Partitioning In Two-phase Systems Research Articles

Partitioning of SWCNT mixtures using amphiphilic carbohydrate-based surfactants

Single-walled carbon nanotubes (SWCNTs) have substantial application potential in many branches of technology. Considering that the spatial order of carbon atoms in SWCNTs determines their properties, the characteristics of a material can be adjusted for a specific application by using only selected types of SWCNTs. Unfortunately, SWCNT purification is challenging as the mechanisms of the currently available sorting techniques are not fully understood. The aqueous two-phase extraction (ATPE) method is a promising approach in this context as it offers SWCNT separation in an aqueous environment, so elucidation of its workings is essential. To come closer to achieving this goal, we synthesized a new range of surfactants based on ionic liquids equipped with amphiphilic carbohydrate moieties. We then examined how the modification of their structure influences SWCNT partitioning in two-phase systems composed of poly(ethylene glycol) and dextran. We concluded that the capacity of the ATPE method for purification of SWCNTs depended upon establishing a sufficient degree of order or disorder in the aqueous medium caused by the introduction of chemical compounds of a kosmotropic and chaotropic nature, respectively.

Tyrosinase from the peels of Musa cavendish: purification, characterization, immobilization and application in phenol biodegradation

Tyrosinase is an enzyme implicated in fruit ripening and the browning of plant parts exposed to mechanical injury. Tyrosinase has numerous reported biotechnological importance. This enzyme was extracted from the waste peels of Musa cavendish, purified via aqueous two-phase partitioning system (ATPS) and QAE-Sephadex ion-exchange chromatography. The purified tyrosinase was biochemically characterized. Tyrosinase was thereafter immobilized, characterized, and applied in the oxidation of phenols in model wastewaters. After purification, the yield and fold obtained were 23% and 8.0 respectively. The molecular weight of the enzyme was estimated to be 43.0 kDa. The purified enzyme had an optimum pH and temperature of 6.5 and 60 °C respectively. The (M−1s−1) obtained for substrates such as L-DOPA, catechol and pyrogallol were respectively. The purified enzyme was stable in organic solvents: acetone > ethanol > methanol > n-butanol. Quercetin, ascorbic acid, l-cysteine and arginine were potent inhibitors. The immobilized tyrosinase was more resistant to thermoinactivation than the free enzyme as evidenced by its kinetic ( and thermodynamic ( characteristics. The immobilized tyrosinase removed 70% of 650-µM phenol after 2 h compared to 50% removal by the free tyrosinase. After six repeated cycles of phenol biodegradation using the immobilized enzyme, about 40% of phenol was removed. The fresh peels of M. cavendish served as a green-based source of relatively thermostable immobilized tyrosinase deployable in large-scale biodegradation of phenol-containing wastewater.

Characterization of low temperature-induced plasma membrane lipidome remodeling combined with gene expression analysis reveals mechanisms that regulate membrane lipid desaturation in Carica papaya

Carica papaya is a cold-sensitive horticultural crop, the mechanisms underlying its cold sensitivity remains unclear which limit its genetic improvements in cold tolerance. In this study, aqueous dextran-polyethylene glycol two-phase partition systems were applied to purify plasma membrane from papaya leaves grown under 28 °C and 10 °C, the plasma membrane total lipids were analyzed by lipidomic approach; meanwhile, the transcription levels of putative fatty acid desaturases were quantified by RT-qPCR. We found that cerebrosides are dominate plasma membrane lipid species, followed by phospholipids and free sterols. Cold treatment increased the mono-unsaturated and di-unsaturated phospholipids, and decreased di-saturated phospholipids, the proportion of total phospholipids was not affected. Cold stress enhanced the transcription of CpFAD4, CpFAD6 and CpFAB2−2. Pathway analysis suggested that the cold induction of CpFAB2−2 transcription and CpFAD2 enzyme activities play important roles for plasma membrane lipidome remodeling under cold stress. Our findings uncovered potential targets to improve papaya cold tolerance.

A new tool for quantification of membrane protein partitioning between different cell membranes

The aqueous two-phase partition system (ATPS) is a method widely used to separate and purify plant and animal membranes carrying bound proteins. However, a common problem of this separation is a mutual contamination of obtained phases. Such contamination adversely affects the accuracy of values of the protein of interest partition between particular membranes when determined by direct measurement. In order to overcome this problem, we have developed a fairly simple mathematical algorithm and found formulas designed to quantify correctly the distribution of the protein of interest between two different membranes. This new tool makes it possible to determine the bias-adjusted ratio of protein distribution between the membranes, regardless of the efficiency of membrane separation in a two-phase system. By means of this algorithm, not only current, but also a number of previously published ATPS-based experiments were (re)analyzed and quantified. The quantitative results of this large-scale analysis of the subcellular localization of various membrane proteins from Arabidopsis, potato, melon, and corn including cytokinin and ethylene receptors, ABCG14 cytokinin transporters, LRR receptor-like protein kinases, and BAK1 co-receptors are presented and discussed here.

