| 1. |
- Andersson, M, et al.
(författare)
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Determination of the pore-size distribution in gels
- 1995
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Ingår i: Bioseparation. - 1573-8272. ; 5:2, s. 65-72
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Tidskriftsartikel (refereegranskat)abstract
- A method for determination of the accessible volume fraction in gels as function of the molecular weight of the solutes is presented. The pore-size distribution is determined by measuring the rate of diffusion of a mixture of solutes into a gel using gel filtration for separation. The solutes, of various sizes, are detected by refractive index measurements. Two marker molecules (blue dextran and glucose) were used to determine the gel void and the amount of liquid adhering to the surface. The technique is simple and can easily be adapted to other systems of a porous nature (membranes, catalyst pellets etc.). The method is applied to an N-isopropylacrylamide gel. This gel is sensitive to temperature changes. A considerable increase in volume is obtained when the temperature is decreased. This makes it suitable for use as a separation agent in gel extraction. In order to assess the performance of this unit operation the pore size distribution for the N-isopropylacrylamide gel was determined at 10 degrees C, 20 degrees C and 30 degrees C, using mixtures of different dextrans as well as different polyethylene glycols.
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| 2. |
- Berggren, K., et al.
(författare)
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Peptide fusion tags with tryptophan and charged residues for control of protein partitioning in PEG-potassium phosphate aqueous two-phase systems
- 2000
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Ingår i: Bioseparation (Dordrecht). - 0923-179X .- 1573-8272. ; 9:2, s. 69-80
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Tidskriftsartikel (refereegranskat)abstract
- A partition study with peptides and recombinant proteins in poly(ethylene glycol)4000-potassium phosphate aqueous two-phase systems has been performed. The aim was to study to what extent the insertion of charged residues could affect protein partition in addition to the already observed effects of tryptophan residues. The model proteins used are based on a staphylococcal protein A derivative, Z, and modified by the insertion of peptide tags close to the C-terminus. The tags differed with respect to their content of both Trp, negatively (Asp) and positively charged (Lys) amino acid residues. The same partitioning trends were observed for the peptides and fusion proteins. The effect of Trp residues was to direct the partitioning towards the PEG phase. The insertion of two negatively charged (Asp) residues into a Trp(4)-tag enhanced the partition towards the PEG phase even more. The introduction of positively charged (Lys) residues in addition to Trp residues, on the other hand, pulled the peptide or protein towards the potassium phosphate phase. The partitioning of peptides gave a good qualitative picture of the effect of the peptide on partitioning when fused to the protein. The efficiencies of the tags were calculated based on partitioning of tags and fusion proteins, and tag efficiencies generally varied between 60 and 85%.
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| 3. |
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| 4. |
- Brumbauer, Anikó, et al.
(författare)
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Fractionation of cellulase and beta-glucosidase in a Trichoderma reesei culture liquid by use of two-phase partitioning
- 1999
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Ingår i: Bioseparation. - 1573-8272. ; 7:6, s. 287-295
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Carlsson, M, et al.
(författare)
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Partitioning in Aqueous Polymer 2-phase Systems .1. Modeling of Affinity Partition
- 1995
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Ingår i: Bioseparation. - 1573-8272. ; 5:3, s. 155-166
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Tidskriftsartikel (refereegranskat)abstract
- Various factors influencing affinity partitioning in aqueous polymer two-phase systems are studied by model calculations using a self-consistent mean-field lattice theory. The latter is an extension of the theory of Scheutjens and Fleer, adapted to take affinity ligand binding into account. The dependence of partition coefficients on ligand concentration is studied for different binding strengths, polymer lengths, and polymer-protein interactions, and the effects of the ligand being attached at different positions of the polymer chain are investigated. The mechanism of the affinity partitioning is discussed. In particular, at saturation the enhanced partitioning by the presence of the ligand substituted polymers should be regarded as an expulsion from the minority phase rather than an attraction to the majority phase. Moreover, the results are put into relation with the multiple-equilibriascheme that has frequently been used to analyse affinity partition data, and comparisons with experimental findings are made. Some important principles for application of affinity partitioning concluded from the modelling results are: i) A higher selectivity can be expected if a polymer which has in general repulsive interactions with proteins is chosen as ligand-carrying polymer, ii) a higher Delta(lnK)(max) is expected for a longer ligand-carrying polymer as compared to a shorter one. With increased polymer length higher ligand concentration is however needed to reach the plateau value. iii) the ligand bound to the end of the polymer gives a higher Delta(lnK)(max) than when bound to the middle of the chain. iv) having both polymer ends carrying ligand should actually lead to a decrease in the Delta(lnK)(max). However, at quite low ligand concentrations the doubly substituted ligand-carrier should be more effective.
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| 6. |
- Farkas, T, et al.
