| dc.contributor.author |
Katsos, NE |
en |
| dc.contributor.author |
Labrou, NE |
en |
| dc.contributor.author |
Clonis, YD |
en |
| dc.date.accessioned |
2014-06-06T06:46:02Z |
|
| dc.date.available |
2014-06-06T06:46:02Z |
|
| dc.date.issued |
2004 |
en |
| dc.identifier.issn |
15700232 |
en |
| dc.identifier.uri |
http://dx.doi.org/10.1016/j.jchromb.2004.04.032 |
en |
| dc.identifier.uri |
http://62.217.125.90/xmlui/handle/123456789/2768 |
|
| dc.subject |
2-(morpholino)ethanesulfonic acid |
en |
| dc.subject |
biomimetic-dye or mimodye/adsorbent |
en |
| dc.subject |
BM |
en |
| dc.subject |
BM1 dye or adsorbent |
en |
| dc.subject |
CB3GA |
en |
| dc.subject |
Cibacron® Blue 3GA |
en |
| dc.subject |
Enzymes |
en |
| dc.subject |
GOX |
en |
| dc.subject |
L-Glutamate oxidase |
en |
| dc.subject |
L-glutamate oxidase |
en |
| dc.subject |
Mes |
en |
| dc.subject |
p-NHBenNHCOCOO-VBAR |
en |
| dc.subject |
Triazine dyes |
en |
| dc.subject |
VBAR |
en |
| dc.subject |
Vilmafix blue A-R |
en |
| dc.subject.other |
Adsorption |
en |
| dc.subject.other |
Aromatic compounds |
en |
| dc.subject.other |
Bacteria |
en |
| dc.subject.other |
Dyes |
en |
| dc.subject.other |
Enzymes |
en |
| dc.subject.other |
Organic acids |
en |
| dc.subject.other |
Affinity adsorbates |
en |
| dc.subject.other |
Batch systems |
en |
| dc.subject.other |
Affinity chromatography |
en |
| dc.subject.other |
adsorbent |
en |
| dc.subject.other |
agarose |
en |
| dc.subject.other |
carboxylase |
en |
| dc.subject.other |
dye |
en |
| dc.subject.other |
glutamate dehydrogenase |
en |
| dc.subject.other |
triazine |
en |
| dc.subject.other |
adsorption |
en |
| dc.subject.other |
adsorption kinetics |
en |
| dc.subject.other |
affinity chromatography |
en |
| dc.subject.other |
alpha chain |
en |
| dc.subject.other |
article |
en |
| dc.subject.other |
biomimetics |
en |
| dc.subject.other |
biotechnology |
en |
| dc.subject.other |
carboxylation |
en |
| dc.subject.other |
enzyme active site |
en |
| dc.subject.other |
enzyme immobilization |
en |
| dc.subject.other |
enzyme inhibition |
en |
| dc.subject.other |
enzyme kinetics |
en |
| dc.subject.other |
enzyme metabolism |
en |
| dc.subject.other |
enzyme purification |
en |
| dc.subject.other |
isotherm |
en |
| dc.subject.other |
ligand binding |
en |
| dc.subject.other |
model |
en |
| dc.subject.other |
molecular interaction |
en |
| dc.subject.other |
nonhuman |
en |
| dc.subject.other |
prediction |
en |
| dc.subject.other |
priority journal |
en |
| dc.subject.other |
protein cross linking |
en |
| dc.subject.other |
protein interaction |
en |
| dc.subject.other |
sequence homology |
en |
| dc.subject.other |
Streptomyces |
en |
| dc.subject.other |
Adsorption |
en |
| dc.subject.other |
Amino Acid Oxidoreductases |
en |
| dc.subject.other |
Amino Acid Sequence |
en |
| dc.subject.other |
Animals |
en |
| dc.subject.other |
Chromatography, Affinity |
en |
| dc.subject.other |
Chromatography, High Pressure Liquid |
en |
| dc.subject.other |
Hydrogen-Ion Concentration |
en |
| dc.subject.other |
Ligands |
en |
| dc.subject.other |
Models, Molecular |
en |
| dc.subject.other |
Molecular Sequence Data |
en |
| dc.subject.other |
Sequence Homology, Amino Acid |
en |
| dc.subject.other |
Triazines |
en |
| dc.subject.other |
Streptomyces |
en |
| dc.title |
Interaction of L-glutamate oxidase with triazine dyes: Selection of ligands for affinity chromatography |
en |
| heal.type |
journalArticle |
en |
| heal.identifier.primary |
10.1016/j.jchromb.2004.04.032 |
en |
| heal.publicationDate |
2004 |
en |
| heal.abstract |
Glutamate oxidase (GOX, EC 1.4.3.11) from Streptomyces catalyses the oxidation of L-glutamate to α-ketoglutarate. Its kinetic constants for L-glutamate were measured equal to 2 mM for Km and 85.8 s -1 for kcat. BLAST search and amino acid sequence alignments revealed low homology to other L-amino acid oxidases (18-38%). Threading methodology, homology modeling and CASTp analysis resulted in certain conclusions concerning the structure of catalytic α-subunit and led to the prediction of a binding pocket that provides favorable conditions of accommodating negatively charged aromatic ligands, such as sulphonated triazine dyes. Eleven commercial textile dyes and four biomimetic dyes or minodyes, bearing a ketocarboxylated-structure as their terminal biomimetic moiety, immobilized on cross-linked agarose gel. The resulted mini-library of affinity adsorbents was screened for binding and eluting L-glutamate oxidase activity. All but Cibacron® Blue 3GA (CB3GA) affinity adsorbents were able to bind GOX at pH 5.6. One immobilized minodye-ligand, bearing as its terminal biomimetic moiety p-aminobenzyloxanylic acid (BM1), displayed the higher affinity for GOX. Kinetic inhibition studies showed that BM1 inhibits GOX in a non-competitive manner with a Ki of 10.5 μM, indicating that the dye-enzyme interaction does not involve the substrate-binding site. Adsorption equilibrium data, obtained from a batch system with BM1 adsorbent, corresponded well to the Freundlich isotherm with a rate constant k of 2.7 mg1/2 ml1/2/g and Freundlich isotherm exponent n of 1. The interaction of GOX with the BM1 adsorbent was further studied with regards to adsorption and elution conditions. The results obtained were exploited in the development of a facile purification protocol for GOX, which led to 335-fold purification in a single step with high enzyme recovery (95%). The present purification procedure is the most efficient reported so far for L-glutamate oxidase. © 2004 Elsevier B.V. All rights reserved. |
en |
| heal.journalName |
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences |
en |
| dc.identifier.issue |
2 |
en |
| dc.identifier.volume |
807 |
en |
| dc.identifier.doi |
10.1016/j.jchromb.2004.04.032 |
en |
| dc.identifier.spage |
277 |
en |
| dc.identifier.epage |
285 |
en |