Sepharose 4B Column Chromatography Research Articles

Overexpression, biochemical characterization, and anticancer activates of L-asparaginase from Bacillus subtilis

L-asparaginases convert L-asparagine into L-aspartate and ammonia. The L-asparaginase from Bacillus subtilis was cloned and expressed in the E. coli strain BL21(DE3)pLysS in the current study. Using glutathione sepharose 4B column chromatography, the L-asparaginase enzyme was uniformly purified 173.34 times, with a final specific activity of 1769.13 IU/mg protein and a yield of 56.14%. The isolated enzyme was identified as a 36 kDa polypeptide chain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immobilized enzyme was placed on top of the Ca alginate beads. The immobilized enzyme is quite stable and retains the majority of its activity at 4 °C (74 percent). The enzymatic and structural characteristics of free and immobilized recombinant L-asparaginase were studied. The activity of the free enzyme peaked after 30 min of incubation at pH 8.0 and 45 °C. After 30 minutes at 50 °C, the immobilized enzyme showed its peak activity at a pH of 8.5. The refined enzyme's amino acid makeup was identified. An enzyme that heals leukemia, Bacillus subtilis L-asparaginase, can be successfully mass-produced using this technique.

Structural features and antioxidant activity of a new galactoglucan from edible mushroom Pleurotus djamor

A new water soluble galactoglucan with apparent molecular weight ~1.61×105Da, was isolated from the edible mushroom Pleurotus djamor by hot water extraction followed by purification through dialysis tubing cellulose membrane and sepharose 6B column chromatography. The sugar analysis showed the presence of glucose and galactose in a molar ratio of nearly 3:1 respectively. The structure of the repeating unit in the polysaccharide was determined through chemical and NMR experiments as: In vitro antioxidant studies showed that the PDPS exhibited hydroxyl radical scavenging activity (EC50=1.681±0.034mg/mL), DPPH radical scavenging activity (EC50=3.83±0.427mg/mL), reducing power (EC50=4.258±0.095mg/mL), and ABTS radical quenching activity (EC50=0.816±0.077mg/mL). So, PDPS should be explored as a natural antioxidant.

Characterization and inhibitory activities on α-amylase and α-glucosidase of the polysaccharide from blue honeysuckle berries

An acidic heteropolysaccharide HEP-2 was obtained from blue honeysuckle berry extracts after purification using Sepharose 6B and Sephadex G-200 column chromatography. The molecular weight of HEP-2, determined with high-performance gel permeation chromatography, was 3.01×106Da. Fourier transform infrared spectroscopy, gas chromatography and nuclear magnetic resonance analyses revealed the presence of α-, β-pyranose ring and six sugar residues in HEP-2. Amorphous aggregates of HEP-2 with irregular shape and lacking the triple helical structure were detected using the Congo red test, scanning electron and atomic force microscopy, and X-ray diffraction studies. Rheological characterization showed typical shear-thinning behavior and viscoelastic property of the polysaccharide. HEP-2 exerted significant inhibitory effects on α-amylase and α-glucosidase and displayed competitive inhibition kinetics. The collective results support the potential utility of HEP-2 as a hypoglycemic agent for enzyme-targeted treatment of diabetes or functional food.

Microwave-assisted extraction of an acidic polysaccharide from Ribes nigrum L.: Structural characteristics and biological activities

Abstract Microwave-assisted extraction of the polysaccharide from blackcurrant (Ribes nigrum L.) fruits (MBP) was optimized using response surface methodology (RSM). The yield of MBP was 10.59 % (w/w) under the optimal conditions of power (414 W), liquid to material ratio (31/1 mL/g), and the time (41 min). After purification using D4006 macroporous resin and Sepharose-6B column chromatography, a water-soluble acidic heteropolysaccharide (PMBP) was obtained with a molecular weight of 1.99 × 106 g/mol. PMBP was thermally stable and exhibited excellent rheological properties. Fourier transform infrared spectroscopy, gas chromatography and nuclear magnetic resonance analysis confirmed the presence of α-, β-pyranose and six main glycosidic linkages in PMBP. Scanning electron microscopy, atomic force microscopy, and X-ray diffraction studies demonstrated that PMBP was amorphous aggregates with irregular shape, porous, and smooth surface. Bioactivity assays revealed the notable antioxidant and hypoglycemic activities of PMBP. Moreover, PMBP was kinetically characterized as a competitive inhibitor for α-amylase and α-glucosidase.

