Sephadex G-100 Column Chromatography Research Articles

Study on an antimicrobial protein produced by Paenibacillus polymyxa JSa-9 isolated from soil

Paenibacillus polymyxa strain JSa-9, a soil isolate showed in vitro inhibitory effects on several pathogens. A protein of about 71.9 kDa, isolated from a culture of JSa-9, exhibited broad-spectrum antibacterial and antifungal activities. The purification procedure consisted of ion-exchange chromatography on DEAE-Sephacel and Sephadex G-100 column chromatography. The purified protein was characterized to be a glycoprotein, which contained 53% of amino acids and 28% of carbohydrate. Its N-terminal sequence was determined as KCATIPVVIKHL and the amino acid composition showed no significant homology with any known antagonistic proteins from P. polymyxa. Because of the excellent antimicrobial activity, this protein may be a good prospect for food preservation and plant disease control.

Purification and characterization of novel manganese peroxidase from Rhizoctonia sp. SYBC-M3

A novel manganese peroxidase of Rhizoctonia sp. SYBC-M3 (R-MnP) was purified by (NH4)2SO4 fractionation, DEAE-cellulose-32 column chromatography, and Sephadex G100 column chromatography. The molecular mass of R-MnP was determined to be approximately 40.4 kDa by SDS-PAGE. The optimum temperature and pH for R-MnP were 55°C and 4.5, respectively. R-MnP was highly stability when the temperature was below 50°C. R-MnP could retain about 60% of its activity when the pH was between 4 and 6.5. However, R-MnP activity was inhibited by Fe3+, Cu2+, and Co3+ as well as increased by Zn2+ and Ca2+. R-MnP demonstrated oxidation of DMP, ABTS, veratryl alcohol, and guaiacol. The K m values of RMnP for H2O2 and Mn2+ were 25.3 and 53.9 μmol/L, respectively.

Isolation, purification and structure of a new water-soluble polysaccharide from Zizyphus jujuba cv. Jinsixiaozao

A novel water-soluble polysaccharide (ZSP3c) was isolated from Zizyphus jujuba cv. Jinsixiaozao and its structure was investigated. ZSP3c was obtained by extraction with hot water followed by purification using DEAE-SepharoseCL-6B anion-exchange, SepharoseCL-6B and Sephadex G-200 column chromatography. It was eluted as a single symmetrical narrow peak on high-performance gel-permeation chromatography (HPGPC) and the average molecular weight (Mw) was estimated to be 1.4 × 10 5 Da. The ZSP3c was composed of l-rhamnose, d-arabinose and d-galactose in a molar ratio of 1:2:8. The structural features of ZSP3c were investigated by partial hydrolysis with acid, methylation analysis and NMR spectroscopy analysis. The results revealed that the main backbone chain of ZSP3c was (1 → 4)- d-galacturonopyranosyl residues interspersed with (1 → 2)- l-rhamnopyranosyl residues and (1 → 2,4)- l-rhamnopyranosyl residues. The neutral side chains of arabinofuranosyl residues and galactopyranosyl residues were attached to the backbone at the O-4 position of rhamnopyranosyl residues.

Purification and characterization of two thermostable laccases with high cold adapted characteristics from Pycnoporus sp. SYBC-L1

The white-rot fungus Pycnoporus sp. SYBC-L1 produced large amount of laccase in submerged fermentation. Two laccase isozymes (LacI and LacII) were purified using (NH 4) 2SO 4 fractionation, DEAE-cellulose and Sephadex G-100 column chromatography. The molecular masses of LacI and Lac II were 55.89 and 63.07 kDa, respectively by SDS-PAGE. Both the laccases showed acidic pH optima and high catalytic activities at low temperature for oxidations of 2,6-dimethoxyphenol (DMP), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonate acid) (ABTS), syringaldazine and guaiacol. LacI and LacII were not only with high cold adaptation, but also fairly stable at high temperature. The half-lives of LacI at 50, 60 and 70 °C were 69.31, 2.58 and 0.13 h, respectively, whereas LacII was more stable with half-lives of 256.72, 21.00 and 2.06 h respectively. The best substrates for the enzymes were both found to be ABTS, in which the K m values of LacI and LacII were 0.0166 and 0.0435 mM and the catalytic efficiencies were 19640.36 and 31172.64 S −1 mM −1, respectively. EDTA and low concentration of Cu 2+ and Mn 2+ almost had non-inhibitions on their activities. LacII with syringaldehyde efficiently decolorized Remazol Brilliant Blue R. The high thermostabilities as well as cold adapted properties made Pycnoporus sp. SYBC-L1 laccases to be excellent candidates in harsh industry.

