Enzyme purification using temperature-induced phase formation.

A new type of aqueous two-phase system composed of an ethylene oxide and propylene oxide random co-polymer, UCON 50-HB-5100, as the upper phase polymer and either dextran or hydroxypropyl starch as the lower phase polymer has been characterized and used to purify 3-phosphoglycerate kinase (EC 2.7.2.3) and hexokinase (EC 2.7.1.1) from bakers' yeast. The UCON 50-HB-5100 polymer has a cloud point of 55 degrees C at which temperature it phase separates from water. This cloud point can be lowered to 40 degrees C by the addition of 0.2 M sodium sulfate salt. The low cloud point of this UCON polymer makes it possible to obtain the target enzymes in a water and buffer solution, and to recover and recycle the UCON 50-HB-5100 polymer. The phase diagrams for the systems UCON 50-HB-5100/Dextran T500 and UCON 50-HB-5100/hydroxypropyl starch have been determined. Yeast homogenate was first partitioned in a system composed of a top phase containing UCON 50-HB-5100 and a bottom phase containing either dextran or hydroxypropyl starch. The top phase containing the enzyme free of cell debris was removed and the temperature increased above the cloud point of the UCON until a new two phase system composed of water as the top phase and a concentrated liquid UCON 50-HB-5100 bottom phase was formed. The water phase containing the enzyme was removed and the bottom phase containing the UCON 50-HB-5100 could be recycled to perform a second extraction.     

Synthesis of dye conjugates of ethylene oxide-propylene oxide copolymers and application in temperature-induced phase partitioning.

Synthesis of conjugates of the ethylene oxide/propylene oxide copolymer UCON 50-HB-5100 and the triazine dyes Cibacron Blue F3G-A and Procion Yellow HE-3G is described. The UCON-dye conjugate of Procion Yellow HE-3G is used as a ligand for affinity partitioning of glucose-6-phosphate dehydrogenase from bakers' yeast. The enzyme is first partitioned in a two-phase system composed of UCON, UCON-ligand and dextran, and the two phases isolated in separate containers. A small amount of salt is then added to the upper phase, which contains the UCON-ligand-enzyme complex, and the temperature increased above the cloud point of the UCON polymer to give a new two-phase system. The new two-phase system consists of an upper salt/water phase containing free enzyme and a lower UCON/water phase containing free UCON-ligand. Temperature-induced phase partitioning is thus seen to be of much assistance in dissociating enzyme-ligand complex, recovering enzyme and recycling UCON-ligand.     

Preparation of benzoyl dextran and its use in aqueous two-phase systems.

The graft modification of dextran with benzoyl groups has been studied. The factors that affect the degree of substitution of benzoyl dextran were investigated. Phase diagrams for aqueous two-phase systems composed of polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran have been determined. Phase separation was also obtained in aqueous solution of two benzoyl dextran polymers with different degrees of substitution. A four-phase system was obtained with a mixture of polyethylene glycol, dextran and two kinds of benzoyl dextrans. The partitioning of methylene blue and a Procion yellow HE-3G dextran derivative were studied in polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran two-phase systems and in systems of two benzoyl dextrans differing in degree of substitution. The proteins bovine serum albumin and glucose-6-phosphate dehydrogenase were partitioned in polyethylene glycol/benzoyl dextran aqueous two-phase systems and the effect of the degree of substitution of benzoyl dextran was studied. Chlorella pyrenoidosa, thylakoid membrane vesicles, plasma membrane vesicles and chloroplasts were partitioned in polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran two-phase systems, and in a polyethylene glycol/dextran/benzoyl dextran four-phase system.     

Partition of polysaccharide-coated liposomes in aqueous two-phase systems

Hydrophobized polysaccharides such as cholesterol-bearing pullulan (CHP), dextran (CHD) and mannan (CHM) effectively coat the liposomal surface. Partition of the hydrophobized polysaccharide-coated liposomes in an aqueous two-phase system (PEO (top)/pullulan (bottom) or PEO (top)/dextran (bottom)) was investigated (PEO = poly(ethylene oxide)). Conventional liposomes without a polysaccharide coat mostly locate at the interface between the two polymer phases. The polysaccharide-coated liposomes, on the other hand, were partly partitioned to the bottom polysaccharide phase depending on the structure of the hydrophobized polysaccharide on the liposomal surface. The affinity between the polysaccharide on the liposomal surface and that in the bulk bottom phase controls the efficiency of partition. The sequence of interaction strength between the two carbohydrates as the following: for the PEO/dextran two-phase system, dextran_(liposome)-dextran_(bulk) > mannan_(liposome)-dextran_(bulk) > pullulan_(liposome)-dextran_(bulk); while for the PEO/pullulan system, the sequence of interaction strength was pullulan_(liposome)-pullulan_(bulk) > dextran_(liposome)-pullulan_(bulk) ≈ mannan_(liposome)-pullulan_(bulk).     

