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At least 883 records · Page 49Linked to original sources
PubMed2001

Acoustic field assisted enhanced demixing of aqueous two-phase systems.

Aqueous two-phase extraction has been recognized as a versatile downstream processing technique for the recovery of biomolecules. A major deterrent to its industrial exploitation is the slow demixing of the two aqueous phases after extraction, due to their similar physical properties. A method to decrease the demixing times of these systems, employing a travelling acoustic wave field, is reported. The effects of phase composition and microbial cells on demixing in a polyethylene glycol/potassium phosphate two-phase system are studied in detail. As phase composition increased, demixing time decreased gradually. Phase volume ratio was found to have a significant effect on demixing time at low phase compositions. However, at intermediate and high phase compositions, only a small effect on demixing time was observed. The effect of phase composition and volume ratio on demixing behavior was explained based on the droplet size of the dispersed phase, which is the resultant effect of the physical properties of the phases. At all the phase compositions studied, the acoustically assisted process decreased the demixing time by 17-60% when compared to demixing under gravity alone. Increasing the cell concentration increased the demixing time markedly in case of yeast cells. However, it remained practically constant in the case of Lactobacillus casei cells. Application of an acoustic field reduced the demixing times up to 60% and 40% in the case of yeast and L. casei cells, respectively. Visual observations indicated that ultrasonication caused mild circulation currents in the phase dispersion enhancing droplet-droplet interaction, which in turn enhanced the rate of coalescence, eventually resulting in an enhanced demixing rate.

Countercurrent Distribution↗
PubMed2002

A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I-hydrophobin I.

Extraction systems for hydrophobically tagged proteins have been developed based on phase separation in aqueous solutions of non-ionic detergents and polymers. The systems have earlier only been applied for separation of membrane proteins. Here, we examine the partitioning and purification of the amphiphilic fusion protein endoglucanase I(core)-hydrophobin I (EGI(core)-HFBI) from culture filtrate originating from a Trichoderma reesei fermentation. The micelle extraction system was formed by mixing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Reppal PES100. The detergent/polymer aqueous two-phase systems resulted in both better separation characteristics and increased robustness compared to cloud point extraction in a Triton X-114/water system. Separation and robustness were characterized for the parameters: temperature, protein and salt additions. In the Triton X-114/Reppal PES100 detergent/polymer system EGI(core)-HFBI strongly partitioned into the micelle-rich phase with a partition coefficient (K) of 15 and was separated from hydrophilic proteins, which preferably partitioned to the polymer phase. After the primary recovery step, EGI(core)-HFBI was quantitatively back-extracted (K(EGIcore-HFBI)=150, yield=99%) into a water phase. In this second step, ethylene oxide-propylene oxide (EOPO) copolymers were added to the micelle-rich phase and temperature-induced phase separation at 55 degrees C was performed. Total recovery of EGI(core)-HFBI after the two separation steps was 90% with a volume reduction of six times. For thermolabile proteins, the back-extraction temperature could be decreased to room temperature by using a hydrophobically modified EOPO copolymer, with slightly lower yield. The addition of thermoseparating co-polymer is a novel approach to remove detergent and effectively releases the fusion protein EGI(core)-HFBI into a water phase.

Cellulase↗
PubMed2002

A new approach based on monitoring of phase formation kinetics for examination of biological particles and cells, using aqueous two phase polymer systems.

An original technique of use of two-phase polymer systems as an analytical research method is described. The technique is based on the absorbance change of two-phase systems in visible spectrum during formation of the phases. Dynamics of this process was demonstrated as the kinetic curves. Addition of studied objects (macromolecules or cells) to the two-phase system modified the shape of the kinetic curve, depending on their individual surface properties. The technique has the following advantages as compared with traditional procedures of the particle surface analysis with the help of two-phase polymer systems: examination of particles with partition coefficients approaching zero; multiple analyses of the same samples; use of interphase as a matrix for study of spontaneous formation of studied particle complexes. The opportunities of the technique were demonstrated in a series of previous authors' works.

Actins↗
PubMed2002

Purification of lactic acid dehydrogenase from crude bovine heart extract by pH-peak focusing counter-current chromatography.

pH-peak focusing counter-current chromatography (CCC) was applied to the purification of lactic acid dehydrogenase (LDH) from a crude bovine heart extract using a cross-axis coil planet centrifuge (CPC). The experiment was performed with two sets of polymer phase systems composed of 16% (w/w) polyethylene glycol (PEG) 1000-12.5% (w/w) potassium phosphate buffer and 15% (w/w) PEG 1540-15% (w/w) ammonium sulfate each at various pH values. The best result was achieved from the PEG 1540-ammonium sulfate polymer phase system by adding a retainer (10 mM acetic acid) to the upper stationary phase and an eluter (100 mM sodium hydroxide) to the lower mobile phase. At a flow-rate of 0.5 ml/min, LDH was eluted as a sharp peak which was well resolved from other proteins. Collected fractions were analyzed by the LDH enzymatic activity and by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis to detect contaminated proteins. LDH was purified directly from crude bovine heart extract in a concentrated state.

Animals↗
PubMed2003

Purification of single-strand DNA binding protein from an Escherichia coli lysate using counter-current chromatography, partition and precipitation.

Single-strand DNA binding protein (SSB) from Escherichia coli lysate was purified by counter-current chromatography (CCC) using the ammonium sulfate precipitation method in a coiled column. About 5 ml of E. coli lysate was separated by CCC using a polymer phase system composed of 16% (w/w) polyethylene glycol (PEG) 1000 and 17% (w/w) ammonium sulfate aqueous polymer two-phase solvent system. The precipitation of proteins in the lysate took place in the CCC column, and the SSB protein was eluted in the fraction 51-56. Many other impurities were either eluted immediately after the solvent front or precipitated in the column. The identities of the proteins in the fractions and in the precipitate were confirmed by SDS-polyacrylamide gel electrophoresis with Coomassie Brilliant Blue staining.

Chemical Precipitation↗
PubMed1995

Chiral separation by high-speed countercurrent chromatography.

Various parameters involved in the chiral separation of (+/-)-DNB-amino acids were investigated using N-dodecanoyl-L-proline-3,5-dimethylanilide as a chiral selector (CS) and two-phase solvent systems composed of hexane/ethyl acetate/methanol/10 mM hydrochloric acid at various volume ratios. The results indicated that increasing the concentration or net amount of the CS in the stationary phase improves both separation factor (alpha) and peak resolution (Rs). The hydrophobicity of the solvent system also increases the alpha value of the racemate while it affects the peak resolution differently according to the partition coefficient of the racemate. Overall results indicated that the best separation of a racemate will be achieved by applying a high CS concentration in the organic phase while adjusting the hydrophobicity of the solvent system so that the partition coefficient of the racemate falls between 0.6 and 0.8. The peak resolution will be further increased by using a longer and/or greater internal diameter coiled column.

Amino Acids↗