Aqueous two-phase extraction of 2,3-butanediol from fermentation broths by isopropanol/ammonium sulfate system

A novel aqueous two-phase system consisted of 2-propanol/ammonium sulfate was used for the extraction of 2,3-butanediol from fermentation broths. The maximum partition coefficient and recovery of 2,3-butanediol reached 9.9 and 93.7%, respectively, and more than 99% of the cells and about 85% of the soluble proteins were removed when 34% (w/w) 2-propanol and 20% (w/w) ammonium sulfate were used. The separated cells could be re-used as inocula for subsequent fermentations. The aqueous two-phase system described in this study may have potential application in the extraction of 2,3-butanediol produced by industrial fermentation processes.     

中药水提物对白念珠菌生物膜抑制作用的研究

目的研究中药水提物对体外白念珠菌生物膜的影响。方法体外构建白念珠菌生物膜,微量稀释法测定中药提取物对白念珠菌浮游菌最低抑菌浓度（M IC）,XTT减低法评价中药提取物对白念珠菌生物膜SM IC（SM IC80,SM IC50）及对白念珠菌黏附能力的影响。结果野菊花、土槿皮和决明子SM IC80为125 mg/m l,其余中药SM IC80为250或500 mg/m l;地肤子SM IC50为7.81 mg/m l,川芎、苏梗、藿香和决明子SM IC50为15.63 mg/m l,其他中药SM IC50在62.5~250 mg/m l。10味中药在100 mg/m l时均能显著抑制白念珠菌细胞的黏附,10 mg/m l时地肤子、苏梗和苦参对黏附仍具抑制作用。结论该10味中药水提物对体外白念珠菌生物膜有较强抑制效应。     

Evaluation of the effects of the parameters involved in the purification of clavulanic acid from fermentation broth by aqueous two-phase systems

The purification of clavulanic acid (CA), which is an important β-lactam antibiotic produced by submerged cultivation of Streptomyces clavuligerus, was studied through the use of phosphate and polyethylene glycol-based aqueous two-phase systems. The parameters’ effect on the yield and purification was evaluated through an experimental design and the preliminary results showed that the polyethylene molecular mass and tie-line length and phase volume ratio exerted the strongest effect on the yield and distribution coefficient in the range tested. In addition, the response surface methodology was used to optimize the distribution coefficient, yield, and purification factor. The optimal conditions of yield and purification factor are in the regions where polyethylene has a low molecular mass, pH close to the isoelectric point, and lower top phase volume. A 100% yield and a 1.5-fold purification factor are obtained when extracting CA by maximizing the conditions of an aqueous two-phase system.     

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.     

Therapeutic effects of Typha elephantina leave’s extract against paracetamol induced renal injury in rabbits

Present study focuses on ameliorative potential of Typha elephantina leave’s aqueous (TE.AQ) extract against Paracetamol (PCM) induced toxicity in rabbits. We fed the male rabbits with 300 mg PCM in alone and in combination with TE.AQ at different doses i.e. (100, 200 and 300 mg/kg body weight) or silymarin (100 mg/kg) daily for 21 days. PCM in alone significantly (P < 0.5) increased serum urea, uric acid, creatinine, total protein, albumin, globulin and blood urea nitrogen. Serum sodium, potassium and magnesium level were high. The glutathione, radical scavenging activity and Thiobarbituric acid reactive substances were significantly reduced. Treatment with TE.AQ at dose rate 300 mg/kg body weight and Silymarin significantly ameliorated all the parameters when compared with PCM administered group. The 100 and 200 mg of TE.AQ showed no significant effects. The histopathological examination confirmed the therapeutic potential of TE.AQ. These results established the presence of natural antioxidants in Typha elephantina leaves.     

Rhodium-catalyzed arylation of α-amido sulfones with arylboronic acids in a water-toluene biphasic system

An efficient method for the synthesis of N-protected diaryl-methyl-amines was developed through a rhodium-catalyzed arylation of α-amido sulfones with arylboronic acids in a water-toluene biphasic system. The use of a base combined with a surfactant played a key role in this biphasic reaction. A diverse range of α-branched amine derivatives bearing different functional groups were obtained within 10 min under the present conditions.     

