Sephadex G-100 Column Research Articles

Purification, Structural Characterization, and Biological Activity of Polysaccharides from Lentinus velutinus

A polysaccharide (LVP) was purified from fruiting body of Lentinus velutinus by ethanol precipitation fractionation and DEAE and Sephadex G-100 column chromatography. The yield of purified polysaccharide was 0.025%. Molecular characteristics of LVP were determined by gel permeation chromatography, FT-IR spectroscopy, and thin-layer chromatography. Our results revealed that LVP is a polysaccharide composed of only glucose units, and has a molecular weight of 336 kDa. Biological activity assays indicated that LVP exhibits both cytotoxic and antioxidant activity. LVP showed specific cytotoxicity against cancer cells (HeLa and HepG2 cells), and alterations in cancer cell morphology were found after LVP treatment.

Structure and anti-tumor activity of a polysaccharide from Bletilla ochracea Schltr.

The polysaccharide fraction of Bletilla ochracea (BOP) was isolated from its tubers, and purified by DEAE-52 Cellulose and Sephadex G-200 column chromatography. The structural features of BOP were analyzed by GC–MS, HPLP, FT-IR, methylation and NMR. The average molecular weight of BOP was approximately 4.9 × 105 Da, and the monosaccharide composition was 7.88:2.12 mix of mannose and glucose. The FT-IR and NMR analyses revealed the following linkage: [→(4-β-D-Man-1)4 → (4-β-D-Glc-1)2 → 4-2-O-acetyl-β-D-Man-1 → 4-β-D-Man-1 → 4-3-O-acetyl-β-D-Man-1 → 4-2-O-acetyl-β-D-Man-1→]. Although BOP had no effect on the proliferation rate of CT26 colon carcinoma cells, it significantly inhibited tumor xenograft growth in vivo by stimulating CD4+ T cell expansion in the spleen of the tumor-bearing mice. Taken together, BOP is a potent immunomodulatory agent that can be considered for anti-tumor therapy.

Characterization of a polysaccharide with antioxidant and anti-cervical cancer potentials from the corn silk cultivated in Jilin province

Corn silk polysaccharides (CSPs) were extracted from the corn silk cultivated in Jilin province, China, where is one of the golden corn belts worldwide. Three fractions (CSP-1, CSP-2 and CSP-3) were obtained by DEAE-52 cellulose and the former two fractions were further purified by Sephadex G-150 column chromatography to obtain CSP-S-1 and CSP-S-2. The molecular weights of CSP-S-1 and CSP-S-2 were calculated to be 586 kDa and 813 kDa, respectively. CSP-S-1 was composed of galactose, arabinose, xylose and rhamnose at a molar ratio of 4.16:1.00:1.01:6.32 and CSP-S-2 was composed of galactose, arabinose, glucose and rhamnose at a molar ratio of 8.71:3.58:0.169:1.00. CSP-S-2 outperformed CSP-S-1 in scavenging DPPH, ABTS and hydroxyl radicals, and significantly inhibited the proliferation of HeLa cells. IR and NMR analysis indicated that CSP-S-2 was pyranose. CSP-S-2 consisted of 1 → 4 and 1 → 6 linkages and exhibited a triple helix configuration. In summary, CSP-S-2 possesses high potential to be developed as a novel antioxidant and anti-cervical cancer agent.

Purifikasi Kolagenase dari Usus Bandeng (Channos channos, Forskal)

Collagenase was purified from intestines of milkfish (Chanos chanos, Forskal) by extraction,ammonium precipitation, ion exchange chromatography on DEAE Sephadex A-50 and gelfiltration on a Sephadex G-100 column. The purification and yields were 114.731 fold and1.26% when compared to those in the starting-crude extract. The molecular mass of milkfishserine collagenase was estimated to be 14.63 kDa and 27.46 kDa.

