Sephadex G-100 Column Chromatography Research Articles

ISOLATION, IDENTIFICATION AND PARTIAL PURIFICATION OF DIHYDROFOLATE SYNTHETASE FROM HYPOTYL STEMS CALLUS OF LETTUSE PLANT ( Lactuca sativa L. )

Callus from hypocotyl explants of lettuce ( Lactuca sativa L.) was initiated on a modified Murashige and Skoog medium containing 1.0 mg/l of benzyladenine (BA) and naphthaleneacetic acid ( NAA ) .Dihydrofolate synthetase (EC.6.3.2.12) was found in lettuce callus and purified about 36 fold by treating with ethanol and chloroform, ammonium sulfate and gel filtration on Sephadex G-200. The molecular weight of dihydrofolate synthetase was found to be around 40 KD. By Sephadex G-200 column chromatography. The optimum pH for the activity was 6.8 . The optimum reaction temperature was 32C°. The Michaelis constants using dihydropetroic acid and adenosinetriphosphate were 0.036x10-3M and 5.263x10-3M respectively.

Purification, characterization and crystallization of an extracellular alkaline protease from Aspergillus nidulans HA‐10

Aspergillus nidulans is a highly potent fungus used in the production of alkaline protease. Extracellular alkaline protease was purified from A. nidulans in a two-step procedure involving ammonium sulphate precipitation and Sephadex G-100 column chromatography. The molecular mass of the enzyme was determined to be 42 kDa by SDS-PAGE. The enzyme activity was also analyzed by zymogram with gelatin. The enzyme was more stable over a wide range of pH (6-10) and the temperatures up to 50 degrees C. It showed optimum enzyme activity at pH 8.0 and a temperature of 35 degrees C. The protease enzyme was completely inhibited by the serine protease inhibitor of phenylmethylsulfonyl fluoride (PMSF). The crystallization of the purified enzyme was performed by hanging drop vapour diffusion method using PEG 6000 as the precipitant. The micro crystals occurred in 40% of PEG 6,000.

Extracellular keratinase from Trichophyton sp. HA-2 isolated from feather dumping soil

A native feather-degrading keratinophilic fungus, Trichophyton sp. HA-2, was isolated from feather dumping soil in India and screened for extracellular keratinase using keratin-agar medium (chicken feathers as substrate). The fungus was grown in mineral salt medium and keratinase production was tested. The highest keratinase production was observed at pH 8.0 and a temperature of 35 °C for a period of 5 weeks. Keratinase secretion was enhanced when cultures were supplemented with a carbon source and keratin source (chicken feathers). This novel keratinase was purified by DEAE-cellulose and sephadex G-100 column chromatography and characterized. The molecular weight of the keratinase was estimated to be 34 kDa by SDS-PAGE. The optimum pH and temperature were determined to be 7.8 and 40 °C, respectively. This enzyme was completely inhibited by phenylmethylsulfonyl fluoride (PMSF), indicating the presence of serine protease.

Components and Antioxidant Activity of the Polysaccharide from Streptomyces virginia H03

A polysaccharide was isolated from the broth of cultured Streptomyces virginia H03 which was treated by ethanol deposition and savage method to remove the protein, and was purified using Sephadex G-150 column chromatography. The components of the polysaccharide were determined by gas chromatography. The purified polysaccharide was made up of mannose, glucose and galactose, in a 2:1:1 proportion. Its average apparent molecular weight was 3.76 x 10(4) Da which was determined by gel permeation chromatography. In addition, several antioxidant assays were adopted to investigate the antioxidant activity of the polysaccharide in vitro. The results indicated that the purified polysaccharide showed significant antioxidant activity against superoxide anion, hydrogen peroxide and 1,1-diphenyl-2-picrylhydrazyl radical, and lipid peroxidation as with standard antioxidants such as vitamin C. Furthermore, the polysaccharide had a better heat stability than vitamin C, which suggested that the polysaccharide might be a potent useful antioxidant.

