Differential extracellular enzymes expression by three Paenibacillus strains using gel-free proteomics análisis
- Autores
- Di Marco, Enzo; Callegari, Eduardo Alberto; Villegas, Liliana Beatriz; Martinez, Maria Alejandra
- Año de publicación
- 2017
- Idioma
- inglés
- Tipo de recurso
- documento de conferencia
- Estado
- versión publicada
- Descripción
- Three Paenibacillus strains, identified according to their 16S rDNA gene sequence and named as AR247, AR460-1 AR489, were selecteddue to their ability to produce glycoside hydrolases (GH) for second generation ethanol and other biotechnological applications. Theassessment of extracellular enzyme production was previously approached by utilizing a mineral-based medium, MM0.2, added withagricultural by-products. These substrates are low-cost and abundantly available carbon sources for biotechnological purposes. Among thetested carbon sources, an alkali pretreated sugarcane bagasse (OH-SCB) was the one that better promoted the production of extracellularxylanases for all strains. The aim of this work was to study the differential extracellular enzymes expression by these strains through gelfree proteomic analysis method.Firstly, crude extracts were obtained by centrifugation after 72 h of cultivation in MM0.2 - OH-SCB 1% medium. Then, samples wereconcentrated by lyophilization and digested by using trypsin for further mass spectrometry analysis. Tryptic peptides obtained wereanalyzed using 2D nano-Ultra Performance Liquid Chromatography, coupled to tandem mass spectrometry . Bioinformatics analysis forprotein identification was performed by searching against Swiss-Prot database, using Mascot server and ProteoIQ v2.8.Peptide summary report provided by Mascot evidenced hemicellulases which are active not only over β-1,4-linkages of xylose unitsbut also on substituents of xylan. An endo-β-1,4-xylanase with 20.1 kDa molecular weight and 9.2 isoelectric point (pI) was foundexclusively in crude extract samples of strain AR247. This enzyme, identified as a GH11, was also detected as a band between 18-20kDa in zymograms and showed a pairwise similarity of ≥ 99 with an homologous from Paenibacillus sp. Y412MC10. In addition, asecond β-1,4-xylanase was identified in the secretome samples, yet not detectable through zymography, from both AR247 and AR489strains. It was of 140.9 kDa, 4.7 pI (potential GH10), and showed to be closely related to a β-1,4-xylanase from P. glucanolyticus.Moreover, additional extracellular xylanase were found, which were related to a GH30 detected in P. favisporus and to two GH25 fromP. polymyxa. Other proteins related to xylan utilization identified were: S?layer protein; sugar ABC transporters and sequences fromsubstrate-binding proteins towards beta glucans. On the other hand, strains AR460-1 and AR489, revealed sequences correspondingto a carbohydrate binding module type CBM54 and an ABC transporter protein. Finally, a chitosanase belonging to GH8 family wasonly found in strain AR489.In conclusion gel-free proteomics analysis proved to be a useful methodology to achieve a widespread knowledge of the enzymaticrepetory contributing to better quality and quantity of results.