Purification, Characterization and De-Staining Potentials of a Thermotolerant Protease Produced by Fusarium oxysporum

Proteases are important industrial enzymes and fungi prove to be good sources of such enzymes. Purification techniques are however necessary for increased specificity in activity and better industrial value. Based on this, a protease produced by a Fusarium oxysporum was purified to homogeneity by Sephadex G-200 column and α–casein agarose chromatography. The enzyme had a molecular weight of 70 kDa in SDS-PAGE. Purified Fusarium oxysporum protease had a specific activity of 93.88 U/mg protein. The purification magnitude was 7.7 and the total yield was 20 %. Purified protease had an optimum pH of 5.0 while the optimum temperature was 40 °C. The enzyme was also thermotolerant (approximately 100 % at 40 °C for 2 h). The enzyme activity was stimulated by surfactants and metal ions like, Tween-20 and Mg2+. Enzyme activity was inhibited in presence of PMSF and EDTA. Casein was found to be the best substrate for protease activity of Fusarium oxysporum FWT1. Protease were tested upon blood stain for de-clotting of blood and was found to exhibit good de-clotting and de-staining activity after 15 minutes treatment time.

Isolation and Biochemical Characterization of Extracellular Proteolityc Enzym From Lactic Acid Bacteria Isolated From Various Indonesian Traditional Fermented Product (Bakasam)

Local isolates of lactic acid bacteria isolated from Bakasam had potential to producing extracellular proteases. The research was carried out with the purpose of knowing the biochemical character of protease produced from isolates of lactic acid bacteria. The results showed extracellular protease isolates BAL 2 (Lactobacillus acidophillus) has a specific activity i.e. of 0.89 U/mg. Biochemical characterization of proteases indicates optimum pH 5.5, and is inhibited by chelating agent EDTA. The result of the purification with Sephadex G-100 column showed a band on SDS-PAGE with the size of molecular weight approximately 90 kD.

Purification of levanase from Enterobacter aerogenes and using it as a prebiotic and antibacterial agent.

A novel strain of Enterobacter aerogenes isolated from rhizosphere soil has an ability to produce intracellular levanase when grown on basal medium containing the levan as substrate. Levanase was purified to homogeneity with a recovery yield of 57.85% and 22.5 fold of purification by using ammonium sulfate at 40% saturation followed by ion exchange chromatography on Sepharose CL-6B column and gel filtration chromatography on Sephadex G-100 column. The purified levanase has a potential prebiotic property of enhancing growth of lactic acid bacteria while apparently inhibiting growth of pathogenic bacteria. Typically, prebiotic properties should not only reduce growth of pathogens but must also promote growth of desirable and beneficial bacteria, such as Lactobacillus. Thus the levanase may be used in animal feed to improve digestibility and support gastrointestinal health.

A novel polysaccharide from Malus halliana Koehne with coagulant activity

A novel polysaccharide in Malus halliana Koehne, named MHP-W, was isolated and purified by DEAE-52 cellulose and Sephadex G-100 columns. Structural features were identified by high performance size-exclusion chromatography (HPSEC), fourier transform infrared (FT-IR) spectrometer, gas chromatography (GC) and (1D & 2D) NMR Spectroscopy. Structural characterization showed that the molecular weight of MHP-W was 353 kDa composed of arabinose, xylose, mannose, glucose and galactose in a molar ratio of 2.59: 0.15: 0.23: 0.25: 9.70. The existence of β-glycosidic bond between the sugar units was confirmed by FT-IR and NMR spectroscopy. The effects of MHP-W on active part thrombin time (APTT), protothrombin time (PT), thrombin time (TT), and fibrinogen (FIB) were screened by a cell-based coagulation activity model. MHP-W could significantly shorten TT (p < 0.001) and increase FIB (p < 0.05) as compared with the control group. The results showed that MHP-W promoted bloodclotting through endogenous and exogenous coagulation pathways as well as increasing fibrinogen content, which indicated that MHP-W had procoagulant activities in vitro.

Synthesis and characterization of natural lanthanum labelled DOTA-Peptides for simulating radioactive Ac-225 labeling

This research aimed to assess the labeling procedure of Lanthanum to DOTATATE and PSMA-617 peptide for simulating Actinium-225 (225Ac) labeling. For this purpose labeling procedure, purification and characterization conditions were optimized. Results showed that for the high reaction yield labeling, microwave heating should be preferred and Sephadex G10 column was found to be more suitable as a purification system compared to Toyopearl column. For the characterization of complex, more precise and reliable results were obtained by the chromatographic system.

