M13 is a non-lytic filamentous bacteriophage (phage). It has been used widely in phage display technology for displaying foreign peptides, and also for studying macromolecule structures and interactions. Traditionally, this phage has been purified by cesium chloride (CsCl) density gradient ultracentrifugation which is highly laborious and time consuming. In the present study, a simple, rapid and efficient method for the purification of M13 based on anion exchange chromatography was established. A pre-packed SepFast Super Q column connected to a fast protein liquid chromatography (FPLC) system was employed to capture released phages in clarified Escherichia coli fermented broth. An average yield of 74% was obtained from a packed bed mode elution using citrate buffer (pH 4), containing 1.5 M NaCl at 1 ml/min flow rate. The purification process was shortened substantially to less than 2 h from 18 h in the conventional ultracentrifugation method. SDS-PAGE revealed that the purity of particles was comparable to that of CsCl gradient density ultracentrifugation method. Plaque forming assay showed that the purified phages were still infectious.
Matched MeSH terms: Chromatography, Ion Exchange/methods*
NP(Δc375) is a truncated version of the nucleocapsid protein of Newcastle disease virus (NDV) which self-assembles into a long helical structure. A packed bed anion exchange chromatography (PB-AEC), SepFastTM Supor Q pre-packed column, was used to purify NP(Δc375) from clarified feedstock. This PB-AEC column adsorbed 76.2% of NP(Δc375) from the clarified feedstock. About 67.5% of the adsorbed NP(Δc375) was successfully eluted from the column by applying 50 mM Tris-HCl elution buffer supplemented with 0.5 M NaCl at pH 7. Thus, a recovery yield of 51.4% with a purity of 76.7% which corresponds to a purification factor of 6.5 was achieved in this PB-AEC operation. Electron microscopic analysis revealed that the helical structure of the NP(Δc375) purified by SepFast(TM) Supor Q pre-packed column was as long as 490 nm and 22-24 nm in diameter. The antigenicity of the purified NP(Δc375) was confirmed by enzyme-linked immunosorbent assay.
Matched MeSH terms: Chromatography, Ion Exchange/instrumentation; Chromatography, Ion Exchange/methods*
The proteome of Naja sumatrana (Equatorial spitting cobra) venom was investigated by shotgun analysis and a combination of ion-exchange chromatography and reverse phase HPLC. Shotgun analysis revealed the presence of 39 proteins in the venom while the chromatographic approach identified 37 venom proteins. The results indicated that, like other Asiatic cobra venoms, N. sumatrana contains large number of three finger toxins and phospholipases A2, which together constitute 92.1% by weight of venom protein. However, only eight of the toxins can be considered as major venom toxins. These include two phospholipases A2, three neurotoxins (two long neurotoxins and a short neurotoxin) and three cardiotoxins. The eight major toxins have relative abundance of 1.6-27.2% venom proteins and together account for 89.8% (by weight) of total venom protein. Other venom proteins identified include Zn-metalloproteinase-disintegrin, Thaicobrin, CRISP, natriuretic peptide, complement depleting factors, cobra venom factors, venom nerve growth factor and cobra serum albumin. The proteome of N. sumatrana venom is similar to proteome of other Asiatic cobra venoms but differs from that of African spitting cobra venom. Our results confirm that the main toxic action of N. sumatrana venom is neurotoxic but the large amount of cardiotoxins and phospholipases A2 are likely to contribute significantly to the overall pathophysiological action of the venom. The differences in toxin distribution between N. sumatrana venom and African spitting cobra venoms suggest possible differences in the pathophysiological actions of N. sumatrana venom and the African spitting cobra venoms, and explain why antivenom raised against Asiatic cobra venom is not effective against African spitting cobra venoms.
The performance of silver-loaded zeolite (HY and HZSM-5) catalysts in the oxidation of butyl acetate as a model volatile organic compound (VOC) was studied. The objective was to find a catalyst with superior activity, selectivity towards deep oxidation product and stability. The catalyst activity was measured under excess oxygen condition in a packed bed reactor operated at gas hourly space velocity (GHSV)=15,000-32,000 h(-1), reaction temperature between 150 and 500 degrees C and butyl acetate inlet concentration of 1000-4000 ppm. Both AgY and AgZSM-5 catalysts exhibited high activity in the oxidation of butyl acetate. Despite lower silver content, AgY showed better activity, attributed to better metal dispersion, surface characteristics and acidity, and its pore system. Total conversion of butyl acetate was achieved at above 400 degrees C. The oxidation of butyl acetate followed a simple power law model. The reaction orders, n and m were evaluated under differential mode by varying the VOC partial pressure between 0.004 and 0.018 atm and partial pressure of oxygen between 0.05 and 0.20 atm. The reaction rate was independent of oxygen concentration and single order with respect to VOC concentration. The activation energies were 19.78 kJ/mol for AgY and 32.26 kJ/mol for AgZSM-5, respectively.
