Functional and structural characterization of an endo-β-1,3-glucanase from Euglena gracilis

Autores
Calloni, Rodrigo Daniel; Muchut, Robertino José; Garay, Alberto Sergio; Arias, Diego Gustavo; Iglesias, Alberto Alvaro; Guerrero, Sergio Adrian
Año de publicación
2023
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Endo-β-1,3-glucanases from several organisms have attracted much attention in recent years because of their capability for in vitro degrading β-1,3-glucan as a critical step for both biofuels production and short-chain oligosaccharides synthesis. In this study, we biochemically characterized a putative endo-β-1,3-glucanase (EgrGH64) belonging to the family GH64 from the single-cell protist Euglena gracilis. The gene coding for the enzyme was heterologously expressed in a prokaryotic expression system supplemented with 3% (v/v) ethanol to optimize the recombinant protein right folding. Thus, the produced enzyme was highly purified by immobilized-metal affinity and gel filtration chromatography. The enzymatic study demonstrated that EgrGH64 could hydrolyze laminarin (KM 23.5 mg ml−1,kcat 1.20 s−1) and also, but with less enzymatic efficiency, paramylon (KM 20.2 mg ml−1,kcat 0.23 ml mg−1 s−1). The major product of the hydrolysis of both substrates was laminaripentaose. The enzyme could also use ramified β-glucan from the baker's yeast cell wall as a substrate (KM 2.10 mg ml−1, kcat 0.88 ml mg−1 s−1). This latter result, combined with interfacial kinetic analysis evidenced a protein's greater efficiency for the yeast polysaccharide, and a higher number of hydrolysis sites in the β-1,3/β-1,6-glucan. Concurrently, the enzyme efficiently inhibited the fungal growth when used at 1.0 mg/mL (15.4 μM). This study contributes to assigning a correct function and determining the enzymatic specificity of EgrGH64, which emerges as a relevant biotechnological tool for processing β-glucans.
Fil: Calloni, Rodrigo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina
Fil: Muchut, Robertino José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Reconquista; Argentina
Fil: Garay, Alberto Sergio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina
Fil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina
Fil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina
Materia
EUGLENOIDS
GH64 PROTEIN
LAMINARIN
PARAMYLON
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:11336/212055
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/212055
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Functional and structural characterization of an endo-β-1,3-glucanase from Euglena gracilisCalloni, Rodrigo DanielMuchut, Robertino JoséGaray, Alberto SergioArias, Diego GustavoIglesias, Alberto AlvaroGuerrero, Sergio AdrianEUGLENOIDSGH64 PROTEINLAMINARINPARAMYLONhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Endo-β-1,3-glucanases from several organisms have attracted much attention in recent years because of their capability for in vitro degrading β-1,3-glucan as a critical step for both biofuels production and short-chain oligosaccharides synthesis. In this study, we biochemically characterized a putative endo-β-1,3-glucanase (EgrGH64) belonging to the family GH64 from the single-cell protist Euglena gracilis. The gene coding for the enzyme was heterologously expressed in a prokaryotic expression system supplemented with 3% (v/v) ethanol to optimize the recombinant protein right folding. Thus, the produced enzyme was highly purified by immobilized-metal affinity and gel filtration chromatography. The enzymatic study demonstrated that EgrGH64 could hydrolyze laminarin (KM 23.5 mg ml−1,kcat 1.20 s−1) and also, but with less enzymatic efficiency, paramylon (KM 20.2 mg ml−1,kcat 0.23 ml mg−1 s−1). The major product of the hydrolysis of both substrates was laminaripentaose. The enzyme could also use ramified β-glucan from the baker's yeast cell wall as a substrate (KM 2.10 mg ml−1, kcat 0.88 ml mg−1 s−1). This latter result, combined with interfacial kinetic analysis evidenced a protein's greater efficiency for the yeast polysaccharide, and a higher number of hydrolysis sites in the β-1,3/β-1,6-glucan. Concurrently, the enzyme efficiently inhibited the fungal growth when used at 1.0 mg/mL (15.4 μM). This study contributes to assigning a correct function and determining the enzymatic specificity of EgrGH64, which emerges as a relevant biotechnological tool for processing β-glucans.Fil: Calloni, Rodrigo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Muchut, Robertino José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Reconquista; ArgentinaFil: Garay, Alberto Sergio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaElsevier France-Editions Scientifiques Medicales Elsevier2023-05info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/212055Calloni, Rodrigo Daniel; Muchut, Robertino José; Garay, Alberto Sergio; Arias, Diego Gustavo; Iglesias, Alberto Alvaro; et al.; Functional and structural characterization of an endo-β-1,3-glucanase from Euglena gracilis; Elsevier France-Editions Scientifiques Medicales Elsevier; Biochimie; 208; 5-2023; 117-1280300-9084CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://linkinghub.elsevier.com/retrieve/pii/S0300908422003418info:eu-repo/semantics/altIdentifier/doi/10.1016/j.biochi.2022.12.016info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-11-26T08:38:05Zoai:ri.conicet.gov.ar:11336/212055instacron: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:34982025-11-26 08:38:05.309CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Functional and structural characterization of an endo-β-1,3-glucanase from Euglena gracilis