Thermoseparating water/polymer system: a novel one-polymer aqueous two-phase system for protein purification

In this study we show that proteins can be partitioned and separated in a novel aqueous two-phase system composed of only one polymer in water solution. This system represents an attractive alternative to traditional two-phase systems which uses either two polymers (e.g., PEG/dextran) or one polymer in high-salt concentration (e.g., PEG/salt). The polymer in the new system is a linear random copolymer composed of ethylene oxide and propylene oxide groups which has been hydrophobically modified with myristyl groups (C(14)H(29)) at both ends (HM-EOPO). This polymer thermoseparates in water, with a cloud point at 14 degrees C. The HM-EOPO polymer forms an aqueous two-phase system with a top phase composed of almost 100% water and a bottom phase composed of 5-9% HM-EOPO in water when separated at 17-30 degrees C. The copolymer is self-associating and forms micellar-like structures with a CMC at 12 microM (0.01%). The partitioning behavior of three proteins (lysozyme, bovine serum albumin, and apolipoprotein A-1) in water/HM-EOPO two-phase systems has been studied, as well as the effect of various ions, pH, and temperature on protein partitioning. The amphiphilic protein apolipoprotein A-1 was strongly partitioned to the HM-EOPO-rich phase within a broad-temperature range. The partitioning of hydrophobic proteins can be directed with addition of salt. Below the isoelectric point (pI) BSA was partitioned to the HM-EOPO-rich phase and above the pI to the water phase when NaClO(4)was added to the system. Lysozyme was directed to the HM-EOPO phase with NaClO(4), and to the water phase with Na-phosphate. The possibility to direct protein partitioning between water and copolymer phases shows that this system can be used for protein separations. This was tested on purification of apolipoprotein A-1 from human plasma and Escherichia coli extract. Apolipoprotein A-1 could be recovered in the HM-EOPO-rich phase and the majority of contaminating proteins in the water phase. By adding a new water/buffer phase at higher pH and with 100 mM NaClO(4), and raising the temperature for separation, the apolipoprotein A-1 could be back-extracted from the HM-EOPO phase into the new water phase. This novel system has a strong potential for use in biotechnical extractions as it uses only one polymer and can be operated at moderate temperatures and salt concentrations and furthermore, the copolymer can be recovered.     

Type-specific separation of animal cells in aqueous two-phase systems using antibody conjugates with temperature-sensitive polymers.

A new type of aqueous two-phase system (ATPS) has been developed in which a temperature-sensitive polymer, poly-N-isopropylacrylamide [poly (NIPAM)] was used as a ligand carrier for the specific separation of animal cells. Monoclonal antibodies were modified with itaconic anhydride and copolymerized with N-isopropylacrylamide, and the ligand-conjugated carriers were added to the polyethylene glycol 8000-dextran T500 aqueous two-phase systems. The antibody-polymer conjugates were partitioned to the top phase in the absence or presence of 0.15 M NaCl. When ligand-conjugated carriers were used, more than 80% of the cells were specifically partitioned to the top phase in the presence of NaCl up to 0.1 M. The cells were partitioned almost completely to the bottom phase at 0.1 M NaCl or above, when no antibody-conjugate was added in the ATPS. As a model system, CD34-positive human acute myeloid leukemia cells (KG-1) were specifically separated from human T lymphoma cells (Jurkat) by applying anti-CD34 conjugated with poly-N-isopropylacrylamide in the aqueous two-phase system. By the temperature-induced precipitation of the polymer, about 90% of the antibody-polymer conjugates were recovered from the top phase, which gave approximately 75% cell separating efficiency in the next cycle of reuse.     

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.     

