Sephadex G-100 Chromatography Research Articles

Optimization for ultrasonic-microwave synergistic extraction of polysaccharides from Cornus officinalis and characterization of polysaccharides

Ultrasonic-microwave synergistic extraction (UMSE) of polysaccharides from Cornus officinalis was optimized by response surface methodology (RSM). The effect of four different factors on the yield of C. officinalis polysaccharides (COP) was studied. RSM results showed that the optimal conditions were extraction time of 31.49823min, microwave power of 99.39769W, and water-to-raw material ratio of 28.16273. The COP yield was 11.38±0.31% using the modified optimal conditions, which was consistent with the value predicted by the model. The crude COP was purified by DEAE-Cellulose 52 chromatography and Sephadex G-100 chromatography. Five fractions, namely, crude COP, COP-1, COP-2, COP-3, and COP-4, were obtained. Monosaccharide composition analysis revealed that the COP was composed of glucose, arabinose, fucose, xylose, mannose, and rhamnose. Preliminary structural characterizations of COP were conducted by scanning electron microscopy and Fourier transform infrared spectroscopy.

PURIFICATION AND CHARACTERIZATION OF HIGH POTENTIAL TYROSINASE FROM MACROFUNGI AND ITS APPLIANCE IN FOOD ENGINEERING

Tyrosinase (EC1.14.18.1) was extracted from oyster mushroom, Pleurotus ostreatus, using 100 mM potassium phosphate buffer (pH 5.8) containing 1 mM of ethylenediaminetetra acetic acid. The enzyme was purified by ammonium sulfate precipitation, followed by Sephadex G-100 and diethylaminoethyl chromatography. The purified enzyme showed a specific activity of 46.4 U/mg with 20.3 % yield. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed single peptide chain with a molecular weight of 75 kDa. The enzyme has optimum activity on pH 6.0 at 35˚ C. The kinetics parameter with L-DOPA (3,4-dihydroxy-L-phenylalanine), with a KM of 0.119 mM and Vmax of 2.97 mM. Thus, purified tyrosinase from P.ostreatus showed similarities with other tyrosinase sources. The results indicate that P.ostreatus can be a novel and better source of tyrosinase extraction due to its higher specific activity. The information offered here should help food industry in developing and using potential tyrosinase desirable efficacy and safety, and for improving food quality.

Isolation, purification, and antioxidant activities of degraded polysaccharides from Enteromorpha prolifera

In the present study, purification and characterization of enzymatic hydrolysates of polysaccharide from Enteromorpha prolifera (HPE) are described. HPE was sequentially purified by DEAE Cellulose-52 chromatography and Sephadex G-100 chromatography to afford three fractions, namely, PHPE1, PHPE2, and PHPE3. Molecular weights of these three fractions were measured to be 103, 45.4, and 9.8kDa, respectively, using high performance gel permeation chromatography (HPGPC). The three fractions were evaluated for their antioxidant activities by determining their ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and superoxide anion radicals. PHPE2 was found to possess the strongest scavenging ability. GC–MS analysis indicates that PHPE2 is mainly composed of mannose, xylose, and glucose.

Characterization of a Solvent-Tolerant Manganese Peroxidase (MnP) from G anoderma Lucidum and Its Application in Fruit Juice Clarification

Manganese peroxidase (MnP) is necessary in fruit juice clarification for the degradation of phenolic compounds. In the present study, MnP was produced using basal medium supplemented with tamarind shell (1% w/v), ethanol (2% v/v) and gallic acid (1 mM) to a maximum activity of 65 U/mL. A 43.0 kDa MnP was purified by ammonium sulfate fractionation, Sephadex G-100 and DEAE-cellulose column chromatography with 6.5-fold and 13% yield. The optimal activity of the purified MnP was achieved at pH 5.5 and temperature of 45C with 2,6-dimethoxyphenol as substrate. The inhibitors sodium azide, cyanide and EDTA inhibited the MnP activity up to 100, 93 and 84%, respectively. MnP showed high activity and stability in the presence of different metal ions, surfactants and organic solvents. Clarification of different fruit juices with MnP showed its potential application in the food and beverage industry. Practical Applications Manganese peroxidase (MnP) is extensively used in various applications, such as pulp delignification, dye decoloration, biotransformation, biosensor applications and fruit juice clarification. Ganoderma lucidum-derived purified MnP is a solvent-tolerant metalloenzyme that showed its potential for industrial application in clarification of various fruit juices. The stability of MnP in the presence of various divalent cations, surfactants and solvents suggests that it could be used in industrial effluent treatments as well as the food and beverage industry.

