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Solvent extraction is an effective method for purufucation of wet process phosphoroc acid. The aim of this study was purification of wet-process phosphoric acid using acid grade phosphorus pentoxide and minimization of fluoride and arsenic. The first step included the pre-purification which was done by discoloration of acid and clarification during which the impurities were reduced. Fluoride reduction rate of about 68% was significant at this step. In the following step, liquid-liquid extraction was used to purify the phosphoric acid from previous stage. The parameters of phase ratio, two-phase contact time and the fluid flow regime in a packed column of the fluidized bed were studied using 2-ethylhexanol as a basic solvent. The results of this part have been used as an evaluation of acid extraction yield and separation factor of impurities using an experiment designed by the Taguchi method. Arsenic, fluoride and phosphorus pentoxide concentration were measured using Inductivity Coupled Plasma (ICP) and Ion Chromatography (IC) analysis. The optimal level of parameters and their effect was obtained. By comparing the optimal and worst level in extraction step, it was found that the amount of acid extracted can vary from 23% to 73%. Furthermore, the removal rate of 49% for fluoride and 67% for arsenic in the worst conditions, can increase to the optimal conditions as much as 72% and 93%, respectively. The overal removal rate of 92% for fluoride and 96% for arsenic in two stages were achieved. Increasing of phase ratio, the time as well as increasing the flow regime created optimal conditions in the extraction section. [ABSTRACT FROM AUTHOR]

Potassium stable isotope compositions (41K/39K ratio) exhibit a wide range of natural variation (∼3‰), whose measurement has been recently achievable thanks to the development of various methods, including collision cell, XHR mode, cold and dry plasma, or dummy bucket MC-ICP-MS. Here, we report the capabilities of a Thermo Scientific new generation MC-ICP-MS, Neoma, to routinely measure the 41K/39K ratio. We found that the effect of potassium concentration mismatch between the sample and standard is comparable to other reported methods. However, the effect of acid concentration mismatch is not perceptible from 0.05 M to 0.5 M HNO3, and the effect of matrix elements on the accuracy of sample-standard bracketing measurements is lower compared to other reported methods up to the element-K normalized ratio of >15%. This relative insensibility to non-spectral matrix effects most probably results from the new design of the introduction system that integrates an iCAP Qnova Series ICP-MS torch and injector assembly. The resulting repeatability (or short-term external precision) and long-term intermediate precision (or long-term reproducibility) are 0.06‰ (2SD, n = 55) and 0.07‰ (2SD, n = 12), respectively. We then use a very simple one step chemical purification of K by ion exchange chromatography and apply the method to the measurement of the δ41K value of geological and biological reference materials, and blood samples of cancer and control patients. The results for reference materials show very good agreement with previously reported values and extend the available data for future interlaboratory comparison studies. Blood samples of cancer and control patients exhibit similar δ41K values despite a suspected isotopic difference between tumour and normal tissues. The overall instrumental robustness observed using the XHR mode remains to be challenged with the precell mass filter and hexapole collision/reaction cell upgrade. [ABSTRACT FROM AUTHOR]

• C-phycocyanin (C-PC) was purified by one ion exchange chromatography (IEC) step. • pH gradient elution yielded better purification results than salt gradient elution. • Different parameters were studied to purify C-PC by IEC with pH gradient elution. • C-PC with purities of 4.2 and 3.5 were obtained with overall recovery of 82.1 %. • A single purification step was used to achieve the maximum C-PC purity. C-phycocyanin (C-PC) is a phycobiliprotein that can be used as a natural blue dye in the food and cosmetic industries, as a biomarker or as an agent in medical treatments, depending on its purity grade. Here we described for the first time a single-step purification process of C-PC extracted from the wet biomass of Spirulina (Arthrospira) platensis LEB-52 using ion exchange chromatography with pH gradient elution. Different conditions varying the elution buffers and volumes, the loading pH and the addition of salt in the elution buffer were studied. The chromatographic condition that resulted in high recovery and purity consisted in equilibration and washing with 0.025 mol/L Tris-HCl buffer pH 6.5 and elution combining a step with 0.08 mol/L NaCl in 0.025 mol/L Tris-HCl buffer pH 6.5 and a pH gradient elution with 0.05 mol/L citrate buffer pH 6.2–3.0. This process resulted in C-PC with purities of 4.2 and 3.5 with recoveries of 32.6 and 49.5 %, respectively, in one purification step. [ABSTRACT FROM AUTHOR]

In recent years, many biological-based products have been developed, representing a significant fraction of income in the pharmaceutical market. Ion exchange chromatography is an important downstream step for the purification of target recombinant proteins present in clarified cell extracts, together with many other unknown impurities. This work develops a robust approach to model and simulate the purification of untagged heterologous proteins, so that the improved conditions to carry out an ion exchange chromatography are identified in a rational basis prior to the real purification run itself. Purification of the pneumococcal surface protein A (PspA4Pro) was used as a case study. This protein is produced by recombinant Escherichia coli and is a candidate for the manufacture of improved pneumococcal vaccines. The developed method combined experimental and computational procedures. Different anion exchange operating conditions were mapped in order to gather a broad range of representative experimental data. The equilibrium dispersive and the steric mass action equations were used to model and simulate the process. A training strategy to fit the model and separately describe the elution profiles of PspA4Pro and other proteins of the cell extract was applied. Based on the simulation results, a reduced ionic strength was applied for PspA4Pro elution, leading to increases of 14.9% and 11.5% for PspA4Pro recovery and purity, respectively, compared to the original elution profile. These results showed the potential of this method, which could be further applied to improve the performance of ion exchange chromatography in the purification of other target proteins under real process conditions. • General approach applied to model untagged protein purification from cell extracts • Experimental strategy to collect wide range of suitable data for modeling developed • Robust parameter estimation strategy comprised in a new approach • Elution profiles of unknown and target proteins in complex mixtures well described • Model simulations enabled increases in target protein recovery and purity [ABSTRACT FROM AUTHOR]

Large amounts of 10 Be are produced at the PSI muon production facility by high-energy proton spallation in polycrystalline graphite. For the extraction of 10 Be out of large amounts of carbon, pyrolysis followed by chemical purification has been performed. Approx. 270 g of graphite from Target E92, which had received a total proton charge of 29 Ah between 2002 and 2005, have been burned at 1000 °C in a stream of oxygen. The volatile radioactive oxidation product 3 H 2 O was trapped in 3 water bubblers connected in series. The remainder, a white hygroscopic solid material mainly consisting of 7 Li 2 O, 9/10 BeO and 10/11 B 2 O 3 , was dissolved in HF and subsequently purified by ion exchange chromatography. Radioactive impurities such as 22 Na, 44 Ti, 54 Mn, 60 Co, 101 Rh, 133 Ba and 172 Hf have been separated from the final product. The purified material represents a mixture of approx. 6.5 mg 9 Be and 3.5 mg (3.3 MBq) 10 Be. It is ready to be used for scientific investigations requiring large amounts of this precious isotope. [ABSTRACT FROM AUTHOR]