Production and Characterization of a Mesophilic Lipase Isolated from Bacillus stearothermophilus AB-1

Using Bacillus stearothermophilus AB-1 isolated from air, the production of lipase was attempted along with its purification and characterization studies. When different carbon and nitrogen sources were supplemented in the culture medium, xylose, tryptophan, alanine, phenylalanine and potassium nitrate were found to be the best. During cultivation, the strain secreted most of its lipase content after 48 h. In particular, the lipase produced in the culture broth showed 300 U mL(-1) when cultivated at optimal temperature and pH of 35 degrees C and 7.5, respectively. The enzyme was purified using 60% ammonium sulfate precipitation and sephadex G200 column chromatography. The enzyme was stable up to 40 degrees C and in the range of pH 7-8. This research reports for the first time the characterization of mesophilic lipase from Bacillus stearothermophilus AB-1 isolated from air.

Production, purification and characterization of mid-redox potential laccase from a newly isolated Trichoderma harzianum WL1

A new strain of Trichoderma harzianum WL1 was isolated from the Western Ghats region of Tamilnadu, India and assayed for laccase activity by ABTS oxidation. Supplementation of CuSO 4 (1 mM) in liquid medium yielded high amounts of laccase (4.36 U ml −1) at an incubation period of 4 days. Laccase enzyme produced by T. harzianum in cultures supplemented with copper sulphate has been purified by ultrafiltration, Sephadex G-100 column chromatography and Concanavalin-A affinity chromatography with a final purification fold of 151.7 and a yield of 0.39%. The purified enzyme was identified as a glycoprotein with a molecular mass of 79 kDa by SDS gel electrophoresis. The UV–vis spectrum of the purified laccase had a peak at 608 and 325 nm suggesting the presence of types I and III Cu centers. The redox potential of the enzyme was found to be 692 mV for the type 1 Cu (T1) site. The optimum pH and temperature for enzyme activity were 4.5 and 35 °C, respectively. Laccase was stable for 24 h at 35 °C and had half-life of 60 min at 65 °C. Purified laccase showed K m values of 180 and 60 μM, respectively, and V max values of 3.95 and 1.42 U mg −1 protein, respectively, for the substrates ABTS and guaiacol. The susceptibility of laccase towards several putative inhibitors and metal cations was also assessed. The enzyme activity was completely inhibited by sodium azide (NaN 3) at the concentration of 20 μM. Laccase activity was also inhibited by several metal cations, especially Hg 2+.

Antioxidant activity of whey protein fractions isolated by gel exclusion chromatography and protease treatment

Whey proteins were isolated from whey powder by a combination of gel exclusion chromatography and protease (pepsin or trypsin) treatment. Whey solution (6 g/dl) was applied to Sephadex G-200 column chromatography and three fractions were obtained. Gel electrophoresis (SDS-PAGE) was used to identify the fractions; the first one contained immunoglobulins and bovine serum albumin, the second contained β-lactoglobulin and α-lactalbumin whereas the third fraction contained small peptides. We have also subjected the whey filtrate to proteases (pepsin and trypsin). Treatment with proteases showed that β-lactoglobulin can be obtained after hydrolysis of the second fraction with pepsin. When the whey filtrate was treated with pepsin and then applied to Sephadex G-200 column chromatography three fractions were obtained; the first one was bovine serum albumin, the second was β-lactoglobulin and the third fraction contained small peptides. After trypsin treatment only two fractions were obtained; the first one was serum albumin and the second fraction was an α-lactalbumin rich fraction. We have determined the antioxidant activity of the fractions using an assay based on the measurement of superoxide radical scavenging activity. Our results showed that among the three fractions, the first fraction had the highest superoxide radical scavenging activity. Also, protease treatment of the second fraction resulted in an increase in the antioxidant activity.

Production, purification, and characterization of thermostable alkaline protease under solid state fermentation conditions for application in bio-detergent technology