Purification, preliminary characterization and bioactivities of polysaccharides from Ostrea rivularis Gould

In this study, purification, preliminary characterization and biological activities of water-soluble polysaccharides from Ostrea rivularis Gould (ORP) were investigated. Firstly, crude ORP was extracted by enzyme-assisted extraction and then sequentially purified by chromatography of DEAE-52 and Sephadex G-100, producing one main purified fractions of ORPp. Furthermore, the preliminary characterization of ORPp was studied, and its antioxidant and spermatogenesis activities were evaluated. Experimental results showed that ORPp was mainly composed of glucose (76.3%) and galactose (23.7%). The average molecular weight of ORPp was 118kDa. Besides, ORPp showed strong antioxidant activities in vitro. For the experiments of antioxidant activities in vivo, ORPp can significantly inhibited the formation of MDA in rats’ serums, and raised the activities of antioxidant enzymes and the level of total antioxidant capacity (TAOC). Furthermore, ORPp could significantly increase the weights of male rats’ sexual organs, promote sperm motility and raise epididymal sperm counts. These results suggest that ORPp could be a new source of natural antioxidants and spermatogenic agent with its potential usage in developing novel supplements and medicines.

Purification and characterization of a glucan from Bacillus Calmette Guerin and the antitumor activity of its sulfated derivative

A water-soluble glucan, BCG-PASW, with a molecular weight of 2.10×104Da, was separated from polysaccharide nucleic acid fraction of Bacillus Calmette Guerin (BCG-PSN) using DEAE-52 cellulose and Sephadex G-200 chromatography. Based on gas chromatography–mass spectrometry (GC–MS), fourier transform infrared (FT-IR) spectra, 1D and 2D nuclear magnetic resonance (NMR) spectroscopy techniques (COSY, HSQC and HMBC), BCG-PASW was found to be an α-d-glucan composed of α-d-(1→4)-linked glucopyranosyl residues, with branches at O-6 consisting of non-reducing terminal α-d-Glcp approximately every eight residues. In vitro antitumor activity by MTS method, its sulfated derivative with a substitution degree of 0.59, could inhibite C666-1 nasopharyngeal carcinoma cells growth significantly. The results indicated that the sulfate content play a decisive role in the bioactivities of the polysaccharides.

Characterization of a novel endoglucanase from Ganoderma lucidum.

We evaluated the production and characterization of endoglucanase from Ganoderma lucidum using different lignocellulose biomasses. We purified a novel carboxymethyl cellulose (CMC) hydrolyzing endoglucanase from the white-rot fungus G. lucidum when the medium was supplemented with 1% (w/v) wheat bran. Endoglucanase was purified 12.5-fold via ammonium sulfate fractionation, Sephadex G-100, and Q-Sepharose column chromatography with a final yield of 15%. SDS-PAGE analysis revealed that the endoglucanase had a molecular mass of 64.0 kDa. The optimal activity of purified endoglucanase was at pH 5.0 and 35 °C, though it was stable between pH 4.0-7.0 and temperatures of 30-60 °C. The purified enzyme was specific to CMC as a suitable substrate. The metal ions Hg(2+), Fe(2+), and Cr(2+) inhibited enzyme activity, while Ca(2+), Mg(2+), and Mn(2+) enhanced enzyme activity. The endoglucanase showed high activity and stability in the presence of different surfactants and non-polar hydrophobic organic solvents. This endoglucanase is tolerant to high temperature, metal ions, surfactants, and solvents, suggesting that it is appropriate for use in biomass conversion for biofuel production under harsh environmental conditions.