Recombinant protein-based virus-like particles (VLPs) are steadily gaining in importance as innovative vaccines against cancer and infectious diseases. Multiple VLPs are currently evaluated in clinical phases requiring a straightforward and rational process design. To date, there is no generic platform process available for the purification of VLPs. In order to accelerate and simplify VLP downstream processing, there is a demand for novel development approaches, technologies, and purification tools. Membrane adsorbers have been identified as promising stationary phases for the processing of bionanoparticles due to their large pore sizes. In this work, we present the potential of two strategies for designing VLP processes following the basic tenet of ‘quality by design’: High-throughput experimentation and process modeling of an anion-exchange membrane capture step. Automated membrane screenings allowed the identification of optimal VLP binding conditions yielding a dynamic binding capacity of 5.7 mg/mL for human B19 parvovirus-like particles derived from Spodoptera frugiperda Sf 9 insect cells. A mechanistic approach was implemented for radial ion-exchange membrane chromatography using the lumped-rate model and stoichiometric displacement model for the in silico optimization of a VLP capture step. For the first time, process modeling enabled the in silico design of a selective, robust and scalable process with minimal experimental effort for a complex VLP feedstock. The optimized anion-exchange membrane chromatography process resulted in a protein purity of 81.5%, a DNA clearance of 99.2%, and a VLP recovery of 59%. [ABSTRACT FROM AUTHOR]

Age of individual uranium-plutonium (U/Pu) mixed particles with various U/Pu atomic ratios (1-70) were determined by inductively coupled plasma mass spectrometry. Micron-sized particles were prepared from U and Pu certified reference materials. The Pu reference was stored for 4-6 years since the last purification (July 14, 2008). The Pu purification age was obtained from the Am/Pu ratio which was calculated from the product of three measured ratios of Pu and Am isotopes in the eluted fractions. These ratios were measured by a high-resolution inductively coupled plasma mass spectrometer equipped with a desolvation system. Femto-gram to pico-gram quantities of Am, U, and Pu in a sample solution were sequentially separated on a small anion-exchange column. The Am/Pu ratio was accurately determined by spiking pure Am into the sample solution. The average determined age for the particles for the five independent U/Pu ratios was in good agreement with the expected age with high accuracy (difference age 0.27 years) and high precision (standard deviation 0.44 years). The described analytical technique can serve as an effective tool for nuclear safeguards and environmental radiochemistry. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Three kinds of polysaccharides, namely, BSP1A, BSP2A, and BSP3B, were isolated from raw bamboo shoot (Dendrocalamus latiflorus) after purification and classification by DEAE cellulose-52 (ion-exchange chromatography) and Sephadex G-50. The molecular weights of BSP1A, BSP2A, and BSP3B were 10.2, 17.0 and 20.0 kDa, respectively, which were measured through GPC (gel performance chromtatography) methods. BSP1A contained arabinose, glucose, and galactose in a molar ratio of 1.0:40.6:8.7. BSP2A and BSP3B contained arabinose, xylose, glucose, and galactose in molar ratios of 6.6:1.0:5.2:10.4 and 8.5:1.0:5.1:11.1, respectively. The existence of the O-glycopeptide bond in BSP1A, BSP2A, and BSP3B was demonstrated by β-elimination reaction. FTIR spectra of the three polysaccharides showed that both BSP2A and BSP3B contained β-D-pyranose sugar rings. However, BSP1A exhibited both β-D-pyranose and α-D-pyranose sugar rings. Congo red test indicated that BSP1A and BSP2A displayed triple helix structures, but BSP3B did not. NMR spectroscopy revealed that BSP1A may exhibit a β-1,6-Glucan pyran type as the main link, and few 1,6-glycosidic galactose pyranose and arabinose bonds were connected; BSP2A mainly demonstrated →5)β-Ara(1→and→3)β-Gal(1→connection. Furthermore, BSP3B mainly presented →3)β-Glu(1→and→3)β-Gal(1→connection and may also contain few other glycosidic bonds. [ABSTRACT FROM AUTHOR]

Sr was produced via Y(n, p) Sr using yttria as target in Fast Breeder Test Reactor (FBTR), Kalpakkam, India. A radiochemical procedure has been developed for the separation of bulk yttrium using TBP by solvent extraction followed by purification of Sr source by ion exchange chromatography using the cation exchange resin Dowex 50WX8 (100-200 mesh) and nitric acid of variable molarity as eluant. The present study establishes the purification of Sr source from the other radionuclidic impurities like Y, Zn, Mn, Co, Rb, Ir, Ru, Sn, Ce, Tb, Eu etc. produced during the irradiation of yttria by using the complexing agent tri-sodium tri-meta phosphate (SMP) in nitric acid medium instead of nitric acid alone as an eluant. The purification was achieved by using 0.1 M SMP as complexing agent which was optimized based on the distribution ratio data and final elution of Sr fraction was obtained in nitric acid medium. This resulted into a faster purification of Sr source in a smaller volume of eluant. Purity of Sr source from the cross contamination of the complexing agent SMP was also ensured. [ABSTRACT FROM AUTHOR]

This report describes the purification and characterization of an enzyme that exhibits cellulase activity produced by the wood degrading bacteria, Bacillus circulans. The enzyme was purified by ion-exchange chromatography using CM-Sepharose CL-6B, and shown to exhibit hydrolytic activity on carboxymethylcellulose. The molecular weight of the purified enzyme was determined to be 43 KDa by means of SDS-PAGE. The kinetic parameters, and the effects of pH and temperature on the purified enzyme were determined. The enzyme was 4.37 fold and showed a specific activity of 29.13 μg of glucose produced/min/mg protein. The apparent Km value for the hydrolysis of carboxymethylcellulose was 1.061 ± 1.17 mg/ml with a Vmax of 13.75 ± 1.51 μg of glucose produced/ml/min. The enzyme showed an optimum pH value of 9.0 and the optimum temperature was 50 °C. Alkalophilicity and moderate thermostability of this enzyme are some of its essential characteristics that may make it suitable for industrial and biotechnological applications. [ABSTRACT FROM AUTHOR]

We report a method for the chemical purification of Pt from geological materials by ion-exchange chromatography for subsequent Pt stable isotope analysis by multiple-collector inductively coupled plasma mass spectrometry (MC-ICPMS) using a 196Pt–198Pt double-spike to correct for instrumental mass bias. Double-spiking of samples was carried out prior to digestion and chemical separation to correct for any mass-dependent fractionation that may occur due to incomplete recovery of Pt. Samples were digested using a NiS fire assay method, which pre-concentrates Pt into a metallic bead that is readily dissolved in acid in preparation for anion-exchange chemistry. Pt was recovered from anion-exchange resin in concentrated HNO3 acid after elution of matrix elements, including the other platinum group elements (PGE), in dilute HCl and HNO3 acids. The separation method has been calibrated using a precious metal standard solution doped with a range of synthetic matrices and results in Pt yields of ≥90% with purity of ≥95%. Using this chemical separation technique, we have separated Pt from 11 international geological standard reference materials comprising of PGE ores, mantle rocks, igneous rocks and one sample from the Cretaceous–Paleogene boundary layer. Pt concentrations in these samples range from ca. 5ngg−1 to 4μgg−1. This analytical method has been shown to have an external reproducibility on δ 198Pt (permil difference in the 198Pt/194Pt ratio from the IRMM-010 standard) of ±0.040 (2sd) on Pt solution standards (Creech et al., 2013, J. Anal. At. Spectrom. 28, 853–865). The reproducibility in natural samples is evaluated by processing multiple replicates of four standard reference materials, and is conservatively taken to be ca. ±0.088 (2sd). Pt stable isotope data for the full set of reference materials have a range of δ 198Pt values with offsets of up to 0.4‰ from the IRMM-010 standard, which are readily resolved with this technique. These results demonstrate the potential of the Pt isotope system as a tracer in geochemical systems. [ABSTRACT FROM AUTHOR]