The L-amino acid oxidase (EC 1. 4. 3. 2) from King cobra (Ophiophagus hannah) venom was purified to electrophoretic homogeneity. The molecular weight of the enzyme was determined to be 140000 when examined by gel filtration and 68000 by SDS-polyacrylamide gel electrophoresis. The enzyme had an isoelectric point of 4.5 and an intravenous LD50 of 5 micrograms/g in mice. It is a glycoprotein and contains two moles of FAD per mole of enzyme. The enzyme exhibited unusual thermal stability and unlike most other venom L-amino acid oxidases, it was stable in alkaline solution and was not inactivated by freezing.
We investigate the dynamic adsorption of anionic surfactant C14 - 16 alpha olefin sulfonate on Berea sandstone cores with different surface wettability and redox states under high temperature that represents reservoir conditions. Surfactant adsorption levels are determined by analyzing the effluent history data with a dynamic adsorption model assuming Langmuir isotherm. A variety of analyses, including surface chemistry, ionic composition, and chromatography, is performed. It is found that the surfactant breakthrough in the neutral-wet core is delayed more compared to that in the water-wet core because the deposited crude oil components on the rock surface increase the surfactant adsorption via hydrophobic interactions. As the surfactant adsorption is satisfied, the crude oil components are solubilized by surfactant micelles and some of the adsorbed surfactants are released from the rock surface. The released surfactant dissolves in the flowing surfactant solution, thereby resulting in an overshoot of the produced surfactant concentration with respect to the injection value. Furthermore, under water-wet conditions, changing the surface redox potential from an oxidized to a reduced state decreases the surfactant adsorption level by 40%. We find that the decrease in surfactant adsorption is caused not only by removing the iron oxide but also by changing the calcium concentration after the core restoration process (calcite dissolution and ion exchange as a result of using EDTA). Findings from this study suggest that laboratory surfactant adsorption tests need to be conducted by considering the wettability and redox state of the rock surface while recognizing how core restoration methods could significantly alter the ionic composition during surfactant flooding.
The results of many previous studies on low salinity/controlled ions water (CIW) flooding suggest that future laboratory and modeling investigations are required to comprehensively understand and interpret the achieved observations. In this work, the aim is co-optimization of the length of the injected slug and soaking time in the CIW flooding process. Furthermore, the possibility of the occurrence of several governing mechanisms is studied. Therefore, the experimental results were utilized to develop a compositional model, using CMG GEM software, in order to obtain the relative permeability curves by history matching. It was concluded that CIW slug injection, concentrated in the potential-determining ion, can increase oil recovery under a multi ion exchange (MIE) mechanism. The wettability of the carbonate rocks was changed from a mixed or oil wet state toward more water wetness. However, there is a CIW slug length, beyond which extending the length does not significantly improve the rock wettability, and consequently, the oil production, which is known as the optimum slug size. This implies that the optimization of the injection process, by minimizing the slug size, can decrease the need for the CIW supply, therefore lowering the process expenditure. Moreover, if the exposure time of the rock and CIW is increased (soaking), a higher level of ion substitution is probable, leading to more oil detachment and production. Rock dissolution/precipitation (leading to a pH change) was found to have a negligible contribution.
This investigation aims at the reclamation of Cr(VI) from synthetic electroplating industrial effluent by electroextraction process namely electrochemical ion exchange (EIX). An electrochemical ion exchange reactor of desired dimensions was fabricated with the help of ion-permeable membranes, stainless steel cathode and PbO₂ coated Ti expanded mesh anode. The performance of the reactor was studied in batch recirculation mode, continuous flow mode at different experimental conditions. The influence of various experimental factors, for instance, initial metal ion concentration (20, 300, 1000 mg/L of Cr(VI)), applied voltages (2.5 V, 5 V, 7.5 V, 10 V) and flow rates of the process stream (2, 4, 6, 8, 10, 12 and 14 ml/min) on removal/reclamation efficiency was deliberated. For comparison purposes, an electrodialysis process was conducted at the same optimal conditions. It was found that the EIX process with three compartments has more removal efficiency at optimum experimental conditions than the electrodialysis process. The continuous flow process of the reactor with 300 mg/L of Cr(VI) as inlet concentration has studied to predict the breakeven point of the reactor. It was noted that Cr(VI) ion concentration in the treated wastewater is almost zero up to the discharge of 20 liters of treated rinse water.