Kinetics Study of Extracellular Detergent Stable Alkaline Protease from Rhizopus oryzae

ABSTRACT In this study, extracellular alkaline protease was produced from Rhizopus oryzae in submerged fermentation using dairy waste (whey) as a substrate. Fermentation kinetics was studied and various parameters were optimized. The strain produced maximum protease at initial medium pH of 6.0 medium depth of 26 mm, inoculum size of 2% at incubation temperature of 35 o C for 168 h of fermentation. Alkaline protease was purified to homogeneity by ammonium sulphate fractionation followed by sephadex G-100 chromatography. The molecular mass of alkaline protease was 69 kDa determined by 10% SDS-PAGE. The optimum pH and temperature of alkaline protease was 9.0 and 40 o C, respectively. Metal profile of the enzyme showed that the enzyme was non-metallic in nature. The K m , K cat , V max and K cat /K m values of purified protease were 7.0 mg/mL, 3.8 x10 2 S -1 , 54.30 µmol/min and 54.28 s -1 mg -1 .mL respectively, using casein as substrate. The purified alkaline protease had stability with commercial detergents.

Chemical constituents and structural characterization of polysaccharides from four typical bamboo species leaves.

In order to find bamboo leaves with high contents of bioactive polysaccharides, 32 samples were chosen to analyze their polysaccharide content by GC and sulfuric acid-anthrone colorimetric assays. Purified polysaccharides (BLPS) were separated from the four varieties P. nigra (Lodd.) Munro (PN), P. vivax McClure (PV), Chimonobambusa quadrangularis (Fenzi) Makino (CQ), and P. bambussoides cv. Tanakae (PB) by ultrasound extraction, solution precipitation, ion exchange resin, DEAE-52 and Sephadex G-100 chromatography. BLPS structural characterization was accomplished by HPLC-GPC, Fourier transform infra-red spectroscopy (FTIR) and NaIO4-HIO4 oxidation reactions. The results showed that the total polysaccharides of the bamboo leaves in samples 1–32 ranged between 1.4% and 5.4%, Samples No. 29–No. 32 (PN, PV, CQ, and PB) contained 2–3 fold more polysaccharides than No. 1~No. 28 among the 32 different species, particularly the content of galactose was in a range of 21.5%–34.1% for these four typical bamboo species leaves, which was also more than 2–3 fold higher than in No. 1–No. 28. Sugar analysis indicated that PN-PBLPS-1, PV-PBLPS-1, CQ-PBLPS-1 and PB-PBLPS-1 from the four varieties were homogeneous polysaccharides with molecular weights of 2.04 × 104, 1.15 × 104, 8.75 × 104 and 1.48 × 104 Da, respectively. PB-PBLPS-1 was a mixture of α-galactopyranose and β-d-glucopyranose linkages with α-(1→6) or β-(1→6)glycosidic bonds, while PN-PBLPS-1, PV-PBLPS-1, and CQ-PBLPS-1 had α galactopyranose linkages with α-(1→6) glycosidic bonds.

Complex Enzyme-Assisted Extraction, Purification, and Antioxidant Activity of Polysaccharides from the Button Mushroom, Agaricus bisporus (Higher Basidiomycetes).

Agaricus bisporus polysaccharides (ABP) were extracted by complex enzyme-assisted extraction methodology. The following were optimal conditions for the extraction of crude ABP: complex enzyme amount, 2.2%; temperature, 62°C; time, 3 h; and pH, 4. Under these conditions, the experimental yield of crude ABP was 6.87%. The crude ABP was purified by diethylaminoethyl-cellulose 52 chromatography and Sephadex G-100 chromatography, and one fraction-namely, ABP-1-was produced. The ABP-1 contained 93.67% carbohydrate, 1.46% protein, and 0.62% uronic acid. The constituent monosaccharides were predominantly glucose, galactose, mannose, and xylose. The antioxidant activities of ABP-1 were investigated by measuring its scavenging ability on 2,2-diphenyl-1-picrylhydrazyl and hydroxyl radicals, its ferric-reducing activity power, and the reducing power assay. At a concentration of 1.2 mg/mL, ABP-1 seemed to possess good free radical scavenging activity, with a scavenging value of about 56%. The results indicate that ABP-1 has good antioxidant activity.

