Continuous laccase concentration in an aqueous two-phase system

An approach to describe continuous partitioning of Cerrena unicolor laccase in a PEG 6000—phosphate aqueous two-phase system was proposed. The laccase was separated from crude supernatant of C. unicolor-submerged culture, and all the experiments were carried out in 25 °C and pH 7 conditions. Masses of both phases and their compositions at phase equilibrium, as well as laccase activity concentrations at different mixing points, were measured in batch experiments. An empirical short-cut method was developed which allows for calculation of mass and volume fractions of the phases, laccase concentration factors, and laccase recoveries. Theoretical predictions were verified by several experiments carried out in a special mixer-settler unit with automatic substrate feed and continuous collection of separated phases. Required concentration of the laccase was possible to achieve in a one-step extraction process in the mixer-settler unit. The predictions of the short-cut method were compared to the results of experimental measurements of phase compositions, phase volume fractions, concentration factors and enzymatic yields at steady-state operation of the extraction unit. The values of experimental results lay well within the 10% error range of the predicted values.     

Phosphodiesterase production in an aqueous two-phase system by Nicotiana tabacum 1507

Studies were conducted on the production of phosphodiesterases by Nicotiana tabacum 1507 cell suspension in an aqueous two-phase system formed by adding 4% polyethylene glycol (mol wt 20,000) and 7.5% dextran (mol wt 70,000) to the medium. The time course of growth, biosynthesis, secretion, and partitioning of phosphodiesterases was followed in comparison with N. tabacum 1507 cultivation as a free suspension. Partitioning of phosphodiesterases took place mainly in the bottom dextran phase, and a possibility was revealed for obtaining an enzyme preparation with high phosphodiesterase activity.     

Electroenzymatic synthesis of chiral alcohols in an aqueous–organic two-phase system

The reduction of acetophenone to (R)-phenylethanol catalysed by the alcohol dehydrogenase from Lactobacillus brevis in combination with electrochemical regeneration of NADPH mediated by a rhodium complex is reported. The reaction in buffer solution was optimised with regard to high productivity (up to 14 gL⁻¹d⁻¹) and enantioselectivity (>99.9%). Enzyme stability under the reaction conditions was increased either by addition of bovine serum albumin as a sacrificial protein or by immobilisation, leading to full conversion and enzyme ttn’s of up to 75,000. To improve the utilisation of cofactor and mediator as well as to broaden the substrate spectrum to more hydrophobic substrates, we introduced an organic phase of methyl tert-butyl ether. This is the first reported two-phase approach for electrochemical cofactor regeneration, which yielded mediator and cofactor ttn’s twice as high as in the one-phase approach. Furthermore, a concentrated product solution of 180 mM enantiopure (R)-phenylethanol was obtained, facilitating product work-up.     

Droplet-based microextraction in the aqueous two-phase system

This report is about microfluidic extraction systems based on droplets of aqueous two-phase system. Mass transfer between continuous phase and dispersed droplet is demonstrated by microextraction of ruthenium red in a microfluidic device. Droplets are generated with electrohydrodynamic method in the same device. By comparing brightness in the digital image of a solution with known concentrations of ruthenium red and those of a droplet in the microextraction, ruthenium red concentration was measured along the microextraction channel, resulting in good agreement with a simple diffusion model. The maximum partition coefficient was 9.58 in the experiment with the 70-mm-long-channel microextractor. The method is usable for terminating microextraction by electrohydrodynamic manipulation of droplet movement direction. Droplets of different ruthenium red concentration, 0.12 and 0.24% (w/w) in this experiment, can be moved to desired place of microfluidic system for further reaction through respectively branched outlets. In this study droplet-based microextraction is demonstrated and the mass transport is numerically analyzed by solving the diffusion–dissolution model.     