Optimization of aqueous two-phase partitioning of Aureobasidium pullulans α-amylase via response surface methodology and investigation of its thermodynamic and kinetic properties

Industrial enzymes such as α-amylase must be thermostable and also easily purified/concentrated. Hence, aqueous two-phase partitioning systems (ATPS) was exploited for the partitioning of α-amylase from Aureobasidium pullulans due to its numerous advantages over conventional purification strategy. A. pullulans α-amylase was partially purified using ATPS via response surface methodology (RSM). The potentials of the ATPS-purified enzyme for possible industrial application such as resistance to thermal inactivation was investigated in comparison with the crude enzyme. PEG-6000 was the polymer of choice for ATPS as it resulted in higher purification factor (PF), %yield (Y), and partition coefficient (PC). At optimum levels (% w/v) of 20, 12 and 7.5 for PEG-6000, sodium citrate and sodium chloride respectively, maximum PF, Y and PC of 4.2, 88%, and 9.9 respectively were obtained. The response model validation and reliability were established based on the closeness between the experimented and predicted values. The kinetic and thermodynamic parameters such as Q10, t1/2, kd, D − value, Ed, ΔHd∗,ΔGd∗andΔSd∗ of the ATPS-purified α-amylase indicated that it was thermostable at 50 to 60 °C compared to the crude α-amylase. A thermodynamically stable and ATPS-purified α-amylase from A. pullulans has properties easily applicable for most industrial processes.

Use of Aqueous Two-Phase and Three-Phase Partitioning Systems for Purification of Lipase Obtained in Solid-State Fermentation by Rhizopus arrhizus

Background: Purification of enzymes by conventional methods such as precipitation and chromatographic techniques is a costly and time-consuming procedure and may lead to low yields of enzyme activity. Alternative liquid-liquid extraction methods such as Aqueous Two-Phase Systems (ATPS) and Three Phase Partitioning (TPP) are characterized by the high enzyme yields and purification degree. Objective: The objective of this study was the application of partitioning systems ATPS and TPP for purification of lipase produced in solid-state fermentation by Rhizopus arrhizus. Methods: ATPS and TPP were used for purification of lipase, obtained by solid state cultivation of Rhizopus arrhizus. Results: Lipase was isolated with PEG4000/potassium sodium tartrate ATPS and the effect of the system composition, including PEG 4000 and potassium sodium tartrate concentrations on lipase partitioning was studied. When using 30% PEG4000/21% potassium sodium tartrate, lipase was distributed in the top phase, and the highest recovery yield of 217% and purification fold of 6.1 were achieved. It was found that at PEG4000 concentration of or higher than 15%, the enzyme was present in the top polymer-rich phase with a partitioning yield of over 90%. Upon application of TPP for lipase isolation, the effect of t-butanol concentration, ammonium sulfate concentration and pH on enzyme partitioning was investigated. The highest lipase recovery yield of 71% and 19.1-fold purification were achieved in the interfacial phase in the presence of 30% ammonium sulfate saturation with 1.0:0.5 crude extract/t-butanol ratio at pH 7 in a single step. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymographic analysis showed significant purification of lipase by TPP and the presence of two multiple forms of the enzyme. Conclusion: ATPS (PEG4000/ Potassium sodium tartrate) and TPP (1.0:0.5 crude extract/t-butanol ratio, 30% ammonium sulfate saturation, pH 7) proved to be rapid methods for the isolation and purification of lipase and they can be used in downstream processing for industrial preparation of the enzyme.

Partial characterization of Bacillus pumilus catalase partitioned in poly(ethylene glycol)/sodium sulfate aqueous two-phase systems

Aqueous two-phase partitioning system (ATPS) was used to extract and purify catalase from Bacillus pumilus. The system parameters for effective purification of catalase were optimized. The best catalase recovery (123%) with a 4.6-fold purification was obtained in the bottom phase of ATPS including the mixture of 15% (w/w) PEG4000, 10% (w/w) Na2SO4 and 3% (w/w) NaCl at pH 5.0. The purified enzyme was characterized regarding its activity and stability. The highest enzyme activity was observed at pH 7.0 and 37 °C on hydrogen peroxide. The enzyme was quite stable at temperatures between 30 and 55 °C and a pH range of 7.0–9.0. The Km and Vmax values were determined from Lineweaver–Burk plot as 11 mM and 1667 µmole ml−1 min−1, respectively. Overall, it can be said that ATPS is a rapid, reasonable, straightforward and cost-effective process for catalase purification in comparison to the chromatographic methods.