Highlights: [•] Retention behavior of monoclonal IgG1 was investigated on IEC and HIC media. [•] Dynamics of both processes was dominated by kinetic effects. [•] A short-cut method was proposed for mathematical modeling. [•] A dynamic model was used for optimization of the integrated process IEC/HIC. [ABSTRACT FROM AUTHOR]

Highlights: [•] Monoclonal anybody IgG1 was isolated out of a CHO derived supernatant. [•] Different purification techniques were used based on IEC, HIC and ATPE. [•] The best performance was achieved by integrating IEC and HIC. [•] The on-column dilution was used for feed loading in the integrated process. [ABSTRACT FROM AUTHOR]

This paper describes the purification and characterization of a novel acid stable and pH tolerant α-amylase from a Aspergillusoryzae JGI 21 isolated from Mangalore. The enzyme displayed a molecular weight of 22 kDa and it was stable over a broad range of acidic and alkaline pH with maximum activity and stability at 6.5. The optimum temperature of enzyme stability was found to be around 24+/-2°C. The purification of α-amylase by ammonium sulphate precipitation and ion-exchange chromatography resulted in 23.56 fold increase in its activity (100.38 U/mg protein). Considering its promising properties, this enzyme can find potential applications in the food industry as well as in laundry detergents. [ABSTRACT FROM AUTHOR]

Abstract: This work is focused on the experimental evaluation and mathematical modeling of ion exchange expanded-bed chromatography for the purification of C-phycocyanin from crude fermentative broth containing Spirulina platensis cells. Experiments were carried out in different expansion degree to evaluate the process performance. The experimental breakthrough curves were used to estimate the mass transfer and kinetics parameters of the proposed model, using the Particle Swarm Optimization algorithm (PSO). The proposed model satisfactorily fitted the experimental data. The results from the model application pointed out that the increase in the initial bed height does not influence the process efficiency, however enables the operation of expanded-bed column at high volumetric flow rates, improving the productivity. It was also shown that the use of mathematical modeling was a good and promising tool for the optimization of chromatographic processes. [Copyright &y& Elsevier]

Trichosporon cutaneum phenylalanine ammonia lyase was selected as a model to investigate the dual substrate activity of this family of enzymes. Sequencing of the PAL gene identified an extensive intron region at the N-terminus. Five amino acid residues differing from a prior report were identified. Highest Phe : Tyr activities (1.6 ± 0.3 : 0.4 ± 0.1 μmol/h g wet weight) were induced by Tyr. The enzyme has a temperature optimum of 32°C and a pH optimum of 8-8.5 and shows no metal cofactor dependence. Michaelis-Menten kinetics (Phe, Km 5.0 ± 1.1 mM) and positive allostery (Tyr, K1 2.4 ± 0.6 mM, Hill coefficient 1.9 ± 0.5) were observed. Anion exchange chromatography gave a purification fold of 50 with 20% yield. The His-Gln motif (substrate selectivity switch region) indicates the enzyme's ability to act on both substrates. [ABSTRACT FROM AUTHOR]

The article presents the findings of a research conducted on examining the purification process of an acidic α-glucan-protein complex from the mushroom Pleurotus sajor-caju. It discusses the methodology used in conducting the research including anion exchange chromatography, gel filtration chromatography and statistical analysis. The research reveals that both xylanase and cellulase play important role in the hydrolysis of the P. sajor-caju cell walls.

Abstract: Screening of crustacean proteins in relation to molting cycle revealed a 48kDa protein which is cross reactive to Gas7 antibody and expressed predominantly in the intermolt stage where the cells remain quiescent. This 48kDa protein of Penaeus monodon has been purified to homogeneity using ammonium sulphate precipitation, ion-exchange chromatography and electro elution. MALDI-TOF analysis of the purified protein revealed the presence of ANK repeat and LEM domain regions homologous to those of mouse. RT-PCR revealed 684bp in all molting stages with varied expression. Bioinformatic analysis showed that the ankyrin repeat domain in the 48kDa protein of P. monodon is highly conserved among Homo sapiens, Mus musculus, Rattus rattus, Danio rerio and Drosophila mojavensis. Secondary structure prediction revealed a commonly conserved region between mouse Gas7 and the ANK repeat region of P. monodon. Thus, we concluded that the isolated 48kDa protein from P. monodon is a novel, Gas7-like protein with ANK repeat and LEM domain involved in molting. [Copyright &y& Elsevier]

Screening for cyclodextrin glycosyltransferase (CGTase)-producing alkaliphilic bacteria from samples collected from hyper saline soda lakes (Wadi Natrun Valley, Egypt), resulted in isolation of potent CGTase producing alkaliphilic bacterium, termed NPST-10. 16S rDNA sequence analysis identified the isolate as Amphibacillus sp. CGTase was purified to homogeneity up to 22.1 fold by starch adsorption and anion exchange chromatography with a yield of 44.7%. The purified enzyme was a monomeric protein with an estimated molecular weight of 92 kDa using SDS-PAGE. Catalytic activities of the enzyme were found to be 88.8 U mg-1 protein, 20.0 U mg-1 protein and 11.0 U mg-1 protein for cyclization, coupling and hydrolytic activities, respectively. The enzyme was stable over a wide pH range from pH 5.0 to 11.0, with a maximal activity at pH 8.0. CGTase exhibited activity over a wide temperature range from 45 °C to 70 °C, with maximal activity at 50 °C and was stable at 30 °C to 55 °C for at least 1 h. Thermal stability of the purified enzyme could be significantly improved in the presence of CaCl2. Km and Vmax values were estimated using soluble starch as a substrate to be 1.7 ± 0.15 mg/mL and 100 ± 2.0 μmol/min, respectively. CGTase was significantly inhibited in the presence of Co2+, Zn2+, Cu2+, Hg2+, Ba2+, Cd2+, and 2-mercaptoethanol. To the best of our knowledge, this is the first report of CGTase production by Amphibacillus sp. The achieved high conversion of insoluble raw corn starch into cyclodextrins (67.2%) with production of mainly β-CD (86.4%), makes Amphibacillus sp. NPST-10 desirable for the cyclodextrin production industry. [ABSTRACT FROM AUTHOR]

Objective: Some properties of cellulase purified from the culture supernatant of Bacillus coagulans Co4, isolated from cocoa pod dumpsite were investigated for possible biotechnological applications. Methods: The crude cellulase was purified to apparent homogeneity using a combination of acetone precipitation, CM Sepharose CL-6B ion exchange chromatography and gel filtration on Sephadex G-100. The molecular and thermodynamic properties of the purified enzyme were studied following standard procedures. Results: The specific activity of the purified cellulase rose from 0.10 to 47 units/mg of protein, at the end of purification. The molecular weight was found to be 14.5 kDa; and an apparent Km value of 0.18±0.06 mg/ml of carboxylmethylcellulose. The optimum pH and temperature were 7.5 and 60°C respectively. The activation energy for carboxylmethylcellulose hydrolysis (Ea) was 16.5 kJ/mol. Na+ and K+ had no effects on its activity at concentrations up to 200 mM, whereas Ca2+ and Mg2+ served as inhibitors at concentrations above 25 and 40 mM respectively. The cellulase retained 40% residual activity when heated at 60°C for 40 minutes. Conclusison: On the basis of these properties, it is concluded that the purified cellulase is moderately thermostable and may have applications in the bioconversion of agricultural wastes into economically useful products. [ABSTRACT FROM AUTHOR]