Purification and characterization of pedioxanthin (carotenoid pigment) produced by Pediococcus pentosaceus N33 strain isolated from pickles

ABSTRACTForty-two lactic acid bacteria were isolated from 28 pickles and yoghurt samples, and screened for their antimicrobial efficacy against certain multidrug resistant (MDR) bacteria using well diffusion method. A strain Pediococcus pentosaceus N33 (MF062087) showed the best inhibitory effects against both Brevibacillus brevis H18 (MG637284) and Lysinibacillus fusiformis R29 (MG637285). Pediococcus pentosaceus N33 strain produced high thermostable antimicrobial compound as indicated by heat treatment between 60 and 100°C for 20 min as well as at 121°C for 15 min. It is a proteinaceous compound highly active at acidic pH (2–6). The antimicrobial compound was extracted and precipitated by 70% ammonium sulfate and finally fractionated using Sephadex G50 column. The molecular weight of pure compound was 10 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The spectroscopic analysis based on UV, IR, NMR and MS analyses were used to characterize and identify the pure antimicrobial compound. From the characterization and identification of this inhibitory compound, it was observed that the target compound contains carotenoid pigment in addition to protein. This carotenoid pigment was identified as pedioxanthin which is the first observed from this strain. Our results evaluated the potential activity of Pediococcus pentosaceus N33 strain as a probiotic agent and its potential contribution for food industry and pharmaceutical applications.

Purification and characterization of recombinant Streptokinase expressed in E.coli from Streptococcus equisimilis with N-terminal methionine

Streptokinase is a extracellular enzyme which is extracted from strains of beta Hemolytic streptococcus. The enzyme is a non-protease plasminogen activator that activates plasminogen to plasmin and degrades fibrin clot through its specific lysine binding site which is used in thrombolytic therapy. Purification of streptokinase produced from S.equisimilis in E.coli with N-terminal methionine was carried out in 3 Chromatography purification steps, 1) CM-Sepharose-FF at pH 4.2 followed by concentration and dialysis over night with Tris-HCl pH 8.0. Partially purified dialyzed enzyme sample was loaded on to 2) DEAE-Sepharose-FF column. The Purified fractions of DEAE column were pooled and applied on to Sephadex G-100 column. Enzyme purity was confirmed by SDS-PAGE and RP-HPLC.Its biological activity is determined by specific streptokinase assay and characterised the enzyme by Peptide mapping, MALDI-TOF, Isoelectric-focusing and RP-HPLC. The isoelectric point (pI) of streptokinase is around 4.98.The results of characterization shows that it contains two forms (Isomers) of streptokinase expressed in E. coli which was analyzed by RP-HPLC and chromogenic assay. The variation is formed by isomer-1 in which 85% of Streptokinase expressed without methionine (85000IU/mg) and Isomer-2 in which 15% of streptokinase expressed with methionine (nil activity) in E. coli. This phenomenon shows that the presence and absence of methionine in isomers of streptokinase varying the catalytic activity of the enzyme.

Structural characterization and anti-inflammatory activity of alkali-soluble polysaccharides from purple sweet potato.

In this study, the structural characterization and anti-inflammation effect of dilute alkali-soluble polysaccharides from purple sweet potato were investigated. Three fractions (F-1, F-2 and F-3) were obtained by purifying crude polysaccharides on DEAE-52 cellulose column. The main fraction (F-1) was further purified on Sephadex G-200 column to afford purified alkali-soluble sweet potato polysaccharide (ASPP). The chemical structure of ASPP was analyzed by gas chromatography, Fourier transform infrared spectroscopy, methylation analysis and nuclear magnetic resonance spectroscopy. Monosaccharide compositional analysis showed ASPP was composed of rhamnose, arabinose, xylose, mannose and glucose in the molar ratio of 2.8:1.9:1.0:7.6:53.3. Moreover, the backbone of ASPP was composed of 1,4-linked Glcp with side chains attached to the O-6 position. The anti-inflammation effect of ASPP was further investigated by in vitro and in vivo experiments. Results showed ASPP could inhibit the levels of nitric oxide, interleukin (IL)-6, IL-1β and TNF-α but increase the production of IL-10 in lipopolysaccharide (LPS)-treated RAW 264.7 macrophage cells. In addition ASPP could reduce the secretion of IL-6, IL-1β and TNF-α in LPS-treated mice. Our results suggest ASPP can be developed as a novel anti-inflammation agent.