The toxic and biological activities of four samples of Trimeresurus purpureomaculatus venom were examined. The lethality, protein composition and biological activities of the four venom samples were similar. Three of the venom samples had LD50 (i.v.) values of 0.9 micrograms/g while the fourth had a lower LD50 (i.v.) of 0.45 micrograms/g. All four venom samples exhibited hemorrhagic, edema-inducing, anticoagulant and thrombin-like activities as well as the usual enzymes found in crotalid venoms. DEAE-Sephacel ion exchange chromatographic fractionation of the venom yielded 10 protein fractions. Only two fractions (fractions A and F) were lethal to mice; the major lethal fraction being fraction F. This fraction had an LD50 (i.v.) of 0.2 micrograms/g and exhibited hemorrhagic, edema-inducing and thrombin-like activity. It also exhibited phospholipase A, arginine ester hydrolase, arginine amidase, protease, 5'-nucleotidase, acetylcholinesterase and alkaline phosphomonoesterase activities. The lethal potency of fraction F is potentiated by fraction G, which exhibited anticoagulant activity as well as hemorrhagic, edema-inducing and enzymatic activities. Fractions F plus G account for almost 100% of the lethal potency of the venom.
Bungarus candidus venom exhibited high hyaluronidase, acetylcholinesterase and phospholipase A activities; low proteinase, 5'-nucleotidase, alkaline phosphomonoesterase and phosphodiesterase activities and moderately high L-amino acid oxidase activity. SP-Sephadex C-50 ion exchange chromatographic fractionation of the venom and Sephadex G-50 chromatography of the major lethal venom fractions indicate that the venom contains at least two highly lethal, basic phospholipases A with LD50 (i.v.) values of 0.02 micrograms/g (F6A) and 0.18 micrograms/g (F4A), respectively; as well as two polypeptide toxins with LD50 (i.v.) values of 0.17 micrograms/g and 0.83 micrograms/g, respectively. The major lethal toxin is the basic lethal phospholipase A, F6A, which accounts for approximately 13% of the venom protein and has a mol. wt of 21,000.
Sumatran pit viper (Trimeresurus sumatranus sumatranus) venom was fractionated by DEAE-Sephacel ion exchange chromatography into seven fractions. Fractions 4, 5 and 6 were lethal to mice and exhibited strong hemorrhagic activity, as well as some enzymatic activities. Fraction 6 also exhibited potent anticoagulant and thrombin-like activities. Analysis of the biological and enzymatic properties of the three lethal fractions suggests that the major lethal component of fractions 4 and 5 may be the hemorrhagic principle, and that the lethality of fraction 6 may be due to the hemorrhagic principle and/or the anticoagulant principle.
Layered double hydroxide (LDH) with Mg/Al molar ratio of 4/1 (MAN-4) was synthesized by co-precipitation and followed by hydrothermal method. The compound was allowed to undergo ion exchange with K2HPO4 for 48 hours to produce MgAlHPO4 (MAHP-4). The solid produced was characterized using X-ray diffraction (XRD) and Fourier Transform Infrared spectroscopy (FTIR). Adsorption of copper solution by MAHP-4 was carried out using batch experiment by mixing the copper solution and the sorbent MAHP-4. The effects of
various parameters such as contact time, pH, adsorbent dosage and initial concentration were investigated. The optimum pH for copper removal was found to be 4 and the optimum time of copper removal was found at 4 hours. The isotherm data was analysed using model isotherm Langmuir with the correlation coefficient of 0.999 was recorded. The maximum adsorption capacity, Qo (mg/g) of 142.8 mg/g was also recorded from the Langmuir isotherm. The remaining copper solution was determined by using EDXRF (Energy Dispersive XRay Fluorescence spectrometry) model MiniPal 4 (PAN analytical). The results in this study indicate that MAHP-4 has potential as an effective adsorbent for removing copper from aqueous solution.