ABSTRACT Peroxidase (POD) from fresh-cut Zizania latifolia was purified using a combination of (NH4)2SO4 fractionation and anion exchange chromatography, resulting in one cationic (PODc) and two anionic fractions (PODa I and II). The cationic fraction, which accounted for 76% of recovered activity, was further purified by gel filtration. The POD activity was purified 61.51-fold with 9.32% recovery. The purified enzyme contains three POD isoenzymes (PODc1-3) as estimated by native polyacrylamide gel electrophoresis and showed molecular weights of 20.6-22.1 kDa on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The optimum pH and temperature were 6.0 and 40C, respectively. The enzyme was stable in the pH range 4.0-7.0 while retained only 2.21% of the activity after 1 min at 60C. The Km for guaiacol and H2O2 were 10 mM and 0.15% (v/v), respectively. Influence of various chemicals on the enzyme activity were studied. These results provide a theoretical basis for inhibiting browning and lignification of fresh-cut Z. latifolia. PRACTICAL APPLICATIONS The marketing of packaged and refrigerated fresh-cut Zizania latifolia is a choice of increasing interest for vegetables processors. However, cut surface browning and tissue lignification in fresh-cut Z. latifolia are two major causes of loss in quality. It has been reported that peroxidase (POD) may be involved in enzymatic browning and lignification. This study investigated the purification and characterization of POD from fresh-cut Z. latifolia. Results showed that heat treatment and/or chemical dip (L-cysteine, N-acetyl-L-cysteine and ascorbic acid) might be suitable techniques for inhibiting browning and lignification of the fresh-cut Z. latifolia. [ABSTRACT FROM AUTHOR]

Abstract: Trypsin was purified from the viscera of barbel by precipitation using ammonium sulphate (0–80%), Sephadex G-100, and Mono Q-Sepharose ion exchange chromatography. The trypsin was purified 27-fold, with 79U/mg specific activity and 31% recovery. The enzyme had a molecular weight of 24kDa; purified trypsin appeared as a single band on native-PAGE. The optimum pH and temperature for enzyme activity were pH 10.0 and 55°C with BAPNA used as a substrate. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECTPYSQ. The Michaelis–Menten constant (K m) and catalytic constant (k cat) values of the enzyme were 0.018mM and 1.21s−1, respectively. The study also investigated the effects of purified trypsin on the recovery of carotenoproteins from shrimp (Parapenaeus longirostris) shells through hydrolysis using 1.0U barbel trypsin/g shrimp shells for 1h at 30°C. The freeze-dried carotenoproteins recovered contained 71.09% protein, 16.47% lipid, 7.78% ash, and 1.79% chitin. [Copyright &y& Elsevier]

Abstract: A novel heme peroxidase from Jatropha curcas, an important source of bio-diesel, was purified to homogeneity using ammonium sulfate fractionation, desalting chromatography and ion exchange chromatography. Molecular mass of this purified enzyme was around 48kDa as detected by SDS-PAGE. Gel filtration analysis revealed that the enzyme was a monomer under native conditions. The purified enzyme had broad substrate specificity with the ideal substrates of guaiacol and o-phenylenediamine. The optimum temperature, pH and K m value of this peroxidase for guaiacol was 60°C, 5.0 and 0.17mM, respectively. In addition, NaCl (2.5M) significantly enhanced the activity of this peroxidase. The purified enzyme was stable under high temperature (70% activity retained after 1h incubation at 70°C), extreme pH environment (93% or more activity retained after 2h incubation under pH 3–12), high NaCl concentration (88% or more activity retained after 2h incubation with 1–4M NaCl) and organic solvents (95% or more activity retained after 54h incubation with various organic solvents). Moreover, this peroxidase was resistant against 20mM hydrogen peroxide, 8M urea, 3M guanidine hydrochloride and 20mM EDTA. However, the peroxidase activity was significantly inhibited by sodium azide, dithiothreitol, CTAB, β-mercaptoethanol, DMSO, toluene and ferrous ion. The enzyme had long shelf life with 180 days at 4°C and 14 days at room temperature. This new robust peroxidase may bring a better understanding for the high anti-adversity property of J. curcas. Meanwhile, the broad substrate specificity, wide stability against high temperature, extreme pH, organic solvent and hydrogen peroxide suggested that the enzyme could be a potential candidate peroxidase source for industrial and biomedical applications. [Copyright &y& Elsevier]

Abstract: An extracellular protease from Pseudomonas aeruginosa A2 grown in media containing shrimp shell powder as a unique source of nutriments was purified and characterized. The enzyme was purified to homogeneity from culture supernatant by ultrafiltration, Sephadex G-100 gel filtration and Sepharose Mono Q anion exchange chromatography, with a 2.23-fold increase in specific activity and 64.3% recovery. The molecular mass of the enzyme was estimated to be 34kDa. Temperature and pH with highest activity were 60°C and 8.0, respectively. The protease activity was inhibited by EDTA suggesting that the purified enzyme is a metalloprotease. The enzyme is stable in the presence of organic solvents mainly diethyl ether and DMSO. The lasB gene, encoding the A2 elastase, was isolated and its DNA sequence was determined. The A2 protease was tested for shrimp waste deproteinization in the process of chitin preparation. The percent of protein removal after 3h hydrolysis at 40°C with an enzyme/substrate (E/S) ratio of 5U/mg protein was about 75%. Additionally, A2 proteolytic preparation demonstrated powerful depilating capabilities of hair removal from bovine skin. Considering its promising properties, P. aeruginosa A2 protease may be considered a potential candidate for future use in several biotechnological processes. [Copyright &y& Elsevier]

Abstract: The paper presents the developed method for purification of ammonium perrhenate solutions by ion-exchange method on strongly acidic cation-exchange resin C160. The developed method is characterized by high, over 95% efficiency of sorption of potassium, sodium, calcium, iron, magnesium and lead ions, and high efficiency of regeneration of the resin with 32% solution of nitric(V) acid. In that way ammonium perrhenate was produced of the following composition: 69.4% Re, <0.0005% Ca, <0.001% K, <0.0005% Mg, <0.0005% Cu, <0.0005% Na, <0.0005% Mo, <0.0005% Ni, <0.0005% Pb, <0.0005% Fe. Some selected results of the experiments into possibilities for application of that specifically purified ammonium perrhenate in production of W–Re–Ni–Fe heavy alloys are presented as well as physical and mechanical properties of the heavy alloys. It was established that the purified ammonium perrhenate is a proper material for production of heavy alloys, and the generated sinters can be used in manufacture of armour-piercing discarding sabots. [Copyright &y& Elsevier]

Neutron irradiation of a natural Sm target is performed to produce Sm. Ion-exchange chromatography is used to separate Sm from Eu radionuclides produced in the process. To prepare a target, SmO powder is dissolved in 0.2 ml of HNO in a quartz vial. Samarium is deposited onto the vial walls by passing nitrogen gas at 120°C. The prepared samarium target is encapsulated in aluminum foil and irradiated in a 5-MW reactor in 5 × 10 n cm s flux. The irradiated target is dissolved in 1 M HCl, and the produced radioisotopes are determined with an HPGe nuclear detector. Finally Sm is separated from Eu radionuclides with Sm recovery yield of more than 66% and purity better than 99.8%. [ABSTRACT FROM AUTHOR]