Hypoglycemic activity and gut microbiota regulation of a novel polysaccharide from Grifola frondosa in type 2 diabetic mice

GFP-N, a novel heteropolysaccharide with a molecular weight of 1.26 × 107 Da, was isolated from maitake mushroom and purified by anion-exchange chromatography on a DEAE cellulose-52 column and gel-filtration chromatography on a Sephadex G-100 column. Its structure was characterized by Fourier transform infrared spectroscopy and one-dimensional (1H- and 13C-) NMR spectra, 1H1H correlation spectroscopy, and 1H13C heteronuclear single-quantum coherence spectroscopy. The structure of GFP-N consisted of L-arabinose, D-mannose and D-glucose and mainly contained three kinds of linkage type units as →2,6)-α-D-Manp-(1 → 4, α-L-Araf-C1→, and →3,6)-β-D-Glcp-(1 → . GFP-N could activate insulin receptor substrate 1, phosphatidylinositol-3-kinase, and glucose transporter 4 and inhibit c-Jun N-terminal kinase 1/2 for hypoglycemic effects in diabetic mouse livers. This is also the first report of the regulatory efficacy of Grifola frondosa polysaccharide on intestinal microflora in vivo using single-molecule real-time sequencing. These results indicated that polysaccharide from maitake mushroom could be as an enhancer to improve type 2 diabetes and a healthy food option to help regulate gut microbiota in diabetic individuals.

Polyextremotolerant Amylase Produced from Novel Enterococcus with Potpourri of Applications

Amylolytic toluene-tolerant Enterococcus faecalis mercadA7 isolated from asbestos contaminated black soil from the Tokat province of Turkey was used for statistically optimized liquid phase fermentation to spawn elevated levels of amylase. Box Behnken technique of Response surface Methodology is followed to optimize three significant factors influencing Amylase production. To purify the amylase, ammonium sulfate fractionation, dialysis and gel filtration on Sephadex G-100 column chromatography were used. Submerged state fermentation at statistically optimized conditions augmented amylase production levels (8467 U ml−1) 8.74-fold compared to the conventional optimization. Purified amylase was found to possess specific activity of 1022 U mg−1, optimal activity at pH 9 and 60 °C, 80% stable at 8.0–10.0 pH range and 90% stable at 45–75 °C for 1 h; with Km value of 8.06 mg ml−1 and a Vmax of 4.36 mmol h−1 ml−1, noteworthy stability up to 43% toluene, and upto 8% NaCl. High titers of Amylase produced from Enterococcus faecalis mercadA7 might find applications in copious industrial processes due to noteworthy stability of the cell-free purified enzyme at elevated temperatures, alkaline, and high toluene or salt concentrated environments.

Purification, characterization and in vitro and in vivo immune enhancement of polysaccharides from mulberry leaves.