Polymer recycling in aqueous two-phase extractions using thermoseparating ethylene oxide–propylene oxide copolymers

This is a study on the recovery and recycling of copolymer in aqueous two-phase systems containing random copolymers of ethylene oxide (EO) and propylene oxide (PO). The random copolymers separate from water solution when heated above the lower critical solution temperature (LCST). The primary phase systems were composed of EOPO copolymer and hydroxypropyl or hydroxyethyl starch. After phase separation the upper EOPO phase was removed and subjected to temperature induced phase separation. Copolymers with different EO/PO compositions have been investigated, EO50PO50 [50% EO and 50% PO (w/w)], EO30PO70 and EO20PO80. The temperature required for thermoseparation decreases when the PO content of the copolymer is increased. The effect on the recovery of copolymer after addition of salts, a second polymer or protein was investigated. The added components increased the recovery of copolymer after thermoseparation, e.g., increased the amount copolymer separated from the water phase after thermoseparation. Recycling of copolymer and measurements of polymer concentrations in the primary top and bottom phases after repeated recycling steps was performed. The fluctuation in polymer concentration of the phases was very small after recycling up to four times. Partitioning of the proteins BSA and lysozyme was studied in primary phase systems after recycling of copolymer. The partition coefficients of total protein and lysozyme was not significantly changed during recycling of copolymer. More than 90% of the copolymer could be recovered in the thermoseparation step by optimising the temperature and time for thermoseparation. In repeated phase partitionings in EOPO–starch systems the EO50PO50 copolymer could be recovered to 77% including losses in primary system and thermoseparation, which is equivalent to a total copolymer reuse of 4.3 times.     

Novel polymer-polymer conjugates for recovery of lactic acid by aqueous two-phase extraction

A new family of polymer conjugates is proposed to overcome constraints in the applicability of aqueous two-phase systems for the recovery of lactic acid. Polyethylene glycol-polyethylenimine (PEI) conjugates and ethylene oxide propylene oxide-PEI (EOPO-PEI) conjugates were synthesized. Aqueous two-phase systems were generated when the conjugates were mixed with fractionated dextran or crude hydrolyzed starch. With 2% phosphate buffer in the systems, phase diagrams with critical points of 3.9% EOPO-PEI-3.8% dextran (DEX) and 3.5% EOPO-PEI-7.9% crude starch were obtained. The phase separation temperature of 10% EOPO-PEI solutions titrated with lactic acid to pH 6 was 35 degrees C at 5% phosphate, and increased linearly to 63 degrees C at 2% phosphate. Lactic acid partitioned to the top conjugate-rich phase of the new aqueous two-phase systems. In particular, the lactic acid partition coefficient was 2.1 in 10% EOPO-PEI-8% DEX systems containing 2% phosphate. In the same systems, the partitioning of the lactic acid bacterium, Lactococcus lactis subsp. lactis, was 0.45. The partitioning of propionic, succinic, and citric acids was also determined in the new aqueous two-phase systems.     

Purification of recombinant and human apolipoprotein A-1 using surfactant micelles in aqueous two-phase systems: recycling of thermoseparating polymer and surfactant with temperature-induced phase separation.

An effective system has been developed for purification of apolipoprotein A-1 from Escherichia coli fermentation solution and human plasma using aqueous two-phase extraction and thermal-phase separation. The system included non-ionic surfactants (Triton or Tween) and as top phase-forming polymer a random copolymer of ethylene oxide (50%) and propylene oxide (50%), Breox PAG 50A 1000, was used. The bottom phase-forming polymer was either hydroxypropyl starch, Reppal PES 100 and PES 200, or hydroxyethyl starch, Solfarex A 85. The top-phase-forming polymer and the surfactants are thermoseparating in water solution, i.e., when heated a water phase and a polymer/surfactant phase are formed. Recombinant apolipoprotein A-1, the Milano variant, was extracted from E. coli fermentation solution in a primary Breox-starch phase system followed by thermal separation of the Breox phase where the target protein was recovered in the water phase. Both in the Breox-starch system and in the water-Breox system Triton X-100 was partitioned to the Breox phase. The addition of non-ionic surfactants to the Breox-starch system had strong effect on the purification and yield of the amphiphilic apolipoprotein A-1. In a system containing 17% Breox PAG 50A 1000, 12% Reppal PES 100 and addition of 1% Triton X-100 the purification factor was 7.2, and the yield 85% after thermal separation of the Breox phase. Recycling of copolymer and surfactant was possible after thermal separation of copolymer phase. Approximately 85% of the copolymer and surfactant could be recycled in each extraction cycle. DNA could be strongly partitioned to the starch phase in the primary-phase system. This resulted in a 1000-fold reduction of E. coli DNA in the apolipoprotein A-1 solution obtained after thermoseparation. In extraction from human plasma containing low concentrations of apolipoprotein A-1, it was possible to reach a purification factor of 420 with 98% yield. By reducing the volume ratio to 0.1 Apo A-1 could be concentrated in a small volume of top phase (concentration factor 10) with a yield of 85% and a purification factor of 110.     