A lectin with anti-microbial and anti proliferative activities from Lantana camara, a medicinal plant

Lectins are known to possess interesting biological properties such as anti microbial, nematicidal, anti tumor and anti viral activities. Lantana camara from verbenaceae family is a medicinal plant known for possessing anti oxidant and anticancer activities. Since anticancer activity is reported in plant lectins, leaves of Lantana camara was used to check the presence of lectin. Here we report the purification, characterization and biological properties of a lectin from Lantana camara (LCL) leaves. LCL was purified by ion exchange chromatography on CM-cellulose column followed by affinity chromatography on mucin coupled Sepharose 4B column and gel filtration chromatography on Superdex G75 column. LCL is a glycoprotein with 10% of the carbohydrate and is blood group non specific. SDS-PAGE analysis of affinity purified LCL showed two proteins with apparent molecular weight of 14.49 kDa and 17.4 kDa which were subsequently separated by Gel filtration chromatography on Superdex G75 column. Hapten inhibition studies of LCL revealed its highest affinity for Chitin, Milibiose, α-D-Methyl galactopyranoside and glycoproteins like mucin, asialomucin. LCL showed strong binding to human colon adenocarcinoma HT29 cells with MFI of 242 which was effectively blocked by 68.1 and 62.5% by both mucin and milibiose. LCL showed dose and time dependent growth inhibitory effects on HT29 cells with IC50 of 3.75 μg/ml at 48 h. LCL has potent antibacterial and anti fungal activity. LCL can be explored for its clinical potential.

Functional Properties of Partially Characterized Polysaccharide from the Medicinal Mushroom Ganoderma neo-japonicum (Agaricomycetes).

This study was conducted to evaluate the mycochemical composition and antiglycemic and antioxidant activities of Ganoderma neo-japonicum hot aqueous extracts, prepared at different boiling durations, and polysaccharides isolated from them. Ground basidiocarps of G. neo-japonicum were double-boiled at 100°C for 0.5, 3, or 4 hours, and the antiglycemic activity was assessed by α-amylase and α-glucosidase enzyme inhibition assays. The antioxidant capacity of the crude hot aqueous extracts (AE-1, AE-2, AE-3) was assessed by DPPH and ABTS radical scavenging and ferric-reducing antioxidant power assays. The total phenolics, protein, and sugar in the crude extracts were also determined. The hot aqueous extract (AE-3) containing a significant amount of total sugar and having enhanced antiglycemic and antioxidant activities was selected for polysaccharide isolation. The isolated crude polysaccharide was separated and purified using diethylaminoethyl-cellulose-52 and Sepharose 6B column chromatography. Fourier transform infrared spectroscopy studies of the purified polysaccharide fraction (PF) showed the presence of typical bands corresponding to polysaccharides. The estimated β-glucan concentration in the PF was 39.26%. In general, the PF exhibited significantly lower antioxidant activity than AE-3. Nevertheless, its potency in inhibiting carbohydratehydrolyzing enzymes may have potential in the management of diabetes mellitus.

Structural characterization and antioxidant activity of a glucan from Meripilus giganteus

A new water soluble glucan (MGPS), with a molecular weight ∼1.48×105Da, was isolated from the fruit bodies of Meripilus giganteus by hot water extraction followed by purification through dialysis tubing cellulose membrane and sepharose 6B column chromatography. Its structural characteristics were investigated by acid hydrolysis, methylation analysis, Smith degradation and 1D/2D NMR experiments. The monosaccharide composition analysis showed that MGPS contain only glucose. The backbone of MGPS was composed of two (1→3)-β-d-glucopyranosyl, two (1→6)-β-d-glucopyranosyl, two (1→6)-α-d-glucopyranosyl, and one (1→4)-α-d-glucopyranosyl residues, out of which one (1→3)-β-d-glucopyranosyl residue was branched at O-6 position with terminal α-d-glucopyranosyl residue and one (1→4)-α-d-glucopyranosyl residue branched at O-6 position with terminal β-d-glucopyranosyl residue. In vitro antioxidant studies showed that the MGPS exhibited hydroxide radical scavenging activity (IC50=390μg/mL), superoxide radical scavenging activity (IC50=70μg/mL), and ferrous ion chelating activity (IC50=290μg/mL).