Mulberry leaf polysaccharide (MLP) was extracted and purified by DEAE-52 cellulose and Sephadex G-100 column chromatography to afford two major purified polysaccharides (MLP-1 and MLP-2). The purified polysaccharides were characterized, and their immune-enhancing properties were investigated. MLP-1 had a molecular weight of 9.31×104 Da and was composed of mannose, rhamnose, glucose, galactose, xylose, and arabinose in a molar ratio of 0.71:1.00:2.76:1.13:3.70:2.81. The molecular weight of MLP-2 was 2.22×106 Da, and its monosaccharide constituents were mannose, rhamnose, glucose, galactose, and arabinose in a molar ratio of 1.31:8.45:6.94:1.00:11.96. Infrared spectroscopy showed that each MLP had a typical absorption peak characteristic of sugars, and ultraviolet (UV) spectroscopy showed that neither MLP contained nucleic acid or protein components. Then, the abilities of these polysaccharides to stimulate spleen lymphocyte proliferation in mice in vitro were compared by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. MLP-2 was more effective than MLP-1; therefore, MLP-2 was chosen for the study of its immune-enhancing effects in vivo. For the in vivo experiments, 14-day-old chickens immunized with Newcastle disease (ND) vaccine were orally administered MLP-2, and Astragalus polysaccharide (APS) was used as the control. Each chicken was orally administered 4 mg or 8 mg of MLP-2 for seven consecutive days starting three days before ND vaccine immunization. MLP-2 significantly improved the ND serum antibody titer and interleukin-2 (IL-2), interferon-γ (IFN-γ) and immunoglobulin A (sIgA) concentrations in tracheal and jejunal wash fluids, and increasing numbers of immune globulin A-positive (IgA+) cells in cecal tonsils and increased body weight. These results indicated that MLP-2 could significantly enhance immune activity and could therefore be utilized as an immunopotentiator drug candidate.

Purification and characterization of alkaline protease produced by Streptomyces flavogriseus and its application as a biocontrol agent for plant pathogens

Background A protease is an enzyme that helps proteolysis: protein catabolism by hydrolysis of peptide bonds. Proteases were the first enzymes to be commercialized, partly on the account of the history of availability and on account of need. Aim The aim of the present work is studying the properties of the partially purified enzyme and some application of the enzyme. Materials and methods In this study, alkaline protease was produced by Streptomyces flavogriseus using the shaking method. The enzyme was partially purified by ammonium sulfate precipitation. The most active fraction was evaluated on Sephadex G-100 column chromatography. The activity of the enzyme was determined at different temperatures and different pH values using various buffers. Results and conclusion In this study, alkaline protease produced by S. flavogriseus was partially purified by ammonium sulfate precipitation at 75% w/v saturation and pooled on Sephadex G-100 with a yield of 60.85% and specific activity of 85.14 U/mg protein. The enzyme was optimally active at 45°C in 0.5 mol/l potassium phosphate buffer (pH 8.0) after 30 min of incubation and was broadly stable at 45°C for 60 min and pH 6–8. The alkaline protease relative activity was increased in the presence Mn2+, Co2+, K+, Na+, Fe2+, and Mg2+ at concentration 1 mmol/l, respectively.

Ultrasonic-assisted enzymatic extraction and characterization of polysaccharides from dandelion (Taraxacum officinale) leaves

Ultrasonic-assisted enzymatic extraction (UAEE) technology was employed to extract the polysaccharides from dandelion leaves (DLP). The extraction conditions were optimized by response surface method (RSM) and Box-Behnken design (BBD). The polysaccharide yield (14.05 ± 0.95%), which well matched with the predicted value of 14.27%, was obtained under the optimum parameters: ultrasound power 500 W, ultrasound time 42 min, cellulase amount 4.90%, and particle size 80 mesh. After purification by AB-8 Macroporous Resin and Sephadex G-100 column, a novel polysaccharide (DLP-I) was isolated and its molecular weight was estimated to be 87,000 g/mol by high performance liquid chromatography (HPLC). Gas chromatography (GC) analysis showed that DLP-I was composed of rhamnose, arabinose, mannose, galactose and glucose in the molar ratio of 1.14:1.00:1.05:4.76:1.52. 1D and 2D NMR spectra confirmed that the backbone of DLP-I was mainly consisted of →4)-α-D-Galp-(1→, →4)-β-D-Manp-(1→, →4)-α-D-Glcp-(1→, →2,4)-α-L-Rhap-(1→, and α-L-Araf-(1→ with branching at O-2 and O-4 of →2,4)-α-L-Rhap-(1→. In addition, the digestion results showed DLP-I could keep its stability during the simulated digestive juices.

