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.     

Production of immobilized penicillin acylase using aqueous polymer systems for enzyme purification and in situ immobilization

A novel approach for the isolation and purification of penicillin acylase (PA), which couples aqueous two-phase partitioning and enzyme immobilization has been investigated.A PA yield of 90% was achieved by treating E. coli cells with 4% butyl acetate, freeze-thawing step, and pressure homogenization. PA purification (93% recovery) was achieved by (1) removing cell debris via precipitation with polyethylene glycol (PEG 2000); (2) aqueous two-phase partitioning using a PEG 2000 + phosphate system (87% recovery).An in situ enzyme immobilization approach, using oxirane acrylic or aldehyde-agarose beads dispersed in the PEG-rich phase, was explored for the conversion of penicillin G to 6-aminopenicillanic acid. An appropriate immobilization reaction time was found. The catalytic performance of the enzyme, when immobilized, was found not to be affected by recycling of the phase-forming components.     

Bioprocess intensification: a potential aqueous two-phase process for the primary recovery of B-phycoerythrin from Porphyridium cruentum

A process for the primary recovery of B-phycoerythrin from Porphyridium cruentum exploiting aqueous two-phase systems (ATPS) was developed in order to reduce the number of unit operations and benefit from an increased yield of the protein product. The evaluation of system parameters such as poly(ethylene glycol) (PEG) molecular mass, concentration of PEG as well as salt, system pH and volume ratio was carried out to determine under which conditions the B-phycoerythrin and contaminants concentrate to opposite phases. PEG 1450-phosphate ATPS proved to be suitable for the recovery of B-phycoerythrin because the target protein concentrated to the top phase whilst the protein contaminants and cell debris concentrated in the bottom phase. An extraction ATPS stage comprising volume ratio (Vr) equal to 1.0, PEG 1450 24.9% (w/w), phosphate 12.6% (w/w) and system pH of 8.0 allowed B-phycoerythrin recovery with a purity of 2.9 (estimated as the relation of the 545-280 nm absorbances). The use of ATPS resulted in a primary recovery process that produced a protein purity of 2.9 +/- 0.2 and an overall product yield of 77.0% (w/w). The results reported demonstrated the practical implementation of ATPS for the design of a primary recovery process as a first step for the commercial purification of B-phycoerythrin produced by P. cruentum.     

海洋真菌Caldariomyces fumago产氯过氧化物酶的双水相提纯条件

采用聚乙二醇(PEG6000)在(NH4)2SO4高饱和度下沉淀夹带蛋白质富集氯过氧化物酶,再利用磷酸盐溶液复溶解共沉淀物形成的双水相萃取体系高浓度回收酶蛋白,最后再经Sephadex G100柱层析纯化获得高纯度酶试样。结果显示:氯过氧化物酶与PEG共沉淀总活力回收率达85.5%,酶在优化的PEG/磷酸盐双水相系统中上下相分配系数k在0.341以下,酶活力回收率达到69.1%,纯度提高了21.57倍,柱层析可使酶纯度进一步提高到24.79倍,总回收率为37.75%。     

A Process for large-scale isolation of beta-galactosidase from E. coli in an aqueous two-phase system

A method suitable for large-scale isolation of beta-galactosidase from a suspension of disintegrated E. coli cells has been developed. In an aqueous two-phase system consiting of PEG 6000 and potassium phosphate, all cell debris and the major part of the proteins and nucleic acids were partitioned to the denser salt phase. Seventy-five percent of the beta-galactosidase was recovered in the lighter PEG phase, giving a purification ratio of about 12.     

Free polymeric bioligands in aqueous two-phase affinity extractions of microbial xylanases and pullulanase

Two reversibly soluble-insoluble polymers (viz. Eudragit S-100 and alginate) were used as free macroaffinity bioligands in polyethylene glycol (PEG)/salt two-phase systems for separation of enzymes. Incorporation of Eudragit S-100 and alginate in the PEG phase led to considerable selectivity in separation of microbial xylanases and pullulanase, respectively. Xylanase from Aspergillus niger was recovered 93% with 56-fold purification, whereas the enzyme from Trichoderma reesei and Bacillus amyloliquefaciens was obtained with 93% activity recovery (31-fold purification) and 90% activity recovery (32-fold purification), respectively. From Bacillus acidopullulyticus pullulanase, 85% enzyme activity recovery with 44-fold purification was obtained. The approach described here shows the potential of developing into a general approach for use of reversibly soluble-insoluble macroaffinity ligand in two-phase affinity extraction.     

Optimisation of aqueous two-phase extraction of human antibodies

The purification of human antibodies in an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) 6000 and phosphate was optimised by surface response methodology. A central composite design was used to evaluate the influence of phosphate, PEG and NaCl concentration and of the pH on the purity and extraction yield of IgG from a simulated serum medium. The conditions that maximise the partition of IgG into the upper phase were determined to be high concentrations of NaCl and PEG, low concentrations of phosphate and low pH values. An ATPS composed of 12% PEG, 10% phosphate, 15% NaCl at pH 6 was further used to purify human monoclonal antibodies from a Chinese Hamster Ovary (CHO) concentrated cell culture supernatant with a recovery yield of 88% in the upper PEG-rich phase and a purification factor of 4.3. This ATPS was also successfully used to purify antibodies from a hybridoma cell culture supernatant with a recovery yield of 90% and a purification factor of 4.1.     

