Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria
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 lo...
| Main Authors: | , , , , , , , , , , |
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| Format: | Artículo |
| Language: | Inglés |
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Elsevier
2019
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| Online Access: | 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 |
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| author | 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_browse | Ballesteros Perdices, Mercedes Campos, Eleonora Cataldi, Angel Adrian Gonzalez, Sergio Alberto Grasso, Daniel Horacio Manzanares Secades, Paloma Negro Alvarez, María José Rorig, Marcela Laura Sabaris Di Lorenzo, Gonzalo Julián Sáez, Felicia Talia, Paola Mónica |
| 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_sort | Campos, Eleonora |
| collection | INTA Digital |
| 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. |
| format | Artículo |
| id | INTA4313 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2019 |
| publishDateRange | 2019 |
| publishDateSort | 2019 |
| publisher | Elsevier |
| publisherStr | Elsevier |
| record_format | dspace |
| spelling | INTA43132019-01-23T11:45:47Z Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria 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 Biocarburante Lignocelulosa Enterobacter Bacterias del Suelo Biofuels Lignocellulose Soil Bacteria Biocombustibles GH43 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 2019-01-23T11:42:11Z 2019-01-23T11:42:11Z 2014-03 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion 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 eng 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) application/pdf Elsevier Microbiological Research 169 (2–3) : 213-220 (February–March 2014) |
| spellingShingle | Biocarburante Lignocelulosa Enterobacter Bacterias del Suelo Biofuels Lignocellulose Soil Bacteria Biocombustibles GH43 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 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 |
| 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_short | 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 |
| topic | Biocarburante Lignocelulosa Enterobacter Bacterias del Suelo Biofuels Lignocellulose Soil Bacteria Biocombustibles GH43 |
| 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 |
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