Enhancing the lateral-flow immunoassay for viral detection using an aqueous two-phase micellar system

Availability of a rapid, accurate, and reliable point-of-care (POC) device for detection of infectious agents and pandemic pathogens, such as swine-origin influenza A (H1N1) virus, is crucial for effective patient management and outbreak prevention. Due to its ease of use, rapid processing, and minimal power and laboratory equipment requirements, the lateral-flow (immuno)assay (LFA) has gained much attention in recent years as a possible solution. However, since the sensitivity of LFA has been shown to be inferior to that of the gold standards of pathogen detection, namely cell culture and real-time PCR, LFA remains an ineffective POC assay for preventing pandemic outbreaks. A practical solution for increasing the sensitivity of LFA is to concentrate the target agent in a solution prior to the detection step. In this study, an aqueous two-phase micellar system comprised of the nonionic surfactant Triton X-114 was investigated for concentrating a model virus, namely bacteriophage M13 (M13), prior to LFA. The volume ratio of the two coexisting micellar phases was manipulated to concentrate M13 in the top, micelle-poor phase. The concentration step effectively improved the M13 detection limit of the assay by tenfold from 5?×?10⁸ plaque forming units (pfu)/mL to 5?×?10⁷ pfu/mL. In the future, the volume ratio can be further manipulated to yield a greater concentration of a target virus and further decrease the detection limits of the LFA.

The study of the partitioning mechanism of methyl orange in an aqueous two-phase system

An aqueous two-phase system of dodecyl triethylammonium bromide (C₁₂NE, cationic surfactant) and sodium dodecyl sulfate (SDS, anionic surfactant) mixture is proposed for the extraction of some dyes and porphyrin compounds. Transparent two phase-systems are formed when the surfactant concentrations and C₁₂NE/SDS ratios are in certain regions. In this study, the aqueous two phase-systems were prepared by mixing 0.1 mol l⁻¹ C₁₂NE and SDS with a molar ratio of 1.7:1.0. The results showed that negatively charged chlorophyll (sodium copper chlorophyllin) and positively charged dye (methyl violet) were efficiently extracted into the upper phase. The negatively charged methyl orange (pH>7) was moved into the upper phase mostly while amphoteric methyl orange (pH<3) was distributed in the two phases uniformly. Except for hydrophobic force, charge interaction between solute and surfactant also play an important role in the extraction process.     

Development of an aqueous two-phase partitioning system for fractionating therapeutic proteins from tobacco extract

In the present study, an aqueous two-phase partitioning system (ATPS) was developed and evaluated as an initial fractionation step for therapeutic antibodies and enzymes from tobacco extracts. A detailed study has been performed to analyze the effect of pH, ionic composition of the system, types of polymers and their molecular weight and concentration, on the partitioning behavior of tobacco proteins and human anti-human immunodeficiency virus (HIV) monoclonal antibody 2F5 (mAb 2F5). A polyethyleneglycol/phosphate (PEG/Pi) aqueous two-phase system composed of 12% (w/w) PEG 1500 and 13% (w/w) phosphate buffer, pH 5, was selected as the system with the highest selectivity of antibody over native tobacco proteins. Under selected conditions, sufficient purification (3-4-fold) with high recovery at the bottom phase (approximately 95%) was achieved for mAb 2F5. In addition, the system allows removal of plant-derived compounds, such as phenolics and toxic alkaloids. The antibody fraction may be directly applied to a Protein A affinity column without any further pre-treatment, thus allowing homogenous antibody preparation. Analysis of the purified antibody fraction by enzyme-linked immunosorbent assay (ELISA) and western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was further demonstrated using additional proteins and enzymes of therapeutic importance, such as neuraminidase (NA) from influenza virus and human anti-HIV monoclonal antibody 2G12 (mAb 2G12), and showed that the system may find wide applicability as an economic extraction strategy for the initial fractionation of biopharmaceuticals from transgenic tobacco plants.     

Enhancing the lateral-flow immunoassay for detection of proteins using an aqueous two-phase micellar system

The lateral-flow (immuno)assay (LFA) has been widely investigated for the detection of molecular, macromolecular, and particle targets at the point-of-need due to its ease of use, rapid processing, and minimal power and laboratory equipment requirements. However, for some analytes, such as certain proteins, the detection limit of LFA is inferior to lab-based assays, such as the enzyme-linked immunosorbent assay, and needs to be improved. One solution for improving the detection limit of LFA is to concentrate the target protein in a solution prior to the detection step. In this study, a novel approach was used in the context of an aqueous two-phase micellar system comprised of the nonionic surfactant Triton X-114 to concentrate a model protein, namely transferrin, prior to LFA. Proteins have been shown to partition, or distribute, fairly evenly between the two phases of an aqueous two-phase system, which in turn results in their limited concentration in one of the two phases. Therefore, larger colloidal gold particles decorated with antibodies for transferrin were used in the concentration step to bind to transferrin and aid its partitioning into the top, micelle-poor phase. By manipulating the volume ratio of the two coexisting micellar phases and combining the concentration step with LFA, the transferrin detection limit of LFA was improved by tenfold from 0.5 to 0.05?μg/mL in a predictive manner. In addition to enhancing the sensitivity of LFA, this universal concentration method could also be used to improve other detection assays.     