Purification, characterization and inhibition studies of α-amylase of Rhyzopertha dominica

Rhyzopertha dominica causes extensive damage to stored wheat grains. α-Amylase, the major insect digestive enzyme, can be an attractive candidate to control the insect damage by inhibiting the enzyme through α-amylase inhibitors. R. dominica α-amylase (RDA) was purified to homogeneity by differential ammonium sulphate fractionation, Sephadex G-25 and Sephadex G-100 column chromatography. The homogenous α-amylase has a molecular weight of 52 kDa. The pH optima was 7.0 and temperature optima was 40 °C. Activation energy of RDA was 3.9 Kcal mol −1. The enzyme showed high activity with starch, amylose and amylopectin whereas dextrins were the poor substrates. The purified enzyme was identified to be α-amylase on the basis of products formed from starch. The enzyme showed Km of 0.98 mg ml −1 for starch as a substrate. Citric acid, oxalic acid, salicylic acid, HgCl 2, tannic acid and α-amylase inhibitors from wheat were inhibitors whereas; Ca 2+ and Mg 2+ were the activators of RDA. Ki values calculated from Dixon graphs with salicylic acid, citric acid, oxalic acid and wheat α-amylase inhibitors were 6.9, 2.6–8.2, 3.2 mM and 0.013–0.018 μM, respectively. The Lineweaver–Burk plots with different inhibitors showed mixed type inhibition. Wheat α-amylase inhibitor showed mainly competitive inhibition with some non-competitive behaviour and other inhibitors showed mainly non-competitive inhibition with some un-competitive behaviour. Feeding trials with salicylic acid, citric acid, oxalic acid and wheat α-amylase inhibitor showed significant effect of salicylic acid and oxalic acid along with wheat α-amylase inhibitor in controlling the multiplication of R. dominica.

Purification and characterization of the -amylase isolated from Penicillium camemberti PL21

The amylase family of enzymes is a great significance due to its wide area of potential application. α-amylase from Penicillium camemberti PL21 obtained from I. N. A. (The technological laboratory of Agriculture National Institute Paris- France) and using orange waste as substrates was produced under optimum conditions, after 168 h of incubation and subjected for purification and characterization. The enzyme was purified by ammonium sulfate precipitation, dialysis, Sephadex G-100 and DEAE-Sepharose CL-6B column chromatography. A trial for the purification of α-amylase resulted in an enzyme with specific activity of (154.2 units/ml/mg protein) with (38.5 folds) purification .The α-amylase activity increased by enzyme concentration rise. The optimum substrate concentration for soluble starch was 1 % (w/v) while the optimum incubation temperature was 30oC. The purified α-amylase enzyme had a maximum activity at pH 6 and the Km value for solublestarch was 0.92 mg/ml. Analyses of this enzyme for molecular mass was carried out by SDS-PAGE electrophoresis, which revealed one band 60. 5 Kda. Key words: Penicillium camemberti PL21, α-amylase, purification, SDS-PAGE electrophoresis.