Abstract: A novel laccase from a white rot fungus Abortiporus biennis was purified following a purification scheme involving ammonium sulfate saturation, ion exchange chromatography on DEAE-cellulose, CM-cellulose and Q-Sepharose, and fast protein liquid chromatography-gel filtration on Superdex 75. The molecular mass of the enzyme was 56kDa estimated by SDS-PAGE. The protein was monomeric with an N-terminal amino acid sequence of AIGPVADLTISNG, which exhibited partial homology to the sequence of laccases from family Polyporaceae, but was different from other families. Its optimal pH was 3.0–4.0, and the optimal temperature was 60°C. The laccase displayed a Km value of 33.5μM with ABTS as a substrate at pH 3.5 and 37°C. It demonstrated anti-proliferative activity against Hep G2 and MCF-7 cells with IC50 values of 12.5μM and 6.7μM, respectively, and exhibited inhibitory activity against HIV-1 reverse transcriptase with an IC50 value of 9.2μM. [Copyright &y& Elsevier]

Abstract: An extracellular alkaline protease (Prot-2) selectively secreted by Botrytis cinerea growing in medium containing Spirulina algae as inducer was purified to homogeneity by a combination of ammonium sulfate precipitation, gel filtration and ion-exchange chromatography, followed by size-exclusion chromatography. Prot-2 presented a single 30-kDa band on SDS-PAGE, which showed proteolytic activity following renaturation. Prot-2 has a monomeric structure, is active in the pH range 5.0–9.0 and shows an optimal temperature of activity at 50°C. Prot-2 is thermostable, and activated by Ca2+. The inhibitory action of reducing agents (dithiothreitol or β-mercaptoethanol) was suppressed by dithiobis-nitrobenzoic acid (DTNB) addition, indicating the role played by disulfide in enzyme activity and/or stability. Prot-2 showed extreme stability towards non-ionic surfactants (5% Tween 20, 5% Triton X-100 and Nonidet P-40). It was relatively stable in 25% aqueous/organic solvent mixtures and was activated by oxidizing agents (H2O2 and sodium perborate). A broad specificity of Prot-2 was tested using a range of natural and synthetic oligopeptide substrates. It was further supported by the hydrolysis profile of the insulin B chain by Prot-2. [Copyright &y& Elsevier]

Abstract: Cation exchange chromatography using conventional resins, having either diffusive or perfusive flow paths, operated in bind-elute mode has been commonly employed in monoclonal antibody (MAb) purification processes. In this study, the performance of diffusive and perfusive cation exchange resins (SP-Sepharose FF (SPSFF) and Poros 50HS) and a convective cation exchange membrane (Mustang S) and monolith (SO3 Monolith) were compared. All matrices were utilized in an isocratic state under typical binding conditions with an antibody load of up to 1000g/L of chromatographic matrix. The dynamic binding capacity of the cation exchange resins is typically below 100g/L resin, so they were loaded beyond the point of anticipated MAb break through. All of the matrices performed similarly in that they effectively retained host cell protein and DNA during the loading and wash steps, while antibody flowed through each matrix after its dynamic binding capacity was reached. The matrices differed, though, in that conventional diffusive and perfusive chromatographic resins (SPSFF and Poros 50HS) demonstrated a higher binding capacity for high molecular weight species (HMW) than convective flow matrices (membrane and monolith); Poros 50HS displayed the highest HMW binding capacity. Further exploration of the conventional chromatographic resins in an isocratic overloaded mode demonstrated that the impurity binding capacity was well maintained on Poros 50HS, but not on SPSFF, when the operating flow rate was as high as 36 column volumes per hour. Host cell protein and HMW removal by Poros 50HS was affected by altering the loading conductivity. A higher percentage of host cell protein removal was achieved at a low conductivity of 3mS/cm. HMW binding capacity was optimized at 5mS/cm. Our data from runs on Poros 50HS resin also showed that leached protein A and cell culture additive such as gentamicin were able to be removed under the isocratic overloaded condition. Lastly, a MAb purification process employing protein A affinity chromatography, isocratic overloaded cation exchange chromatography using Poros 50HS and anion exchange chromatography using QSFF in flow through mode was compared with the MAb''s commercial manufacturing process, which consisted of protein A affinity chromatography, cation exchange chromatography using SPSFF in bind-elute mode and anion exchange chromatography using QSFF in flow through mode. Comparable step yield and impurity clearance were obtained by the two processes. [Copyright &y& Elsevier]

Abstract: Angiotensin converting enzyme 2 (ACE2) is a component of the renin–angiotensin system (RAS) which converts Ang II, a potent vasoconstrictor peptide into Ang 1–7, a vasodilator peptide which may act as a negative feedback hormone to the actions of Ang II. The discovery of this enzyme added a new level of complexity to this system. The mesangial cells (MC) have multiple functions in glomerular physiology and pathophysiology and are able to express all components of the RAS. Despite of being localized in these cells, ACE2 has not yet been purified or characterized. In this study ACE2 from mice immortalized MC (IMC) was purified by ion-exchange chromatography. The purified enzyme was identified as a single band around 60–70kDa on SDS-polyacrylamide gel and by Western blotting using a specific antibody. The optima pH and chloride concentrations were 7.5 and 200mM, respectively. The N-terminal sequence was homologous with many species ACE2 N-terminal sequences as described in the literature. ACE2 purified from IMC was able to hydrolyze Ang II into Ang 1–7 and the K m value for Ang II was determined to be 2.87±0.76μM. In conclusion, we purified and localized, for the first time, ACE2 in MC, which was able to generate Ang 1–7 from Ang II. Ang 1–7 production associated to Ang II degradation by ACE2 may exert a protective effect in the renal hemodynamic. [Copyright &y& Elsevier]

Abstract: Rotavirus VP6 self-assembles into high order macrostructures useful as novel scaffolds for the construction of multifunctional hybrid nanobiomaterials. This application requires large quantities of high quality pure material with strict structural consistency. Strategies for obtaining high quality recombinant VP6 and different characterization techniques are explored and compared in this work. VP6 was expressed in the insect cell–baculovirus system. VP6 assemblies were selectively purified utilizing an ion exchange and size exclusion (SE) chromatography. Purification steps were monitored and characterized by dynamic light scattering (DLS), ELISA, SDS-PAGE, HPLC and Western blot. DLS showed that the initial ultrafiltration step removed small particles, the intermediate anion exchange chromatographic step completely removed the baculovirus, whereas the final size exclusion chromatography permitted the selective recovery of correctly assembled VP6 nanotubes and discrimination of non-assembled VP6, as confirmed by transmission electron microscopy. VP6 assembled into tubular structures with diameter of 75nm and several nanometers in length. The purification yield was 20% of multimeric assemblies with a purity >98%. The resulting material was suitable for the production of functionalized hybrid nanobiomaterials through in situ synthesis of metallic nanoparticles. [Copyright &y& Elsevier]