Aqueous micellar two-phase system composed of hyamine-type hydrophobically modified ethylene oxide and application for cytochrome P450 BM-3 separation

The hydrophobically modified ethylene oxide polymer, HM-EO, was modified with an alkyl halide to prepare a hyamine-type HM-EO, named N-Me-HM-EO, which could be used for forming N-Me-HM-EO/buffer aqueous micellar two-phase system. The critical micelle concentration of N-Me-HM-EO solution and the phase diagrams of N-Me-HM-EO/buffer systems were determined. By using this novel aqueous micellar two-phase system, the separation of cytochrome P450 BM-3 from cell extract was explored. The partitioning behavior of P450 BM-3 in N-Me-HM-EO/buffer systems was measured. The influences of some factors such as total proteins concentration, pH, temperature and salt concentration, on the partitioning coefficients of P450 BM-3 were investigated. Since the micellar aggregates in the N-Me-HM-EO enriched phase were positively charged, it was possible to conduct the proteins with different charges to top or bottom phases by adjusting pH and salt concentration in the system. A separation scheme consisting of two consecutive aqueous two-phase extraction steps was proposed: the first extraction with N-Me-HM-EO/buffer system at pH 8.0, and the second extraction in the same system at pH 6.0. The recovery of P450 BM-3 was 73.3% with the purification factor of 2.5. The results indicated that the aqueous micellar two-phase system composed of hyamine modified polysoap has a promising application for selective separation of biomolecules depending on the enhanced electrostatic interactions between micelles and proteins.     

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.     

Magnetic aqueous two-phase separation: a new technique to increase rate of phase-separation, using dextran-ferrofluid or larger iron oxide particles

A new technique to speed up the phase separation of aqueous two-phase systems is described. The technique is based on the addition of magnetically susceptible additives (ferrofluids or iron oxide particles). In a magnetic field such additives will induce a faster phase separation. In one approach, dextran-stabilized ferrofluid was added to an aqueous two-phase system containing polyethylene glycol and dextran. The ferrofluid was totally partitioned to the dextran phase. After mixing of the two-phase system, it was possible to reduce the separation time by a factor of 35 by applying a magnetic field to the system. Another approach involved the use of 1-micron iron oxide particles instead of ferrofluid. In this case also, the phase-separation time was reduced, by a factor of about 70, when the system was placed in a magnetic field. The addition of ferrofluid and/or iron oxide particles was shown to have no influence on enzyme partitioning or on enzyme activity. The partitioning of chloroplasts, on the other hand, was influenced unless the ferrofluid used had been treated with epoxysilane. A column system comprising 15 magnetic separation stages was constructed and was used for semicontinuous separation of enzyme mixtures.     

Magnetic aqueous two-phase separation in preparative applications.

Magnetic aqueous two-phase separation is a new technique to speed up the separation of aqueous two-phase systems (Anal. Biochem. 1987, 167, 331-339). It is based on the addition of magnetically susceptible material (e.g. 1-micron iron oxide particles) which induces rapid phase separation when a mixed system is placed in a magnetic field. The technique has been applied to a number of two-phase systems. The time for phase separation was decreased by a factor of 5-240,000, with the largest improvement for systems containing high concentrations of protein and for systems with viscous or nearly isopycnic phases. An apparatus for preparative multistage extraction with magnetic separation was constructed and tested on glycolytic enzymes present in a yeast extract using a dextran/Cibacron blue-polyethylene glycol system. The presence of iron oxide particles did not adversely affect the extracted enzymes. An electromagnet-based apparatus for continuous phase separation on a larger scale was also designed. A phase system containing crude dextran and unpurified cell homogenate was effectively processed. The apparatus also allowed effective separation when the phase containing iron oxide particles was only a small fraction (4%) of the total phase system.     