Purification and characterization of extreme alkaline, thermostable keratinase, and keratin disulfide reductase produced by Bacillus halodurans PPKS-2

Two alkaline keratinases-I and II secreted by Bacillus halodurans PPKS-2 were purified and characterized. Both the keratinases were purified using ammonium sulfate, DEAE-Sephadex followed by Sephadex G-200 column chromatography. The purification was 21.5-fold and 11.17% yield for keratinase-I and 23.7-fold with yield 18.46 for keratinase-II and its molecular weights 30 and 66 kDa. Both purified enzymes were relatively stable over a broad pH range 7.0-13.0 and optimally active at pH 11.0 and 60-70 degrees C. Keratinase-II was found to be more stable at 70 degrees C for 3 h and retained 100% of its activity, whereas keratinase-I lost 10% activity. Keratinase-I had high keratin disulfide reductase activity with low keratinase activity whereas keratinase-II had high keratinase activity with low keratin disulfide reductase activity. Keratinase activities of both the enzymes were completely inhibited by PMSF at 1 mM, whereas keratin disulfide reductase activity of keratinase-I was not affected. Enzymes were active and stable in the presence of the surfactants, bleaching agents (20% H(2)O(2)), commercial detergents (1%), and SDS (20%). Both the enzymes were partially sequenced and found that keratinase-I and II had a homology with disulfide reductases and serine type of proteases, respectively.

Chemical characterisation of polysaccharides from Lilium davidii

Lilium davidii var. unicolor Salisb is known for its esculent and exceptionally sweet bulbs. This article reports the isolation and purification of a non-starch polysaccharide from the bulb tissues. The polysaccharide was fractionated by Sephadex G-100 column chromatography, giving two polysaccharide fractions. We collected the main peak polysaccharide, termed Lilium davidii polysaccharide (LDP). The molecular appearance of LDP at different concentrations was observed with atomic force microscopy (AFM) imaging, and the chemical characterisation of LDP was studied by physical, chemical and spectroscopic techniques: for example, differential scanning calorimetry (DSC) analysis, methylation analysis, GC, GC-MS, and NMR. The results demonstrate that the LDP is an amorphous powder, containing three monosaccharide molecules: D-mannose (D-Man), D-glucose (D-Glc) and D-galactose (D-Gal), with approximate molar ratios of 10 : 19 : 1. The morphology of LDP was arranged as irregular crumb-like or island forms (3-D images) when the concentration of the solution was low, and more molecules were entangled as a rugged sugar layer at high concentration. The LDP has a molecular weight of 5.17 × 104 g mol−1. On the basis of methylation and GC-MS analysis, IR, NMR, the likely linkages of sugar components of LDP was described as follows: the main chain of the LDP is primarily made up of a 1,4-linked form for β-Glc and a 1,3-linked form for α-Man with molar ratios of 2 : 1. On average, there is one 1,6-linked form for α-Gal or one 1,3-linked form for α-Man residues which can be substituted at 6-O from among 30 sugar residues. The reduction terminal is β-Glc.

Nonradioactive Iodine-Labeled Antibodies-Inductively Coupled Plasma Mass Spectrometry for Immunoassay

In the present study, the system of nonradioactive iodine-labeled-antibodies linking inductively coupled plasma mass spectrometry for immunoassay was reported. The goat-anti-Escherichia coli and goat anti rabbit were considered as simulant antigen and antibody respectively in order to establish a new method of immunoassay by inductively coupled plasma mass spectrometry which has the advantage of high sensitivity, low detection limit and preferable linearity range. During the experiment, the N-bromosuccinimide, a mild oxidant, was used to oxidize the non-radioactive iodine (127 I) that labeled the protein. The method of nonradioactive iodine labeled protein was established and the best labeling condition was explored. The compound of I was purified by Sephadex G50 column chromatography, then the stability and activity were examined. The results showed that the labeling program was simple, reaction time was within two minutes, the labeling yield achieved 63.12% and none of I shed from the compound after 96 hours. The simulant antigen and antibody reacted on polystyrene microtiter plate and the I was detected by ICP-MS, the detection limit of the method was 0.12 mg x L(-1), relative standard deviation (n = 9) was less than 3% and the linearly dependent coefficient was 0.998 7. This system can also be used in analysis of other protein, nucleic acid and so on.