Social microbial inocula confer functional stability in a methyl tert-butyl ether extractive membrane biofilm bioreactor

Methyl tert-butyl ether (MTBE) degradation technologies based on two-phase partitioning systems such as extractive membrane biofilm reactors (EMBFR) permit separation of biological and contaminant compartments, thus allowing optimization of the biological section. In this study, we set-up an EMBFR with three MTBE-degrading and cooperating strains (termed social biofilm: Agrobacterium sp. MS2, Paenibacillus etheri SH7T and Rhodococcus ruber EE6). The removal efficiency of the social-biofilm EMBFR was 80%, and functional stability was observed in the reactor, i.e. more efficient than previous studies (single-strain inoculated EMBFR, <50% removal efficiency and unstable function). Metabolite tert-butyl alcohol was not observed, and the EC50 values were higher than those observed in single-strain EMBFRs. Comparative analysis of the MTBE enzymatic pathway and the social-biofilm was performed, where the mechanism of cooperation observed within the social-biofilm is likely due to enzymatic redundancy. Functional outcomes were equal to previous batch tests, hence 100% scalability was obtained. Overall, higher functional and stability outcomes are obtained with the use of the social-biofilm in an MTBE-EMBFR.

Further insights into underlying mechanisms for the release of biological nitrification inhibitors from sorghum roots

Sorghum roots release two categories of biological nitrification inhibitors (BNIs) – hydrophilic-BNIs and hydrophobic-BNIs. Earlier research indicated that rhizosphere pH and plasma membrane (PM) H+ATPase are functionally linked with the release of hydrophilic BNIs, but the underlying mechanisms are not fully elucidated. This study is designed to reveal further insights into the regulatory mechanisms of BNIs release in root systems, using three sorghum genetic stocks. Sorghum plants were grown in a hydroponic system with pH of nutrient solutions ranging from 3.0 9.0. Pharmacological agents [(fusicoccin and vanadate) and anion-channel blockers (−niflumic acid (NIF) and anthracene-9-carboxylate (A9C)] were applied to root exudate collection solutions; BNI activity was determined with luminescent Nitrosomonas europaea bioassay. Sorgoleone levels in root exudates and H+ excretion from roots were determined. Two-phase partitioning system is used to isolate root plasma membrane (PM) and H+ ATPase activity was determined. A decrease in rhizosphere pH improved the release of hydrophilic-BNIs from roots of all the three sorghum genotypes, but had no effect on the release of hydrophobic-BNIs. Hydrophobic-BNI activity and sorgoleone levels in root-DCM wash are positively correlated. Fusicoccin promoted H+extrusion and stimulated the release of hydrophilic-BNIs. Vanadate, in contrast, suppressed H+ extrusion and lowered the release of hydrophilic-BNIs. Anion-channel blockers did not inhibit the release of hydrophilic BNIs, but enhanced H+-extrusion and hydrophilic-BNIs release. Rhizosphere pH has a major influence on hydrophilic-BNIs release, but not on the release of hydrophobic-BNIs. The low rhizosphere pH stimulated PM-H+ ATPase activity; H+-extrusion is closely coupled with hydrophilic-BNIs release. Anion-channel blockers stimulated H+ extrusion and hydrophilic-BNIs release. Our results indicate that some unknown membrane transporters are operating the release of protonated BNIs, which may compensate for charge balance when transport of other anions is suppressed using anion-channel blockers. A new hypothesis is proposed for the release of hydrophilic-BNIs from sorghum roots.

Plasma Membrane Protein Profiling in Beta-Amyloid-Treated Microglia Cell Line.