Abstract: Adenovirus type 3 dodecahedric virus-like particles (Ad3 VLP) are an interesting delivery vector. They penetrate animal cells in culture very efficiently and up to 300,000 Ad3 VLP can be observed in one cell. The purification of such particles usually consists of several steps. In these work we describe the method development and optimization for the purification of Ad3 VLP using the Convective Interaction Media analytical columns (CIMac). Results obtained with the CIMac were compared to the already established two-step purification protocol for Ad3 VLP based on sucrose density gradient ultracentifugation and the Q-Sepharose ion-exchange column. Pure, concentrated and bioactive VLP were obtained and characterized by several analytical methods. The recovery of the Ad3 VLP was more than 50% and the purified fraction was almost completely depleted of DNA; less than 1% of DNA was present. The purification protocol was shortened from five days to one day and remarkably high penetration efficacy of the CIMac-purified vector was retained. Additionally, CIMac QA analytical column has proven to be applicable for the final and in-process control of various Ad3 VLP samples. [Copyright &y& Elsevier]

We report novel methods for the chemical purification of Mg from silicate rocks by ion-exchange chromatography, and high-precision analysis of Mg-isotopes by high-resolution multiple collector inductively coupled plasma source mass spectrometry (HR-MC-ICPMS). Using these methods, we have measured the relative and absolute Mg-isotope composition of a number of terrestrial and extraterrestrial materials, including international reference rock standards as well as pure Mg standards, olivine crystals separated from a mantle-derived spinel lherzolite (J12 olivine), one enstatite chondrite, a martian shergottite and sea water samples. Repeated analyses of terrestrial and extraterrestrial samples demonstrate that it is possible to routinely measure the relative Mg-isotope composition of silicate materials with an external reproducibility of 2.5 and 20 ppm for the μ26Mg* and μ25Mg values, respectively (μ notation is the per 106deviation from a reference material). Analyses of bulk mantle-derived rocks as well as a martian shergottite and an enstatite chondrite define a restricted range in μ25Mg of −120 ± 28 ppm (2sd) relative to the DSM-3 reference standard (μ25,26Mg = 0), suggesting that the Mg-isotope composition of inner solar system bulk planetary materials is uniform within the resolution of our analyses. We have determined the absolute Mg-isotope composition of the J12 olivine, two CI chondrites as well as the DSM-3 and Cambridge-1 reference standards using a mixed 26Mg-24Mg double-spike. The differences between the absolute 25Mg/24Mg ratios of the various materials analyzed relative to the DSM-3 standard are in excellent agreement with results obtained by the sample-standard bracketing method. Based on the averages obtained for the J12 olivine separates, we estimate the absolute Mg-isotope composition for Earth''s mantle – and hence that of the bulk silicate Earth – to be 25Mg/24Mg = 0.126896 ± 0.000025 and 26Mg/24Mg = 0.139652 ± 0.000033. Given the restricted range of μ25Mg obtained for bulk planetary material by the sample-standard bracketing technique and the excellent agreement between the data obtained by the relative and absolute methods, we propose that these new values represent the absolute Mg-isotope composition of the bulk inner solar system. Using the absolute Mg-isotope composition of the J12 olivine, we calculate the isotopic abundances of Mg as 24Mg = 0.789548 ± 0.000026, 25Mg = 0.100190 ± 0.000018, and 26Mg = 0.110261 ± 0.000023. Based on this result, we have calculated an atomic weight for Mg of 24.305565 ± 0.000045, which is marginally heavier than previous estimates but a factor of 10 more precise. [ABSTRACT FROM AUTHOR]

Abstract: A bacterial strain LL1 producing κ-carrageenase was isolated from the decayed seaweed collected from Yellow Sea, China and identified as Pseudoalteromonas porphyrae. The extracellular κ-carrageenase in the supernatant of cell culture of the marine bacterium P. porphyrae LL1 was purified to homogeneity with a 202.6-fold increase in specific κ-carrageenase activity as compared to that in the supernatant by ultrafiltration, gel filtration chromatography, and anion-exchange chromatography. According to the data from sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 40.0kDa. The purified enzyme could actively convert κ-carrageenan into tetrasaccharides, but poorly convert λ-carrageenan. The optimal pH and temperature of the purified enzyme were 8.0 and 55°C, respectively. The enzyme was significantly stimulated by Mg2+ and Ba2+. The enzyme was inhibited by phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid, EDTA, EGTA and 1,10-phenanthroline. The K m and V max values of the purified enzyme for κ-carrageenan were 4.4mg/ml and 0.1mg/minml, respectively. The amino acid sequence (NPQPHIAKPGQTWILQEKRS) of N-terminus of the purified enzyme was identical to that of N-terminus of the deduced protein encoded by the gene encoding κ-carrageenase cloned from the marine bacterium. [ABSTRACT FROM AUTHOR]

Abstract: Aspergillus niger RNase belonging to RNase T2 family is purified to homogeneity using ammonium sulfate precipitation and anion-exchange chromatography from A. niger. The enzyme is glycosylated monomer with molecule mass of 28.5kDa. Optimum activity of the enzyme is achieved at pH 3.5 and temperature 55°C. The enzyme shows broad substrate specificity with poly (A), poly (C), poly (G), poly (U) and yeast RNA. The enzyme is highly stable against various metal ions and organic solvents, thus dictates its applicability in various industries. Two peptide sequences, PGGTLLQTQFWDYDP and TLDSYTALSDAGITPSEDATYK obtained by MS/MS analysis and confirmed by BLAST search, show substantial sequence homology with Aspergillus phoenicis, Aspergillus clavatus and P. merneffei. Multiple sequence analysis of the peptide sequences of purified enzyme shows 97%, 75% and 72% identity with A. phoenicis, A. clavatus and P. merneffei, respectively using Clustal W tool. Spectroscopic studies by absorbance, fluorescence, and circular dichroism reveal that the enzyme has α+β type secondary structure with approximately 29% α-helix, 24% β-sheet and 47% random coil by K2D software. [ABSTRACT FROM AUTHOR]

Abstract: Negative chromatography antibody purification (N-CAP) using the weak anion exchanger STREAMLINE™ DEAE to extract impurities while retaining the target antibody is proposed as an effective method for the recovery of antibody from rabbit serum. The effects of pH and initial protein concentration on the removal of albumin were investigated. The optimal pH and initial protein concentration for the efficient removal of albumin from rabbit serum were pH 8.0 and 0.5mg/ml, respectively. Under optimal binding conditions, DEAE successfully removed more than 90% of the albumin from rabbit serum with less than 20% IgG loss. This process offered good polyclonal IgG yield of 80% with a purity of 83% and a purification factor of 5.5. The use of a strong anion exchanger like STREAMLINE™ Q XL for albumin removal was also explored. Under similar optimized conditions, albumin removal by Q XL was as high as 90%. However, IgG recovery and purity were reduced to about 70% and 62%, respectively. Thus, N-CAP using the anion exchanger DEAE removes albumin from rabbit serum and thereby offers an efficient means of purifying polyclonal antibodies. [ABSTRACT FROM AUTHOR]

Poly(hydroxamic acid) resin (PHA) was prepared by modification of polyacrylamide (PAAm) prepared through γ-irradiation technique and used for the first time in separation of Zr(IV) from Y(III) and Sr(II) as a simulation mode for the purification of 89Zr from its parents (Y or Sr). The adsorption behaviors of zirconium, yttrium, and strontium on the prepared PHA resin in different media, namely, hydrochloric acid, acetate buffer, and citrate buffer were studied as a function of pH. In addition, in cation-exchange column chromatography experiments using PHA, three different eluants, namely, 10-5 mol/L HCl, acetate buffer pH 3.5, and 2 mol/L HCl, were employed for elution of Zr(IV), Y(III) and Sr(II), respectively, where Zr(IV), Y(III) and Sr(II) were eluted in amounts of 80%, 99.9%, and 100%, respectively. The purification process of Y(III) from Zr(IV) was carried out using regenerated PHA. [ABSTRACT FROM AUTHOR]