(författare)
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Partitioning of beta-mannanase and alpha-galactosidase from Aspergillus niger in Ucon/Reppal aqueous two-phase systems and using temperature-induced phase separation
- 1996
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Ingår i: Bioseparation. - 1573-8272. ; 6:3, s. 147-157
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Tidskriftsartikel (refereegranskat)abstract
- Enzyme partitioning and recovery with a new aqueous two-phase system based on commercially available hydroxypropyl starch Reppal PES 200 and the thermo-separating polymer Ucon 50-HB-5100 was studied. Ucon is an ethylene oxide-propylene oxide random copolymer. A culture supernatant of Aspergillus niger containing extracellular beta-mannanase and alpha-galactosidase was partitioned in two steps. The primary aqueous two-phase system contained Ucon and Reppal as phase forming polymers. The effect on enzyme partitioning of salt composition, salt concentration, pH and polymer concentration was studied with the aim of obtaining optimal partitioning of target enzymes to the phase containing the thermoseparating Ucon polymer. The partitioning of the enzymes could be strongly influenced by addition of the hydrophobic triethyl ammonium ion and the chaotropic perchlorate ion. Also the effect of cationic surfactant, cetyl trimethyl ammonium bromide, on enzyme partitioning was studied. In the second step, temperature induced phase separation was carried out on the isolated Ucon phase. A water phase and a concentrated aqueous Ucon phase were formed. The enzymes were obtained in the water phase almost free of polymer.
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| 7. |
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| 8. |
- Johansson, Hans-Olof, et al.
(författare)
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Separation of amino acids and peptides by temperature induced phase partitioning. Theoretical model for partitioning and experimental data
- 1999
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Ingår i: Bioseparation. - 1573-8272. ; 7:4-5, s. 259-267
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Tidskriftsartikel (refereegranskat)abstract
- There is a strong interest in use of `smart polymers' in separation systems. These are polymers which can react on external influence, such as temperature or pH change. With such polymers it is possible from the outside to affect the properties of a separation system. Amphiphilic copolymers show drastic changes in solubility properties, such as self-association and phase separation, at e.g. temperature increase. The random copolymers of ethylene oxide and propylene oxide units (EOPO-polymers) can form aqueous two-phase systems above the copolymer cloud point temperature. Two phases are formed, one consisting of 40-60% polymer in water and the other of almost 100% water. Amino acids and peptides can be partitioned in the thermoseparating systems. The partitioning strongly depends on the solute hydrophobicity, where aromatic amino acids and peptides are partitioned to the polymer phase and hydrophilic to the water phase. Salt effects can be used to enhance the partitioning of charged molecules. The thermodynamic driving forces which govern the partitioning of molecules in a thermoseparated aqueous phase system is described with use of the Flory-Huggins theory for polymer solutions. Expressions are derived which show the entropic and enthalpic effects on solute partitioning.
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| 9. |
- Kaul, Rajni, et al.
(författare)
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Secondary purification
- 1992
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Ingår i: Bioseparation. - 1573-8272. ; 3:1, s. 1-26
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Forskningsöversikt (refereegranskat)
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| 10. |
- Lozinsky, Vladimir, et al.
(författare)
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The potential of polymeric cryogels in bioseparation
- 2001
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Ingår i: Bioseparation. - 1573-8272. ; 10:4-5, s. 163-188
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Tidskriftsartikel (refereegranskat)abstract
- This is a review discussing the production and properties of cryogels (from the Greek κριoσ (kryos) meaning frost or ice), immobilization of ligands in cryogels and the application of affinity cryogels in bioseparation. Cryotropic gel formation proceeds in a non-frozen liquid microphase existing in the macroscopically frozen sample. Due to the cryoconcentration of gel precursors in the non-frozen liquid microphase, cryogelation is characterised by a decrease in the critical concentration of gelation and an increase in gelation rates compared with traditional gelation at temperatures above freezing point. Cryogels can be obtained through the formation of both physically and covalently cross-linked heterogeneous polymer networks. Interconnected systems of macropores and sponge-like morphology are typical for cryogels, allowing unhindered diffusion of solutes of practically any size. Most of the water present in spongy cryogels is capillary bound and can be removed mechanically by squeezing. The properties of cryogels can be regulated by the temperature of cryogelation, the time the sample is kept in a frozen state and freezing/thawing rates, by the nature of the solvent and by the use of soluble and insoluble additives. The unique macroporous morphology of cryogels, in combination with osmotic, chemical and mechanical stability, makes them attractive matrices for chromatography of large entities such as protein aggregates, membrane fragments, viruses, cell organells and even whole cells. Special attention is given to immunosorption of viruses on cryogel-based sorbents. As chromatographic materials, cryogels can be used both in bead form and as spongy cylindrical blocks (monoliths) synthesized inside the chromatographic column. The macroporous nature of cryogels is also advantageous for their application as matrices in the immobilization of biocatalysts operating in both aqueous and organic solvents. New potential applications of cryogels are discussed.
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