PURIFICATION AND CHARACTERIZATION OF POLYSACCHARIDE FROM MICROALGAE BTM 11 AS INHIBITOR OF HEPATITIS C VIRUS RNA HELICASE

Hepatitis C virus is one of the causative agents for HCV-related liver disease development with high virulence. Antiviral drugs can be discovered through molecular target-based therapy by finding the inhibitors for RNA helicase that play crucial role in viral replication. An inhibitor can be derived from polysaccharides produced by microalgae. In this study, polysaccharide from microalgae BTM11 which had inhibitory activity against RNA helicase have been purified and characterized.On the other hand, the RNA helicase was produced by E. coli BL21(DE3)pLyss harboring NS3 RNA helicase HCV gene in pET-21b plasmid. This enzyme then was purified by affinity chromatography and this purified enzyme was used for HCV RNA helicase inhibitory assay. Polysaccharide fractions were separated from the extract of BTM 11 using Sepharose 4B column chromatography. Inhibitor activity was measured using colorimetry ATPase assay based on releasing of phosphate inorganic. The results of SDS-PAGE and Western blot showed that the purified RNA helicase had a molecular weight of 54kDa. The highest inhibition activity of HCV RNA helicase (88 ± 2,4726%) was achieved at fraction 10 of purified polysaccharide. The HPLC result showed that compounds of polysaccharide active fraction were maltopentose (R t 4.183) and glucose (R t 5.673). Both of 1 H-NMR and IR spectra showed hydroxyl and carbonyl groups that present in the polysaccharide structure. Key words : Hepatitis C Virus, RNA Helicase, Microalgae BTM11, chromatography, polysaccharide

Purification, antioxidant activity and antiglycation of polysaccharides from Polygonum multiflorum Thunb

Polysaccharides, one of the most important constituents in Polygonum multiflorum Thunb, a famous Chinese medicinal herb, were isolated by DEAE-52, Sepharose 4B and Sephacryl S-300 column chromatography. Two polysaccharides (PMP-1 and PMP-2) were identified as homogeneous in molecular weight with HPLC. The molecular weights were 4.8 × 10(2) and 6.1 × 10(2) kDa, respectively. Antioxidant activity tests were performed with two polysaccharides at concentrations of 0.1-1.5mg/mL. The results indicated that the inhibitory activity on oxidation and glycation exhibited a dose-dependent response. PMP-2 exhibited a much stronger antioxidant capacity against free radical, lipid oxidation and protein glycation. The IC₅₀ values of PMP-2 were 0.47, 0.6 and 0.93 mg/mL for superoxide anion scavenging, hydroxyl radical scavenging, and hydroxyl peroxide scavenging, respectively. The inhibitory ability of PMP-2 on lipid oxidation was most markedly in rat liver, followed by heart and kidney. Meanwhile, PMP-2 also showed satisfactory suppression of AGEs formation. This suggested that the polysaccharides present in PM can contribute to the biological effects.

Preliminary using the lectin from maritima jackbean seeds (Canavalia maritima Aublet) for analysis of IgG from human serum by Lectin-Elisa technique

In recent investigation, we proved specific interaction between lectin ConM from Canavalia maritima of Vietnam and IgG from human serum. In order to using the lectin for analysis IgG from human serum, we carried out the purification of IgG from healthy human serum by Protein A - Sepharose 4B and ConM-Sepharose 4B column chromatography followed to prepare the ConM-ELISA biokit for analyzing IgG from human serum. Results obtained showed that average concentration of IgG from healthy human serum was determined to be 12.7 mg/ml. Using suitable concentration of ConM to be 5 mg/well by ConM-ELISA technique with human serum diluted 200 times.