Screening studies were carried out for one hundred, and fifty-three bacterial isolates with respect to their ability to produce protease(s), after growing on some industrial wastes at 55ºC, and pH 9.Two most potent thermophilic bacterial isolates concerning of alkaline thermostable protease(s) production were identified as Bacillus licheniformis B42 and Geobacillus stearothermophilus B78. Alkaline thermostable proteases productivity by the two most potent bacterial isolates was affected by substrate concentrations (solid substrate), carbon source, nitrogen source, mineral ions, vitamins, amino acid supplements, incubation temperature, incubation period, and inoculum size. Maximum both enzymes production by B. licheniformis B42 and G. stearothermophilus B78 were obtained on slaughter house wastes (SHW) concentrations,1.5,2.5 %;carbon sources; galactose, no tested sugar; nitrogen sources; diammonium hydrogen phosphate, ammonium molybdate; chelating agents; ethylene diamine tetra acetic acid (EDTA) (500 ppm), no tested ions; vitamins; thiamine pyridoxine; amino acids; arginine, DL-tyrosine at 55°C for 72 h. when inoculated by 0.5, and 2.5 ml respectively. The protease yield under all optimal conditions was increased many folds from 563.68 to 17825 U/ml (31 fold) by B. licheniformis and from 23.7 to 7096 U/ml (299 fold) by G. stearothermophilus. The purification fold of B. licheniformis B42 alkaline thermostable protease increased to 394.7 after applying Sephadex G200 and 411.9 times after Sephadex G100 column chromatography techniques. Fifteen amino acids were detected in the partially purified protease. Optimum conditions of the partially purified alkaline thermostable protease on Sephadex G200 followed by Sephadex G100 produced by B. licheniformis B42 were: incubation temperature, 50°C; thermostable between 50-70°C; pH,10; and incubation time,36 h exhibited maximal activity. The purified enzymes exhibited good stability towards chlorine. Compatibility of the purified enzyme with various commercial detergents reported that purified protease was unstable in presence of commercial detergents. Calcium chloride was able to restore and enhance the activity of the partially purified enzyme in the presence of commercial detergents. The alkaline serine proteases secreted by both strains are industrially important in respect of their abilities to act in alkaline pH and to show stability in broad pH ranges in addition to their stabilities toward sodium dodecyl sulphate(SDS), and hydrogen peroxide (H2O2), suggesting that it is a potentially candidate as an additive in the detergents formulations. Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 111-129

Structural analysis of a polysaccharide from Fructus Mori Albae

Fructus Mori Albae polysaccharide (FMAP) was first isolated from Fructus Mori Albae, and purified by Sephadex G-200 column chromatography. Its average molecular weight ( M w = 1.30 × 10 5) was determined by gel permeation chromatography (GPC). The polysaccharide was found to be composed of d-galactose, d-mannose and d-glucose by TLC and GC–MS. According to IR, methylation and GC–MS, the main chain of the polysaccharide consists of (1 → 3)-linked mannan with branches of galactosyl and glucosyl residues at O-6 on mannosyl residues of the backbone.

Purification and characterization of lectin from humoral fluids ofCharybdis feriatus

To search new sources of lectin, an experiment on lectin distribution in humoral serum of crabCharybdis feriatus (in short, CFL) was conducted March, 2002. When adding solid ammonium sulfate into the fluids up to 50% saturation at 4°C, most CFL activity showed precipitates who were then continually extracted by ammonium sulfate of different concentrations. The supernatant, which was called primary CFL fluids, was given a 17.60-fold purification and 45.70% recovery of total activity. Finally, by using Sephadex G-100 column chromatography, the CFL in the primary CFL fluid was highly purified. Compared to the original humoral fluids, the last purified CFL got a 203.90-fold purification and 30.48% recovery of total activity, and demonstrated a single band on SDS-PAGE. In the same time, the purified CFL was detected for agglutination activity with 7 kinds of animal erythrocytes. Other characterization, such as sugar inhibition, and the effect of temperature on the agglutination activity of CFL were also studied. Our results indicate that agglutination activity of CFL was influenced by sugar and temperature.

Activation kinetics of cetylpyridinium chloride on the prophenol oxidase from pupae of blowfly ( Sarcophaga bullata)

Prophenol oxidase (PPO) (EC 1.14.18.1) is isolated from pupae of blowfly ( Sarcophaga bullata) and purified by employing ammonium sulfate fractionation, ion-exchange chromatography on DEAE-cellulose, and gel filtration through Sephadex G-100 column chromatography. The enzyme exists in a latent or inactive state. Cetylpyridinium chloride (CPC), a cationic detergent, is found to activate the PPO activity. The activation of the enzyme by CPC has first been studied by using the kinetic method of the substrate reaction described by Tsou. The results show that the enzyme is activated by a complexing scheme that has not been previously identified. The enzyme first reversibly and quickly binds CPC and then undergoes a slow reversible active course. The activation reaction is a single molecule reaction and the apparent activation rate constant is dependent on the CPC concentration with the function relationship fit with a hyperbola. The micro rate constants of activation and the association constant are determined from the measurements. Substrate binding does not affect the micro rate constants of activation by CPC.