Filamentous fungi such as Fusarium equiseti KR706303 and Penicillium citrinum KR706304
are capable of sequestering heavy metals from aqueous solutions. In the present study, the role
play by various functional groups present in the cell wall of F. equiseti KR706303 and P.
citrinum KR706304 during lead and copper ions biosorption was investigated. The fungal
biomass was chemically treated to modify the functional groups present in their cell wall. These
modifications were studied through biosorption experiments. It was found that an esterification
of the carboxyl and phosphate groups, methylation of the amine groups and extraction of lipids
significantly decrease the biosorption of both lead and copper ions studied. Therefore, the
carbonyl, hydroxyl and amide groups were recognized as important in the biosorption of metal
ions by the tested fungi. The study showed that there was no release of any metal ions from the
biomass after biosorption, indicating that ion exchange may not be a key mechanism in the
biosorption of lead and copper ions by these fungi but complexation of metal ions within the
fungal cell wall.
T1 Lipase is a thermostable secretary protein of Geobacillus zalihae strain previously expressed in a prokaryotic system and purified using three-step purification: affinity 1, affinity 2, and ion exchange chromatography (IEX). This approach is time consuming and offers low purity and recovery yield. In order to enhance the purification strategy of T1 lipase, affinity 2 was removed so that after affinity 1, the cleaved Glutathione S-transferase (GST) and matured T1 lipase could be directly separated through IEX. Therefore, a rational design of GST isoelectric point (pI) was implemented by prediction using ExPASy software in order to enhance the differences of pI values between GST and matured T1 lipase. Site-directed mutagenesis at two locations flanking the downstream region of GST sequences (H215R and G213R) was successfully performed. Double point mutations changed the charge on GST from 6.10 to 6.53. The purified lipase from the new construct GST tag mutant-T1 was successfully purified using two steps of purification with 6,849 U/mg of lipase specific activity, 33% yield, and a 44-fold increase in purification. Hence, the increment of the pI values in the GST tag fusion T1 lipase resulted in a successful direct separation through IEX and lead to successful purification.
For every ton of biodiesel produced, about 100 kg of glycerol is also generated as a by-product. The traditional method of removing glycerol is mainly by gravity separation or centrifugation. This method generates crude glycerol, which may still contain impurities such as methanol, oil, soap, salt, and other organic materials at ppm levels. The effective usage of crude glycerol is important to improve the economic sustainability of the biodiesel industry while reducing the environmental impacts caused by the generated waste. The application and value of crude glycerol can be enhanced if these impurities are removed or minimized. Thus, it is important to develop a method which can increase the economic and applicable value of crude glycerol. Therefore, in the present study, the dual step purification method comprised of acidification and ion exchange techniques has been used to purify the crude glycerol and convert it into higher-value products. The acidification process started with the pH adjustment of the crude glycerol, using phosphoric acid to convert soap into fatty acid and salts. Then, the pretreated glycerol was further purified by ion exchange with a strong cation H+ resin. Gas chromatography (GC) was used to analyze both crude and purified glycerol and expressed as the weight percentage of glycerol content. A maximum glycerol purity of 98.2% was obtained after the dual step purification method at the optimized conditions of 60% of solvent, the flow rate of 15 mL/min and 40 g of resin. Further, the glycerol content measured being within the accepted amount of BS 2621:1979. Therefore, this study has proven that the proposed crude glycerol purification process is effective in improving the glycerol purity and could enhance the applicability of glycerol in producing value-added products which bring new revenue to the biodiesel industry.
Whole blood samples from patients with various forms of alpha- and beta- thalassaemia were incubated with 14C-Leucine to determine the relative rates of production of the alpha and beta chains by their reticulocytes. The labelled globin chains were fractionated by CM-Cellulose Chromatography in 8M Urea and the incorporated activity determined. The relative rates of synthesis of alpha and beta chains in some cases of alpha and beta- thalassaemia were established and the chain synthetic ratios were compared with similar ratios in normal individuals. The results show that it is possible to identify from the relative rates of in-vitro synthesis of the alpha and beta chains, the presence of the common thalassaemia slates in particular beta-thal trait, beta-thal homozygotes, Hb H disease and alpha0-thal trait. The presence of transfused blood does not affect the result. This study indicates that an abnormal alpha/beta chain synthesis ratio is useful in defining alpha and beta-thalassaemia variants.