Fil: Di Marco, Enzo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina
Fil: Callegari, Eduardo Alberto. University of South Dakota; Estados Unidos
Fil: Villegas, Liliana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Química de San Luis. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Química de San Luis; Argentina
Fil: Martinez, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnología; Argentina
XII Congreso Argentino de Microbiología General
San Miguel de Tucumán
Argentina
Sociedad Argentina de Microbiologia General - Materia
-
Paenibacillus
Proteomics
Xylan - Nivel de accesibilidad
- acceso abierto
- Condiciones de uso
- https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
- Repositorio
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/243523
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Differential extracellular enzymes expression by three Paenibacillus strains using gel-free proteomics análisisDi Marco, EnzoCallegari, Eduardo AlbertoVillegas, Liliana BeatrizMartinez, Maria AlejandraPaenibacillusProteomicsXylanhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Three Paenibacillus strains, identified according to their 16S rDNA gene sequence and named as AR247, AR460-1 AR489, were selecteddue to their ability to produce glycoside hydrolases (GH) for second generation ethanol and other biotechnological applications. Theassessment of extracellular enzyme production was previously approached by utilizing a mineral-based medium, MM0.2, added withagricultural by-products. These substrates are low-cost and abundantly available carbon sources for biotechnological purposes. Among thetested carbon sources, an alkali pretreated sugarcane bagasse (OH-SCB) was the one that better promoted the production of extracellularxylanases for all strains. The aim of this work was to study the differential extracellular enzymes expression by these strains through gelfree proteomic analysis method.Firstly, crude extracts were obtained by centrifugation after 72 h of cultivation in MM0.2 - OH-SCB 1% medium. Then, samples wereconcentrated by lyophilization and digested by using trypsin for further mass spectrometry analysis. Tryptic peptides obtained wereanalyzed using 2D nano-Ultra Performance Liquid Chromatography, coupled to tandem mass spectrometry . Bioinformatics analysis forprotein identification was performed by searching against Swiss-Prot database, using Mascot server and ProteoIQ v2.8.Peptide summary report provided by Mascot evidenced hemicellulases which are active not only over β-1,4-linkages of xylose unitsbut also on substituents of xylan. An endo-β-1,4-xylanase with 20.1 kDa molecular weight and 9.2 isoelectric point (pI) was foundexclusively in crude extract samples of strain AR247. This enzyme, identified as a GH11, was also detected as a band between 18-20kDa in zymograms and showed a pairwise similarity of ≥ 99 with an homologous from Paenibacillus sp. Y412MC10. In addition, asecond β-1,4-xylanase was identified in the secretome samples, yet not detectable through zymography, from both AR247 and AR489strains. It was of 140.9 kDa, 4.7 pI (potential GH10), and showed to be closely related to a β-1,4-xylanase from P. glucanolyticus.Moreover, additional extracellular xylanase were found, which were related to a GH30 detected in P. favisporus and to two GH25 fromP. polymyxa. Other proteins related to xylan utilization identified were: S?layer protein; sugar ABC transporters and sequences fromsubstrate-binding proteins towards beta glucans. On the other hand, strains AR460-1 and AR489, revealed sequences correspondingto a carbohydrate binding module type CBM54 and an ABC transporter protein. Finally, a chitosanase belonging to GH8 family wasonly found in strain AR489.In conclusion gel-free proteomics analysis proved to be a useful methodology to achieve a widespread knowledge of the enzymaticrepetory contributing to better quality and quantity of results.Fil: Di Marco, Enzo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Callegari, Eduardo Alberto. University of South Dakota; Estados UnidosFil: Villegas, Liliana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Química de San Luis. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Química de San Luis; ArgentinaFil: Martinez, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnología; ArgentinaXII Congreso Argentino de Microbiología GeneralSan Miguel de TucumánArgentinaSociedad Argentina de Microbiologia GeneralSociedad Argentina de Microbiologia General2017info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectCongresoBookhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/243523Differential extracellular enzymes expression by three Paenibacillus strains using gel-free proteomics análisis; XII Congreso Argentino de Microbiología General; San Miguel de Tucumán; Argentina; 2017; 153-153CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://samige.org.ar/libros/2017.pdfNacionalinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:38:10Zoai:ri.conicet.gov.ar:11336/243523instacron: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 09:38:10.523CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse |
dc.title.none.fl_str_mv |
Differential extracellular enzymes expression by three Paenibacillus strains using gel-free proteomics análisis |
title |
Differential extracellular enzymes expression by three Paenibacillus strains using gel-free proteomics análisis |
spellingShingle |
Differential extracellular enzymes expression by three Paenibacillus strains using gel-free proteomics análisis Di Marco, Enzo Paenibacillus Proteomics Xylan |
title_short |
Differential extracellular enzymes expression by three Paenibacillus strains using gel-free proteomics análisis |
title_full |
Differential extracellular enzymes expression by three Paenibacillus strains using gel-free proteomics análisis |
title_fullStr |
Differential extracellular enzymes expression by three Paenibacillus strains using gel-free proteomics análisis |
title_full_unstemmed |
Differential extracellular enzymes expression by three Paenibacillus strains using gel-free proteomics análisis |
title_sort |
Differential extracellular enzymes expression by three Paenibacillus strains using gel-free proteomics análisis |
dc.creator.none.fl_str_mv |
Di Marco, Enzo Callegari, Eduardo Alberto Villegas, Liliana Beatriz Martinez, Maria Alejandra |
author |
Di Marco, Enzo |
author_facet |
Di Marco, Enzo Callegari, Eduardo Alberto Villegas, Liliana Beatriz Martinez, Maria Alejandra |
author_role |
author |
author2 |
Callegari, Eduardo Alberto Villegas, Liliana Beatriz Martinez, Maria Alejandra |
author2_role |
author author author |
dc.subject.none.fl_str_mv |
Paenibacillus Proteomics Xylan |
topic |
Paenibacillus Proteomics Xylan |
purl_subject.fl_str_mv |
https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
dc.description.none.fl_txt_mv |
Three Paenibacillus strains, identified according to their 16S rDNA gene sequence and named as AR247, AR460-1 AR489, were selecteddue to their ability to produce glycoside hydrolases (GH) for second generation ethanol and other biotechnological applications. Theassessment of extracellular enzyme production was previously approached by utilizing a mineral-based medium, MM0.2, added withagricultural by-products. These substrates are low-cost and abundantly available carbon sources for biotechnological purposes. Among thetested carbon sources, an alkali pretreated sugarcane bagasse (OH-SCB) was the one that better promoted the production of extracellularxylanases for all strains. The aim of this work was to study the differential extracellular enzymes expression by these strains through gelfree proteomic analysis method.Firstly, crude extracts were obtained by centrifugation after 72 h of cultivation in MM0.2 - OH-SCB 1% medium. Then, samples wereconcentrated by lyophilization and digested by using trypsin for further mass spectrometry analysis. Tryptic peptides obtained wereanalyzed using 2D nano-Ultra Performance Liquid Chromatography, coupled to tandem mass spectrometry . Bioinformatics analysis forprotein identification was performed by searching against Swiss-Prot database, using Mascot server and ProteoIQ v2.8.Peptide summary report provided by Mascot evidenced hemicellulases which are active not only over β-1,4-linkages of xylose unitsbut also on substituents of xylan. An endo-β-1,4-xylanase with 20.1 kDa molecular weight and 9.2 isoelectric point (pI) was foundexclusively in crude extract samples of strain AR247. This enzyme, identified as a GH11, was also detected as a band between 18-20kDa in zymograms and showed a pairwise similarity of ≥ 99 with an homologous from Paenibacillus sp. Y412MC10. In addition, asecond β-1,4-xylanase was identified in the secretome samples, yet not detectable through zymography, from both AR247 and AR489strains. It was of 140.9 kDa, 4.7 pI (potential GH10), and showed to be closely related to a β-1,4-xylanase from P. glucanolyticus.Moreover, additional extracellular xylanase were found, which were related to a GH30 detected in P. favisporus and to two GH25 fromP. polymyxa. Other proteins related to xylan utilization identified were: S?layer protein; sugar ABC transporters and sequences fromsubstrate-binding proteins towards beta glucans. On the other hand, strains AR460-1 and AR489, revealed sequences correspondingto a carbohydrate binding module type CBM54 and an ABC transporter protein. Finally, a chitosanase belonging to GH8 family wasonly found in strain AR489.In conclusion gel-free proteomics analysis proved to be a useful methodology to achieve a widespread knowledge of the enzymaticrepetory contributing to better quality and quantity of results. Fil: Di Marco, Enzo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina Fil: Callegari, Eduardo Alberto. University of South Dakota; Estados Unidos Fil: Villegas, Liliana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Química de San Luis. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Química de San Luis; Argentina Fil: Martinez, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnología; Argentina XII Congreso Argentino de Microbiología General San Miguel de Tucumán Argentina Sociedad Argentina de Microbiologia General |