In the responsiveness of microglia to toxic stimuli, plasma membrane proteins play a key role. In this study we treated with a synthetic beta amyloid peptide murine microglial cells metabolically differently labelled with stable isotope amino acids (SILAC). The plasma membrane was selectively enriched by a multi-stage aqueous two-phase partition system. We were able to identify by 1D-LC-MS/MS analyses 1577 proteins, most of them are plasma membrane proteins according to the Gene Ontology annotation. An unchanged level of amyloid receptors in this data set suggests that microglia preserve their responsiveness capability to the environment even after 24-h challenge with amyloid peptides. On the other hand, 14 proteins were observed to change their plasma membrane abundance to a statistically significant extent. Among these, we proposed as reliable biomarkers of the inflammatory microglia phenotype in AD damaged tissues MAP/microtubule affinity-regulating kinase 3 (MARK3), Interferon-induced transmembrane protein 3 (IFITM3), Annexins A5 and A7 (ANXA5, ANXA7) and Neuropilin-1 (NRP1), all proteins known to be involved in the inflammation processes and in microtubule network assembly rate.

Aluminum-Enhanced Proton Release Associated with Plasma Membrane H+-Adenosine Triphosphatase Activity and Excess Cation Uptake in Tea (Camellia sinensis) Plant Roots

Cultivated tea (Camellia sinensis) plants acidify the rhizosphere, but the mechanisms responsible for rhizosphere acidification associated with aluminium (Al) have not been fully elucidated. The present study examined the effect of Al on root-induced rhizosphere acidification, plasma membrane H+-ATPase activity and the cation–anion balance of tea plants. The H+ exudation from tea plant roots with or without Al treatment was visualized using an agar sheet with bromocresol purple. Al strongly enhanced H+ exudation and the acidification of tea plant roots by Al was closely associated with plasma membrane H+-ATPase activity. Detection of H+-ATPase hydrolytic activity associated with plasma membranes of roots under different Al treatments using the two-phase partition system indicated that plasma membrane H+-ATPase activity increased with Al concentration. The Al content, amount of proton release and H+-ATPase activity in the roots of tea plants were quantified after 7 days with or without Al. The Al content, amount of proton release and H+-ATPase activity were significantly higher in roots with Al treatment than those without Al treatment. The value for the cation–anion balance showed that all treatments had an excess of cations relative anions, and the amount of excess cations uptake increased with the increasing Al concentration. The results suggested that Al-enhanced proton release was associated with plasma membrane H+-ATPase activity and excess cation uptake. Taken together, these findings provide insights into the contributing factors of soil acidification in tea plantation.

Separation of catalase from Amsonia orientalis with single step by aqueous two-phase partitioning system (ATPS)

ABSTRACT Catalase from Amsonia orientalis was purified by ATPS, and its efficiency was compared against hydrophobic interaction chromatography. Activity recovery and purification fold of purified catalase by ATPS were examined under varying experimental conditions. The effects of various factors such as type of phase-forming salts, (PEG) mass, with their different concentrations, pH and temperature effects on partitioning were investigated. The highest activity recovery (156%) and purification fold (8.67) of catalase were obtained in the ATPS system containing 10% (g/g) PEG4000, 15% (g/g) Na2SO4 at pH 6.0 and room temperature. In hydrophobic interaction chromatography, the enzyme has been purified 12.54-fold with 57.5% recovery. The molecular weight of catalase was determined as 75 kDa by SDS-PAGE.

Remediation of PAH-contaminated soil at a gas manufacturing plant by a combined two-phase partition system washing and microbial degradation process.

The aim of this study was to design a remediation technique using both soil washing and microbial degradation to remove polycyclic aromatic hydrocarbons (PAHs) from contaminated soil. PAH biodegradation by inoculation of Mycobacterium sp. was first tested. The effectiveness of washing agents (Tween 80 solution, biodiesel, and a two-phase partition system (TPPS)) was then evaluated with column experiments. Third, the combination of TPPS washing and microbial degradation was studied. PAH bioavailability before and after biodegradation and the joint remediation was also assessed using hydroxypropyl-β-cyclodextrin (HPCD) extraction. Only phenanthrene and anthracene were noticeably biodegradable when the soil was inoculated with Mycobacterium sp. TPPS containing 2% (v/v) biodiesel and 2.5% (w/v) Tween 80 was used as the washing agent for the joint remediation test because it gave higher PAH extractions than Tween 80 solution with lower doses, and there was less residue in the soil. Joint TPPS washing and microbial degradation gave a total PAH removal of 92.6%, which was much higher than the results from either the biodegradation or washing experiments alone. Removals of all high molecular weight (HMW) PAHs were improved. Bioavailable concentrations of all PAHs decreased significantly after the joint remediation process, indicating that there were reduced risks from all PAHs. The results demonstrate that the combination of TPPS washing and microbial degradation is a useful and innovative process for remediation of PAH-contaminated soils.

