Application of polyethylene glycol-based aqueous biphasic reactive extraction to the catalytic oxidation of cyclic olefins

Glutaric acid and 1,2,3,4-butanetetracarboxylic acid (BTCA) have been synthesized by sodium tungstate catalyzed oxidation of the cyclic olefins: cyclopentene and 1,2,3,6-tetrahydrophthalic anhydride (THPA), using hydrogen peroxide in a polyethylene glycol (PEG)-2000/NaHSO(4) aqueous biphasic system (PEG-ABS). The production of glutaric acid and BTCA was found to increase from the monophasic to the biphasic regimes, and was found to be greatest at short tie-line lengths (TLLs), close to the system's critical point, yielding glutaric acid and BTCA in 73.1 and 82.5% yield, respectively. The results imply that mutual mixing or contact of the components is important, because the product dicarboxylic acids were found to increase from the monophasic side to the critical point and decrease from the critical point to more divergent regimes. The two reactions were compared with adipic acid synthesis from cyclohexene in terms of the cyclic olefin structure, and the partitioning of the dicarboxylic acid product in the ABS.     

Liquid–liquid extraction of an extracellular alkaline protease from fermentation broth using aqueous two-phase and reversed micelles systems

Summary The study of recovery of an extracellular alkaline protease from fermentation broth produced by Norcadiopsis sp, was carried out with liquid–liquid extraction through sodium di-(2-ethylhexyl) sulphosuccinate/isooctane reversed micelles systems and aqueous two-phase systems (polyethylene glycol/potassium phosphate). The best conditions for extraction and back-extraction with the reversed micelles system was obtained at pH 9.0 and pH 5.0, respectively, showing a yield of protein of 6.16%, a specific activity of 4.10 U/ml and a purification factor of 1.80. The studies using aqueous two-phase systems of polyethylene glycol/potassium phosphate at pH 10.0 showed purification factors of 2 and 5, and protein yield of 11 and 4%, respectively, for polyethylene glycol 550/potassium phosphate and polyethylene glycol 8000/potassium phosphate. The results indicate that the aqueous two-phase systems are more attractive as a first step in the isolation and purification processes.     

Preparation of carboxymethyl chitosan in aqueous solution under microwave irradiation

Carboxymethyl chitosan was prepared by reacting chitosan with chloroacetic acid in water under microwave irradiation. The effect of the reaction conditions was investigated and optimal conditions were identified. The influence of mass ratio of chloroacetic acid to chitosan, microwave power and pH on the degree of substitution or intrinsic viscosity were further studied. The degree of substitution of the carboxymethyl chitosan synthesized exceeded 0.85.     

Effect of amino acids on the eutectic behavior of NaCl solutions studied by DSC

The effect of a series of amino acids on the eutectic behavior of NaCl solutions at isotonic concentration has been studied by differential scanning calorimetry. The inclusion of different amino acids had different effects on eutectic formation. The amino acids were grouped into four categories based on their effect on eutectic formation: category C were amino acids that had no effect on eutectic formation; category D amino acids inhibited eutectic formation; category T amino acids shifted the melting of the eutectic to a lower temperature; category E amino acids caused the formation of a new eutectic with a melting temperature approximately -5 degrees C. The mechanism of these different effects on eutectic behavior is discussed, based on the chemical structure of the amino acids.     

Physical and rheological characterisation of polyethylene glycol-cashew-nut tree gum aqueous two-phase systems

The characterisation of the polyethylene glycol-cashew-nut tree gum aqueous two-phase system is described. Factors which affect the phase diagram including polymer molecular mass, pH and temperature were analysed. The physico-chemical properties of the system such as density, viscosity, volume ratio and phase separation times were also described. The characteristics of the system studied indicate it to be very attractive as a separation technique.     

Human aqueous humor proteome in cataract,glaucoma, and pseudoexfoliation syndrome

Twenty‐nine human aqueous humor samples from patients with eye diseases such as cataract and glaucoma with and without pseudoexfoliation syndrome were characterized by LC–high resolution MS analysis. In total, 269 protein groups were identified with 1% false discovery rate including 32 groups that were not reported previously for this biological fluid. Since the samples were analyzed individually, but not pooled, 36 proteins were identified in all samples, comprising the constitutive proteome of the fluid. The most dominant molecular function of aqueous humor proteins as determined by GO analysis is endopeptidase inhibitor activity. Label‐free protein quantification showed no significant difference between glaucoma and cataract aqueous humor proteomes. At the same time, we found decrease in the level of apolipoprotein D as a marker of the pseudoexfoliation syndrome. The data are available from ProteomeXchange repository (PXD002623).     

One-step purification of proteins from chicken egg white using counter-current chromatography

Proteins present in chicken egg white are separated by counter-current chromatography (CCC) in one step using a cross-axis coil planet centrifuge (X-axis CPC). The separation was performed with an aqueous polymer two-phase system composed of 16% (w/w) poly(ethylene glycol) 1000 and 12.5% (w/w) dibasic potassium phosphate by eluting the lower phase at a flow-rate of 1.0 ml/min. From about 20 g of the crude egg white solution, lysozyme, ovalbumin, and ovotransferrin were resolved within 5.5 h. Each component was identified by 12% SDS gel electrophoresis with Coomassie brilliant blue staining.     

