Purification of an l-arabinose isomerase from Enterococcus faecium DBFIQ E36 employing a biospecific affinity strategy

Autores
Torres, Pedro; Manzo, Ricardo Martín; Rubiolo, Amelia Catalina; Batista Viera, Francisco; Mammarella, Enrique José
Año de publicación
2014
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
l-Arabinose isomerase is an intracellular enzyme that can convert l-arabinose to l-ribulose and d-galactose to d-tagatose, a promising but rare nutraceutical. Most of l-arabinose isomerases purified upto date employed the combination between DNA recombinant technology and affinity chromatographybased on poly-histidine tail recognition, but few of the enzymes were obtained and purified in a non-recombinant way. For these reasons, a specific affinity bioadsorbent containing l-arabitol as ligand, acompetitive inhibitor of the enzyme, was designed and synthesized for achieving pure preparations ofthe enzyme l-arabinose isomerase from wild-type Enterococcus faecium DBFIQ E36 strain, isolated fromraw cow milk.The two-step purification procedure consisted in fractionation by ammonium sulphate precipitationfollowed by affinity chromatography with obtained bioadsorbent, allowing the purification, to elec-trophoretical homogeneity, of target enzyme. Characterization studies were performed with purifiedl-arabinose isomerase in order to increase knowledge of their physicochemical properties. In this sense,enzyme exhibited an optimum temperature of 50?C and optimum pH of 7.0, maintaining good sta-bility in the ranges 20–45?C and pH 6.5–8. Kiwere calculated, employing d-galactose as substrate, forl-arabitol and l-ribitol, achieving values of 7.9 mM and 183 mM, respectively. Kmand Vmaxvalues obtainedwere 35 mM and 81 U mg-1at 50?C, respectively. Mass spectrometry assay revealed a 48 kDa monomerwhereas gel permeation chromatography achieved a 187 kDa molecular weight for native enzyme. Finally,2D-electrophoresis and isoelectrofocusing analysis revealed an isoelectric point value of 3.80. Resultshave unveiled both an acidic nature and promising properties for l-arabinose isomerase isolated from E.faecium DBFIQ E36.
Fil: Torres, Pedro. Universidad de la Republica; Uruguay
Fil: Manzo, Ricardo Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentina
Fil: Rubiolo, Amelia Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentina
Fil: Batista Viera, Francisco. Universidad de la Republica; Uruguay
Fil: Mammarella, Enrique José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentina
Materia
L-Arabinose Isomerase
D-Tagatose D-Galactos
Affinity Chromatography
Enterococcus Faecium Strain
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:11336/9194
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/9194
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Purification of an l-arabinose isomerase from Enterococcus faecium DBFIQ E36 employing a biospecific affinity strategyTorres, PedroManzo, Ricardo MartínRubiolo, Amelia CatalinaBatista Viera, FranciscoMammarella, Enrique JoséL-Arabinose IsomeraseD-Tagatose D-GalactosAffinity ChromatographyEnterococcus Faecium Strainhttps://purl.org/becyt/ford/2.9https://purl.org/becyt/ford/2l-Arabinose isomerase is an intracellular enzyme that can convert l-arabinose to l-ribulose and d-galactose to d-tagatose, a promising but rare nutraceutical. Most of l-arabinose isomerases purified upto date employed the combination between DNA recombinant technology and affinity chromatographybased on poly-histidine tail recognition, but few of the enzymes were obtained and purified in a non-recombinant way. For these reasons, a specific affinity bioadsorbent containing l-arabitol as ligand, acompetitive inhibitor of the enzyme, was designed and synthesized for achieving pure preparations ofthe enzyme l-arabinose isomerase from wild-type Enterococcus faecium DBFIQ E36 strain, isolated fromraw cow milk.The two-step purification procedure consisted in fractionation by ammonium sulphate precipitationfollowed by affinity chromatography with obtained bioadsorbent, allowing the purification, to elec-trophoretical homogeneity, of target enzyme. Characterization studies were performed with purifiedl-arabinose isomerase in order to increase knowledge of their physicochemical properties. In this sense,enzyme exhibited an optimum temperature of 50?C and optimum pH of 7.0, maintaining good sta-bility in the ranges 20–45?C and pH 6.5–8. Kiwere calculated, employing d-galactose as substrate, forl-arabitol and l-ribitol, achieving values of 7.9 mM and 183 mM, respectively. Kmand Vmaxvalues obtainedwere 35 mM and 81 U mg-1at 50?C, respectively. Mass spectrometry assay revealed a 48 kDa monomerwhereas gel permeation chromatography achieved a 187 kDa molecular weight for native enzyme. Finally,2D-electrophoresis and isoelectrofocusing analysis revealed an isoelectric point value of 3.80. Resultshave unveiled both an acidic nature and promising