两相体系中不对称催化加氢研究进展

综述了近年来水／有机两相催化体系中的催化不对称加氢研究进展，以及两相体系不对称加氢反应中对映选择性下降与水作为溶剂的关系、水的作用和提高对映选择性的方法。引用文献39篇。     

Concentration dependence of the interfacial tension for aqueous two-phase polymer solutions of dextran and polyethylene glycol

We studied the interfacial tension between coexisting phases of aqueous solutions of dextran and polyethylene glycol. First, we characterized the phase diagram of the system and located the binodal. Second, the tie lines between the coexisting phases were determined using a method that only requires measuring the density of the coexisting phases. The interfacial tension was then measured by a spinning drop tensiometer over a broad range of polymer concentrations close to and above the critical point. In this range, the interfacial tension increases by 4 orders of magnitude with increasing polymer concentration. The scaling exponents of the interfacial tension, the correlation length, and order parameters were evaluated and showed a crossover behavior depending on the distance to the critical concentration. The scaling exponent of the interfacial tension attains the value 1.50 ± 0.01 further away from the critical point, in good agreement with mean field theory, but the increased value 1.67 ± 0.10 closer to this point, which disagrees with the Ising value 1.26. We discuss possible reasons for this discrepancy. The composition and density differences between the two coexisting phases, which may be taken as two possible order parameters, showed the expected crossover from mean field behavior to Ising model behavior as the critical point is approached. The crossover behavior of aqueous two-phase polymer solutions with increasing concentration is similar to that of polymer solutions undergoing phase separation induced by lowering the temperature.     

Partitioning of alkali halides in aqueous dextran-ficoll two-phase system

Partitioning of alkali halides MeX in aqueous dextran-Ficoll two-phase system of fixed polymer and ionic composition was studied. It is found that NaCl and LiX concentrate in the Ficoll-rich phase, while all KX, RbX, and CsX concentrate in the dextran-rich phase. Partitioning of KX, RbX, and CsX in the presence of a large excess of NaCl is found to depend on the type of the halide anion X^–.     

Semicontinuous production of cellulolytic enzymes withTrichoderma reesei Rutgers C30 in an aqueous two-phase system

Cellulolytic enzyme production was studied in an aqueous twophase system, PEG 8000 5%-Dextran 7%, withTrichoderma reesei Rutgers C30 in a 7L fermentor. In batch cultivations, an average of 2.5 filter paper units (FPU)/mL were obtained in the top phase. In cultiva tions in regular media without polymers, the same enzyme concen tration was obtained. The enzyme yield was 205 FPU/g cellulose in the phase system, and 259 FPU/g cellulose in the regular medium. An extractive fed-batch cultivation was maintained in the aqueous twophase system for 360 h. The enzyme containing top phase was with drawn after phase separation. New cellulose substrate and nutrients were added with the new top phase. The enzyme extraction was started after 120 h of cultivation, and was repeated every 72 h. The total substrate concentration was 40 g/L. A maximum enzyme concentration of 4.8 FPU/mL was obtained in the withdrawn cell-free top phase. The enzyme yield was 148 FPU/g cellulose.     

Phase equilibrium and macrolide antibiotics partitioning in real water samples using a two-phase system composed of the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate and an aqueous solution of an inorganic salt

An ionic liquid-salt aqueous two-phase system (ILATPS) based on the hydrophilic ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF₄) and inorganic salt was developed for direct separation and analysis of macrolide antibiotics coupled with molecular fluorescence spectrophotometry. Liquid–liquid equilibria of [Bmim]BF₄-salt aqueous two-phase systems were studied for different salts and temperatures. It was found that the salting-out ability of different salts may be related to the Gibbs energy of hydration of the ions, and the two-phase area was expanded with a decrease in temperature. The partition coefficients as well as extraction efficiencies of azithromycin and mydecamycin in [Bmim]BF₄-salt aqueous two-phase system were influenced by the types of salts, concentration of salt, and the extracting temperature. Under the optimum conditions, the average partition coefficient of azithromycin in [Bmim]BF₄-Na₂CO₃ ILATPS was 162, and that of mydecamycin in [Bmim]BF₄- NaH₂PO₄ ILATPS was 90.9. This method has been satisfactorily applied to the determination of azithromycin and mydecamycin in real water samples with detection limits of 0.059 µg mL^?1 and 0.019 µg mL^?1. This extraction method is a simple, non-toxic and effective sample pretreatment technique with promise also for the separation of other small biomolecules.     