Partial Purification and Characterization of A Novel Histidine Decarboxylase from Enterobacter aerogenes DL-1

Histidine decarboxylase (HDC) from Enterobacter aerogenes DL-1 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 52.4 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for HDC activity was 6.5, and the enzyme was stable between pH 4 and 8. Enterobacter aerogenes HDC had optimal activity at 40°C and retained most of its activity between 4 and 50°C. HDC activity was reduced in the presence of numerous tested compounds. Particularly with SDS, it significantly (p < 0.01) inhibited enzyme activity. Conversely, Ca2+ and Mn2+ showed prominent activation effects (p < 0.01) with activity increasing to 117.20% and 123.42%, respectively. The Lineweaver–Burk plot showed that K m and V max values of the enzyme for L-histidine were 0.21 mM and 71.39 µmol/min, respectively. In comparison with most HDCs from other microorganisms and animals, HDC from E. aerogenes DL-1 displayed higher affinity and greater reaction velocity toward L-histidine.

Production and Partial Characterization of Extracellular Protease from Bacillus sp. GJP2 Isolated from a Hot Spring

In the present investigation extracellular protease production is reported from a moderately thermotolerant bacterial strain GJP2, isolated from a hot spring. The enzyme production conditions were optimized by determining maltose as the best carbon source and a combination of yeast extract and peptone as the most suitable nitrogen sources at pH 8.5 and temperature of 50 °C. A total of 31.7 fold enzyme purification was achieved by precipitating the culture supernatant with 70 % (NH4)2SO4 followed by Sephadex G-100 and ion exchange chromatography. Electrophoresis analysis of the purified enzyme revealed its molecular weight approximately as 64 kDa. The purified enzyme showed maximum activity at pH and temperature of 8.5 and 50 °C, respectively. Based on the partial 16S rRNA sequencing and biochemical properties the bacterium was identified as a Bacillus sp.

Extraction optimization, preliminary characterization and immunological activity of polysaccharides from figs

Complex enzyme extraction (CEE), purification, characterization of fig polysaccharides (FPs) from dried figs were investigated. Orthogonal experiment was used to optimize the concentration of cellulose, pectinase and papain. Response surface methodology (RSM) was employed to optimize extraction conditions. The optimum extraction conditions were: enzyme concentration of 1.5%, 1.5%, 0.5% (wt%) of pectinase, papain, cellulose, ratio of water to raw material 40.3mL/g, extraction time 54.1min, temperature 34.15°C and pH 3.8. Under these conditions, the experimental yield was 7.98±0.17%. Two homogeneous heteropolysaccharides (FPs-1-1, FPs-2-1) were purified by DEAE-Sepharose and Sephadex G-200 chromatography, which were composed of rhamnose, arabinose, xylose, mannose, glucose and galactose with molecular weight of 1.52×106 and 4.75×105Da, respectively. The bioactivity assay showed that FPs-1-1 and FPs-2-1 could more significantly enhance splenic lymphocyte proliferation, phagocytosis and NO production of macrophages, could be explored as potential immunopotentiating agent for use in functional food or medicine.

I - insulin transfer to mitochondria.

The aim of this study was to determine if insulin is transferred to mitoplasts by insulin-degrading enzyme (IDE).Hepatic mitochondria were isolated and controlled by electron microscopy. IDE was obtained from rats muscle by successive chromatography steps. Insulin accumulation in mitoplasts and outer membrane + intermembrane space (OM + IMS) was studied with (125)I-insulin. Mitochondrial insulin accumulation and degradation was assayed with Sephadex G50 chromatography, insulin antibody and 5 % TCA. Mitoplasts and OM + IMS were isolated with digitonin. Insulin accumulation was studied at 25 °C at different times, without or with IDE, Bacitracin, 2,4-dinitrophenol, apyrase or sodium succinate + adenosine diphosphate. Insulin accumulation in mitoplasts and OM + IMS after mitochondrial cross-linking was studied with electrophoresis in SDS-PAGE, immunoblots of IDE, insulin or TIM23 (inner mitochondrial transporter) and autoradiography.The studies showed that addition of IDE increased insulin transfer from OM + IMS to mitoplasts, and the insulin accumulation in mitoplast was IDE dependent. Bacitracin and 2,4-dinitrophenol decreased this transfer. The [Insulin-IDE] complex and [Mitoplasts] was studied as a bimolecular reaction following a second order reaction. The constant "k" (liter.mol⁻¹ s⁻¹) showed that IDE increased and Bacitracin or 2,4-dinitrophenol decreased the velocity of insulin transfer. SDS-PAGE and immunoblots studies showed bands and radioactivity coincident with IDE, insulin and TIM23. Non degraded insulin was demonstrated in immunoblot after IDE immunoprecipitation from mitoplasts. Confocal studies showed mitochondrial colocalization of IDE and insulin.The results showed that insulin at 25 °C were transferred from OM + IMS to mitoplasts by IDE or that the enzyme facilitates this transfer, and they reach the matrix together.

