Engineering the L-Arabinose Isomerase from Enterococcus Faecium for D-Tagatose Synthesis

Autores
de Souza, Marylane; Manzo, Ricardo Martín; Garcia, Jose Luis; Mammarella, Enrique José; Gonçalves, Luciana; Pessela, Benevides
Año de publicación
2017
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N-His-l-AI and C-His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C-His-l-AI was preferentially hexameric in solution, whereas N-His-l-AI was mainly monomeric. The specific activity of the N-His-l-AI at acidic pH was higher than that of C-His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg−1, respectively. However, C-His-l-AI was more active and stable at alkaline pH than N-His-l-AI. N-His-l-AI follows a Michaelis-Menten kinetic, whereas C-His-l-AI fitted to a sigmoidal saturation curve.
Fil: de Souza, Marylane. Universidade Federal de Ceará;
Fil: Manzo, Ricardo Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina
Fil: Garcia, Jose Luis. Higher Council for Scientific Research; España
Fil: Mammarella, Enrique José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina
Fil: Gonçalves, Luciana. Universidade Federal de Ceará;
Fil: Pessela, Benevides. Higher Council for Scientific Research; España
Materia
L-ARABINOSE ISOMERASE
RECOMBINANT DNA
AFFINITY PURIFICATION
D-TAGATOSE
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/33578
Acceder
id CONICETDig_35b6e65762ed7b13a63024f22d8cb30d
oai_identifier_str oai:ri.conicet.gov.ar:11336/33578
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Engineering the L-Arabinose Isomerase from Enterococcus Faecium for D-Tagatose Synthesisde Souza, MarylaneManzo, Ricardo MartínGarcia, Jose LuisMammarella, Enrique JoséGonçalves, LucianaPessela, BenevidesL-ARABINOSE ISOMERASERECOMBINANT DNAAFFINITY PURIFICATIOND-TAGATOSEhttps://purl.org/becyt/ford/2.9https://purl.org/becyt/ford/2l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N-His-l-AI and C-His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C-His-l-AI was preferentially hexameric in solution, whereas N-His-l-AI was mainly monomeric. The specific activity of the N-His-l-AI at acidic pH was higher than that of C-His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg−1, respectively. However, C-His-l-AI was more active and stable at alkaline pH than N-His-l-AI. N-His-l-AI follows a Michaelis-Menten kinetic, whereas C-His-l-AI fitted to a sigmoidal saturation curve.Fil: de Souza, Marylane. Universidade Federal de Ceará;Fil: Manzo, Ricardo Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Garcia, Jose Luis. Higher Council for Scientific Research; EspañaFil: Mammarella, Enrique José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Gonçalves, Luciana. Universidade Federal de Ceará;Fil: Pessela, Benevides. Higher Council for Scientific Research; EspañaMolecular Diversity Preservation International2017-12info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/33578Garcia, Jose Luis; Mammarella, Enrique José; Gonçalves, Luciana; Manzo, Ricardo Martín; de Souza, Marylane; Pessela, Benevides; et al.; Engineering the L-Arabinose Isomerase from Enterococcus Faecium for D-Tagatose Synthesis; Molecular Diversity Preservation International; Molecules; 22; 12; 12-2017; 21641420-3049CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://www.mdpi.com/1420-3049/22/12/2164info:eu-repo/semantics/altIdentifier/doi/10.3390/molecules22122164info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:22:58Zoai:ri.conicet.gov.ar:11336/33578instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-22 11:22:58.451CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Engineering the L-Arabinose Isomerase from Enterococcus Faecium for D-Tagatose Synthesis
title Engineering the L-Arabinose Isomerase from Enterococcus Faecium for D-Tagatose Synthesis
spellingShingle Engineering the L-Arabinose Isomerase from Enterococcus Faecium for D-Tagatose Synthesis
de Souza, Marylane
L-ARABINOSE ISOMERASE
RECOMBINANT DNA
AFFINITY PURIFICATION
D-TAGATOSE