两相分配法制备南极红酵母质膜

用葡聚糖T-500(Dextran T-500)和聚乙二醇(PEG-3350)两相体系制备南极红酵母(Rhodotorula sp.)菌株 NJ298的质膜。首先在2 mmol/L KCl浓度下, 选用5种不同的聚合物浓度(5.6%、5.8%、6.0%、6.2%、6.4 %, W/W), 研究了NJ298质膜在两相体系中的分配情况, 在此基础上进一步研究了KCl浓度(2 mmol/L、4 mmol/L、6 mmol/L、8 mmol/L、10 mmol/L)对NJ298质膜的纯度及得率的影响。结果表明, 选用6.0%聚合物浓度, 4 mmol/L KCl的两相分配体系, 分离3次可得到相对纯度在78.2%的南极红酵母质膜组分, 标志酶鉴定及磷钨酸染色电镜检测均表明获得了高纯度密实的正向型的质膜囊泡。这为进一步研究该菌株的南极极端环境适应机制奠定了基础。     

两相分配法制备南极红酵母质膜

用葡聚糖 T-500(Dextran T-500)和聚乙二醇(PEG-3350)两相体系制备南极红酵母(Rhodotorula sp.)菌株 NJ298 的质膜.首先在 2 mmol/L KCl 浓度下,选用5种不同的聚合物浓度(5.6%、5.8%、6.0%、6.2%、6.4%,W/W),研究了 NJ298 质膜在两相体系中的分配情况,在此基础上进一步研究了 KCl 浓度(2 mmol/L、4 mmol/L、6 mmol/L、8 mmol/L、10 mmol/L)对 NJ298 质膜的纯度及得率的影响.结果表明,选用6.0%聚合物浓度,4 mmol/LKCl 的两相分配体系,分离3次可得到相对纯度在 78.2%的南极红酵母质膜组分,标志酶鉴定及磷钨酸染色电镜检测均表明获得了高纯度密实的正向型的质膜囊泡.这为进一步研究该菌株的南极极端环境适应机制奠定了基础.     

Downstream processing of human antibodies integrating an extraction capture step and cation exchange chromatography

In this paper we explore an alternative process for the purification of human antibodies from a Chinese hamster ovary (CHO) cell supernatant comprising a ligand-enhanced extraction capture step and cation exchange chromatography (CEX). The extraction of human antibodies was performed in an aqueous two-phase system (ATPS) composed of dextran and polyethylene glycol (PEG), in which the terminal hydroxyl groups of the PEG molecule were modified with an amino acid mimetic ligand in order to enhance the partition of the antibodies to the PEG-rich phase. This capture step was optimized using a design of experiments and a central composite design allowed the determination of the conditions that favor the partition of the antibodies to the phase containing the PEG diglutaric acid (PEG-GA) polymer, in terms of system composition. Accordingly, higher recovery yields were obtained for higher concentrations of PEG-GA and lower concentrations of dextran. The highest yield experimentally obtained was observed for an ATPS composed of 5.17% (w/w) dextran and 8% (w/w) PEG-GA. Higher purities were however predicted for higher concentrations of both polymers. A compromise between yield and purity was achieved using 5% dextran and 10% PEG-GA, which allowed the recovery of 82% of the antibodies with a protein purity of 96% and a total purity of 63%, determined by size-exclusion chromatography. ATPS top phases were further purified by cation exchange chromatography and it was observed that the most adequate cation exchange ligand was carboxymethyl, as the sulfopropyl ligand induced the formation of multi-aggregates or denatured forms. This column allowed the elution of 89% of the antibodies present in the top phase, with a protein purity of 100% and a total purity of 91%. The overall process containing a ligand-enhanced extraction step and a cation exchange chromatography step had an overall yield of 73%.     

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

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

Factors influencing the yield and purity of goat sperm plasma membranes isolated by means of an aqueous two-phase polymer system

An improved aqueous two-phase polymer method has been developed for the isolation of sperm plasma membranes by manipulating various parameters that influence markedly the purity as well as yield of the membrane. The method consists of hypotonic shock of intact spermatozoa with 1.25 mM EDTA to dissociate the plasma membrane and dispersion of these cells to a two-phase polymer system consisting of 5.5% 252-Kd dextran and 4.2% 20-Kd polyethylene glycol prior to centrifugation at 9700 X g for 30 min when the two polymer phases are separated; the membrane fraction sediments at the interphase. The resulting membrane fraction was purified further by repeating the two-phase fractionation step. The yield of the membranes was approx. 35-40%, based on the recovery of the membrane-bound marker enzymes alkaline phosphatase and 5'-nucleotidase. The isolated membranes showed a high degree of purity as evidenced by phase contrast and electron microscopic studies and analyses of marker enzymes characteristic of cellular organelles. The yield and purity of the membranes have been found to be markedly dependent on the conditions of the hypotonic shock, obtained as a function of, EDTA concentration and on the molecular sizes of the dextran and polyethylene glycol that constitute the two-phase polymer system, as well as on the centrifugal force used for the sedimentation of the membrane.     