description |
Three Paenibacillus strains, identified according to their 16S rDNA gene sequence and named as AR247, AR460-1 AR489, were selecteddue to their ability to produce glycoside hydrolases (GH) for second generation ethanol and other biotechnological applications. Theassessment of extracellular enzyme production was previously approached by utilizing a mineral-based medium, MM0.2, added withagricultural by-products. These substrates are low-cost and abundantly available carbon sources for biotechnological purposes. Among thetested carbon sources, an alkali pretreated sugarcane bagasse (OH-SCB) was the one that better promoted the production of extracellularxylanases for all strains. The aim of this work was to study the differential extracellular enzymes expression by these strains through gelfree proteomic analysis method.Firstly, crude extracts were obtained by centrifugation after 72 h of cultivation in MM0.2 - OH-SCB 1% medium. Then, samples wereconcentrated by lyophilization and digested by using trypsin for further mass spectrometry analysis. Tryptic peptides obtained wereanalyzed using 2D nano-Ultra Performance Liquid Chromatography, coupled to tandem mass spectrometry . Bioinformatics analysis forprotein identification was performed by searching against Swiss-Prot database, using Mascot server and ProteoIQ v2.8.Peptide summary report provided by Mascot evidenced hemicellulases which are active not only over β-1,4-linkages of xylose unitsbut also on substituents of xylan. An endo-β-1,4-xylanase with 20.1 kDa molecular weight and 9.2 isoelectric point (pI) was foundexclusively in crude extract samples of strain AR247. This enzyme, identified as a GH11, was also detected as a band between 18-20kDa in zymograms and showed a pairwise similarity of ≥ 99 with an homologous from Paenibacillus sp. Y412MC10. In addition, asecond β-1,4-xylanase was identified in the secretome samples, yet not detectable through zymography, from both AR247 and AR489strains. It was of 140.9 kDa, 4.7 pI (potential GH10), and showed to be closely related to a β-1,4-xylanase from P. glucanolyticus.Moreover, additional extracellular xylanase were found, which were related to a GH30 detected in P. favisporus and to two GH25 fromP. polymyxa. Other proteins related to xylan utilization identified were: S?layer protein; sugar ABC transporters and sequences fromsubstrate-binding proteins towards beta glucans. On the other hand, strains AR460-1 and AR489, revealed sequences correspondingto a carbohydrate binding module type CBM54 and an ABC transporter protein. Finally, a chitosanase belonging to GH8 family wasonly found in strain AR489.In conclusion gel-free proteomics analysis proved to be a useful methodology to achieve a widespread knowledge of the enzymaticrepetory contributing to better quality and quantity of results. |
publishDate |
2017 |
dc.date.none.fl_str_mv |
2017 |
dc.type.none.fl_str_mv |
info:eu-repo/semantics/publishedVersion info:eu-repo/semantics/conferenceObject Congreso Book http://purl.org/coar/resource_type/c_5794 info:ar-repo/semantics/documentoDeConferencia |
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publishedVersion |
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conferenceObject |
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http://hdl.handle.net/11336/243523 Differential extracellular enzymes expression by three Paenibacillus strains using gel-free proteomics análisis; XII Congreso Argentino de Microbiología General; San Miguel de Tucumán; Argentina; 2017; 153-153 CONICET Digital CONICET |
url |
http://hdl.handle.net/11336/243523 |
identifier_str_mv |
Differential extracellular enzymes expression by three Paenibacillus strains using gel-free proteomics análisis; XII Congreso Argentino de Microbiología General; San Miguel de Tucumán; Argentina; 2017; 153-153 CONICET Digital CONICET |
dc.language.none.fl_str_mv |
eng |
language |
eng |
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info:eu-repo/semantics/altIdentifier/url/https://samige.org.ar/libros/2017.pdf |
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info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-nc-sa/2.5/ar/ |
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openAccess |
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https://creativecommons.org/licenses/by-nc-sa/2.5/ar/ |
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Nacional |
dc.publisher.none.fl_str_mv |
Sociedad Argentina de Microbiologia General |
publisher.none.fl_str_mv |
Sociedad Argentina de Microbiologia General |
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reponame:CONICET Digital (CONICET) instname:Consejo Nacional de Investigaciones Científicas y Técnicas |
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Consejo Nacional de Investigaciones Científicas y Técnicas |
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CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas |
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dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar |
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