Purification of papain by metal affinity partitioning in aqueous two-phase polyethylene glycol/sodium sulfate systems.

A simple and inexpensive aqueous two-phase affinity partitioning system using metal ligands was introduced to improve the selectivity of commercial papain extraction. Polyethylene glycol 4000 was first activated using epichlorohydrin, then it was covalently linked to iminodiacetic acid. Finally, the specific metal ligand Cu(2+) was attached to the polyethylene glycol-iminodiacetic acid. The chelated Cu(2+) content was measured by atomic absorption spectrometry as 0.88 mol/mol (polyethylene glycol). The effects on the purification at different conditions, including polyethylene glycol molecular weight (2000, 4000, and 6000), concentration of phase-forming components (polyethylene glycol 12-20% w/w and sodium sulfate 12-20%, w/w), metal ligand type, and concentration, system pH and the commercial papain loading on papain extraction, were systematically studied. Under optimum conditions of the system, i.e. 18% w/w sodium sulfate, 18% w/w polyethylene glycol 4000, 1% w/w polyethylene glycol-iminodiacetic acid-Cu(2+) and pH 7, a maximum yield of 90.3% and a degree of purification of 3.6-fold were obtained. Compared to aqueous two phase extraction without ligands, affinity partitioning was found to be an effective technique for the purification of commercial papain with higher extraction efficiency and degree of purification.

Identification of prohibitin 1 as a potential prognostic biomarker in human pancreatic carcinoma using modified aqueous two-phase partition system combined with 2D-MALDI-TOF-TOF-MS/MS.

Membrane proteins are an important source of potential targets for anticancer drugs or biomarkers for early diagnosis. In this study, we used a modified aqueous two-phase partition system combined with two-dimensional (2D) matrix-assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS, 2D-MALDI-TOF-TOF-MS/MS) analysis to isolate and identify membrane proteins in PANC-1 pancreatic cancer cells. Using this method, we identified 55 proteins, of which 31 (56.4%) were membrane proteins, which, according to gene ontology annotation, are associated with various cellular processes including cell signal transduction, differentiation, and apoptosis. Immunohistochemical analysis showed that the expression level of one of the identified mitochondria membrane proteins, prohibitin 1 (PHB1), is correlated with pancreatic carcinoma differentiation; PHB1 is expressed at a higher level in normal pancreatic tissue than in well-differentiated carcinoma tissue. Further studies showed that PHB1 plays a proapoptotic role in human pancreatic cancer cells, which suggests that PHB1 has antitumorigenic properties. In conclusion, we have provided a modified method for isolating and identifying membrane proteins and demonstrated that PHB1 may be a promising biomarker for early diagnosis and therapy of pancreatic (and potentially other) cancers.

Biodegradation of Endocrine Disruptors in Solid-Liquid Two-Phase Partitioning Systems by Enrichment Cultures

Naturally occurring and synthetic estrogens and other molecules from industrial sources strongly contribute to the endocrine disruption of urban wastewater. Because of the presence of these molecules in low but effective concentrations in wastewaters, these endocrine disruptors (EDs) are only partially removed after most wastewater treatments, reflecting the presence of these molecules in rivers in urban areas. The development of a two-phase partitioning bioreactor (TPPB) might be an effective strategy for the removal of EDs from wastewater plant effluents. Here, we describe the establishment of three ED-degrading microbial enrichment cultures adapted to a solid-liquid two-phase partitioning system using Hytrel as the immiscible water phase and loaded with estrone, estradiol, estriol, ethynylestradiol, nonylphenol, and bisphenol A. All molecules except ethynylestradiol were degraded in the enrichment cultures. The bacterial composition of the three enrichment cultures was determined using 16S rRNA gene sequencing and showed sequences affiliated with bacteria associated with the degradation of these compounds, such as Sphingomonadales. One Rhodococcus isolate capable of degrading estrone, estradiol, and estriol was isolated from one enrichment culture. These results highlight the great potential for the development of TPPB for the degradation of highly diluted EDs in water effluents.