Recovery of the proteose peptone component 3 from cheese whey in Reppal PES 100/polyethylene glycol aqueous two-phase systems

Recovery of the proteose peptone component 3 from cheese whey was optimal using a 16% (w/w) Reppal PES 100 – 24% (w/w) PEG 600 aqueous two-phase system, at pH 7, giving a mass recovery yield of 99% and a purity of 83% for proteose peptone component 3 in the upper phase. Using the above system a partition coefficient of 30.7 and a purification factor of 6.9 were achieved.     

Application of a PEG/salt aqueous two-phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract

Aqueous two-phase partition systems (ATPS) have been widely used for the separation of a large variety of biomolecules. In the present report, the application of a polyethylene glycol/phosphate (PEG/phosphate) ATPS for the separation of anti-HIV monoclonal antibodies 2G12 (mAb 2G12) and 4E10 (mAb 4E10) from unclarified transgenic tobacco crude extract was investigated. Optimal conditions that favor opposite phase partitioning of plant debris/mAb as well as high recovery and purification were found to be 13.1% w/w (PEG 1500), 12.5% w/w (phosphate) at pH 5 with a phase ratio of 1.3 and 8.25% w/w unclarified tobacco extract load. Under these conditions, mAb 2G12 and mAb 4E10 were partitioned at the bottom phosphate phase with 85 and 84% yield and 2.4- and 2.1-fold purification, respectively. The proposed ATPS was successfully integrated in an affinity-based purification protocol, using Protein A, yielding antibodies of high purity and yield. In this study, ATPS was shown to be suitable for initial protein recovery and partial purification of mAb from unclarified transgenic tobacco crude extract.     

A two-phase polymer method for isolation of maturing goat sperm plasma membrane

An aqueous two-phase polymer method originally developed for the isolation of plasma membrane from mature goat epididymal spermatozoa (Rana, A.P.S. and Majumder, G.C., Prep. Biochem., 17, 261, 1987) has been found to be unsuitable for the maturing spermatozoa derived from caput and corpus epididymides because of significant contamination of the isolated membrane with intact cells. A modified method has been developed by manipulating the centrifugal force (required for membrane sedimentation) for the isolation of maturing sperm plasma membrane of high yield (approximately 55%) and purity as judged by marker enzyme assays and phase contrast and electron microscopic analyses. The method consists of treatment of intact spermatozoa with 1.25 mM EDTA, dispersion of these cells to a two-phase polymer system comprising 5.5% 252-Kd dextran and 4.2% 20-Kd polyethylene glycol compound and subsequent centrifugation at 12,000 X g for 30 min when the two phases separate out and membranes sediment at the interphase. The repeatation of the two-phase fractionation step yielded greater purity of the plasma membrane.     

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.     

Liquid-liquid extraction of a Schistosoma mansoni recombinant protein from an Escherichia coli extract in 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 composed of polyethylene glycol (PEG) and potassium phosphate. 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 were determined by ELISA. The system composed of PEG 3550 (19.7% w/w) and potassium phosphate (17.7% w/w) led to a yield of 59% and an antigen purification factor of 3 in the PEG-rich phase. It was observed that the antigen partition in ATPS was strongly affected by the pH value and tie line length. In addition, it was possible in a single step, to remove the cell debris, that precipitated at the interface of the 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.     

Purification and aqueous two-phase partitioning properties of recombinant Vitreoscilla hemoglobin.

Soluble recombinant Vitreoscilla hemoglobin was purified from E. coli lysate by sequential two-phase extraction techniques. Extraction of lysate containing VHb in PEG/dextran gave a 3.6-fold increase in VHb purity in the PEG-rich phase via a size exclusion mechanism. Further extraction of the recovered PEG phase in PEG/sodium sulfate gave an additional 2.0-fold increase in purity in the PEG-rich phase due to an electrostatic mechanism. Final extraction of the PEG phase in PEG/magnesium sulfate gave an additional 1.3-fold increase in VHb purity in the magnesium sulfate-rich phase. The final yield from the extractive purification was 47% with purity of VHb estimated to be greater than 95%. Yields from the sulfate salt extractions are essentially quantitative due to the extreme partitioning behavior of VHb in these systems. VHb partition coefficients as large as 46 in PEG/sodium sulfate and as small as 0.06 in PEG/magnesium sulfate were observed. Similar small partition coefficients were obtained with PEG/manganese sulfate extractions. This dramatic effect of divalent cation content on the partition coefficient of VHb in PEG/sulfate salt systems was investigated by pH and magnesium ion titration experiments. Results show the effect to be largest and nearly constant for pH values greater than 6.0 and diminished at lower pH values. A model based on magnesium ion binding to negatively charged amino acids is shown to correlate with the data well. Based on model formulation and the partitioning behavior of contaminant proteins, the observed effect is expected to be applicable to other proteins.     

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.