properties for l-arabinose isomerase isolated from E.faecium DBFIQ E36.Fil: Torres, Pedro. Universidad de la Republica; UruguayFil: Manzo, Ricardo Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); ArgentinaFil: Rubiolo, Amelia Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); ArgentinaFil: Batista Viera, Francisco. Universidad de la Republica; UruguayFil: Mammarella, Enrique José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); ArgentinaElsevier Science2014-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/9194Torres, Pedro; Manzo, Ricardo Martín; Rubiolo, Amelia Catalina; Batista Viera, Francisco; Mammarella, Enrique José; Purification of an l-arabinose isomerase from Enterococcus faecium DBFIQ E36 employing a biospecific affinity strategy; Elsevier Science; Journal Of Molecular Catalysis B: Enzymatic; 102; 3-2014; 99-1051381-1177enginfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.molcatb.2014.01.023info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S1381117714000344info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:33:21Zoai:ri.conicet.gov.ar:11336/9194instacron: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:34982025-09-29 09:33:22.155CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Purification of an l-arabinose isomerase from Enterococcus faecium DBFIQ E36 employing a biospecific affinity strategy
title Purification of an l-arabinose isomerase from Enterococcus faecium DBFIQ E36 employing a biospecific affinity strategy
spellingShingle Purification of an l-arabinose isomerase from Enterococcus faecium DBFIQ E36 employing a biospecific affinity strategy
Torres, Pedro
L-Arabinose Isomerase
D-Tagatose D-Galactos
Affinity Chromatography
Enterococcus Faecium Strain
title_short Purification of an l-arabinose isomerase from Enterococcus faecium DBFIQ E36 employing a biospecific affinity strategy
title_full Purification of an l-arabinose isomerase from Enterococcus faecium DBFIQ E36 employing a biospecific affinity strategy
title_fullStr Purification of an l-arabinose isomerase from Enterococcus faecium DBFIQ E36 employing a biospecific affinity strategy
title_full_unstemmed Purification of an l-arabinose isomerase from Enterococcus faecium DBFIQ E36 employing a biospecific affinity strategy
title_sort Purification of an l-arabinose isomerase from Enterococcus faecium DBFIQ E36 employing a biospecific affinity strategy
dc.creator.none.fl_str_mv Torres, Pedro
Manzo, Ricardo Martín
Rubiolo, Amelia Catalina
Batista Viera, Francisco
Mammarella, Enrique José
author Torres, Pedro
author_facet Torres, Pedro
Manzo, Ricardo Martín
Rubiolo, Amelia Catalina
Batista Viera, Francisco
Mammarella, Enrique José
author_role author
author2 Manzo, Ricardo Martín
Rubiolo, Amelia Catalina
Batista Viera, Francisco
Mammarella, Enrique José
author2_role author
author
author
author
dc.subject.none.fl_str_mv L-Arabinose Isomerase
D-Tagatose D-Galactos
Affinity Chromatography
Enterococcus Faecium Strain
topic L-Arabinose Isomerase
D-Tagatose D-Galactos
Affinity Chromatography
Enterococcus Faecium Strain
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 is an intracellular enzyme that can convert l-arabinose to l-ribulose and d-galactose to d-tagatose, a promising but rare nutraceutical. Most of l-arabinose isomerases purified upto date employed the combination between DNA recombinant technology and affinity chromatographybased on poly-histidine tail recognition, but few of the enzymes were obtained and purified in a non-recombinant way. For these reasons, a specific affinity bioadsorbent containing l-arabitol as ligand, acompetitive inhibitor of the enzyme, was designed and synthesized for achieving pure preparations ofthe enzyme l-arabinose isomerase from wild-type Enterococcus faecium DBFIQ E36 strain, isolated fromraw cow milk.The two-step purification procedure consisted in fractionation by ammonium sulphate precipitationfollowed by affinity chromatography with obtained bioadsorbent, allowing the purification, to elec-trophoretical homogeneity, of target enzyme. Characterization studies were performed with purifiedl-arabinose isomerase in order to increase knowledge of their physicochemical properties. In this sense,enzyme exhibited an optimum temperature of 50?C and optimum pH of 7.0, maintaining good sta-bility in the ranges 20–45?C and pH 6.5–8. Kiwere calculated, employing d-galactose as substrate, forl-arabitol and l-ribitol, achieving values of 7.9 mM and 183 mM, respectively. Kmand Vmaxvalues obtainedwere 35 mM and 81 U mg-1at 50?C, respectively. Mass spectrometry assay revealed a 48 kDa monomerwhereas gel permeation chromatography achieved a 187 kDa molecular weight for native enzyme. Finally,2D-electrophoresis and isoelectrofocusing analysis revealed an isoelectric point value of 3.80. Resultshave unveiled both an acidic nature and promising properties for l-arabinose isomerase isolated from E.faecium DBFIQ E36.