Antioxidant activities of different fractions of polysaccharide purified from Gynostemma pentaphyllum Makino

The water-soluble crude polysaccharide GM, obtained from the Gynostemma pentaphyllum Makino by boiling-water extraction and ethanol precipitation, was fractionated by DEAE–Sepharose CL-6B column chromatography, and purified by Sephadex G-100 column chromatography, giving three polysaccharide fractions termed GMA, GMB and GMC. The monosaccharide components of them were studied by PC and GC. On the basis of superoxide radical assay, hydroxyl radical assay and self-oxidation of 1,2,3-phentriol assay, the antioxidant activities of GM, GMA, GMB and GMC were investigated. Among these contents, GMC had the higher scavenging effects on superoxide radicals and inhibitory effects on self-oxidation of 1,2,3-phentriol, and so should be explored as a novel potential antioxidant.

Purification and characterization of enzyme responsible for N-myristoylation of octapeptide in aqueous solution without ATP and coenzyme a from Pseudomonas aeruginosa

The enzyme that catalyzes N-acyl linkage between myristic acid and the NH(2)-terminal glycine residue of the octapeptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg-NH(2) in aqueous solution without ATP and coenzyme A was found in Pseudomonas aeruginosa. The enzyme was purified from cell-free crude extract using DEAE-Cellulose, Sephadex G-200, CM-Sephadex C-50, and hydroxyapatite column chromatographies, and then purified approximately 1900-fold with about 1.5% recovery of enzyme activity from the crude extract. Finally, the purified enzyme showed a main band on SDS polyacrylamide gel electrophoresis after staining with Coomassie Brilliant Blue. The band corresponded to a molecular mass of approximately 60 kDa. The K(m)s of the purified enzyme for the substrate myristic acid and the octapeptide were 0.36 and 2.6 mM, respectively. When myristoyl-CoA instead of myristic acid was used as the substrate for the enzyme reaction, myristoyl octapeptide could be synthesized as observed in the case of myristic acid. The K(m) of myristoyl-CoA was 0.17 mM.

Purification and characterization of laccase produced by a white rot fungus Pleurotus sajor-caju under submerged culture condition and its potential in decolorization of azo dyes

An extracellular laccase was isolated and purified from Pleurotus sajor-caju grown in submerged culture in a bioreactor, and used to investigate its ability to decolorize three azo dyes. The extracellular laccase production was enhanced up to 2.5-fold in the medium amended with xylidine (1 mM). Purification was carried out using ammonium sulfate (70% w/v), DEAE-cellulose, and Sephadex G-100 column chromatography. The enzyme was purified up to 10.3-fold from the initial protein preparation with an overall yield of 53%. The purified laccase was monomeric with an apparent molecular mass of 61.0 kDa. The purified enzyme exerted its optimal activity with 2,2-azino-bis(3-ethylbenzo-thiazoline-6-sulfonate (ABTS) and oxidized various lignin-related phenols. The catalytic efficiencies kcat/Km determined for ABTS and syringaldazine were 9.2x10(5) and 8.7x10(5), respectively. The optimum pH and temperature for the purified enzyme was 5.0 and 40 degrees C, respectively. Sodium azide completely inhibited the laccase activity. The absorption spectrum revealed type 1 and type 3 copper signals. The purified enzyme decolorized azo dyes such as acid red 18, acid Black 1, and direct blue 71 up to 90, 87, and 72%, respectively. Decolorization ability of P. sajor-caju laccase suggests that this enzyme could be used for decolorization of industrial effluents.