Extraction of proteins from biological fluids by use of an ionic liquid/aqueous two-phase system

An ionic liquid/aqueous two-phase system based on the hydrophilic ionic liquid 1-butyl-3-methylimidazolium chloride (BmimCl) and K(2)HPO(4) has been employed for direct extraction of proteins from human body fluids for the first time. Proteins present at low levels were quantitatively extracted into the BmimCl-rich upper phase with a distribution ratio of about 10 between the upper and lower phase and an enrichment factor of 5. Addition of an appropriate amount of K(2)HPO(4) to the separated upper phase results in a further phase separation, giving rise to an improved enrichment factor of 20. FTIR and UV spectroscopy demonstrated that no chemical (bonding) interactions between the ionic liquid and the protein functional groups were identifiable, while no alterations of the natural properties of the proteins were observed. The partitioning of proteins in the two-phase system was assumed to have been facilitated by the electrostatic potential difference between the coexisting phases, as well as by salting out effects. The system could be applied successfully for the quantification of proteins in human urine after on-line phase separation in a flow system. The use of an ionic liquid, as a green solvent, offers clear advantages over traditional liquid-liquid extractions, in which the use of toxic organic solvents is unavoidable.     

Distribution behavior of some drug enantiomers in an aqueous two-phase system using counter-current extraction with protein

Summary The partial resolution of some drug enantiomers by counter-current extraction in an aqueous two-phase system using bovine serum albumin (BSA) or ovomucoid (OVM) as chiral discriminator is described. The phase system was prepared with dextran 40, polyethylene glycol and a protein. Ofloxacin (OFLX) enantiomer was well recognized by the BSA system, whereas the enantiomers of carvedilol (-blocker) and DG-5128 (a new oral hypoglycaemic drug) were hardly recognized by the OVM system. The distribution coefficients of (S)-, (R)- and rac-OFLX varied with pH. Good enantioselectivity for OFLX was obtained at relatively high pH ( pH 9). The crude optically enriched OFLX enantiomers were extracted from the aqueous fractions and finally purified by thin-layer chromatography.     

Temperature-sensitive aqueous surfactant two-phase system formation in cationic-anionic surfactant systems

Temperature-induced aqueous surfactant two-phase system (T-ASTP), which was found to be of generic importance, was investigated in a series of conventional mixed cationic-anionic surfactant systems. On the basis of the investigations of turbidity, dynamic light scattering, transmission electron microscopy, and fluorescence resonance energy transfer, the formation of T-ASTP can be attributed to temperature-induced vesicle aggregation. Aggregated vesicles existed in the upper part, while the separated vesicles existed in the lower part. The phase separation temperature can be regulated by varying the surfactant composition or adding additives, such as d-sorbitol, urea, or NaBr. The hydrophobic interaction and cooperative effect between cationic and anionic surfactants played a significant role in the formation of T-ASTP.     

聚乙二醇双水相萃取光度法测定镍

用水溶性高聚物、表面活性剂及有机物与无机盐形成的双水相体系萃取分离色素、蛋白质及测定金属离子已有报道[1-5].     

Selective separation and enrichment of proteins in aqueous two-phase extraction system

A simple aqueous two-phase extraction system(ATPS) of PEG/phosphate was proposed for selective separation and enrichment of proteins.The combination of ATPE with HPLC was applied to identify the partition of proteins in two phases.Five proteins (bovine serum albumin,Cytochrome C,lysozyme,myoglobin,and trypsin) were used as model proteins to study the effect of phosphate concentration and pH on proteins partition.The PEG/phosphate system was firstly applied to real human saliva and plasma samples,some pro...     

成相无机盐种类对PEG双水相相平衡影响

不同种类无机盐分别与PEG1000、PEG2000、PEG,4000和PEG6000构成双水相体系,在298.15K下,采用浊点滴定法研究双水相体系相平衡,通过Merchuk经验方程拟合实验点确定双水相体系的双节线,进而考察成相无机盐种类对PEG双水相相平衡的影响.结果表明:具有相同阴离子,易于形成双水相的无机盐顺序为...