Aqueous two-phase extraction as a platform in the biomanufacturing industry: economical and environmental sustainability

The biotech industry is, nowadays, facing unparalleled challenges due to the enhanced demand for biotechnology-based human therapeutic products, such as monoclonal antibodies (mAbs). This has led companies to improve substantially their upstream processes, with the yield of monoclonals increasing to titers never seen before. The downstream processes have, however, been overlooked, leading to a production bottleneck. Although chromatography remains the workhorse of most purification processes, several limitations, such as low capacity, scale-related packing problems, low chemical and proteolytic stability and resins' high cost, have arisen. Aqueous two-phase extraction (ATPE) has been successfully revisited as a valuable alternative for the capture of antibodies. One of the important remaining questions for this technology to be adopted by the biotech industries is, now, how it compares to the currently established platforms in terms of costs and environmental impact. In this report, the economical and environmental sustainability of the aqueous two-phase extraction process is evaluated and compared to the currently established protein A affinity chromatography. Accordingly, the ATPE process was shown to be considerably advantageous in terms of process economics, especially when processing high titer cell culture supernatants. This alternative process is able to purify continuously the same amount of mAbs reducing the annual operating costs from 14.4 to 8.5 million (US$/kg) when cell culture supernatants with mAb titers higher than 2.5 g/L are processed.     

Separation and purification of laccases from two different fungi using aqueous two-phase extraction

Selective purification still poses a challenge in the downstream processing of biomolecules such as proteins and especially enzymes. In this study a polyethylene glycol 3000 (PEG 3000)–phosphate aqueous two-phase system at 25 °C and pH 7 was successfully used for laccase purification and separation. Initially, the effect of phase forming components on enzyme activities in homogenous systems was studied. In the course of the extraction experiments tie lines, enzyme source, initial enzyme activities, phase ratio and sodium chloride concentrations were varied and their influence on the activity partitioning was determined. Partitioning results were validated using clear-native-PAGE and isoelectric focusing. Based on these results, the separation of laccases from Trametes versicolor and Pleurotus sapidus was investigated using the principle of superposition. Sodium chloride was used to adjust laccase partitioning in the applied aqueous two-phase system (ATPS). Finally, two modes of operation are proposed depending on the aim of the purification task. One mode with 0.133 g g⁻¹ of PEG3000, 0.063 g g⁻¹ of phosphate and without sodium chloride separates P. sapidus laccases from T. versicolor laccases with clearance factors of 5.23 and 6.45, respectively. The other mode of operation with 0.124 g g⁻¹ of PEG3000, 0.063 g g⁻¹ of phosphate and 0.013 g g⁻¹ of sodium chloride enables a partitioning of both laccases into the bottom phase of the ATPS resulting in a purification factor of 2.74 and 96% activity recovery.     

PEG/NaPA aqueous two-phase systems for the purification of proteases expressed by Penicillium restrictum from Brazilian Savanna

The partitioning of proteases expressed by Penicillium restrictum from Brazilian Savanna in an inexpensive aqueous two-phase system composed of poly (ethylene glycol) (PEG) and sodium polyacrylate (NaPA) was studied. The effects of PEG molecular weight and concentration, as well as NaPA concentration and the concentration of fermented broth on protease partitioning were studied. Partitioning into the top PEG-rich phase was increased in systems with smaller PEG-molecular weight, higher NaPA concentration and lower PEG concentration. For most systems studied, purification has been achieved by directing the biomolecule partition to the opposite phase of the other proteins, providing the enzyme purification. The highest partition coefficient was obtained using 20 wt% NaPA, 4 wt% PEG 2000 g mol⁻¹ and 45 wt% fermented broth, leading to a purification factor of 1.98 and partition coefficient of 37.73. The system showed high mass balances and yield, indicating enzyme stability and applicability for industrial processes. The partitioning results using the PEG/NaPA/NaCl system show that this method could be used to purify or concentrate protease from fermented broth.     

Dynamic and extensional properties of starch in aqueous dimethylsulfoxide

The effect of starch composition and concentration on the rheological properties of starch in a mixed solvent, water–DMSO, was investigated in dynamic shear and extensional mode. High amylose corn starch containing 70% amylose and 30% amylopectin, common corn starch containing 25% amylose and 75% amylopectin, and waxy corn starch containing about 99% amylopectin were used in this study. Concentrations of 2, 4, 6, and 8% (w/v) in 10% water-90% DMSO (v/v) were used for each starch type. An increase in the amylopectin content of starch from 30 to 99% caused a change in behavior from semidilute solution to viscoelastic solid at a concentration of 8% (w/v). At a concentration of 2%, an increase in the amylopectin content of starch from 30 to 99% caused a change from Newtonian to incipient gel-like behavior. Behavior at intermediate concentrations of 4 and 6% (w/v) varied from semidilute to critical gel-like with increasing amylopectin content. A power-law relaxation was observed for all concentrations of common and waxy corn starches with the slope decreasing with increase in concentrations. A 2% waxy corn starch solution displayed extension thinning behavior, while a 2% high amylose corn starch solution displayed Newtonian behavior.