English

Ascorbate peroxidase (EC 1.11.1.11; APX) was purified from ripe ber ( Ziziphus mauritiana L.) fruits var. Illaichi using conventional techniques of ammonium sulphate fractionation, gel filtration through Sephadex G-100 and ion-exchange chromatography on DEAE-cellulose. The enzyme was purified about 47.4 fold with 34.6% recovery. The molecular weight as determined by gel filtration was found to be 58.08 kDa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) yielded a single major protein band with molecular weight of 29.79 kDa indicating that the enzyme was a homodimer. Native PAGE revealed a single prominent band suggesting that enzyme was purified to near homogeneity. The optimum pH for APX was found to be 7.8. It exhibited the Michaelis-Menten kinetics with Km values for ascorbate and H 2 O 2 of 1.82 and 2.85 mM, respectively. Mn 2+ , NO 3 - , SO 4 2- and Co 2+ were found to be potent inhibitors of APX while K + , Na + , Ca 2+ and Cl - stimulated the enzyme activity. Diethylpyrocarbonate (DEPC), dithiothreitol (DTT), NaBH 4 and mercaptoethanol inhibited the enzyme activity while iodoacetate and 5, 5’-dithiobis-2-nitrobenzene (DTNB) had no inhibitory effect. Based on the inhibition studies, histidine and tryptophan have been suggested to be present at the active site. Keywords. Fruit ripening, Ziziphus mauritiana, ascorbate peroxidase, purification African Journal of Biotechnology , Vol 13(31) 3323-3331

Purification, characterisation and protective effects of polysaccharides from alfalfa on hepatocytes

The objective of this study was to determine the preliminary characteristics and protective effects of alfalfa polysaccharides (APS) on hepatocytes in vitro. The crude APS was purified by DEAE-cellulose and Sephadex G-100 chromatography, resulting in the four purified fractions: APS-1, APS-2, APS-3 and APS-4. The results indicated that APS-3 had higher carbohydrate and uronic acid contents and that APS-4 had a more complicated monosaccharide composition compared to the other purified fractions. The average molecular weights of APS-1, APS-2, APS-3 and APS-4 were 48,536, 6,221, 66,559 and 13,076Da, respectively. Furthermore, APS (crude and its purified fractions) restored the activities of antioxidant enzymes and increased the total antioxidant capacity of hepatocytes subjected to H2O2-induced oxidative stress. Furthermore, APS treatment counteracted the increases in lactic dehydrogenase and malonaldehyde in the culture supernatant. These results clearly demonstrate that APS possesses a protective effect against oxidative injury in hepatocytes.

Polysaccharides from Inonotus obliquus sclerotia and cultured mycelia stimulate cytokine production of human peripheral blood mononuclear cells in vitro and their chemical characterization.

