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; Sabaris Di Lorenzo, Gonzalo Julián; Gonzalez, Sergio Alberto; Rorig, Marcela Laura; Talia, Paola Mónica; Grasso, Daniel Horacio; Sáez, Felicia; Manzanares Secades, Paloma; Ballesteros Perdices, Mercedes; Cataldi, Angel Adrian
Año de publicación
2014
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 2.92 mM and 1.32 seg−1, respectively. Activity was also demonstrated on xylobiose (X2), with Km 17.8 mM and Kcat 380 s−1. These results demonstrated that Xyl43 is a functional beta-xylosidase and it is the first evidence of this activity for Enterobacter sp.
Instituto de Biotecnología
Fil: Campos, Eleonora. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina
Fil: Negro Alvarez, María José. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT). Unidad Biocarburantes; España
Fil: Sabaris Di Lorenzo, Gonzalo Julián. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina
Fil: Gonzalez, Sergio Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina
Fil: Rorig, Marcela Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Suelos; Argentina
Fil: Talia, Paola Mónica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina
Fil: Grasso, Daniel Horacio. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Suelos; Argentina
Fil: Sáez, Felicia. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT). Unidad Biocarburantes; España
Fil: Manzanares Secades, Paloma. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT). Unidad Biocarburantes; España
Fil: Ballesteros Perdices, Mercedes. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT). Unidad Biocarburantes; España
Fil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina
Fuente
Microbiological Research 169 (2–3) : 213-220 (February–March 2014)
Materia
Biocarburante
Lignocelulosa
Enterobacter
Bacterias del Suelo
Biofuels
Lignocellulose
Soil Bacteria
Biocombustibles
GH43
Nivel de accesibilidad
acceso abierto
Condiciones de uso
http://creativecommons.org/licenses/by-nc-sa/4.0/
Repositorio
INTA Digital (INTA)
Institución
Instituto Nacional de Tecnología Agropecuaria
OAI Identificador
oai:localhost:20.500.12123/4313
Acceder
id INTADig_ec9557b22df9e36df4c8539e6754f21a
oai_identifier_str oai:localhost:20.500.12123/4313
network_acronym_str INTADig
repository_id_str l
network_name_str INTA Digital (INTA)
spelling Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteriaCampos, EleonoraNegro Alvarez, María JoséSabaris Di Lorenzo, Gonzalo JuliánGonzalez, Sergio AlbertoRorig, Marcela LauraTalia, Paola MónicaGrasso, Daniel HoracioSáez, FeliciaManzanares Secades, PalomaBallesteros Perdices, MercedesCataldi, Angel AdrianBiocarburanteLignocelulosaEnterobacterBacterias del SueloBiofuelsLignocelluloseSoil BacteriaBiocombustiblesGH43The 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 2.92 mM and 1.32 seg−1, respectively. Activity was also demonstrated on xylobiose (X2), with Km 17.8 mM and Kcat 380 s−1. These results demonstrated that Xyl43 is a functional beta-xylosidase and it is the first evidence of this activity for Enterobacter sp.Instituto de BiotecnologíaFil: Campos, Eleonora. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Negro Alvarez, María José. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT). Unidad Biocarburantes; EspañaFil: Sabaris Di Lorenzo, Gonzalo Julián. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Gonzalez, Sergio Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Rorig, Marcela Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Suelos; ArgentinaFil: Talia, Paola Mónica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Grasso, Daniel Horacio. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Suelos; ArgentinaFil: Sáez, Felicia. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT). Unidad Biocarburantes; EspañaFil: Manzanares Secades, Paloma. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT). Unidad Biocarburantes; EspañaFil: Ballesteros Perdices, Mercedes. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT). Unidad Biocarburantes; EspañaFil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaElsevier2019-01-23T11:42:11Z2019-01-23T11:42:11Z2014-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfhttps://www.sciencedirect.com/science/article/pii/S0944501313000906http://hdl.handle.net/20.500.12123/43130944-50131618-0623https://doi.org/10.1016/j.micres.2013.06.004Microbiological Research 169 (2–3) : 213-220 (February–March 2014)reponame:INTA Digital (INTA)instname:Instituto Nacional de Tecnología Agropecuariaenginfo:eu-repo/semantics/openAccesshttp://creativecommons.org/licenses/by-nc-sa/4.0/Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)2025-09-29T13:44:33Zoai:localhost:20.500.12123/4313instacron:INTAInstitucionalhttp://repositorio.inta.gob.ar/Organismo científico-tecnológicoNo correspondehttp://repositorio.inta.gob.ar/oai/requesttripaldi.nicolas@inta.gob.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:l2025-09-29 13:44:34.067INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuariafalse
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
Biocarburante
Lignocelulosa
Enterobacter
Bacterias del Suelo
Biofuels
Lignocellulose
Soil Bacteria
Biocombustibles
GH43
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
Sabaris Di Lorenzo, Gonzalo Julián
Gonzalez, Sergio Alberto
Rorig, Marcela Laura
Talia, Paola Mónica
Grasso, Daniel Horacio
Sáez, Felicia
Manzanares Secades, Paloma
Ballesteros Perdices, Mercedes
Cataldi, Angel Adrian
author Campos, Eleonora
author_facet Campos, Eleonora
Negro Alvarez, María José
Sabaris Di Lorenzo, Gonzalo Julián
Gonzalez, Sergio Alberto
Rorig, Marcela Laura
Talia, Paola Mónica
Grasso, Daniel Horacio
Sáez, Felicia
Manzanares Secades, Paloma
Ballesteros Perdices, Mercedes
Cataldi, Angel Adrian
author_role author
author2 Negro Alvarez, María José
Sabaris Di Lorenzo, Gonzalo Julián
Gonzalez, Sergio Alberto
Rorig, Marcela Laura
Talia, Paola Mónica
Grasso, Daniel Horacio
Sáez, Felicia
Manzanares Secades, Paloma
Ballesteros Perdices, Mercedes
Cataldi, Angel Adrian
author2_role author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Biocarburante
Lignocelulosa
Enterobacter
Bacterias del Suelo
Biofuels
Lignocellulose
Soil Bacteria
Biocombustibles
GH43
topic Biocarburante
Lignocelulosa
Enterobacter
Bacterias del Suelo
Biofuels
Lignocellulose
Soil Bacteria
Biocombustibles
GH43
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 2.92 mM and 1.32 seg−1, respectively. Activity was also demonstrated on xylobiose (X2), with Km 17.8 mM and Kcat 380 s−1. These results demonstrated that Xyl43 is a functional beta-xylosidase and it is the first evidence of this activity for Enterobacter sp.
Instituto de Biotecnología
Fil: Campos, Eleonora. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina
Fil: Negro Alvarez, María José. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT). Unidad Biocarburantes; España
Fil: Sabaris Di Lorenzo, Gonzalo Julián. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina
Fil: Gonzalez, Sergio Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina
Fil: Rorig, Marcela Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Suelos; Argentina
Fil: Talia, Paola Mónica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina
Fil: Grasso, Daniel Horacio. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Suelos; Argentina
Fil: Sáez, Felicia. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT). Unidad Biocarburantes; España
Fil: Manzanares Secades, Paloma. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT). Unidad Biocarburantes; España
Fil: Ballesteros Perdices, Mercedes. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT). Unidad Biocarburantes; España
Fil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). 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 2.92 mM and 1.32 seg−1, respectively. Activity was also demonstrated on xylobiose (X2), with Km 17.8 mM and Kcat 380 s−1. These results demonstrated that Xyl43 is a functional beta-xylosidase and it is the first evidence of this activity for Enterobacter sp.
publishDate 2014
dc.date.none.fl_str_mv 2014-03
2019-01-23T11:42:11Z
2019-01-23T11:42:11Z
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 https://www.sciencedirect.com/science/article/pii/S0944501313000906
http://hdl.handle.net/20.500.12123/4313
0944-5013
1618-0623
https://doi.org/10.1016/j.micres.2013.06.004
url https://www.sciencedirect.com/science/article/pii/S0944501313000906
http://hdl.handle.net/20.500.12123/4313
https://doi.org/10.1016/j.micres.2013.06.004
identifier_str_mv 0944-5013
1618-0623
dc.language.none.fl_str_mv eng
language eng
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
eu_rights_str_mv openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv Microbiological Research 169 (2–3) : 213-220 (February–March 2014)
reponame:INTA Digital (INTA)
instname:Instituto Nacional de Tecnología Agropecuaria
reponame_str INTA Digital (INTA)
collection INTA Digital (INTA)
instname_str Instituto Nacional de Tecnología Agropecuaria
repository.name.fl_str_mv INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuaria
repository.mail.fl_str_mv tripaldi.nicolas@inta.gob.ar
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score 12.559606

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