Purification and characterization of trypsin from the pyloric caeca of brownstripe red snapper (Lutjanus vitta)

Abstract Trypsin was purified from the pyloric caeca of brownstripe red snapper (Lutjanus vitta) by ammonium sulphate (40–60% saturation) precipitation, soybean trypsin inhibitor (SBTI)-Sepharose 4B column and DEAE-Sephacel column chromatography. Purified trypsin showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) and native-PAGE. A yield of 4.9% with the purification-fold of 20 was obtained. Trypsin had an apparent molecular weight of 23 kDa. SBTI and N-ρ-tosyl- l -lysine-chloromethylketone (TLCK) showed a strong inhibitory effect on the purified trypsin, while other protease inhibitors exhibited negligible inhibition. Trypsin had maximal activity at pH 8.5 and 60 °C for the hydrolysis of α-N-benzoyl- dl -arginine-ρ-nitroanilide (BAPNA). It was stable within the temperature range of 25–55 °C and pH range of 7.0–10.0. Purified trypsin had a Michaelis–Menten constant (Km) and catalytic constant (kcat) of 0.507 mM and 4.71 s−1, respectively, when BAPNA was used as the substrate. For the hydrolysis of α-N-ρ-tosyl- l -arginine methyl ester (TAME), Km and kcat were 0.328 mM and 112 s−1, respectively.

Purification and identification of polysaccharide derived from Chlorella pyrenoidosa

The conditions for extracting and purifying polysaccharides from Chlorella pyrenoidosa, including intensity and duration of ultrasound, the temperature and incubation time, and ethanol concentration, were investigated through an orthogonal design of L16(45) in this work. High performance liquid chromatography (HPLC) and gas chromatography (GC) were used to characterize the compounds in C. pyrenoidosa. The highest yield of 44.8 g kg−1 was achieved at 400 W of ultrasound for 800 s and then followed by incubation in water bath at 100 °C for 4 h in 80% ethanol. Two polysaccharide fractions (S1 and S2) were separated from the extracts of C. pyrenoidosa using Sepharose 4B column chromatography. The average molecular weights (Mw) of S1 and S2 were 81,877 Da and 1749 Da, respectively. Gas chromatographic (GC) traces of the hydrolyzed polysaccharides showed that most of the majority of monosaccharide in both fractions was mannose (78.0% and 76.5% of relative mass from S1 and S2, respectively) with low levels of glucose (13.2% and 8.4% of relative mass from S1 and S2, respectively). The Fourier-transform infrared spectra (FT-IR) of S1 and S2 revealed typical characteristics of polysaccharides. Both samples had the characteristics of hydroxyl groups, weak C–H band and α-pyranoses; however, only S2 had a carboxyl group.

Characterization of the brown pigment in sautéed onion by metal-chelating Sepharose 6B column chromatography and microbial decolorization

The brown pigment in sautéed onion was characterized by a microbiological procedure (involving microbial decolorization) and a chemical procedure (involving metal-chelating chromatography with a Sepharose 6B column). The suitability of the chemical procedure as a method for classifying brown pigments was also examined. The pigment from sautéed onion was about 40% decolorized by Coriolus versicolor IFO 30340, as were the model pigments caramel and model melanoidin (MM). The phenol-type model pigments (PTMP) were approximately 40–60% decolorized by Paecilomyces canadensis NC-1, but the sautéed-onion pigment, the MM and the caramel were merely 5% decolorized by it. The pigment from sautéed onion was separated into two components by Cu2+, Fe2+, or Zn2+-chelating chromatography, as were caramel and MM; the PTMP was not separated into multiple components by either Cu2+ or Fe2+-chelating chromatography. The results from both methods indicate that the pigment from sautéed onion is similar to MM and caramel but not the phenol-type pigment.