title_short Engineering the L-Arabinose Isomerase from Enterococcus Faecium for D-Tagatose Synthesis
title_full Engineering the L-Arabinose Isomerase from Enterococcus Faecium for D-Tagatose Synthesis
title_fullStr Engineering the L-Arabinose Isomerase from Enterococcus Faecium for D-Tagatose Synthesis
title_full_unstemmed Engineering the L-Arabinose Isomerase from Enterococcus Faecium for D-Tagatose Synthesis
title_sort Engineering the L-Arabinose Isomerase from Enterococcus Faecium for D-Tagatose Synthesis
dc.creator.none.fl_str_mv de Souza, Marylane
Manzo, Ricardo Martín
Garcia, Jose Luis
Mammarella, Enrique José
Gonçalves, Luciana
Pessela, Benevides
author de Souza, Marylane
author_facet de Souza, Marylane
Manzo, Ricardo Martín
Garcia, Jose Luis
Mammarella, Enrique José
Gonçalves, Luciana
Pessela, Benevides
author_role author
author2 Manzo, Ricardo Martín
Garcia, Jose Luis
Mammarella, Enrique José
Gonçalves, Luciana
Pessela, Benevides
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv L-ARABINOSE ISOMERASE
RECOMBINANT DNA
AFFINITY PURIFICATION
D-TAGATOSE
topic L-ARABINOSE ISOMERASE
RECOMBINANT DNA
AFFINITY PURIFICATION
D-TAGATOSE
purl_subject.fl_str_mv https://purl.org/becyt/ford/2.9
https://purl.org/becyt/ford/2
dc.description.none.fl_txt_mv l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N-His-l-AI and C-His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C-His-l-AI was preferentially hexameric in solution, whereas N-His-l-AI was mainly monomeric. The specific activity of the N-His-l-AI at acidic pH was higher than that of C-His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg−1, respectively. However, C-His-l-AI was more active and stable at alkaline pH than N-His-l-AI. N-His-l-AI follows a Michaelis-Menten kinetic, whereas C-His-l-AI fitted to a sigmoidal saturation curve.
Fil: de Souza, Marylane. Universidade Federal de Ceará;
Fil: Manzo, Ricardo Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina
Fil: Garcia, Jose Luis. Higher Council for Scientific Research; España
Fil: Mammarella, Enrique José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina
Fil: Gonçalves, Luciana. Universidade Federal de Ceará;
Fil: Pessela, Benevides. Higher Council for Scientific Research; España
description l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N-His-l-AI and C-His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C-His-l-AI was preferentially hexameric in solution, whereas N-His-l-AI was mainly monomeric. The specific activity of the N-His-l-AI at acidic pH was higher than that of C-His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg−1, respectively. However, C-His-l-AI was more active and stable at alkaline pH than N-His-l-AI. N-His-l-AI follows a Michaelis-Menten kinetic, whereas C-His-l-AI fitted to a sigmoidal saturation curve.
publishDate 2017
dc.date.none.fl_str_mv 2017-12
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/33578
Garcia, Jose Luis; Mammarella, Enrique José; Gonçalves, Luciana; Manzo, Ricardo Martín; de Souza, Marylane; Pessela, Benevides; et al.; Engineering the L-Arabinose Isomerase from Enterococcus Faecium for D-Tagatose Synthesis; Molecular Diversity Preservation International; Molecules; 22; 12; 12-2017; 2164
1420-3049
CONICET Digital
CONICET
url http://hdl.handle.net/11336/33578
identifier_str_mv Garcia, Jose Luis; Mammarella, Enrique José; Gonçalves, Luciana; Manzo, Ricardo Martín; de Souza, Marylane; Pessela, Benevides; et al.; Engineering the L-Arabinose Isomerase from Enterococcus Faecium for D-Tagatose Synthesis; Molecular Diversity Preservation International; Molecules; 22; 12; 12-2017; 2164
1420-3049
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://www.mdpi.com/1420-3049/22/12/2164
info:eu-repo/semantics/altIdentifier/doi/10.3390/molecules22122164
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Molecular Diversity Preservation International
publisher.none.fl_str_mv Molecular Diversity Preservation International
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
_version_ 1846781753061015552
score 12.982451

Publicaciones similares