Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465
Autor(a) principal: | |
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Data de Publicação: | 2022 |
Outros Autores: | , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Brazilian Journal of Biology |
Texto Completo: | http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1519-69842022000100184 |
Resumo: | Abstract Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% β-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme. |
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Brazilian Journal of Biology |
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Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465amylaseGeobacilluscloningpropertiesin silicoAbstract Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% β-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.Instituto Internacional de Ecologia2022-01-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S1519-69842022000100184Brazilian Journal of Biology v.82 2022reponame:Brazilian Journal of Biologyinstname:Instituto Internacional de Ecologia (IIE)instacron:IIE10.1590/1519-6984.239449info:eu-repo/semantics/openAccessAl-Amri,A.Al-Ghamdi,M. A.Khan,J. A.Altayeb,H. N.Alsulami,H.Sajjad,M.Baothman,O. A.Nadeem,M. S.eng2021-06-01T00:00:00Zoai:scielo:S1519-69842022000100184Revistahttps://www.scielo.br/j/bjb/https://old.scielo.br/oai/scielo-oai.phpbjb@bjb.com.br||bjb@bjb.com.br1678-43751519-6984opendoar:2021-06-01T00:00Brazilian Journal of Biology - Instituto Internacional de Ecologia (IIE)false |
dc.title.none.fl_str_mv |
Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465 |
title |
Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465 |
spellingShingle |
Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465 Al-Amri,A. amylase Geobacillus cloning properties in silico |
title_short |
Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465 |
title_full |
Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465 |
title_fullStr |
Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465 |
title_full_unstemmed |
Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465 |
title_sort |
Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465 |
author |
Al-Amri,A. |
author_facet |
Al-Amri,A. Al-Ghamdi,M. A. Khan,J. A. Altayeb,H. N. Alsulami,H. Sajjad,M. Baothman,O. A. Nadeem,M. S. |
author_role |
author |
author2 |
Al-Ghamdi,M. A. Khan,J. A. Altayeb,H. N. Alsulami,H. Sajjad,M. Baothman,O. A. Nadeem,M. S. |
author2_role |
author author author author author author author |
dc.contributor.author.fl_str_mv |
Al-Amri,A. Al-Ghamdi,M. A. Khan,J. A. Altayeb,H. N. Alsulami,H. Sajjad,M. Baothman,O. A. Nadeem,M. S. |
dc.subject.por.fl_str_mv |
amylase Geobacillus cloning properties in silico |
topic |
amylase Geobacillus cloning properties in silico |
description |
Abstract Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% β-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme. |
publishDate |
2022 |
dc.date.none.fl_str_mv |
2022-01-01 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1519-69842022000100184 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1519-69842022000100184 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1590/1519-6984.239449 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
text/html |
dc.publisher.none.fl_str_mv |
Instituto Internacional de Ecologia |
publisher.none.fl_str_mv |
Instituto Internacional de Ecologia |
dc.source.none.fl_str_mv |
Brazilian Journal of Biology v.82 2022 reponame:Brazilian Journal of Biology instname:Instituto Internacional de Ecologia (IIE) instacron:IIE |
instname_str |
Instituto Internacional de Ecologia (IIE) |
instacron_str |
IIE |
institution |
IIE |
reponame_str |
Brazilian Journal of Biology |
collection |
Brazilian Journal of Biology |
repository.name.fl_str_mv |
Brazilian Journal of Biology - Instituto Internacional de Ecologia (IIE) |
repository.mail.fl_str_mv |
bjb@bjb.com.br||bjb@bjb.com.br |
_version_ |
1752129888797589504 |