Structural analysis of anti-tumor heteropolysaccharide GFPS1b from the cultured mycelia of Grifola frondosa GF9801

A 21-kDa heteropolysaccharide, coded as GFPS1b, was obtained from the cultured mycelia of Grifola frondosa GF9801 by hot-water extraction, ethanol precipitation, and fractioned by DEAE Sepharose Fast-flow, followed by the purification with Sephadex G-100 column chromatography using an AKTA purifier. It exhibited more potent anti-proliferative activity on MCF-7 cells than other polysaccharide fractions. GFPS1b was an acidic polysaccharide with approximately 16.60% protein and 4.3% uronic acid. Gas chromatography of absolute acid hydrolysate of GFPS1b suggested that it was composed of d-glucose, d-galactose, and l-arabinose with a molar ratio of 4:2:1. Periodate oxidation, Smith degradation, partial acid hydrolyzation, methylation analysis, FT-IR, and 1H, 13C NMR spectroscopy analysis revealed that GFPS1b had a backbone consisting of α-(1 → 4)-linked d-galacopyranosyl and α-(1 → 3)-linked d-glucopyranosyl residues substituted at O-6 with glycosyl residues composed of α- l-arabinose–(1 → 4)-α- d-glucose (1→ linked residues.

Characterization of Major Stable Proteins in Chardonnay Wine

Chardonnay wine produced in the conventional manner contained 69.8 to 108mg/L soluble proteins including nondialyzable polypeptides ; with an average of 87.2mg/L. Proteins were separated by precipitation with ammonium sulfate, and fractionated into two fractions, F1 (mainly invertase and proteoglycans) and F2 (glycoproteins), by Sephadex G-100 column chromatography. The fractions were further separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) [isoelectric focusing and SDS-polyacrylamide gel electrophoresis]. More than 160 and 150 protein spots were detected on the 2-D PAGE maps of F1 and F2, respectively. The major proteins or polypeptides were determined to be fruit proteins, such as thaumatin- and osmotin-like proteins, invertase, lipid transfer protein (LTP), and their hydrolysis products. This is first report on the existence of LTP (or its hydrolysis product) and the hydrolysis products of major grape proteins (invertase, osmotin-like protein, and thaumatin-like protein) in wine.

Insensitivity of acetylcholinesterase in a field strain of the fall armyworm, Spodoptera frugiperda (J.E. Smith)

Acetylcholinesterase (AChE) was purified from adult heads of the fall armyworm ( Spodoptera frugiperda) by using a two-step procedure involving gel filtration on a Sephadex G-200 column and affinity chromatography on a procainamide–ECH Sephadex 4B column. Both susceptible and field strains possessed two AChE isozymes, namely, AChE-1 and AChE-2, with subunit molecular weights of 63.7 and 66.1 kDa. The purified AChE had an apparent K m value of 33.5 μM and a V max of 7.07 μmol/min/mg protein in the susceptible strain. The apparent K m and the V max were 2.2- and 2.0-fold higher, respectively, in the field strain than in the susceptible strain. The purified AChE from the field strain was 17- to 345-fold less sensitive than that from the susceptible strain to inhibition by carbamates (carbaryl, eserine, methomyl, and bendiocarb) and organophosphates (methyl paraoxon and paraoxon), insensitivity being highest toward carbaryl. The results further support the notion that insensitive AChE played an important role in the insecticide resistance observed in the field strain.

Epitaxial deposition of calcium oxalate on uric acid rich stone matrix is induced by a 29 kDa protein

Background Association of macromolecules particularly the role of proteins in urolithiasis has been studied for last few centuries, but still a complete profile of stone matrix proteins that mediate co-precipitation of uric acid and calcium oxalate has not been characterized. We isolated and characterize proteins from uric acid rich stone matrix, which have oxalate binding activity. Methods Matrix proteins were isolated from uric acid rich stone matrix using EDTA as a demineralizing agent. The radiolabelled solubilized proteins were fractionated with increasing ionic concentration by DEAE cellulose column chromatography to identify the oxalate binding protein. It was purified using Sephadex G-200 column chromatography. Amino acid composition was determined and monoclonal antibody was produced against the oxalate binding uric acid rich stone matrix protein. Urinary uric acid binding proteins were isolated from stone formers urine, their oxalate binding activity assayed and cross reactivity with the produced monoclonal antibody were checked using ELISA and Western blotting. Results Matrix on DEAE column chromatography elution yielded 3 protein peaks and they were named as fraction I, II and III among which fraction I had higher oxalate binding activity which was further purified with Sephadex G-200 column which yielded 2 protein peaks designated as Ia and Ib. Fraction Ib with molecular weight 29 kDa exhibited the maximum oxalate binding activity. Forty percent of this 29 kDa protein is comprised of basic amino acids. Monoclonal antibody (IgG 1) was produced against the 29 kDa stone matrix protein. Urinary uric acid binding proteins were isolated from stone formers, 4 protein peaks were obtained named as fraction I to IV. Among them, fraction IV having molecular weight of ∼29 kDa cross reacted up to 85.6% with 29 kDa stone matrix protein. Moreover, urinary 29 kDa protein exhibited oxalate binding activity of 94.16 ± 6.08 pmol/mg protein at pH 5.5. Conclusion The 29 kDa protein isolated from uric acid rich stone matrix and urine are one and the same, thereby insinuating that 29 kDa protein might play a major role in epitaxial deposition of calcium oxalate over uric acid core, consequently favoring the lithogenic events like uric acid and calcium oxalate nucleation, aggregation and retention.