Inonotus obliquus is an edible and medicinal mushroom to treat many diseases. In the present study, polysaccharides and fractions were isolated and purified by DEAE-52 and Sephadex G-200 chromatography from I. obliquus wild sclerotia, culture broth and cultured mycelia under submerged fermentation. The extracts and fractions could significantly induce the secretion of TNF-α, IFN-γ, IL-1β, and IL-2 in human peripheral blood mononuclear cells (PBMCs) and showed no toxicity to PBMCs. The stimulation effect of the six extracts and eight fractions on the four-cytokine production was dose-dependent. Sclerotial polysaccharides were more effective in the four-cytokine production at 150 μg/ml while exopolysaccharides and endopolysacchrides showed a much better effect on IL-1β production at 30 μg/ml. Purified fractions from exopolysaccharides and endopolysaccharides were more effective than the fraction from sclerotia in most cytokine production. These heteropolysaccharide-protein conjugates mainly contained glucose, galactose, and mannose. Protein content, molecular weight, monosaccharide molar ratio, and anomeric carbon configuration differed from each other and had effects on the cytokine induction activity of the polysaccharides to some extent.

A polysaccharide from Salvia miltiorrhiza Bunge improves immune function in gastric cancer rats

A neutral polysaccharide fraction (SMPA) prepared from the roots of Salvia miltiorrhiza by DEAE-cellulose and Sephadex G-100 chromatography was tested for its immune enhancing function in N-methyl-N′-nitro-nitrosoguanidine (MNNG)-induced gastric cancer rats by intragastric administration. SMPA (200mg/kg) treatment not only increased the body weight, but also improved the immune organ indices. Furthermore, studies of various immunological activities in gastric cancer rats revealed that SMPA significantly stimulated splenocyte proliferation, promoted anti-inflammatory cytokines (IL-2, IL-4 and IL-10) production, inhibited pro-inflammatory cytokine (IL-6 and TNF-α) secretion, augmented the killing activity of natural killer (NK) cells and cytotoxic T lymphocytes (CTL), and increased phagocytotic function of macrophages in gastric cancer rats. In addition, SMPA administration evidently elevated total intracellular granzyme-B and IFN-γ levels produced by splenocytes in gastric cancer rats. Taken together, these results suggested that SMPA could act as an effective immunomodulator and might be explored as a potential supplemental source for gastric cancer therapy.

Isolation, purification, and structural features of a polysaccharide from Phellinus linteus and its hypoglycemic effect in alloxan-induced diabetic mice.

Phellinus linteus is a medicinal mushroom that has been used in Oriental countries for centuries for its antitumor, antioxidant, immunomodulatory, and biological activity on hyperglycemia. A water-soluble crude polysaccharide was extracted using hot water from P. linteus mycelia grown under submerged culture. An orthogonal experiment was used to optimize the extraction conditions of P. linteus mycelia polysaccharides (PLP). The crude polysaccharide was purified using DEAE Sephadex A-50 and Sephadex G-200 chromatography. Fourier transform infrared (FT-IR) spectroscopy and nuclear magnetic resonance ((1) H NMR) spectroscopy were used to investigate the structure of the purified P. linteus polysaccharide (PLP-I), revealing that it was mainly a branched-type glycan with both α- and β-linkages and a pyranoid sugar ring conformation. PLP orally administered at 100 mg/kg body weight/d could significantly reduce the blood glucose level by 35.60% in alloxan-induced diabetic mice. The results of an oral glucose tolerance test (OGTT) revealed that PLP had an effect on glucose disposal after 28 d of treatment. The result revealed that PLP from a submerged culture of P. linteus mycelia possessed potent hypoglycemic properties. The polysaccharide may be useful as a functional food additive and a hypoglycemic agent.

Sulfated modification of longan polysaccharide and its immunomodulatory and antitumor activity in vitro

A water-soluble polysaccharide fraction (LP1) was prepared from Dimocarpus longan Lour. by hot water extraction, DEAE-cellulose and Sephadex G-100 chromatography. Its sulfated derivative (LP1-S) was prepared by the sulfuric acid method. Preliminary tests in vitro showed LP1 and LP1-S could stimulate murine lymphocytes proliferation, increase pinocytic activity of murine macrophages and production of nitric oxide (NO), interleukin 6 (IL-6), IL-1β and tumor necrosis factor-alpha (TNF-α) in macrophages. Furthermore, LP1-S exhibited higher antiproliferative activity against human nasopharyngeal carcinoma HONE1 cells in vitro than LP1, which might be caused by the sulfate group in its structures. These results indicated that the LP1-S might be useful for developing safe antitumor drugs or health food.