Abstract: A straightforward one-step Hf purification protocol for geological samples using common anion-exchange chromatography and isotopic measurements by multiple collector inductively coupled plasma mass spectrometry (MC-ICP-MS) is described in this paper. The sample is dissolved in concentrated HF–HNO3 mixtures with round-bottom Savillex™ Teflon screw-top capsules or high-pressure stainless steel jacketed Teflon bombs. After complete dissolution, the chemical separation between Hf and isobarically interfering elements such as Lu, Yb, and matrix elements like Ti, is performed and achieved simultaneously using a common one-step anion exchange resin (AG1-X8). The Hf yields are >90% and total procedural blanks are lower than 20pg. This protocol not only avoids using multiple-stage ion exchange resins, but also eliminates perchloric acid to break down fluorides after HF treatment of the sample. In addition to whole rock, this protocol is also practical for zircon and baddeleyite Hf purification prior to isotopic measurement. New Hf isotopic data for zircon 91500, Temora, CN92-1 and FM0411, together with Phalaborwa baddeleyite are presented for this method. Multiple analyses of the international geological reference materials and standard zircon and baddeleyite used in U–Pb geochronology demonstrate that this method is simple, economic, efficient and reproducible. [ABSTRACT FROM AUTHOR]

Abstract: An organic solvent-tolerant lipase from newly isolated Pseudomonas aeruginosa LX1 has been purified by ammonium sulfate precipitation and ion-exchange chromatography leading to 4.3-fold purification and 41.1% recovery. The purified lipase from P. aeruginosa LX1 was homogeneous as determined by SDS-PAGE, and the molecular mass was estimated to be 56kDa. The optimum pH and temperature for lipase activity were found to be 7.0 and 40°C, respectively. The lipase was stable in the pH range 4.5–12.0 and at temperatures below 50°C. Its hydrolytic activity was found to be highest towards p-nitrophenyl palmitate (C16) among the various p-nitrophenol esters investigated. The lipase displayed higher stability in the presence of various organic solvents, such as n-hexadecane, isooctane, n-hexane, DMSO, and DMF, than in the absence of an organic solvent. The immobilized lipase was more stable in the presence of n-hexadecane, tert-butanol, and acetonitrile. The transesterification activity of the lipase from P. aeruginosa LX1 indicated that it is a potential biocatalyst for biodiesel production. [ABSTRACT FROM AUTHOR]

The present study examined the overloading of ion-exchange membrane adsorbers, a form of frontal chromatography, as the final purification step in the production of mAbs (monoclonal antibodies) produced from CHO (Chinese-hamster ovary) cells. Preferential binding of impurities over antibody product was exploited using commercially available cation- and anion-exchange membranes. Three different antibody feedstreams previously purified over Protein A and ion-exchange column chromatography were tested. Feedstream conductivity and pH were adjusted to induce product and impurity adsorption. Membranes were then overloaded in a normal flow mode, resulting in retention of impurities and breakthrough of purified antibody. Although some amount of the product also binds to the membranes (usually ≤30 g mAb/l membrane), yields of ≥99% were achieved by marginalizing the losses, typically by loading more than 3 kg mAb/l membrane. Analyses of the purified pools show consistent removal of impurities despite strong mAb–ligand interactions and high membrane loadings. The clearance of host cell proteins was affected by pH and conductivity, but was unaffected by flow rate, membrane properties or scale. The importance of the present study lies in our demonstration of an alternative use of ion-exchange membranes for fast, effective and high yielding purification of mAbs. [ABSTRACT FROM AUTHOR]

Abstract: Aminopeptidase from a solvent tolerant strain Pseudomonas aeruginosa PseA was purified and studied for its biochemical and molecular characteristics. Ion-exchange chromatography resulted in 11.9-fold purification and 38% recovery of the 56kDa enzyme. The enzyme was found to be stable over a pH range of 6.0–8.0 and appreciably thermostable up to 70°C. PseA aminopeptidase exhibited K m of 3.02mM and V max of 6.71μmol/mg/min towards l-Leu-p-nitroanilide. Remarkable stability in both hydrophilic and hydrophobic solvents makes PseA aminopeptidase unique. Partial N-terminal sequence of enzyme showed exact match with probable aminopeptidase of P. aeruginosa PAO1, coded by gene pepB. Polymerase chain reaction amplified the 1611-bp open reading frame encoding a 57.51kDa, 536 amino acid PseA PepB polypeptide. The deduced PseA PepB protein sequence contained a 24-residue signal peptide (2.57kDa) followed by a 1.28kDa propeptide and a mature product of 500 residues. Search for conserved domain in PseA aminopeptidase explored its place in zinc-metallopeptidase family. Primary sequence analysis showed the hydrophobic inclination of the protein; and the 3D structure modeling elucidated the presence of a high content of hydrophobic residues on its surface probably imparting solvent stability to it. The enzyme might find potential applications in non-aqueous enzymology due to its marked thermostability and striking solvent stability. [Copyright &y& Elsevier]

Abstract: A protease with a molecular mass of 28kDa, designated as hmsp, was isolated from fresh fruiting bodies of the edible mushroom Hypsizigus marmoreus. The purification protocol entailed ion exchange chromatography on DEAE-cellulose, CM-cellulose, and FPLC-gel filtration on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on CM-cellulose. hmsp was thermolabile, and exhibited a temperature optimum at 50°C and a pH optimum at pH 7.5. The activity of the protease was adversely affected by PMSF, EGTA and aprotinin, indicating that it is a serine protease. Based on the N-terminal sequence, the cDNA of hmsp was cloned by using RACE combined with the TAIL-PCR method. The deduced protease sequence contained a signal peptide with 19 amino acids, a pro-region with 82 amino acids, and a mature protease with 285 amino acids and a molecular mass of 28.07kDa. It possessed the three active sites characteristic of the subtilisin family (S8A). hmsp demonstrated 63%, 57% and 44% identity in amino acid sequence respectively to Absp1, Absp2, and Gf-spr1, which are serine proteases from Agaricus bisporus and Grifola frondosa. [Copyright &y& Elsevier]

Abstract: A two-step purification strategy comprising of polyethylene glycol (PEG) precipitation and anion-exchange chromatography was developed for a panel of monoclonal immunoglobulin M (IgM) (pI 5.5–7.7) produced from hybridoma cultures. PEG precipitation was optimized with regards to concentration, pH and mixing. For anion-exchange chromatography, different resins were screened of which Fractogel EMD, a polymer grafted porous resin had the highest capacity. Despite its significantly slower mass transfer, the binding capacity was still higher compared to a convection driven resin (monolith). This purification strategy was successfully demonstrated for all 9 IgMs in the panel. In small scale most antibodies could be purified to >95% purity with the exception of two which gave a lower final purity (46% and 85%). The yield was dependent on the different antibodies ranging from 28% to 84%. Further improvement of recovery and purity was obtained by the digestion of DNA present in the hybridoma supernatant using an endonuclease, benzonase. So far this strategy has been applied for the purification of up to 2l hybridoma supernatants. [Copyright &y& Elsevier]