In vitro reduction of trypsin inhibitor by purified NADPH/thioredoxin system from sprouts of sweet potato (Ipomoea batatas (L) Lam.) storage roots

Sweet potato ( Ipomoea batatas (L.) Lam) storage roots were sprouted in the dark for two months, and the sprouts were used as source materials for purification of thioredoxin reductase successively via the DE-52, Sephadex G-75, and 2′,5′-ADP Sepharose 4B column chromatography. Thioredoxin reductases with a molecular mass of ca 33 kDa were obtained. The purification was 813-fold and the yield was 2.5%. The effect of thioredoxin reductase on trypsin inhibitor activity (TIA) was analyzed with a TIA assay method. Native, oxidized form of trypsin inhibitors (TIs) inhibited trypsin activity to 24% of its original value. Trypsin activity recovered to 38 and 46% of its original value, respectively, when TI was reduced in advance by NTR system and DTT. The data indicate that NTR-reduced TIs lost the capacity to inhibit trypsin activity in vitro. mBBr/SDS-PAGE was used to analyze the reduction of TIs on gels. The reduced TIs were more sensitive to heat denaturation, and exhibited increased digestibility by partially purified proteases from sweet potato sprouts. Based on these results, we conclude that the NADPH/thioredoxin system is likely associated with the reduction of TIs and initiation of degradation and re-mobilization of stored TIs during sprouting of sweet potato storage roots. Meanwhile we may expect sprouts to be a better protein source for consumption than SP storage root itself.

Purification and properties of betaine aldehyde dehydrogenase from Avena sativa.

Betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8) is the enzyme that catalyzes the second step in the synthesis of the osmoprotectant, glycine betaine. NAD-dependent BADH was purified from Avena sativa shoots by DEAE Sephacel, hydroxyapatite, 5'-AMP Sepharose 4B, Mono Q and TSK-GEL column chromatographies to homogeneity by the criterion of native PAGE, and the properties of BADH were compared with those of aminoaldehyde dehydrogenase purified to homogeneity from A. sativa. The molecular mass estimated by both gel filtration using TSK-GEL column and Sephacryl S-200 was 120 and 115, kDa, respectively. The enzyme is a homodimer with a subunit molecular mass of 61 kDa as shown by SDS-PAGE. The pI value of the enzyme was found to be 6.3. The purified enzyme catalyzed not only the oxidation of betaine aldehyde (BAL), but also that of aminoaldehydes, 3-aminopropionaldehyde (APAL), 4-aminobutyraldehyde (ABAL), and 4-guanidinobutyraldehyde (GBAL). The K(m) values for BAL, APAL, ABAL and GBAL were 5x10(-6), 5.4x10(-7), 2.4x10(-5) and 5x10(-5) M, respectively. APAL showed substrate inhibition at a concentration of 0.1 mM. A fragment of BADH cleaved by V8 protease shared homology with other plant BADHs.

Entamoeba histolytica cysteine proteinases disrupt the polymeric structure of colonic mucin and alter its protective function.

The adherent mucous gel layer lining the colonic epithelium is the first line of host defense against invasive pathogens, such as Entamoeba histolytica. The mucous layer prevents the attachment of amoeba to the colonic epithelium by trapping and aiding in the expulsion of the parasite. Disruption of the mucous layer is thought to occur in invasive amebiasis, and the mechanism by which the parasite overcomes this barrier is not known. The aim of this study was to characterize the specific interactions occurring between E. histolytica secreted cysteine proteinases and colonic mucin as a model to examine the initial events of invasive amebiasis. E. histolytica secreted products were examined for mucinase activity utilizing mucin metabolically labeled with [(35)S]cysteine as a substrate. Cysteine proteinases degraded mucin in a time- and dose-dependent manner. A significant reduction (>50%) in high-molecular-weight mucin with altered buoyant density was observed when degraded mucin was analyzed by Sepharose 4B column chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, and CsCl density gradient centrifugation. Mucinase activity was eliminated by the specific cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane and was independent of glycosidase activity. Moreover, the degraded mucin was 38% less effective than native mucin at inhibiting amebic adherence to target epithelial cells. These results are the first to show that E. histolytica cysteine proteinases alter the protective function of the mucous barrier by disrupting the structure of the MUC2 polymer. Mechanistically, the parasite achieves this via proteolytic degradation of the terminal cysteine-rich domains.