Purification, Composition Analysis, and Antioxidant Activity of Exopolysaccharides from Mycelial Culture of Paecilomyces cicadae (Miq.) Samson (Ascomycetes)

The biomass growth and exopolysaccharide (EPS) production by a strain of Paecilomyces cicadae (Miq.) Samson ZJ001, which achieved a high concentration of biomass (5.6−6 g/L at day 4), was studied. The crude EPS (containing 68.9% sugar and about 26.2% protein) was fractionated by DEAE-52 column chromatography and puri fied by Sephadex G-100 column chromatography to produce the puri fied EPS (PEPS). Its primary structural features and molecular weight were characterized by Fourier infrared spectrometry (FTIR), gel permeation chromatography (GPC), and gas chromatography (GC). The results indicated that the PEPS was a protein-bound polysaccharide (2.3% protein) composed of rhamnose, arabinose, fucose, xylose, mannose, glucose, and galactose in the molar ratio of 0.63: 13.1:1.30:21.3:46.5:6.0:10.22, with a molecular weight of 167 kDa. The antioxidant activities of the puri fied EPS were evaluated in vitro by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay, the ferric-reducing antioxidant power assay, the superoxide radical assay, and the hydroxyl radical assay. The puri fied EPS was revealed to have strong reductive power (672.3 μmol/L), moderate scavenging activity against DPPH radical (64.1%) and superoxide radicals (58.3%), and potent inhibiting power for hydroxyl radical (92.2%).

A possible role of chorion protease in shell membrane degradation during development of quail embryos

In the eggs of the quail Coturnix japonica, the limiting membrane demarcates the shell membrane at the interface with the albumen and decreases in width during the hatching process. This study was done to identify agents that affect the width of this limiting membrane. Zymography tests on extracts from extraembryonic tissues, yolk sacs, or chorioallantoic membranes, or all three, showed proteolytic activities during d 4 to 10 of incubation. Localization experiments on these activities, performed on d 5 eggs, indicated that they were located in an avascular chorion. Electron microscopic analysis showed there were secretory cells specifically located in the avascular chorion. After partial purification of d 5 avascular chorion extracts using QA52 and Sephadex G-200 column chromatography, the proteolytic activity of 20 kDa was isolated. The protease showed a high level of activity toward succinyl-Gly-Pro-Leu-Gly-Pro-4-methylcoumaryl-7-amide. It had an optimal pH of 9 and digested the limiting membrane. These enzymatic activities were inhibited moderately by EDTA and strongly by leupeptin and aprotinin. It was concluded that it is the 20-kDa protease, showing collagenase-like activity produced by the avascular chorion, that affects the limiting membrane.

Studies on the chemical structure and antitumor activity of an exopolysaccharide from Rhizobium sp. N613

The molecular structure of the rhizobium exopolysaccharide (REPS) was analyzed by enzymolysis, periodate oxidation, and Smith degradation, and by IR and NMR spectroscopy. The results indicated that REPS was a β-glucan with a backbone of β- d-(1→4)-linked glucose residues and branches of β- d-(1→6)-linked glucose residues. The branch was attached to the main chain at the 6-O-position. The molar ratio of 1→4 and 1→6 was 2:1. The terminal C3 of the (1→6)-Glc branch had an O-acetyl group. The molecular weight was estimated to be 35 kDa by Sephadex G-100 column chromatography. The antitumor activity of REPS was evaluated in mice bearing sarcoma 180, hepatoma 22, and Ehrlich ascites carcinoma tumor, respectively. At doses of 10–60 mg/kg, it was observed that tumor formation decreased significantly ( P <0.01), but the relative spleen and thymus weight, the phagocytic function of monocytes, lymphocyte proliferation, and serum hemolysis antibody increased significantly ( P <0.05). Results of these studies demonstrated that the REPS polysaccharide possessed antitumor activity.