Analytical partitioning of poly(ethylene glycol)-modified proteins

Covalently grafting proteins with varying numbers (n) of poly(ethylene glycol) molecules (PEGs) often enhances their biomedical and industrial usefulness. Partition between the phases in aqueous polymer two-phase systems can be used to rapidly characterize polymer-protein conjugates in a manner related to various enhancements. The logarithm of the partition coefficient (K) approximates linearity over the range 0<n<x. However, x varies with the nature of the conjugate (e.g., protein molecular mass) and such data analysis does not facilitate the comparison of varied conjugates. The known behavior of surface localized PEGs suggests a better correlation should exist between log K and the weight fraction of polymer in PEG-protein conjugates. Data from four independent studies involving three proteins (granulocyte-macrophage colony stimulation factor, bovine serum albumin and immunoglobulin G) has been found to support this hypothesis. Although somewhat simplistic, ‘weight fraction’ based analysis of partition data appears robust enough to accommodate laboratory to laboratory variation in protein, polymer and phase system type. It also facilitates comparisons between partition data involving disparate polymer-protein conjugates.     

Pyridine nucleotide-dependent ferricyanide reduction associated with isolated plasma membranes of maize (Zea mays L.) roots

Summary Plasma membranes (PM) from maize roots (Zea mays L.) were isolated by aqueous two-phase partitioning. The isolated membrane fraction showed a 4.6-fold enrichment in specific activity of the PM marker enzyme vanadate-sensitive, Mg²⁺-ATPase over a microsomal pellet collected at 50,000 × g. Activities of marker enzymes for mitochondria, endoplasmic reticulum, tonoplast, and Golgi apparatus were low or not detectable in the PM fraction. Quantitative morphometric analysis using the PM-specific silicotungstic acid stain showed the fraction to be > 92% PM vesicles. Using detergent stimulation of ATPase activity as a measure of structurally linked latency, greater than 90% of the PM vesicles were oriented with the cytoplasmic surface inside.An electron transport activity was investigated in the PM fraction. The rate of NADH oxidation in the absence of an artificial electron acceptor was < 167pkat·mg protein^–1; however, NADH catalysed the reduction of a variety of artificial electron acceptors including ferricyanide (2.6 nkat·mg protein^–1), cytochromec (0.8 nkat·mg protein^–1), a tetrazolium derivative (0.6 nkat·mg protein^–1) and dichlorophenol indophenol (0.4 nkat·mg protein^–1). While the NADH-dependent ferricyanide and dichlorophenol indophenol reductases were stimulated 6-fold by 0.025% (v/v) Triton X-100, the cytochromec and INT reductases were not greatly stimulated. Washing membranes with high salt significantly decreased the NADH-dependent, and eliminated the NADPH-dependent, ferricyanide reductase activity measured in the absence of detergent. These results suggest that NADH was oxidized on the extracytoplasmic surface of the membrane; however, a significant portion of this activity was extrinsic and may have originated from cytoplasmic contamination during isolation. The greater portion of the PM-associated NAD(P)H oxidation and/or ferricyanide reduction was catalyzed on sites not exposed to the outer surface of the membrane.Abbreviations BTP 1,3-bis[tris(hydroxymethyl)-methylamino]-propane - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate dihydrate - cytc cytochromec - DCIP 2,6-dichlorophenol indolphenol - INT 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride - kat mole·s^–1 - Mes 2-(N-morpholino)ethanesulfonic acid - MF microsomal fraction - PM plasma membrane - STA silicotungstic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol The mention of vendor or product does not imply that they are endorsed or recommended by U.S. Department of Agriculture over vendors of similar products not mentioned.     

Characterization and partial purification of multiple electron transport activities in plasma membranes from maize (Zea mays) roots

The plasma membrane of eukaryotic cells contains endogenous, integral electron transport proteins. In the maize ( Zea mays L. cv. Golden Cross Bantam) root plasma membrane, these activities include NAD(P)H-ferricyanide reductase. NAD(P)H-duroquinone reductase (1.6.5.1) and NAD(P)H-ascorbate free-radical reductase (EC 1.6.5.4). Differences in degree of stimulation upon vesicle rupture with detergent and in specificities for pyridine nucleotides suggest that these activities constitute distinct components in the membranes. Solubilization of reductase activities was examined using Triton X-100 over a wide range of retergent-to-protein ratios. The Triton-solubilized enzymes were purified using dye-ligand affinity chromatography on Cibacron blue 3G-A agarose utilizing biospecific elution with NADH. Resolution of the redox activities was accomplished upon differential elution with 0.1.1.0 and 10 m M NADH. The distinctive characteristics of the enzymes and the differential chromatographic behavior of the respective activities provided evidence for the presence of separate enzymatic redox components in maize root plasma membranes with implications for an electron transfer chain.