Purification and characterization of a lectin from Xanthomonas campestris NCIM 5028

1996 ◽  
Vol 42 (6) ◽  
pp. 609-612 ◽  
Author(s):  
Bhagyashree Joshi ◽  
Jayant M. Khire ◽  
Hephzibah SivaRaman ◽  
M. Islam Khan
Keyword(s):  
Molecular Mass ◽  
Host Plant ◽  
Xanthomonas Campestris ◽  
Simple Procedure ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Ammonium Sulphate Precipitation ◽  
Molecular Masses ◽  
Lectin Purification

A lectin was isolated from culture filtrates of Xanthomonas campestris NCIM 5028, by a simple procedure of hydrophobic chromatography on phenyl-Sepharose after ammonium sulphate precipitation. The lectin was a heterodimer, with subunit molecular masses of 30 000 and 28 000. Gel filtration on S-300 column, calibrated with markers, showed its molecular mass to be approximately 70 000. Its isoelectric point was 7.2. The agglutination of the rabbit erythrocytes by the lectin was inhibited by fetuin glycopeptides and host plant (Brassica oleracea) extracts.Key words: Xanthomonas campestris, lectin, purification.

Fermentative Production, Purification and Characterization of Nisin

2008 ◽  
Vol 4 (5) ◽  
Author(s):  
Swapnali S Gujarathi ◽  
Sandip B. Bankar ◽  
Laxmi A. Ananthanarayan
Keyword(s):  
Ammonium Sulphate ◽  
Hydrophobic Interaction ◽  
Orthogonal Array ◽  
Gel Filtration ◽  
Statistical Design ◽  
Temperature Stability ◽  
Gel Chromatography ◽  
Purification And Characterization ◽  
Ammonium Sulphate Precipitation

Bacteriocins are bactericidal or bacteriostatic in action and active against closely related species. Nisin is the bacteriocin produced by the lactic acid bacteria (LAB). Nisin is a small (3353 Da), cationic, hydrophobic, and 34-amino acid peptide. It is used in products such as pasteurized processed cheese, salad dressing, and liquid whole eggs to inhibit the growth of Gram-positive microorganisms including Listeria monocytogenes. The objective of the present work was to study production, purification and characterization of nisin. The production of nisin was carried out using Lactococcus lactis subsp lactis MTCC 440 by one-factor-at-a-time method and statistical design (Orthogonal array). Purification was carried out using ammonium sulphate precipitation followed by hydrophobic interaction and gel chromatography. Characterization was done for pH and temperature stability. The activity of nisin after one factor at a time optimization was found to be 5120 AU/ml. The activity of the nisin increased to 6800 AU/ml after optimization by Orthogonal Array design. Ammonium sulphate precipitation gives good yield of nisin with 60 to 80% saturation with 2.52 fold purity. The overall purification by hydrophobic interaction and gel filtration chromatography was 10.87 fold with 50.84% yield and 8.8 fold with 49.65% yield as compared to crude broth respectively.

Purification and characterization of fructans with β‐2, 1‐ and β‐2, 6‐glycosidic linkages suitable for enzyme studies

2020 ◽  
Author(s):  
GD BONNETT ◽  
Ian Sims ◽  
JA ST. JOHN ◽  
RJ SIMPSON
Keyword(s):  
Molecular Mass ◽  
Small Proportion ◽  
Gel Filtration ◽  
Triticum Aestivum L ◽  
High Molecular Mass ◽  
Enzyme Assays ◽  
Purification And Characterization ◽  
Linear Molecules ◽  
Low Levels

Fructan pentasaccharides were purified, in quantities suitable for use as substrates for enzyme assays, from Neosugar‐p‐(Meijj Seika Kaisha Ltd. Japan), tubers of Helianthus tuberosus L., L., and stems and leaf sheaths of Triticum aestivum L by a combination of gel‐filtration and RP‐HPLC. Fructan of higher molecular mass (mean DP = 30) was purified from Leaves of Lolium rigidum Gaud, that had been induced to accumulate fructan and characterized along; with the commercially available fructan from Cichorium intybus L. (Sigma, St Louis, USA) (mean DP = 33). The fructan pentasaccharide purified from H. tuberosus was found to contain exclusively 2, 1‐linked fructose and terminal fructose and terminal glucose, and was identified as (1, 1, 1)‐kestopentatise. The fructan pentasaccharide purified from Neosugar‐P also contained (1,1,1)‐kestopentaose. although the presence of fructan Klinked glucose and 1 % 2, 6‐linked fructose indicated that a small proportion of other kestopentaoses were present, The fructan pentasaccharide purified from T aestivum consisted of almost exclusively 2,6‐linked fructose and terminal glucose and terminal fructose and was considered to contain predominantly (6,6,6)‐kestopentaose. The presence of 1 % 2,1,6)‐linked fructose indicated the sample also contained a small proportion of branched kestopentanse. The high molecular mass fructan from C. intybus was found to comprise linear molecules containing only 2,1‐linked fructose, terminal glucose and terminal fructose‐ High molecular mass fructan from L. rigidum contained predominantly 2. h‐linked fructose, had predominantly internal glucose, indicated by 2 %, 1.6‐linked glucose, low levels of branching, indicated 2 % 2,1,6‐linked fructose residues; and 1% of the residues were 2,1 ‐linked fructose. Copyright © 1994, Wiley Blackwell. All rights reserved