Microwave–assisted extraction and identification of polysaccharide from Lycoris aurea

Three polysaccharide fractions (LAP-1, LAP-2, and LAP-3) were separated from the extract of Lycoris aurea using DEAE-52 and Sephadex G-150 column chromatography. FT-IR spectroscopy revealed the typical characteristics of polysaccharide fractions. The molecular weights of three polysaccharides were characterized by HPGFC, which were 31.9 kDa, 35.7 kDa, and 72.7 kDa, respectively. Their chemical compositions were also studied. The conditions for microwave-assisted extraction (MAE) of polysaccharide from L. aurea were investigated through an orthogonal design of L9 (33) in this work.

Composition analysis and antioxidant activity of polysaccharide from Dendrobium denneanum

Three polysaccharide fractions (DDP1-1, DDP2-1 and DDP3-1) were successfully purified from the crude polysaccharide of Dendrobium denneanum by DEAE-Cellulose and Sephadex G-200 column chromatography. The average molecular weights (Mws) of these fractions were 51.5, 26.1 and 6.95 kDa, respectively. Monosaccharide components analysis indicated that DDP1-1 and DDP2-1 were composed of arabinose, xylose, mannose, glucose and galactose in a molar ratio of 1.00:2.82:57.11:140.82:7.76 and 1.00:1.62:1.18:77.5:7.79. DDP3-1 was composed of arabinose, mannose, glucose and galactose in a molar ratio of 1.00:1.03:8.84:2.00. On the basis of antioxidant test in vitro, DDP2-1 exhibited the highest antioxidant ability among these samples.

Soluble proteins of guinea pig seminal vesicle lumen: purification and partial characterization.

Luminal contents of guinea pig seminal vesicle were extracted with 0.154 M NaCl and the soluble protein fraction was studied by DEAE-cellulose and Sephadex G-100 column chromatography; anionic, cationic, and SDS-polyarcylamide disc gel electrophoresis; isoelectric focusing; molecular weight determinations by SDS-polyacrylamide disc gel electrophoresis; analysis for neutral carbohydrate; sucrose density-gradient centrifugation for determination of sedimentation coefficients; and amino acid analyses. Four protein fractions (referred to as proteins 1, 2, 3, and 4 in order to elution) were obtained by DEAE-cellulose column chromatography. Proteins 1, 2, and 4 gave single major bands in multiple disc gel electrophoretic systems. Protein 3 gave one major band and one minor component believed to be a fragment of a primary vesicular protein. Protein 1 is very basic and probably represents the clotting protein of guinea pig semen. The three nonclotting proteins could not be detected in serum and are probably intrinsic to seminal vesicle epithelium. All four proteins were found in animals weighing 700 g or more; only two of the three nonclotting proteins were found in more than half of the younger animals. This makes it even more likely that one or more of the nonclotting proteinc could serve as a useful gene marker in studies of androgen mechanisms.

Ram proacrosin. A simple method for isolation of proacrosin free of inhibitors, and proacrosin autoactivation studies.

Two partially purified proacrosin forms have been obtained from acid extracts of ram ejaculated spermatozoa by Sephadex G-100 column chromatography in 0.001 M HCl. The form obtained from the extracts of whole ejaculates with apparently higher molecular mass was free of any acrosin inhibitor and was used for autoactivation studies. The autoactivation followed a classical S-shaped activation curve and was a two-step process. The presence of calcium ions slowed down the autoactivation process and stabilized the active acrosin formed. The activity of latter rapidly decreased when the pH of the fully activated mixture was adjusted to pH 3. The activity was not restored by re-adjusting the pH to 8. A proacrosin with apparently lower molecular mass was isolated from the extract of washed spermatozoa. Both proacrosins, however, showed the same molecular mass Mr approximately 58 000 daltons, when examined by gel filtration in 0.1 M NaCl (pH 3).

