Two phase aqueous extraction of whey proteins in a polyethylene glycol - arabinogalactan system

The whey protein separation potential of aqueous two-phase systems of arabinogalactan [AG] (Lonza FiberAidTM) and polyethylene glycol [PEG], buffered with 10 mmol/g phosphate or citrate buffer, was studied. 100 mmol/g potassium chloride [KCl] was added as required. Previously-published phase equilibrium results were verified and the absorbance of whey protein isolate [WPI] in an AG-PEG solution was measured. The effect of pH, KCl concentration, initial WPI concentrations and upper to lower phase mass ratios on whey partitioning was studied. The best separation system contained 17.20% (w/w) AG, 7.20% (w/w) PEG, 10 mmol citrate buffer (pH 5.4) and 100 mmol KCl per gram of total system. The upper to lower phase mass and volume ratios were 1:1 and 16:11 respectively. Approximately 12 mg (mainly α-lactalbumin) of the 20 mg WPI added partitioned into the AG-rich upper phase. This system has potential to reduce chromatographic requirements in large scale separation of protein mixtures

Electrophoretic aqueous two-phase partition of proteins

An electrical field was applied to an aqueous two-phase system. The integration of these techniques in one step is used to improve the partition of biological molecules. Experiments were performed for an individual protein - BSA - and for a mixture of proteins - lysozyme and egg albumin - at different polarities and opposite charges of the biomolecules for the aqueous two-phase system Reppal PES - PEG8000. The separation between those two proteins improved more than two-fold

Physicochemical characterization of the PEG8000-Na2SO4 aqueous two-phase system

The polyethylene glycol-sodium sulfate aqueous two-phase system has been characterized at 23 °C. Tielines for the phase diagram were obtained experimentally. Phases in equilibrium were characterized by means of the solvatochromic parameters π*, α, and β, which provide a measurement of the polarity/polarizability and the H-bond donor and acceptor abilities, respectively. The ability of the phases to participate in hydrophobic interactions was characterized by means of the free energy of transfer of a methylene group between the conjugated phases, using the partition of a homologous series of dinitrophenylated amino acids. The results show the effect of the presence of polymer and salt in the aqueous phase, and a comparison of both phases with pure water is made.LSRE-PortoUniversidade Católica PortuguesaEscola Superior de Biotecnologia do PortoFundação para a Ciência e a Tecnologia (FCT

Physical characterisations of a single-stage Kühni-type aqueous two-phase extraction column

The main parameters which influence the behaviour of phase separation in a single-stage Kühni-type aqueous two-phase extraction column containing polyethylene (PEG) and di-potassium hydrogen phosphate were characterised. Two aqueous two-phase system (ATPS) composed of 12% (w/w) PEG 1450 and 12% (w/w) di-potassium hydrogen phosphate (designated as 12/12) and 12% (w/w) PEG 1450 and 11% (w/w) di-potassium hydrogen phosphate (designated as 12/11) were chosen in this study. The hold-up D increased with increasing impeller speeds and mobile phase flow rates. Phase separation for the 12/11 system was slower than that for the 12/12 system, which resulted in higher dispersed phase hold-up values for the 12/11 system. For 12/12 system, mass transfer of plasmid DNA (pDNA) from the dispersed mobile phase to the stationary phase increased rapidly with increasing impeller speeds of 130, 160 and 200 rpm which was reflected in the decreased values for CT/CTo. The degree of back-mixing quantified by the axial dispersion coefficient Dax was estimated to be 2.7 × 10−6 m2 s−1

Separation of endo-polygalacturonase using aqueous two-phase partitioning

The partitioning of endo-polygalacturonase (endo-PG) in polyethylene glycol (PEG)–polyvinyl alcohol (PVA10 000) and PEG–hydroxypropyl starch (Reppal PES100) aqueous two-phase systems was studied, and revealed the possibility of using aqueous two-phase extraction to purify and concentrate endo-PG from its clarified fermentation broth. For the PEG8000– PVA10 000 system, endo-PG presented in the fermentation broth (at concentration that is more than 40% of total protein) mainly dominates in the top phase with a partitioning coefficient of 6, while total protein concentrates in the bottom phase. A separation scheme consisting of two consecutive aqueous two-phase extraction steps was proposed: a first extraction in polyethylene glycol (PEG8000)–polyvinyl alcohol system, followed by a second extraction in PEG8000–(NH4)2SO4 system. This allowed the separation of endo-PG from polymer and the recycling of PEG polymer, since endo-PG was very strongly partitioned into the bottom phase of the PEG8000–(NH4)2SO4 system. Laboratory-scale experiments were performed to test the efficiency of this scheme. It was found that enzyme recovery was up to 91% with a total purification factor of about 1.9 and a concentration factor of more than 5. About 90% of the total PEG added into the systems can be recovered, and no reduction was obtained in the purification factor using recycled PEG.Fundação para a Ciência e a Tecnologia (FCT) – PRAXIS XXI.CEC (INCO-DC) - contract number ERB IC18 CT97 0182

Resolution of 2-bromo-arylacetic acid ester by Yarrowia lipolytica lipase in water/supercritical CO2 two-phase systems

A mutated lipase from Yarrowia lipolytica was used in aqueous phase/scCO2 two-phase systems to perform the enzymatic resolution of (R, S) 2-bromophenyl acetic octyl ester. A solution of phosphate salt (1000 mmol/L) was added to buffer the aqueous phase in contact with CO2 and resulting pH values around 6 were measured in a high pressure cell using a solvatochromic probe. Thus, an acceptable conversion rate and good enantioselectivity could be obtained but kinetics were shown to remain slower compared to an aqueous phase/decane two-phase system. Moreover, increasing pressure was shown to further slowdown the kinetics. This was hypothesized to be related to the mechanism of opening of the active site of the lipase which requires interfacial contact with a hydrophobic solvent phase. This condition is suspected not to be met in the case of scCO2 in contact with an aqueous phase because the amount of water dissolved in the supercritical phase diminishes its hydrophobicity

Camomile autofermentation in polyethylene glycol/dextran two-phase system

The objective of this study was the investigation of the extractive bioconversion of apigenin-7-O-β-glucoside in camomile ligulate flowers into apigenin by autofermentation in polyethylene glycol 6000/dextran 200000 two-phase system. In 22.5% polyethylene glycol/14% dextran aqueous two-phase system the obtained yield of apigenin in the top phase was 96.5%. In the presence of plant material that partiotioned to the interphase, the yield of apigenin in the top phase was 3.5 times higher in comparison to the model system