scholarly journals Purification and characterization of an extracellular (1 → 6)-β-glucanase from the filamentous fungus Acremonium persicinum

1996 ◽  
Vol 316 (3) ◽  
pp. 841-846 ◽  
Author(s):  
Stuart M. PITSON ◽  
Robert J. SEVIOUR ◽  
Barbara M. McDOUGALL ◽  
Bruce A. STONE ◽  
Maruse SADEK
Keyword(s):  
Molecular Mass ◽  
Filamentous Fungus ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Eisenia Bicyclis ◽  
Ph Optimum ◽  
Hydrolysis Products ◽  
Sds Page ◽  
Hydrolysis Of

An endo-(1 → 6)-β-glucanase has been isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. SDS/PAGE of the purified enzyme gave a single band with an apparent molecular mass of 42.7 kDa. The enzyme is a non-glycosylated, monomeric protein with a pI of 4.9 and pH optimum of 5.0. It hydrolysed (1 → 6)-β-glucans (pustulan and lutean), initially yielding a series of (1 → 6)-β-linked oligoglucosides, consistent with endo-hydrolytic action. Final hydrolysis products from these substrates were gentiobiose and gentiotriose, with all products released as β-anomers, indicating that the enzyme acts with retention of configuration. The purified enzyme also hydrolysed Eisenia bicyclis laminarin, liberating glucose, gentiobiose, and a range of larger oligoglucosides, through the apparent hydrolysis of (1 → 6)-β- and some (1 → 3)-β-linkages in this substrate. Km values for pustulan, lutean and laminarin were 1.28, 1.38, and 1.67 mg/ml respectively. The enzyme was inhibited by N-acetylimidazole, N-bromosuccinimide, dicyclohexylcarbodi-imide, Woodward's Regent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4 and some metal ions, whereas D-glucono-1,5-lactone and EDTA had no effect.

scholarly journals Human liver N-acetylgalactosamine 6-sulphatase. Purification and characterization

1991 ◽  
Vol 279 (2) ◽  
pp. 515-520 ◽  
Author(s):  
J Bielicki ◽  
J J Hopwood
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Chloride Ions ◽  
Lysosomal Degradation ◽  
Purification And Characterization ◽  
Keratan Sulphate ◽  
Native Molecular Mass ◽  
Sds Page ◽  
Molecular Masses ◽  
Column Procedure

Human N-acetylgalactosamine 6-sulphatase (EC 3.1.6.14), which is involved in the lysosomal degradation of the glycosaminoglycans keratan sulphate and chondroitin 6-sulphate, was purified more than 130,000-fold in 2.8% yield from liver by an eight-step column procedure. One major form was identified with a pI of 5.7 and a native molecular mass of 62 kDa by gel filtration. When analysed by SDS/PAGE, dithioerythritol-reduced enzyme contained polypeptides of molecular masses 57 kDa, 39 kDa and 19 kDa, whereas non-reduced enzyme contained a major polypeptide of molecular mass 70 kDa. It is proposed that active enzyme contains either the 57 kDa polypeptide or disulphide-linked 39 kDa and 19 kDa polypeptides. Minor amounts of other enzyme forms separated during the chromatofocusing step and the Blue A-agarose step were not further characterized. Purified N-acetylgalactosamine 6-sulphatase was inactive towards 4-methylumbelliferyl sulphate, but was active, with pH optima of 3.5-4.0, towards 6-sulphated oligosaccharide substrates. Km values of 12.5 and 50 microM and Vmax. values of 1.5 and 0.09 mumol/min per mg were determined with oligosaccharide substrates derived from chondroitin 6-sulphate and keratan sulphate respectively. Sulphate, phosphate and chloride ions were inhibitors of enzyme activity towards both substrates, with 50 microM-Na2SO4 giving 50% inhibition towards the chondroitin 6-sulphate trisaccharide substrate.