A novel bacteriocin with a broad inhibitory spectrum produced by Lactobacillus sake C2, isolated from traditional Chinese fermented cabbage

By excluding the effects of organic acids and hydrogen peroxide, strain C2 producing a bacteriocin strongly inhibited Staphylococcus aureus ATCC 63589 and Escherichia coli ATCC 25922 was selected from 300 lactic acid bacteria isolated from traditional Chinese fermented cabbage. Strain C2 was identified as Lactobacillus sakei by phenotypical and physiological tests and 16S rDNA identification. This bacteriocin, which was designated sakacin C2, was purified by cold ethanol and Sephadex G50 column chromatography for further studies. The molecular weight of sakacin C2 was 5.5 kDa by Tris-Tricine SDS–PAGE, which was the largest among all known sakacins. Sakacin C2 exhibited a wide range of antimicrobial activity, strong heat stability (15 min at 121 °C) and pH stability (pH 3.0–8.0). Sakacin C2 was sensitive to protease but insensitive to lipase, α-amylase and β-amylase. These characters suggested that sakacin C2 was a novel Class II bacteriocin with a broad inhibitory spectrum and might broaden the application range of bacteriocins from LAB in the food industry.

Purification, Structural Characterization, and Antioxidant Activity of Antioxidant Substance from the Red SeaweedGloiopeltis tenax

An antioxidant substance (AOS) obtained from an enzymatic extract of the red seaweed Gloiopeltis tenax was purified by DEAE-Sephadex CL-6B and Sephadex G-100 column chromatography. The purification yield of AOS was 14.3%. The AOS predominantly contained fucose, mannose, and galactose but also contained a sulfate group. The structure of AOS was investigated by periodate oxidation, desulfation, Fourier transform-infrared spectroscopy, and nuclear magnetic resonance spectroscopy. AOS was mainly composed of alternating units of beta-D-Glc(1 --> 2)alpha-D-Man(1 --> 4)beta-D-Gal(1 --> 4)alpha-D-Man(1 --> 4)beta-D-Gal alpha-D-Man (1 --> 4) beta-D-Glc (or Xyl)- and branched linkage of alpha-D-Man(1 --> 3) alpha-D-Fuc. In addition, the fucose residues were shown to be 2-O- and 4-O-sulfated and, therefore, were either terminal or 3-linked. The antioxidative activity of AOS was measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and the beta-carotene-linoleate assay systems and was compared with those of butylated hydroxytoluene and ascorbic acid (AscA). The results showed that AOS exhibited higher antioxidative activity than AscA in the DPPH assay model and in the beta-carotene-linoleate assay system at all of the four concentration levels tested (from 50 to 200 microg/mL). These results suggested that AOS from the red seaweed G. tenax is an efficient novel antioxidant.

Purification and Characterization of a Novel Heparin Degrading Enzyme from Aspergillus flavus (MTCC-8654)

A heparinase-producing fungus was isolated, and the strain was taxonomically characterized as Aspergillus flavus by morphophysiological and 26S rRNA gene homology studies. The culture produced intracellular heparinase enzyme, which was purified 40.5-fold by DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-100 column chromatography. Specific activity of the purified enzyme was found to be 44.6 IU/microg protein and the molecular weight of native as well as reduced heparinase was 24 kDa, showing a monomeric unit structure. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. The enzyme activity was maximum at 30 degrees C in the presence of 300 mM NaCl at pH 7.0. In the presence of Co2+, Mn2+ ions, and reducing agents (beta-mercaptoethanol, dithiothreitol), enzyme activity was enhanced and inhibited by iodoacetic acid. These observations suggested that free sulfohydryl groups of cysteine residues were necessary for catalytic activity of the enzyme. The enzyme was also inhibited by histidine modifier, DEPC, which suggests that along with cysteine, histidine may be present at its active site. The enzyme showed a high affinity for heparin as a substrate with K (m) and V (max) as 2.2 x 10(-5 )M and 30.8 mM min(-1), respectively. The affinity of the enzyme for different glycosaminoglycans studied varied, with high substrate specificity toward heparin and heparin-derived polysaccharides. Depolymerization of heparin and fractionation of the oligosaccharides yielded heparin disaccharides as main product.

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.