Abstract: A new thermostable Cu/Zn SOD from a thermotolerant yeast strain Kluyveromyces marxianus NBIMCC 1984 has been purified and characterized. The purification procedure comprises thermal treatment and dialysis, ion-exchange chromatography and chromatofocusing. The methodology is a rapid, efficient and highly specific, generating pure preparation (specific activity 996Umg of protein−1) with a yield of 53%. The purified enzyme is a homodimer with Mw of 34034Da and has high N-terminal homology with other yeasts’ Cu/Zn SOD enzymes. The protein is characterized with some unique features such as—thermostability (t 1/2 at 70°C=30min), pH stability in the alkaline range (7.5–8.5) and resistance to inhibitors and variety of chemicals. These characteristics reveal possibilities for wide practical application of K. marxianus Cu/Zn SOD enzyme. [Copyright &y& Elsevier]

Abstract: A new l-amino acid oxidase (designated as DRS-LAAO) was purified from Daboia russellii siamensis venom by ion-exchange, gel filtration and affinity chromatographies. DRS-LAAO is a homodimeric enzyme with a molecular weight of 120.0kDa as measured by size exclusion chromatography and the monomeric molecular weight of 58.0kDa as measured by SDS-PAGE under both non-reducing and reducing conditions. The N-terminal amino acid sequence (ADDKNPLEECFREDD) of DRS-LAAO shares high identity with other snake venom l-amino acid oxidases, especially with those isolated from viperid venoms. The enzyme displayed high specificity towards hydrophobic l-amino acids. The best substrate of DRS-LAAO was L-Leu followed by L-Phe and L-Ile, while five substrates — L-Pro, L-Asn, L-Gly, L-Ser and L-Cys were not oxidized. Optimal pH of DRS-LAAO was 8.8. The enzyme showed no hemorrhagic activity even at a dosage of 55.0μg. DRS-LAAO dose-dependently inhibited platelet aggregation induced by ADP (83.33μM) and TMVA (55.0nM) with an IC50 value of 32.8μg/ml and 32.3μg/ml, respectively. The minimum inhibitory concentrations (MICs) of DRS-LAAO against Staphylococci aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) were 9.0, 144.0 and 288.0μg/ml, respectively. The minimum bactericidal concentrations (MBCs) of the enzyme for these strains were twice of the MIC values. These results showed that DRS-LAAO had the strongest antimicrobial activity against S. aureus among these three international standard stains. Antibacterial-activities of DRS-LAAO against eight clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were also tested. The MICs of DRS-LAAO against these isolates ranged from 4.5 to 36.0μg/ml. And the MBCs of the enzyme against these isolates ranged from 9.0 to 72.0μg/ml. [Copyright &y& Elsevier]

Abstract: Intrinsically disordered proteins (IDPs) is a term used to describe proteins that do not have a well-defined tertiary structure. IDPs have many roles such as in cell cycle control (p53), neuronal signal transmission (myelin basic protein), and protein stability (dehydrins). Producing recombinant IDPs in bacteria for nuclear magnetic resonance (NMR) studies is problematic because the lack of stable tertiary structure makes them excellent substrates for bacterial proteases, which will cause loss in yield. We have developed a two-step method to produce the grape dehydrin K2 and YSK2 using Escherichia coli. Dehydrins are expressed by certain plants in response to dehydration, increased salinity, or low temperatures. Purification of 10mg/L (K2) and 15mg/L (YSK2) was performed by boiling bacterial pellets to lyse the cells, remove most of the contaminating proteins, and denature bacterial proteases. This resulted in protein purity comparable to that produced by sonication and nickel affinity chromatography. Boiling was followed by cation exchange chromatography to remove the remaining trace contaminants. The sample was shown to be more than 95% pure by reversed-phase high-performance liquid chromatography. The method presented here can easily be adapted to the purification of other IDPs and heat-stable proteins without requiring multiple chromatography steps or the use of protease inhibitors. [Copyright &y& Elsevier]

Summary: A protease-producing bacterium was isolated from slaughterhouse waste samples, Hyderabad, India. It was related to Bacillus cereus on the basis of 16S rRNA gene sequencing and biochemical properties. The protease was purified to homogeneity using ammonium sulfate precipitation, and ion exchange chromatography with a fold purification of 1.8 and a recovery of 49%. The enzyme had a relative molecular weight of 28kDa, pH and temperature optima for this protease were 10 and 60°C. The activity was stable between a pH range of 7.0 and 12.0. The activity was inhibited by EDTA and enhanced (four-fold) by Cu2+ ions indicating the presence of metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents and anionic surfactants. The enzyme also showed stability in the presence of organic solvents. [Copyright &y& Elsevier]

Abstract: A platelet aggregation inhibitor, named snake venom platelet aggregation dissociator (SV-PAD)-1, with a dissociative reaction of ADP-induced platelet aggregation, was purified from the venom of Protobothrops elegans (Sakishima-habu) by gel-filtration employing Sephadex G-100, and ion-exchange chromatographies using DEAE-Sepharose Fast Flow, CM-Sepharose Fast Flow, and Mono S. By this procedure, about 1.5mg of purified protein was obtained from 1.0g of P. elegans venom. The purified protein showed a single protein band and the molecular weight was about 110kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The pI of purified protein showed four-bands of 7.7, 7.8, 7.95, and 8.15. This protein strongly inhibited ADP-induced platelet aggregation in rabbit platelet-rich plasma (PRP), and its IC50 was about 58nM. It inhibited ristocetin-induced platelet aggregation in rabbit PRP (IC50: 100nM), but hardly blocked collagen-induced platelet aggregation. This protein promptly dissociated platelet aggregation in rabbit PRP stimulated by high-concentration ADP. [Copyright &y& Elsevier]

Abstract: Invertase is a major protein of grape juice and wine. Accordingly, in order to study the biochemical and structural characteristics of this protein and for a better understanding of its physico-chemical properties, large amounts of the pure protein are needed. A simple method for the purification of the grape vacuolar invertase in a preparative-scale is described in this work. The grape protein was isolated and purified from must by ultrafiltration and anion exchange chromatography. The identification and purity determination of the grape invertase fraction were assessed by SDS-PAGE, and were then confirmed using nanoLC-chip-MS/MS analysis. The laboratory fractionation procedure presented in this work generated large quantities of pure grape vacuolar invertase from must. [Copyright &y& Elsevier]

Abstract: A procedure based on BIA Separations CIM DEAE anion-exchange chromatography was developed to separate double-stranded (ds) RNA of hypovirus infecting phytopathogenic fungus Cryphonectria parasitica. Using a linear gradient of 25mM 4-morpholinepropanesulfonic acid (MOPS), pH 7.0 as a binding buffer, and 25mM MOPS, 1.5M NaCl, 0.1mM EDTA, 15% isopropanol (v/v), pH 7.0 as an elution buffer, hypoviral dsRNA was additionally purified from nucleic acid species present in preparations partially purified by standard CF-11 cellulose chromatography. Moreover, crude phenol/chloroform extracts of the fungal tissue were also applied to monolithic supports and CIM DEAE chromatograms revealed clear evidence for hypoviral presence without CF-11 chromatography, nucleic acid precipitation, and electrophoresis. [Copyright &y& Elsevier]