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
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/243523

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oai_identifier_str oai:ri.conicet.gov.ar:11336/243523
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling 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
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Book
http://purl.org/coar/resource_type/c_5794
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status_str publishedVersion
format conferenceObject
dc.identifier.none.fl_str_mv 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
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://samige.org.ar/libros/2017.pdf
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https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
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