Purification of hyperthermophilic archaeal amylolytic enzyme (MJA1) using thermoseparating aqueous two-phase systems

Purification of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii, was investigated in the ethylene oxide-propylene oxide random copolymer (PEO-PPO)/(NH(4))(2)SO(4), and poly(ethylene glycol) (PEG)/(NH(4))(2)SO(4) aqueous two-phase systems. MJA1 partitioned in the top polymer-rich phase, while the remainder of proteins partitioned in the bottom salt-rich phase. It was found that enzyme recovery of up to 90% with a purification factor of 3.31 was achieved using a single aqueous two-phase extraction step. In addition, the partition behavior of pure amyloglucosidase in polymer/salt aqueous two-phase systems was also evaluated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. This work is the first reported application of thermoseparating polymer aqueous two-phase systems for the purification of extremophile enzymes.     

Choice of an aqueous polymer two-phase system for cell partition

Partition of human erythrocytes in aqueous two-phase polymer systems produced by Ficoll and different molecular weight fractions of dextran and polyethylene glycol and the influence of the ionic composition on the cells' partition in the systems was studied. It is found that the Ficoll-dextran-40 system is characterized by a number of advantages as compared with the common dextran-polyethylene glycol system or the others systems under study. The main advantage of the system appears to be that it is possible to concentrate the red cells in the top phase or in the bottom phase of the system, depending on the system ionic composition. The influence of the nature and the concentration of salt additives on this two-phase system formation is examined.     

Fractionation of cellulase and beta-glucosidase in a Trichoderma reesei culture liquid by use of two-phase partitioning

An aqueous two-phase system based on the two polymers poly(ethylene glycol) and dextran has been used for the fractionation of cellulase enzymes present in culture liquid obtained by fermentation with Trichoderma reesei. The activities of beta-glucosidase and glucanases were separated to high degree by using the two-phase systems for a counter-current distribution process in nine transfer steps. While the glucanases had high affinity to the poly(ethylene glycol) rich top phase the beta-glucosidase was enriched in the dextran-containing bottom phase. Multiple counter-current distribution performed indicates the heterogeneity of beta-glucosidase activities assuming at least four isoenzyme forms. One step concentration of beta-glucosidase by using system with 46:1 phase volume ratio resulted in 16 times higher enzyme activity.     

Aqueous two-phase system formed by thermoreactive vinyl imidazole/vinyl caprolactam copolymer and dextran for partitioning of a protein with a polyhistidine tail

A new type of aqueous two-phase system (ATPS) has been developed for application combining two attractive concepts in downstream processing: the immobilised metal affinity partitioning and the use of thermoprecipitating polymers. ATPS consisting of the thermoprecipitating copolymer of N-vinyl caprolactam/1-vinyl imidazole loaded with Cu ions (Cu-poly-VI-VCL) in the top phase and dextran T70 in the bottom phase was used for purification of recombinant lactate dehydrogenase carrying an affinity tag of 6 histidine residues (His-LDH ) from a crude E. coli extract. The enzyme partitioned preferentially into the top Cu-poly-VI-VCL-rich phase. After phase separation, the latter was mixed with EDTA. Temperature increase to 45°C resulted in thermoprecipitation of VCL/VI-polymer, which could subsequently be recycled. His-LDH remained solubilized in the aqueous phase resulting in 8-fold purification and 80 % recovery in a single step.     

Enzymatic hydrolysis of cellulose in aqueous two-phase systems. I. Partition of cellulases from Trichoderma reesei

The partitioning of endo-beta-glucanase, exo-beta-glucanase, and beta-glucosidase from Trichoderma reesei QM 9414 in aqueous two-phase systems has been studied with the object of designing a phase system for continuous bioconversion of cellulose. The partitioning of the enzymes in two-phase systems composed of various water soluble polymeric compounds were studied. Systems based on dextran and polyethylene glycol (PEG) were optimal for one-sidedly partitioning the enzymes to the bottom phase. The influence of polymer molecular weights, polymer concentration, ionic composition of the medium, pH, temperature, and adsorption of the enzymes to cellulose on the enzyme partition coefficients (K) were studied. By combining the effects of polymer molecular weight and adsorption to cellulose, K values could be reduced for endo-beta-glucanase to 0.02 and for beta-glucosidase to 0.005 at 20 degrees C in a phase system of Dextran 40-PEG 40000 in the presence of excess cellulose, At 50 degrees C, K values were increased by a factor of two. In a phase system based on inexpensive crude dextran and PEG, the partition coefficient for endo-beta-glucanase was 0.16 and for beta-glucosidase was 0.14 at 20 degrees C with excess cellulose present.     

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