Alterations in band 3 protein and anion exchange in red blood cells of renal failure patients

The precise nature of band 3 protein and its involvement in oxalate exchange in the red blood cells (RBCs) of renal failure patients has not been studied in detail. Therefore, here we studied the oxalate exchange and binding by band 3 protein in RBCs of humans with conditions of acute and chronic renal failure (ARF and CRF). The RBCs of ARF and CRF patients exhibited abnormal red cell morphology and an increased resistance to osmotic hemolysis. Further, an increase in the cholesterol content and decrease in the activities of Na(+)-K(+)-, Ca(2+)-, and Mg(2+)-ATPases of membranes were observed in the RBCs of ARF and CRF patients. A decrease in the oxalate flux was observed in the RBCs of ARF and CRF patients. The oxalate-binding activities of the RBC membranes were significantly lower in ARF (20 pmoles/mg protein) and CRF (5.3 pmoles/mg protein) patients as compared to that in the normal subjects (36 pmoles/mg protein). DEAE-cellulose and Sephadex G-200 column chromatography purification profiles revealed a distinctive shift in oxalate-binding activity of band 3 protein of RBCs of ARF and CRF patients as compared to that of the normal subjects. It was also observed from the binding studies with a fluorescent dye, eosin-5-maleimide, which specifically binds to band 3 protein, that the RBCs of ARF and CRF patients exhibited only 53 and 32% of abundance of band 3 protein, respectively, as compared to that in the RBCs of the normal subjects, thus revealing a decrease in the band 3 protein content in ARF and CRF patients. These results for the first time showed a decrease in the oxalate exchange in RBCs of patients with ARF and CRF, which was also concomitant with the low levels of abundance of band 3 protein.

NAD deamidation ?a new reaction? by an enzyme from Aspergillus terreus DSM 826

NAD deamidation is a non-previously recognized reaction. This reaction has been found to be catalyzed by extracts of Aspergillus terreus DSM 826. Conversion of NAD to the biosynthetic intermediate, deamido NAD, by these extracts, at the optimum pH and temperature did not exceed about 55 of the amount of the substrate added. Completion of the reaction was achieved when the extracts were pre-heated at 50 degrees C for 15 min in absence of the substrate. In a very similar manner, the extracts catalyzed hydrolytic cleavage of the amide linkages of different biomolecules such as nicotinamide, nicotinamide riboside, nicotinamide mononucleotide, L-glutamine, L-asparagine and acetamide. Polyacrylamide was also deamidated under the same conditions. In addition, complete dephosphorylation of the dinucleotide molecule was also effected by the same extracts. Separation of the NAD deamidating enzyme from the NAD dephosphorylating enzyme was achieved on using either DEAE - Sephadex A-25 or Sephadex G-200 column chromatography. The obtained phosphohydrolase-free-deamidase showed optimum activity at pH 8 of 0.1 M phosphate buffer and 50 degrees C. It exhibited broad substrate specificity and hyperbolic substrate saturation kinetics. It was isosterically inhibited by the product of its activity and this inhibition was prevented by heating the extracts at 50 degrees C for 15 min. Its activity was not affected in presence of sodium fluoride, partially inhibited in presence of magnesium chloride and was retained in the freezer for some months.