Two ascorbate peroxidases from broccoli: identification, expression and characterization of their recombinant proteins

Two distinct clones having high nucleotide identity to the sequences encoding ascorbate peroxidase (APX) were isolated from broccoli ( Brassica oleracea L. var. italica ). Deduced amino acid sequences of both cDNAs, BO-APX 1 (accession number AB078599) and BO-APX 2 (accession number AB078600), shared identity of 92.8% and there was more than 80% identity between BO-APX s and other plant cytosolic APXs at the protein level. Gene expression and protein levels of BO-APX 1 and BO-APX 2 were investigated in various parts of broccoli after harvest. Transcript levels of BO-APX 2 gradually increased in florets, while those of BO-APX 1 decreased in florets after harvest. BO-APX 1 and BO-APX 2 were expressed in Escherichia coli as a fusion protein with glutathione S -transferase (GST) and purified to homogeneity by glutathione sepharose 4B column chromatography. Both proteins of BO-APX 1 and BO-APX 2 appeared as a single major band on SDS-PAGE corresponding to a mass of 25 kDa and reacted with polyclonal antibodies raised against recombinant BO-APX 1 . Both enzymes showed high specificities for ascorbate and hydrogen peroxide. The km values of recombinant BO-APX 1 and BO-APX 2 for ascorbate were 395 and 526 μM and those for hydrogen peroxide were 15 and 7 μM, respectively. The role of APX was discussed in relation to ascorbate breakdown in broccoli florets during senescence.

Purification and properties of aminoaldehyde dehydrogenase from Avena sativa

NAD-dependent aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.-) from Avena shoots was purified by DEAE Sephacel, hydroxyapatite, 5'-AMP Sepharose 4B, Mono Q, and TSK-GEL column chromatographies to homogeneity by the criterion of native PAGE. SDS-PAGE yielded a single band at a molecular mass of 55 kDa. IEF studies showed a band with a p I value of 5.3. In contrast to AMADHs from other species, the TSK-GEL chromatography showed that AvenaAMADH exists as a monomer in the native state. The purified enzyme catalyzed the oxidations of 3-aminopropionaldehyde (APAL), 4-aminobutyraldehyde (ABAL) N-(3-aminopropyl)-4-aminobutyraldehyde (APBAL), and 4-guanidinobutyraldehyde (GBAL), but not of betaine aldehyde or indoleacetaldehyde. The K(m) values for APAL, ABAL, and GBAL were 1.5x10(-6), 2.2x10(-6), and 1.3x10(-5) M, respectively. Although N-terminal amino acid sequence of Avena AMADH could not be determined due to a modification of the amino residue, the sequence of the fragment of AMADH cleaved by V8 protease showed greater similarity to the barley BADH than to the pea AMADH.

Comparative Studies of Three Type II Ribosome-Inactivating Proteins from the Seeds of Three Species of the Genus Cinnamomum

Bodinierin, one of the major proteins in the kernels of camphor tree (Cinnamomum Bodinieri), has been identified as a novel type II ribosome-inactivating protein. Using sepharose-4B column chromatography followed by acid precipitation and ammonium sulfate precipitation, bodinierin has been purified to be homogeneous as characterized by SDS-PAGE. Bodinierin was composed of two chains (A- and B-chain) with the molecular weight of 31 and 34 kDa respectively. The reduced bodinierin showed strong inhibitory activity to protein synthesis in the rabbit reticulocyte lysate and the RNA N-glycosidase activity. The bodinierin also displayed the carbohydrate binding activity to agglutinate rabbit erythrocyte. A comparison of bodinierin with cinnamomin and porrectin that were isolated from the kernels of other species of the same genus (Cinnamomum) demonstrated that they had similar structure and biological activities, which provided phylogenetic evidence to the three species.

Serine alkaline protease from a newly isolated Bacillus sp. SSR1

Abstract An extracellular serine alkaline protease produced by Bacillus sp. SSR1 was purified to homogeneity by Sephadex A-50 and Sepharose 6B column chromatography. The enzyme is a monomeric protein with a molecular weight of 29 and 35 kDa as estimated by SDS–PAGE and native PAGE respectively. The purified enzyme is stable in the alkaline pH range (8.0–11.0) and retains 100% activity at its optimum temperature of 40°C even after 300 min of incubation. The presence of CaCl 2 shifts the optimum temperature to 45°C and produces a 1.3-fold increase in its activity. The enzyme remains active and stable in various detergents and is strongly activated by metal ions (Fe 3+ , Ca 2+ , Na + )