Purification and characterization of fructans with β‐2, 1‐ and β‐2, 6‐glycosidic linkages suitable for enzyme studies

2020 ◽  
Author(s):  
GD BONNETT ◽  
Ian Sims ◽  
JA ST. JOHN ◽  
RJ SIMPSON
Keyword(s):  
Molecular Mass ◽  
Small Proportion ◽  
Gel Filtration ◽  
Triticum Aestivum L ◽  
High Molecular Mass ◽  
Enzyme Assays ◽  
Purification And Characterization ◽  
Linear Molecules ◽  
Low Levels

Fructan pentasaccharides were purified, in quantities suitable for use as substrates for enzyme assays, from Neosugar‐p‐(Meijj Seika Kaisha Ltd. Japan), tubers of Helianthus tuberosus L., L., and stems and leaf sheaths of Triticum aestivum L by a combination of gel‐filtration and RP‐HPLC. Fructan of higher molecular mass (mean DP = 30) was purified from Leaves of Lolium rigidum Gaud, that had been induced to accumulate fructan and characterized along; with the commercially available fructan from Cichorium intybus L. (Sigma, St Louis, USA) (mean DP = 33). The fructan pentasaccharide purified from H. tuberosus was found to contain exclusively 2, 1‐linked fructose and terminal fructose and terminal glucose, and was identified as (1, 1, 1)‐kestopentatise. The fructan pentasaccharide purified from Neosugar‐P also contained (1,1,1)‐kestopentaose. although the presence of fructan Klinked glucose and 1 % 2, 6‐linked fructose indicated that a small proportion of other kestopentaoses were present, The fructan pentasaccharide purified from T aestivum consisted of almost exclusively 2,6‐linked fructose and terminal glucose and terminal fructose and was considered to contain predominantly (6,6,6)‐kestopentaose. The presence of 1 % 2,1,6)‐linked fructose indicated the sample also contained a small proportion of branched kestopentanse. The high molecular mass fructan from C. intybus was found to comprise linear molecules containing only 2,1‐linked fructose, terminal glucose and terminal fructose‐ High molecular mass fructan from L. rigidum contained predominantly 2. h‐linked fructose, had predominantly internal glucose, indicated by 2 %, 1.6‐linked glucose, low levels of branching, indicated 2 % 2,1,6‐linked fructose residues; and 1% of the residues were 2,1 ‐linked fructose. Copyright © 1994, Wiley Blackwell. All rights reserved

scholarly journals Purification and Characterization of Sepiapterin Deaminase from the Silkworm, Bombyx mori

Pteridines ◽  
1998 ◽  
Vol 9 (1) ◽  
pp. 18-21 ◽  
Author(s):  
Hiroshi Sawada ◽  
Motoki Kanekatsu ◽  
Motoko Nakagoshi ◽  
Kenjiro Dohke ◽  
Teruhiko Iino ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Bombyx Mori ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Native Form ◽  
Sds Page ◽  
Column Chromatographic ◽  
Silkworm Bombyx Mori

Summary Sepiapterin deaminase has been purified approximately 6,000-told from the larval integument of the lemon mutant of the silkworm by several column chromatographic procedures. Sepiapterin and isosepiapterin were active substrates among various pteridines tested. The molecular mass of this enzyme was estimated to be 74 kDa by SDS-PAGE and 70 kDa by gel filtration, suggesting that the native form of the enzyme is monomeric protein . All silkworm strains, to the best of our knowledge, had an activity of the enzyme and the enzyme was widely distributed in the larval tissues. Sepiapterin deaminase may have an important function on the silkworm.

Purification and partial characterization of peptidase-1, a sex-linked enzyme inPleurodeles waltl(urodele amphibian)

1997 ◽  
Vol 75 (6) ◽  
pp. 803-806 ◽  
Author(s):  
Etienne Rudolf ◽  
Jean-Michel Girardet ◽  
Anne-Marie Bautz ◽  
Christian Dournon
Keyword(s):  
Ethylene Glycol ◽  
Molecular Mass ◽  
Subcellular Fraction ◽  
Gel Filtration ◽  
Tetraacetic Acid ◽  
Optimal Activity ◽  
Isoelectric Points ◽  
Molecular Masses ◽  
Pleurodeles Waltl

Peptidase-1 is a sex-linked enzyme, which can be purified from the liver of the amphibian urodele Pleurodeles waltl. We estimated its apparent molecular mass as 170 kDa by gel filtration chromatography. The enzyme is composed of two subunits with apparent molecular masses of 90 and 99 kDa. It is strongly inhibited by ethylenediaminotetraacetic acid, ethylene glycol bis( beta -aminoethyl ether)-N,N-tetraacetic acid, and 1,10-phenanthroline, indicating that peptidase-1 is a metallopeptidase. Peptidase-1 has optimal activity at 55°C and pH 8.5. This acidic enzyme displays two apparent isoelectric points, at 4.9 and 5.2, and is essentially located in the cytosolic subcellular fraction.Key words: peptidase-1, amphibian, purification, characterization, sex-linked enzyme.

