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 kinetic characterization of pickerel liver alcohol dehydrogenase with dual coenzyme specificity

1993 ◽  
Vol 71 (9-10) ◽  
pp. 421-426 ◽  
Author(s):  
Loola S. Al-Kassim ◽  
C. Stan Tsai
Keyword(s):  
Alcohol Dehydrogenase ◽  
Gel Filtration ◽  
Kinetic Studies ◽  
Resonance Spectroscopy ◽  
Coenzyme Specificity ◽  
Liver Alcohol Dehydrogenase ◽  
Steady State Kinetic ◽  
Dehydrogenase Isozyme ◽  
State Kinetic

A major alcohol dehydrogenase isozyme that displays dual coenzyme specificity has been purified from pickerel liver by ion-exchange, gel filtration, and affinity chromatographic procedures. The purified enzyme is chromatographically and electrophoretically homogeneous. It is dimeric and possesses common physical properties shared by other liver alcohol dehydrogenases. Phosphorus-31 nuclear magnetic resonance spectroscopy demonstrates that NADP+ binds to two coenzyme sites of the pickerel enzyme. Steady-state kinetic studies suggest that pickerel liver alcohol dehydrogenase catalyzes NAD(P)+-linked ethanol oxidation via a random pathway. While the NADP+ reduction involves the formation of an abortive complex at high NADP+ concentrations, the NAD+ reduction at low NAD+ concentrations follows an ordered Bi-Bi mechanism with NAD+ being the leading reactant.Key words: purification, pickerel liver, alcohol dehydrogenase.

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.

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 Partial Purification and Characterization of Catalase from Banana Peels

2016 ◽  
Vol 13 (2) ◽  
pp. 392-398
Author(s):  
Baghdad Science Journal
Keyword(s):  
Gel Filtration ◽  
Maximum Velocity ◽  
Kinetic Studies ◽  
Maximal Activity ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Km Value ◽  
Almost All ◽  
Kinetics Of

Catalase (EC 1.11.1.6) is a well known enzyme which exists in almost all living creatures exposing to oxygen (such as plants, bacteria, and animals). It is a very necessary enzyme to protect the cell from oxidative detriment by reactive oxygen species (ROS). The aim of this study is the partial purification and characterization of Catalase enzyme from Banana peels. In this study, fresh banana peels are treated with 70 % ethanol ,further separated with chloroform ,water and ethyl acetate respectively .The supernatant of the enzymatic sample which is treated with chloroform is loaded into gel filtration column with Sephadex G-100 (1.0 x 90 cm) equilibrated with pH7 buffer media (phosphate buffer 0.1 M). Kinetic studies of the purified enzyme activity are measured and characterized .The maximal activity (26.04 units/mg) of catalase is observed with chloroform buffer extraction. The kinetics of catalase; Michalis constant Km and maximum velocity Vmax is determined using Linweaver- Burk plot, The Km value for catalase (434.7mM), Vmax (100 m mole min -1). Characterization results demonstrate that the optimal pH for activity is (7.6). And the optimal temperature for activity is 30?C .The present study indicates that Banana peels is a good source of catalase enzyme.

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.

scholarly journals Purification and characterization of 5-ketofructose reductase from Erwinia citreus

1988 ◽  
Vol 253 (2) ◽  
pp. 511-516 ◽  
Author(s):  
J L Schrimsher ◽  
P T Wingfield ◽  
A Bernard ◽  
R Mattaliano ◽  
M A Payton
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Single Species ◽  
Kinetic Mechanism ◽  
Relative Molecular Mass ◽  
Terminal Sequence ◽  
Purification And Characterization ◽  
Steady State Kinetic ◽  
State Kinetic

5-Ketofructose reductase [D(-)fructose:(NADP+) 5-oxidoreductase] was purified to homogeneity from Erwinia citreus and demonstrated to catalyse the reversible NADPH-dependent reduction of 5-ketofructose (D-threo-2,5-hexodiulose) to D-fructose. The enzyme appeared as a single species upon analyses by SDS/polyacrylamide-gel electrophoresis and isoelectric focusing with an apparent relative molecular mass of 40,000 and an isoelectric point of 4.4. The amino acid composition of the enzyme and the N-terminal sequence of the first 39 residues are described. The steady-state kinetic mechanism was an ordered one with NADPH binding first to the enzyme and then to 5-ketofructose, and the order of product release was D-fructose followed by NADP+. The reversible nature of the reaction offers the possibility of using this enzyme for the determination of D-fructose.

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