Physicochemical properties of polysaccharides separated from Camellia oleifera Abel seed cake and its hypoglycemic activity on streptozotocin-induced diabetic mice

Two homogeneous polysaccharide fractions named SCP-1 (7.16 × 106 Da) and SCP-2 (2.00 × 104 Da) were purified by DEAE-52 cellulose and Sephadex G-200 column chromatography successively from Camellia oleifera Abel seed cake. They were characterized by Fourier transformed infrared (FT-IR), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) spectroscopy and differential scanning calorimetry (DSC). The monosaccharide compositions of SCP-1 were d‑mannose, d‑glucose and l‑xylose with a molar ratio of 1.77:0.93:1 and that of the SCP-2 were d‑mannose, l‑rhamnose, d‑glucose and l‑xylose with a molar ratio of 5.27:1.21:0.16:1. Animal experiments suggested that the plasma glucose levels in hyperglycemia mice were reduced by 11.34%, 30.70%, 46.83% after administration of high, medium and low doses of SCP-1, and reduced by 16.67%, 23.93% and 33.00% after administration of high, medium and low doses of SCP-2, respectively. SCP-1 and SCP-2 also increased the activities of glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD), as well as decreased the content of malondialdehyde (MDA) in the hyperglycemia mice. These results suggested that SCP-1 and SCP-2 possessed strong hypoglycemic activities in streptozotocin-induced model mice. In addition, the hypoglycemic activity of SCP-1 was stronger than that of SCP-2.

1H NMR-based metabonomics of the hypoglycemic effect of polysaccharides from Cordyceps militaris on streptozotocin-induced diabetes in mice

The crude polysaccharide was extracted from Cordyceps militaris. Material ratio of powder and water was 1:10. The polysaccharide was successively purified by Sevag and chromatography on Sephadex G-100 column to produce a polysaccharide fraction termed CBPS-II. The average molecular weight of CBPS-II was 1.273 × 103 kDa. The study was conducted to investigate the hypoglycemic effect of Cordyceps militaris polysaccharide on diabetic mice. Analysis of the clinical chemistry of the serum samples included serum creatinine (CRE), urea nitrogen (BUN), triglyceride (TG) and total cholesterol (TC). Results revealed that a certain dose of polysaccharide can alleviate the symptoms of metabolic disorders of diabetes, contributing to the body to restore the normal levels. The metabolic profiling method was adopted to find the related biomarkers and the metabolic pathway of diabetes. Moreover, results showed that 100 mg·kg−1 of Cordyceps polysaccharides can effectively reduce the blood glucose level of diabetic mice, thus regulating the metabolism of their energy, amino acids and intestinal microbes. The biomarkers noted in their metabolism were glucose, lactic acid, 3-hydroxy butyric acid, creatine, glutamate, valine, leucine, isoleucine and very low density lipoprotein (VLDL).

Relationship between structural properties and antitumor activity of Astragalus polysaccharides extracted with different temperatures

This study investigated the effects of different temperatures on structural characterization and antitumor activity of polysaccharides from Astragalus membranaceus. APS4 and APS90 were extracted at 4°C and 90°C, respectively, and purified by Sephadex G-200 column. APS4-90 were obtained from APS4 after treatment at 90°C for 6h. MTT results showed that APS4 possessed the highest inhibitory effects on MGC-803, A549 and HepG2 cells. HPGPC analysis showed that the average molecular weights of these polysaccharides were approximately 1.5×106Da, while the asymmetrical peak of APS4-90 suggested heat degradation and configuration changes of APS4. GC, NMR and methylation results showed that these three polysaccharides had similar monosaccharide components (mainly contain glucose), and their backbones were composed of (1→2)‑α‑d‑Glcp. However, APS4 showed higher content of (1→2,6)‑α‑d‑Glcp compared to APS4-90 and APS90, which indicated that higher branched degree would be responsible for the stronger in vitro antitumor activity in APS4. These results were also confirmed by specific rotation and SEM analysis. Our study suggested that APS4 had the potential application for cancer treatment.