Extractive fermentation using aqueous two-phase systems for integrated production and purification of extracellular lipase derived from Burkholderia pseudomallei

An extractive fermentation using aqueous two-phase partitioning system was developed in this study for the simultaneous cell cultivation and downstream processing of extracellular lipase derived from Burkholderia pseudomallei ( B. pseudomallei). The B. pseudomallei growth and the enzyme production in different types of two-phase partitioning systems were investigated. An aqueous two-phase system (ATPS), which is composed of 9.6% (w/w) polyethylene glycol (PEG) 8000 and 1.0% (w/w) Dextran T500, provided the best conditions for the extractive lipase production. In this integrated process, biomass was accumulated in the bottom phase whereas the lipase was extracted to the top phase. High yield of 92.1% was recorded in the single batch operation. Repetitive batch of fermentation was progressively carried out by continuous replacement of the top phase every 24 h, which resulted in an average enzyme concentration of 16.5 U/ml for seven extractive batch over the duration of 168 h. The extractive fermentation in aqueous two-phase partitioning system is an attractive approach for the combination of the lipase production and the purification process, owing to the repeated use of the two-phase fermentation system and the ease of lipase recovery.

Application of a PEG/salt aqueous two‐phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract

Aqueous two-phase partition systems (ATPS) have been widely used for the separation of a large variety of biomolecules. In the present report, the application of a polyethylene glycol/phosphate (PEG/phosphate) ATPS for the separation of anti-HIV monoclonal antibodies 2G12 (mAb 2G12) and 4E10 (mAb 4E10) from unclarified transgenic tobacco crude extract was investigated. Optimal conditions that favor opposite phase partitioning of plant debris/mAb as well as high recovery and purification were found to be 13.1% w/w (PEG 1500), 12.5% w/w (phosphate) at pH 5 with a phase ratio of 1.3 and 8.25% w/w unclarified tobacco extract load. Under these conditions, mAb 2G12 and mAb 4E10 were partitioned at the bottom phosphate phase with 85 and 84% yield and 2.4- and 2.1-fold purification, respectively. The proposed ATPS was successfully integrated in an affinity-based purification protocol, using Protein A, yielding antibodies of high purity and yield. In this study, ATPS was shown to be suitable for initial protein recovery and partial purification of mAb from unclarified transgenic tobacco crude extract.

New downstream processing strategy for the purification of monoclonal antibodies from transgenic tobacco plants

Affinity chromatography on immobilized Protein A is the current method of choice for the purification of monoclonal antibodies (mAbs). Despite its widespread use it presents certain drawbacks, such as ligand instability, leaching, toxicity and high cost. In the present work, we report a new procedure for the purification of two human monoclonal anti-HIV (human immunodeficiency virus) antibodies (mAbs 2G12 and 4E10) from transgenic tobacco plants using stable and low cost chromatographic materials. The first step of the mAb 2G12 purification procedure is comprised of an aqueous two-phase partition system (ATPS) for the removal of polyphenols while providing an essential initial purification boost (2.01-fold purification). In the second step, mAb 2G12 was purified using cation-exchange chromatography (CEX) on S-Sepharose FF, by elution with 20 mM sodium phosphate buffer pH 7.5, containing 0.1 M NaCl. The eluted mAb was directly loaded onto an immobilized metal affinity chromatography column (IMAC, Zn 2+–iminodiacetic acid–Sepharose 6B) and eluted by stepwise pH gradient. The proposed method offered 162-fold purification with 97.2% purity and 63% yield. Analysis of the antibody preparation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme immunosorbent assay (ELISA) and western blot showed that the mAb 2G12 was fully active and free of degraded variants, polyphenols and alkaloids. The effectiveness of the present purification protocol was evaluated by using a second transgenic human monoclonal anti-HIV mAb 4E10. The results showed that the same procedure can be successfully used for the purification of mAb 4E10. In the case of mAb 4E10, the proposed method offered 148-fold purification with 96.2% purity and 36% yield. Therefore, the proposed protocol may be of generic use for the purification of mAbs from transgenic tobacco plants.

In situ extraction of polar product of whole cell microbial transformation with polyethylene glycol‐induced cloud point system

A novel polyethylene glycol-induced cloud point system (PEG-CPS) was developed for in situ extraction of moderate polar product by setting a microbial transformation of benzaldehyde into L-phenylacetylcarbinol (L-PAC) with Saccharomyces cerevisiae (baker's yeast) as a model reaction. The biocompatibility of the microorganism in PEG-CPS was comparatively studied with a series of water-organic solvent two-phase partitioning systems. The tolerance of microorganism to the toxic substrate benzaldehyde was increased and the moderate polar product L-PAC was extracted into the surfactant-rich phase in the PEG-CPS. The novel PEG-CPS fills the gap of in situ extraction of polar product in microbial transformation left by water-organic solvent two-phase partitioning system. At the same time, the application of PEG-CPS in a microbial transformation also avoids expensive solvent when compared with that of aqueous two-phase system or CPS.