Cloning, purification and biochemical characterization of metallic-ions independent and thermoactive l-arabinose isomerase from the Bacillus stearothermophilus US100 strain.
Identifieur interne : 000838 ( Main/Exploration ); précédent : 000837; suivant : 000839Cloning, purification and biochemical characterization of metallic-ions independent and thermoactive l-arabinose isomerase from the Bacillus stearothermophilus US100 strain.
Auteurs : Moez Rhimi [Tunisie] ; Samir BejarSource :
- Biochimica et biophysica acta [ 0006-3002 ] ; 2006.
Descripteurs français
- KwdFr :
- Aldose-ketose isomerases (génétique), Aldose-ketose isomerases (isolement et purification), Chromatographie en phase liquide à haute performance, Cinétique, Clonage moléculaire, Cobalt (pharmacologie), Concentration en ions d'hydrogène, Données de séquences moléculaires, Geobacillus stearothermophilus (enzymologie), Geobacillus stearothermophilus (génétique), Manganèse (pharmacologie), Protéines recombinantes (isolement et purification), Stabilité enzymatique, Séquence d'acides aminés, Séquence nucléotidique, Température élevée.
- MESH :
- enzymologie : Geobacillus stearothermophilus.
- génétique : Aldose-ketose isomerases, Geobacillus stearothermophilus.
- isolement et purification : Aldose-ketose isomerases, Protéines recombinantes.
- pharmacologie : Cobalt, Manganèse.
- Chromatographie en phase liquide à haute performance, Cinétique, Clonage moléculaire, Concentration en ions d'hydrogène, Données de séquences moléculaires, Stabilité enzymatique, Séquence d'acides aminés, Séquence nucléotidique, Température élevée.
English descriptors
- KwdEn :
- Aldose-Ketose Isomerases (genetics), Aldose-Ketose Isomerases (isolation & purification), Amino Acid Sequence, Base Sequence, Chromatography, High Pressure Liquid, Cloning, Molecular, Cobalt (pharmacology), Enzyme Stability, Geobacillus stearothermophilus (enzymology), Geobacillus stearothermophilus (genetics), Hot Temperature, Hydrogen-Ion Concentration, Kinetics, Manganese (pharmacology), Molecular Sequence Data, Recombinant Proteins (isolation & purification).
- MESH :
- chemical , genetics : Aldose-Ketose Isomerases.
- chemical , isolation & purification : Aldose-Ketose Isomerases, Recombinant Proteins.
- chemical , pharmacology : Cobalt, Manganese.
- enzymology : Geobacillus stearothermophilus.
- genetics : Geobacillus stearothermophilus.
- Amino Acid Sequence, Base Sequence, Chromatography, High Pressure Liquid, Cloning, Molecular, Enzyme Stability, Hot Temperature, Hydrogen-Ion Concentration, Kinetics, Molecular Sequence Data.
Abstract
The araA gene encoding L-arabinose isomerase from Bacillus stearothermophilus US100 strain was cloned, sequenced and over-expressed in E. coli. This gene encodes a 496-amino acid protein with a calculated molecular weight of 56.161 kDa. Its amino acid sequence displays the highest identity with L-AI from Thermus sp. IM6501 (98%) and that of Geobacillus stearothermophilus T6 (97%). According to SDS-PAGE analysis, under reducing and non-reducing conditions, the recombinant enzyme has an apparent molecular weight of nearly 225 kDa, composed of four identical 56-kDa subunits. The L-AI US100 was optimally active at pH 7.5 and 80 degrees C. It was distinguishable by its behavior towards divalent ions. Indeed, the L-AI US100 activity and thermostability were totally independent for metallic ions until 65 degrees C. At temperatures above 65 degrees C, the enzyme was also independent for metallic ions for its activity but its thermostability was obviously improved in presence of only 0.2 mM Co2+ and 1 mM Mn2+. The V(max) values were calculated to be 41.3 U/mg for L-arabinose and 8.9 U/mg for D-galactose. Their catalytic efficiencies (k(cat)/K(m)) for l-arabinose and D-galactose were, respectively, 71.4 and 8.46 mM(-1) min(-1). L-AI US100 converted the d-galactose into D-tagatose with a high conversion rate of 48% after 7 h at 70 degrees C.