This study considered the temporal variations in rainfall and water level patterns as governing factors, which influence the geochemical process of coastal aquifer around Pondicherry, South India. Rainfall and water level data were collected from 2006 to 2016, which showed that the amount of rainfall from 2006 to 2011 was higher than that of 2011 to 2016. To understand the geochemical process governing groundwater, samples were collected during 2006 (n = 54), followed by 2011 (n = 93), and during 2016 (n = 63) as part of continuous observation. The major ions and stable isotopes (δ18O and δD) were analyzed in the samples to determine the geochemical variations. The predominant types were noted as Na-HCO3 and Na-Cl; Ca-HCO3 and Ca-Mg-Cl; and Na-Cl and Ca-Mg-Cl in 2006, 2011, and 2016, respectively. Saturation states of sulfate and carbonate minerals were compared for the study periods and it indicates that the saturation index (SI) values were increased from 2006 to 2011, but decreased from 2011 to 2016. PHREEQC inverse modeling revealed the predominance for the dissolution and leaching of carbonate minerals during increased rainy periods, and the increase of halite saturation during lesser rainfall period. AQUACHEM mixing studies suggested that geochemical signatures of 2006 and 2011 were preserved in samples of 2016 in different proportions. Considering the major factors, the main processes prevailing in the study area were inferred to be dissolution and leaching during 2006~2011 years and seawater intrusion along with ion exchange during 2011~2016 years. In all these periods of study, anthropogenic impact was also identified in the groundwater samples. Hence, this study revealed that the rainfall and water level gave a significant variation in the geochemical process of groundwater in the coastal aquifer system.
Hepatitis B core antigen (HBcAg) is an important serological marker used in the diagnosis of hepatitis B virus (HBV) infections. In the current study, a fast and efficient preparative purification protocol for truncated HBcAg from Escherichia coli disruptate was developed. The recombinant HBcAg was first captured by anion exchange expanded bed adsorption chromatography integrated with a cell disruption process. This online capture process has shortened the process time and eliminated the "hold-up" period that may be detrimental to the quality of target protein. The eluted product from the expanded bed adsorption chromatography was subsequently purified using size-exclusion chromatography. The results showed that this novel purification protocol achieved a recovery yield of 45.1% with a product purity of 88.2%, which corresponds to a purification factor of 4.5. The recovered HBcAg is still biologically active as shown by ELISA test.
Matched MeSH terms: Chromatography, Ion Exchange/methods*
Poly(oligo(ethylene glycol) methacrylate) (POEGMA), an inert polymer was grafted onto an anion exchange adsorbent for the exclusion of relatively larger hepatitis B virus-like particles (HB-VLPs) from the anion exchange ligand (Q) and at the same time this process allowed the selective adsorption of smaller size Escherichia coli host cell proteins (HCPs). The chain lengths of the POEGMA grafted were modulated by varying the amount of monomers used in the polymer grafting. The purification factor and yield of the HB-VLPs obtained from the flow-through of negative chromatography were 2.3 and 66.0±3.1%, respectively, when shorter chain length of POEGMA (SQ) was grafted. Adsorbent grafted with longer chain of POEGMA (LQ) excluded some HCPs that are larger in size together with the HB-VLPs, reducing the purity of the recovered HB-VLPs. Further heat-treatment of the flow-through pool from SQ followed by centrifugation increased the purity of heat stable HB-VLPs to 87.5±1.1%. Heat-treatment of the flow through sample resulted in thermal denaturation and aggregation of HCPs, while the heat stable HB-VLPs still remained intact as observed under a transmission electron microscope. The performance of the negative chromatography together with heat treatment in the purification of HB-VLPs is far better than the reported bind-and-elute techniques.
Matched MeSH terms: Chromatography, Ion Exchange/methods
This paper describes a refinement in the purification step that facilitated the downstream recovery of high purity BmR1 recombinant protein, which is a protein used as a test reagent in the commercialized rapid tests for detection of lymphac filariasis i.e. Brugia Rapid™ and panLF rapid™. Purification was performed by immobilized metal affinity chromatography (IMAC), followed by ion exchange chromatography (IEX). Results showed that a total of 10.27 mg of BmR1 was obtained when IMAC was performed using 20 mM of imidazole and 5 column volume of wash buffer containing 500 mM of NaCl. Purity of the target protein was enhanced when buffer at pH 5.8 was used during the IEX. Two proteins that recurrently appeared below the BmR1 recombinant protein were identified by mass-spectrometry analysis as the same protein, thus they were probably degradation products of BmR1. These strategies improve purity of the target protein to be used in applications such as production of aptamers and monoclonal antibodies.
Matched MeSH terms: Chromatography, Ion Exchange/methods*