuronidase (rGUS), from transgenic tobacco. Screening experiments by fractional factorial designs showed that PEG concentration and ionic strength of the system significantly affected the partitioning of native tobacco proteins and GUS. Response surface methodology was used to determine an optimized aqueous two-phase system for the purification of rGUS from transgenic tobacco. In a 13.4% (w/w) PEG 3400/18% (w/w) potassium phosphate system, 74% of the rGUS was recovered in the top PEG-rich phase while more than 90% of the native tobacco proteins were removed in the interphase and the bottom phase. A purification factor of about 20 was achieved in this process. The most important impurity from tobacco, Rubisco, was largely removed from the rGUS in the recovered phase.     

Rotavirus-like particles primary recovery from insect cells in aqueous two-phase systems

Virus-like particles have a wide range of applications, including vaccination, gene therapy, and even as nanomaterials. Their successful utilization depends on the availability of selective and scalable methods of product recovery and purification that integrate effectively with upstream operations. In this work, a strategy based on aqueous two phase system (ATPS) was developed for the recovery of double-layered rotavirus-like particles (dlRLP) produced by the insect cell-baculovirus expression system. Polyethylene glycol (PEG) molecular mass, PEG and salt concentrations, and volume ratio (Vr, volume of top phase/volume of bottom phase) were evaluated in order to determine the conditions where dlRLP and contaminants concentrated to opposite phases. Two-stage ATPS consisting of PEG 400-phosphate with a Vr of 13.0 and a tie-line length (TLL) of 35% (w/w) at pH 7.0 provided the best conditions for processing highly concentrated crude extract from disrupted cells (dlRLP concentration of 5 microg/mL). In such conditions intracellular dlRLP accumulated in the top phase (recovery of 90%), whereas cell debris remained in the interface. Furthermore, dlRLP from culture supernatants accumulated preferentially in the interface (recovery of 82%) using ATPS with a Vr of 1.0, pH of 7.0, PEG 3350 (10.1%, w/w) and phosphate (10.9%, w/w). The purity of dlRLP from culture supernatant increased up to 55 times after ATPS. The use of ATPS resulted in a recovery process that produced dlRLP with a purity between 6 and 11% and an overall product yield of 85% (w/w), considering purification from intracellular and extracellular dlRLP. Overall, the strategy proposed in this study is simpler than traditional methods for recovering dlRLP, and represents a scalable and economically viable alternative for production processes of vaccines against rotavirus infection with significant scope for generic commercial application.     

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.     

Extractive cultivation of Lactococcus lactis using a polyethylene glycol/MgSO4 · 7H2O aqueous two-phase system to produce nisin

To relieve lactic acid inhibition, an aqueous two-phase system (ATPS) was used to grow Lactococcus lactis. Its composition was 11% (w/v) PEG 20000/3.5% (w/v) MgSO₄ 7H₂O. In this ATPS medium, the cells were completely partitioned in the bottom phase, and lactic acid had the biggest partition coefficient of the eight ATPS media tested. The cell biomass in this medium was 0.64 mg ml^–1, only 60% of that of the control medium, but nisin production (803 IU ml^–1) was enhanced by 33%. The increase in nisin was explained as a result of extraction of lactic acid from the bottom phase to the top one. The changes of tie-line length and phase volume ratio for the identical tie line could affect cell growth and nisin accumulation.     

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.     

用双水相系统从大肠杆菌细胞内释放L-天门冬酰胺酶

聚乙二醇－磷酸盐双水相系统可以释放大肠杆菌ＡＴＣＣ１１３０３细胞内的Ｌ－天门冬酰胺酶。研究了聚乙二醇、磷酸氢二钾的浓度对酶和蛋白质的释放及分配的影响。在双水相系统中加入适量的盐酸胍和ＴｒｉｔｏｎＸ１００可以提高酶的释放量。实验表明,用新的下相代替富含酶的下相,上相能够重复使用几次。这种方法将酶的释放和萃取结合为一个步骤,使酶的纯化更加简单和有效