DOI: 10.1016/j.bbagen.2005.11.007
PubMed: 16386851
Affiliations:
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Le document en format XML
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Bejar</name></author></analytic><series><title level="j">Biochimica et biophysica acta</title><idno type="ISSN">0006-3002</idno><imprint><date when="2006" type="published">2006</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Aldose-Ketose Isomerases (genetics)</term><term>Aldose-Ketose Isomerases (isolation & purification)</term><term>Amino Acid Sequence</term><term>Base Sequence</term><term>Chromatography, High Pressure Liquid</term><term>Cloning, Molecular</term><term>Cobalt (pharmacology)</term><term>Enzyme Stability</term><term>Geobacillus stearothermophilus (enzymology)</term><term>Geobacillus stearothermophilus (genetics)</term><term>Hot Temperature</term><term>Hydrogen-Ion Concentration</term><term>Kinetics</term><term>Manganese (pharmacology)</term><term>Molecular Sequence Data</term><term>Recombinant Proteins (isolation & purification)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Aldose-ketose isomerases (génétique)</term><term>Aldose-ketose isomerases (isolement et purification)</term><term>Chromatographie en phase liquide à haute performance</term><term>Cinétique</term><term>Clonage moléculaire</term><term>Cobalt (pharmacologie)</term><term>Concentration en ions d'hydrogène</term><term>Données de séquences moléculaires</term><term>Geobacillus stearothermophilus (enzymologie)</term><term>Geobacillus stearothermophilus (génétique)</term><term>Manganèse (pharmacologie)</term><term>Protéines recombinantes (isolement et purification)</term><term>Stabilité enzymatique</term><term>Séquence d'acides aminés</term><term>Séquence nucléotidique</term><term>Température élevée</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Aldose-Ketose Isomerases</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Aldose-Ketose 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xml:lang="en"><term>Amino Acid Sequence</term><term>Base Sequence</term><term>Chromatography, High Pressure Liquid</term><term>Cloning, Molecular</term><term>Enzyme Stability</term><term>Hot Temperature</term><term>Hydrogen-Ion Concentration</term><term>Kinetics</term><term>Molecular Sequence Data</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Chromatographie en phase liquide à haute performance</term><term>Cinétique</term><term>Clonage moléculaire</term><term>Concentration en ions d'hydrogène</term><term>Données de séquences moléculaires</term><term>Stabilité enzymatique</term><term>Séquence d'acides aminés</term><term>Séquence nucléotidique</term><term>Température élevée</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The araA gene encoding L-arabinose isomerase from Bacillus stearothermophilus US100 strain was cloned, sequenced and over-expressed in E. coli. This gene encodes a 496-amino acid protein with a calculated molecular weight of 56.161 kDa. Its amino acid sequence displays the highest identity with L-AI from Thermus sp. IM6501 (98%) and that of Geobacillus stearothermophilus T6 (97%). According to SDS-PAGE analysis, under reducing and non-reducing conditions, the recombinant enzyme has an apparent molecular weight of nearly 225 kDa, composed of four identical 56-kDa subunits. The L-AI US100 was optimally active at pH 7.5 and 80 degrees C. It was distinguishable by its behavior towards divalent ions. Indeed, the L-AI US100 activity and thermostability were totally independent for metallic ions until 65 degrees C. At temperatures above 65 degrees C, the enzyme was also independent for metallic ions for its activity but its thermostability was obviously improved in presence of only 0.2 mM Co2+ and 1 mM Mn2+. The V(max) values were calculated to be 41.3 U/mg for L-arabinose and 8.9 U/mg for D-galactose. Their catalytic efficiencies (k(cat)/K(m)) for l-arabinose and D-galactose were, respectively, 71.4 and 8.46 mM(-1) min(-1). L-AI US100 converted the d-galactose into D-tagatose with a high conversion rate of 48% after 7 h at 70 degrees C.</div></front></TEI><affiliations><list><country><li>Tunisie</li></country></list><tree><noCountry><name sortKey="Bejar, Samir" sort="Bejar, Samir" uniqKey="Bejar S" first="Samir" last="Bejar">Samir Bejar</name></noCountry><country name="Tunisie"><noRegion><name sortKey="Rhimi, Moez" sort="Rhimi, Moez" uniqKey="Rhimi M" first="Moez" last="Rhimi">Moez Rhimi</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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