Fil: Torres, Pedro. Universidad de la Republica; Uruguay
Fil: Manzo, Ricardo Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentina
Fil: Rubiolo, Amelia Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentina
Fil: Batista Viera, Francisco. Universidad de la Republica; Uruguay
Fil: Mammarella, Enrique José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentina
description l-Arabinose isomerase is an intracellular enzyme that can convert l-arabinose to l-ribulose and d-galactose to d-tagatose, a promising but rare nutraceutical. Most of l-arabinose isomerases purified upto date employed the combination between DNA recombinant technology and affinity chromatographybased on poly-histidine tail recognition, but few of the enzymes were obtained and purified in a non-recombinant way. For these reasons, a specific affinity bioadsorbent containing l-arabitol as ligand, acompetitive inhibitor of the enzyme, was designed and synthesized for achieving pure preparations ofthe enzyme l-arabinose isomerase from wild-type Enterococcus faecium DBFIQ E36 strain, isolated fromraw cow milk.The two-step purification procedure consisted in fractionation by ammonium sulphate precipitationfollowed by affinity chromatography with obtained bioadsorbent, allowing the purification, to elec-trophoretical homogeneity, of target enzyme. Characterization studies were performed with purifiedl-arabinose isomerase in order to increase knowledge of their physicochemical properties. In this sense,enzyme exhibited an optimum temperature of 50?C and optimum pH of 7.0, maintaining good sta-bility in the ranges 20–45?C and pH 6.5–8. Kiwere calculated, employing d-galactose as substrate, forl-arabitol and l-ribitol, achieving values of 7.9 mM and 183 mM, respectively. Kmand Vmaxvalues obtainedwere 35 mM and 81 U mg-1at 50?C, respectively. Mass spectrometry assay revealed a 48 kDa monomerwhereas gel permeation chromatography achieved a 187 kDa molecular weight for native enzyme. Finally,2D-electrophoresis and isoelectrofocusing analysis revealed an isoelectric point value of 3.80. Resultshave unveiled both an acidic nature and promising properties for l-arabinose isomerase isolated from E.faecium DBFIQ E36.
publishDate 2014
dc.date.none.fl_str_mv 2014-03
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/11336/9194
Torres, Pedro; Manzo, Ricardo Martín; Rubiolo, Amelia Catalina; Batista Viera, Francisco; Mammarella, Enrique José; Purification of an l-arabinose isomerase from Enterococcus faecium DBFIQ E36 employing a biospecific affinity strategy; Elsevier Science; Journal Of Molecular Catalysis B: Enzymatic; 102; 3-2014; 99-105
1381-1177
url http://hdl.handle.net/11336/9194
identifier_str_mv Torres, Pedro; Manzo, Ricardo Martín; Rubiolo, Amelia Catalina; Batista Viera, Francisco; Mammarella, Enrique José; Purification of an l-arabinose isomerase from Enterococcus faecium DBFIQ E36 employing a biospecific affinity strategy; Elsevier Science; Journal Of Molecular Catalysis B: Enzymatic; 102; 3-2014; 99-105
1381-1177
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1016/j.molcatb.2014.01.023
info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S1381117714000344
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
application/pdf
dc.publisher.none.fl_str_mv Elsevier Science
publisher.none.fl_str_mv Elsevier Science
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
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score 13.070432

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