PURIFICATION AND CHARACTERIZATION OF CAPRINE PROLACTIN

1974 ◽  
Vol 60 (2) ◽  
pp. 359-367 ◽  
Author(s):  
A. S. McNEILLY ◽  
P. ANDREWS
Keyword(s):  
Simple Procedure ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Product Yield ◽  
Pituitary Hormones ◽  
Major Product ◽  
Purification And Characterization ◽  
Pituitary Glands ◽  
Ovine Prolactin

SUMMARY Prolactin was isolated from frozen goat pituitary glands by a simple procedure involving gel filtration and chromatography on DEAE-cellulose. The major product (yield, 2·5 mg/g pituitary tissue) had high pigeon crop sac-stimulating activity (27 i.u./mg) and was free of growth hormone and other pituitary hormones. The molecular weight was similar to that of ovine prolactin. Caprine prolactin was immunologically indistinguishable from ovine prolactin in radioimmunoassays, in which ovine prolactin antiserum and either ovine or caprine prolactin labelled with 125I were used. The results indicate that caprine and ovine prolactin are closely related and that radioimmunoassay for ovine prolactin may be used to estimate caprine prolactin in serum.

scholarly journals Overexpression, Purification, and Characterization of the Cloned Metallo-β-Lactamase L1 fromStenotrophomonas maltophilia

1998 ◽  
Vol 42 (4) ◽  
pp. 921-926 ◽  
Author(s):  
Michael W. Crowder ◽  
Timothy R. Walsh ◽  
Linda Banovic ◽  
Margaret Pettit ◽  
James Spencer
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Kinetic Studies ◽  
Laser Desorption Ionization ◽  
Purification And Characterization ◽  
Flight Mass Spectrometry ◽  
Steady State Kinetic ◽  
Cephalosporin Antibiotics ◽  
State Kinetic

ABSTRACT The metallo-β-lactamase L1 from Stenotrophomonas maltophilia was cloned, overexpressed, and characterized by spectrometric and biochemical techniques. Results of metal analyses were consistent with the cloned enzyme having 2 mol of tightly bound Zn(II) per monomer. Gel filtration chromatography demonstrated that the cloned enzyme exists as a tightly held tetramer with a molecular mass of ca. 115 kDa, and matrix-assisted laser desorption ionization and time-of-flight mass spectrometry indicated a monomeric molecular mass of 28.8 kDa. Steady-state kinetic studies with a number of diverse penicillin and cephalosporin antibiotics demonstrated that L1 effectively hydrolyzes all tested compounds, withk cat/Km values ranging between 0.002 and 5.5 μM−1 s−1. These characteristics of the recombinant enzyme are contrasted to those previously reported for metallo-β-lactamases isolated directly fromS. maltophilia.

Purification and Characterization of Tissue-Type Plasminogen Activator in Maxillary Mucosa with Chronic Inflammation

1985 ◽  
Vol 54 (02) ◽  
pp. 485-489 ◽  
Author(s):  
Yukiyoshi Hamaguchi ◽  
Masuichi Ohi ◽  
Yasuo Sakakura ◽  
Yasuro Miyoshi
Keyword(s):  
Plasminogen Activator ◽  
Chronic Inflammation ◽  
Gel Filtration ◽  
Potassium Acetate ◽  
Tissue Type ◽  
Purification And Characterization ◽  
Tissue Type Plasminogen Activator ◽  
Urokinase Inhibitor ◽  
Type Plasminogen Activator

SummaryTissue-type plasminogen activator (TPA) was purified from maxillary mucosa with chronic inflammation and compared with urokinase. Purification procedure consisted of the extraction from delipidated mucosa with 0.3M potassium acetate buffer (pH 4.2), 66% saturation of ammonium sulfate, zinc chelate-Sepharose, concanavalin A-Sepharose and Sephadex G-100 gel filtration chromatographies.The molecular weight of the TPA was approximately 58,000 ± 3,000. Its activity was enhanced in the presence of fibrin and was quenched by placental urokinase inhibitor, but not quenched by anti-urokinase antibody. The TPA made no precipitin line against anti-urokinase antibody, while urokinase did.All these findings indicate that the TPA in maxillary mucosa with chronic inflammation is immunologically dissimilar to urokinase and in its affinity for fibrin.

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