Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria

Autores
Campos, Eleonora; Negro Alvarez, María José; Sabarís Di Lorenzo, Gonzalo Julián; González, Sergio Alberto; Rorig, Marcela; Talia, Paola; Grasso, Daniel Hector; Sáez, Felicia; Manzanares Secades, Paloma; Ballesteros Perdices, Mercedes; Cataldi, Ángel Adrián
Año de publicación
2013
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
The use of lignocellulosic biomass for second generation biofuels requires optimization of enzymatic breakdown of plant cell walls. In this work, cellulolytic bacteria were isolated from a native and two cultivated forest soil samples. Amplification of glycosyl hydrolases was attempted by using a low stringency-degenerate primer PCR strategy, using total soil DNA and bulk DNA pooled from positive colonies as template. A set of primers was designed based on Acidothermus cellulolyticus genome, by search of conserved domains of glycosyl hydrolases (GH) families of interest. Using this approach, a fragment containing an open reading frame (ORF) with 98% identity to a putative GH43 beta-xylosidase coding gene from Enterobacter cloacae was amplified and cloned. The full protein was expressed in Escherichia coli as N-terminal or C-terminal His-tagged fusions and purified under native conditions. Only N-terminal fusion protein, His-Xyl43, presented beta-xylosidase activity. On pNPX, optimal activity was achieved at pH 6 and 40°C and Km and Kcat values were 2.92mM and 1.32seg-1, respectively. Activity was also demonstrated on xylobiose (X2), with Km 17.8mM and Kcat 380s-1. These results demonstrated that Xyl43 is a functional beta-xylosidase and it is the first evidence of this activity for Enterobacter sp.
Fil: Campos, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina
Fil: Negro Alvarez, María José. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; España
Fil: Sabarís Di Lorenzo, Gonzalo Julián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina
Fil: González, Sergio Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina
Fil: Rorig, Marcela. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Suelos; Argentina
Fil: Talia, Paola. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina
Fil: Grasso, Daniel Hector. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Suelos; Argentina
Fil: Sáez, Felicia. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; España
Fil: Manzanares Secades, Paloma. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; España
Fil: Ballesteros Perdices, Mercedes. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; España
Fil: Cataldi, Ángel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina
Materia
BETA-XYLOSIDASE
ENTEROBACTER
GH43
LIGNOCELLULOSIC BIOFUELS
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:11336/85402
Acceder
id CONICETDig_b9d4c8f50d45b65a9f78e8ce47127eb5
oai_identifier_str oai:ri.conicet.gov.ar:11336/85402
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteriaCampos, EleonoraNegro Alvarez, María JoséSabarís Di Lorenzo, Gonzalo JuliánGonzález, Sergio AlbertoRorig, MarcelaTalia, PaolaGrasso, Daniel HectorSáez, FeliciaManzanares Secades, PalomaBallesteros Perdices, MercedesCataldi, Ángel AdriánBETA-XYLOSIDASEENTEROBACTERGH43LIGNOCELLULOSIC BIOFUELShttps://purl.org/becyt/ford/2.9https://purl.org/becyt/ford/2The use of lignocellulosic biomass for second generation biofuels requires optimization of enzymatic breakdown of plant cell walls. In this work, cellulolytic bacteria were isolated from a native and two cultivated forest soil samples. Amplification of glycosyl hydrolases was attempted by using a low stringency-degenerate primer PCR strategy, using total soil DNA and bulk DNA pooled from positive colonies as template. A set of primers was designed based on Acidothermus cellulolyticus genome, by search of conserved domains of glycosyl hydrolases (GH) families of interest. Using this approach, a fragment containing an open reading frame (ORF) with 98% identity to a putative GH43 beta-xylosidase coding gene from Enterobacter cloacae was amplified and cloned. The full protein was expressed in Escherichia coli as N-terminal or C-terminal His-tagged fusions and purified under native conditions. Only N-terminal fusion protein, His-Xyl43, presented beta-xylosidase activity. On pNPX, optimal activity was achieved at pH 6 and 40°C and Km and Kcat values were 2.92mM and 1.32seg-1, respectively. Activity was also demonstrated on xylobiose (X2), with Km 17.8mM and Kcat 380s-1. These results demonstrated that Xyl43 is a functional beta-xylosidase and it is the first evidence of this activity for Enterobacter sp.Fil: Campos, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Negro Alvarez, María José. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; EspañaFil: Sabarís Di Lorenzo, Gonzalo Julián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: González, Sergio Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Rorig, Marcela. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Suelos; ArgentinaFil: Talia, Paola. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Grasso, Daniel Hector. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Suelos; ArgentinaFil: Sáez, Felicia. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; EspañaFil: Manzanares Secades, Paloma. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; EspañaFil: Ballesteros Perdices, Mercedes. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; EspañaFil: Cataldi, Ángel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaElsevier Gmbh2013-05info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/85402Campos, Eleonora; Negro Alvarez, María José; Sabarís Di Lorenzo, Gonzalo Julián; González, Sergio Alberto; Rorig, Marcela; et al.; Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria; Elsevier Gmbh; Microbiological Research; 169; 2-3; 5-2013; 213-2200944-5013CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0944501313000906info:eu-repo/semantics/altIdentifier/doi/10.1016/j.micres.2013.06.004info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T09:59:21Zoai:ri.conicet.gov.ar:11336/85402instacron: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:34982025-09-03 09:59:21.775CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria
title Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria
spellingShingle Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria
Campos, Eleonora
BETA-XYLOSIDASE
ENTEROBACTER
GH43
LIGNOCELLULOSIC BIOFUELS
title_short Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria
title_full Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria
title_fullStr Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria
title_full_unstemmed Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria
title_sort Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria
dc.creator.none.fl_str_mv Campos, Eleonora
Negro Alvarez, María José
Sabarís Di Lorenzo, Gonzalo Julián
González, Sergio Alberto
Rorig, Marcela
Talia, Paola
Grasso, Daniel Hector
Sáez, Felicia
Manzanares Secades, Paloma
Ballesteros Perdices, Mercedes
Cataldi, Ángel Adrián
author Campos, Eleonora
author_facet Campos, Eleonora
Negro Alvarez, María José
Sabarís Di Lorenzo, Gonzalo Julián
González, Sergio Alberto
Rorig, Marcela
Talia, Paola
Grasso, Daniel Hector
Sáez, Felicia
Manzanares Secades, Paloma
Ballesteros Perdices, Mercedes
Cataldi, Ángel Adrián
author_role author
author2 Negro Alvarez, María José
Sabarís Di Lorenzo, Gonzalo Julián
González, Sergio Alberto
Rorig, Marcela
Talia, Paola
Grasso, Daniel Hector
Sáez, Felicia
Manzanares Secades, Paloma
Ballesteros Perdices, Mercedes
Cataldi, Ángel Adrián
author2_role author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv BETA-XYLOSIDASE
ENTEROBACTER
GH43
LIGNOCELLULOSIC BIOFUELS
topic BETA-XYLOSIDASE
ENTEROBACTER
GH43
LIGNOCELLULOSIC BIOFUELS
purl_subject.fl_str_mv https://purl.org/becyt/ford/2.9
https://purl.org/becyt/ford/2
dc.description.none.fl_txt_mv The use of lignocellulosic biomass for second generation biofuels requires optimization of enzymatic breakdown of plant cell walls. In this work, cellulolytic bacteria were isolated from a native and two cultivated forest soil samples. Amplification of glycosyl hydrolases was attempted by using a low stringency-degenerate primer PCR strategy, using total soil DNA and bulk DNA pooled from positive colonies as template. A set of primers was designed based on Acidothermus cellulolyticus genome, by search of conserved domains of glycosyl hydrolases (GH) families of interest. Using this approach, a fragment containing an open reading frame (ORF) with 98% identity to a putative GH43 beta-xylosidase coding gene from Enterobacter cloacae was amplified and cloned. The full protein was expressed in Escherichia coli as N-terminal or C-terminal His-tagged fusions and purified under native conditions. Only N-terminal fusion protein, His-Xyl43, presented beta-xylosidase activity. On pNPX, optimal activity was achieved at pH 6 and 40°C and Km and Kcat values were 2.92mM and 1.32seg-1, respectively. Activity was also demonstrated on xylobiose (X2), with Km 17.8mM and Kcat 380s-1. These results demonstrated that Xyl43 is a functional beta-xylosidase and it is the first evidence of this activity for Enterobacter sp.
Fil: Campos, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina
Fil: Negro Alvarez, María José. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; España
Fil: Sabarís Di Lorenzo, Gonzalo Julián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina
Fil: González, Sergio Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina
Fil: Rorig, Marcela. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Suelos; Argentina
Fil: Talia, Paola. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina
Fil: Grasso, Daniel Hector. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Suelos; Argentina
Fil: Sáez, Felicia. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; España
Fil: Manzanares Secades, Paloma. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; España
Fil: Ballesteros Perdices, Mercedes. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; España
Fil: Cataldi, Ángel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina
description The use of lignocellulosic biomass for second generation biofuels requires optimization of enzymatic breakdown of plant cell walls. In this work, cellulolytic bacteria were isolated from a native and two cultivated forest soil samples. Amplification of glycosyl hydrolases was attempted by using a low stringency-degenerate primer PCR strategy, using total soil DNA and bulk DNA pooled from positive colonies as template. A set of primers was designed based on Acidothermus cellulolyticus genome, by search of conserved domains of glycosyl hydrolases (GH) families of interest. Using this approach, a fragment containing an open reading frame (ORF) with 98% identity to a putative GH43 beta-xylosidase coding gene from Enterobacter cloacae was amplified and cloned. The full protein was expressed in Escherichia coli as N-terminal or C-terminal His-tagged fusions and purified under native conditions. Only N-terminal fusion protein, His-Xyl43, presented beta-xylosidase activity. On pNPX, optimal activity was achieved at pH 6 and 40°C and Km and Kcat values were 2.92mM and 1.32seg-1, respectively. Activity was also demonstrated on xylobiose (X2), with Km 17.8mM and Kcat 380s-1. These results demonstrated that Xyl43 is a functional beta-xylosidase and it is the first evidence of this activity for Enterobacter sp.
publishDate 2013
dc.date.none.fl_str_mv 2013-05
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/11336/85402
Campos, Eleonora; Negro Alvarez, María José; Sabarís Di Lorenzo, Gonzalo Julián; González, Sergio Alberto; Rorig, Marcela; et al.; Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria; Elsevier Gmbh; Microbiological Research; 169; 2-3; 5-2013; 213-220
0944-5013
CONICET Digital
CONICET
url http://hdl.handle.net/11336/85402
identifier_str_mv Campos, Eleonora; Negro Alvarez, María José; Sabarís Di Lorenzo, Gonzalo Julián; González, Sergio Alberto; Rorig, Marcela; et al.; Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria; Elsevier Gmbh; Microbiological Research; 169; 2-3; 5-2013; 213-220
0944-5013
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0944501313000906
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.micres.2013.06.004
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
dc.publisher.none.fl_str_mv Elsevier Gmbh
publisher.none.fl_str_mv Elsevier Gmbh
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 12.885934

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