title Functional and structural characterization of an endo-β-1,3-glucanase from Euglena gracilis
spellingShingle Functional and structural characterization of an endo-β-1,3-glucanase from Euglena gracilis
Calloni, Rodrigo Daniel
EUGLENOIDS
GH64 PROTEIN
LAMINARIN
PARAMYLON
title_short Functional and structural characterization of an endo-β-1,3-glucanase from Euglena gracilis
title_full Functional and structural characterization of an endo-β-1,3-glucanase from Euglena gracilis
title_fullStr Functional and structural characterization of an endo-β-1,3-glucanase from Euglena gracilis
title_full_unstemmed Functional and structural characterization of an endo-β-1,3-glucanase from Euglena gracilis
title_sort Functional and structural characterization of an endo-β-1,3-glucanase from Euglena gracilis
dc.creator.none.fl_str_mv Calloni, Rodrigo Daniel
Muchut, Robertino José
Garay, Alberto Sergio
Arias, Diego Gustavo
Iglesias, Alberto Alvaro
Guerrero, Sergio Adrian
author Calloni, Rodrigo Daniel
author_facet Calloni, Rodrigo Daniel
Muchut, Robertino José
Garay, Alberto Sergio
Arias, Diego Gustavo
Iglesias, Alberto Alvaro
Guerrero, Sergio Adrian
author_role author
author2 Muchut, Robertino José
Garay, Alberto Sergio
Arias, Diego Gustavo
Iglesias, Alberto Alvaro
Guerrero, Sergio Adrian
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv EUGLENOIDS
GH64 PROTEIN
LAMINARIN
PARAMYLON
topic EUGLENOIDS
GH64 PROTEIN
LAMINARIN
PARAMYLON
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Endo-β-1,3-glucanases from several organisms have attracted much attention in recent years because of their capability for in vitro degrading β-1,3-glucan as a critical step for both biofuels production and short-chain oligosaccharides synthesis. In this study, we biochemically characterized a putative endo-β-1,3-glucanase (EgrGH64) belonging to the family GH64 from the single-cell protist Euglena gracilis. The gene coding for the enzyme was heterologously expressed in a prokaryotic expression system supplemented with 3% (v/v) ethanol to optimize the recombinant protein right folding. Thus, the produced enzyme was highly purified by immobilized-metal affinity and gel filtration chromatography. The enzymatic study demonstrated that EgrGH64 could hydrolyze laminarin (KM 23.5 mg ml−1,kcat 1.20 s−1) and also, but with less enzymatic efficiency, paramylon (KM 20.2 mg ml−1,kcat 0.23 ml mg−1 s−1). The major product of the hydrolysis of both substrates was laminaripentaose. The enzyme could also use ramified β-glucan from the baker's yeast cell wall as a substrate (KM 2.10 mg ml−1, kcat 0.88 ml mg−1 s−1). This latter result, combined with interfacial kinetic analysis evidenced a protein's greater efficiency for the yeast polysaccharide, and a higher number of hydrolysis sites in the β-1,3/β-1,6-glucan. Concurrently, the enzyme efficiently inhibited the fungal growth when used at 1.0 mg/mL (15.4 μM). This study contributes to assigning a correct function and determining the enzymatic specificity of EgrGH64, which emerges as a relevant biotechnological tool for processing β-glucans.
Fil: Calloni, Rodrigo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina
Fil: Muchut, Robertino José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Reconquista; Argentina
Fil: Garay, Alberto Sergio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina
Fil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina
Fil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina
description Endo-β-1,3-glucanases from several organisms have attracted much attention in recent years because of their capability for in vitro degrading β-1,3-glucan as a critical step for both biofuels production and short-chain oligosaccharides synthesis. In this study, we biochemically characterized a putative endo-β-1,3-glucanase (EgrGH64) belonging to the family GH64 from the single-cell protist Euglena gracilis. The gene coding for the enzyme was heterologously expressed in a prokaryotic expression system supplemented with 3% (v/v) ethanol to optimize the recombinant protein right folding. Thus, the produced enzyme was highly purified by immobilized-metal affinity and gel filtration chromatography. The enzymatic study demonstrated that EgrGH64 could hydrolyze laminarin (KM 23.5 mg ml−1,kcat 1.20 s−1) and also, but with less enzymatic efficiency, paramylon (KM 20.2 mg ml−1,kcat 0.23 ml mg−1 s−1). The major product of the hydrolysis of both substrates was laminaripentaose. The enzyme could also use ramified β-glucan from the baker's yeast cell wall as a substrate (KM 2.10 mg ml−1, kcat 0.88 ml mg−1 s−1). This latter result, combined with interfacial kinetic analysis evidenced a protein's greater efficiency for the yeast polysaccharide, and a higher number of hydrolysis sites in the β-1,3/β-1,6-glucan. Concurrently, the enzyme efficiently inhibited the fungal growth when used at 1.0 mg/mL (15.4 μM). This study contributes to assigning a correct function and determining the enzymatic specificity of EgrGH64, which emerges as a relevant biotechnological tool for processing β-glucans.
publishDate 2023
dc.date.none.fl_str_mv 2023-05
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/11336/212055
Calloni, Rodrigo Daniel; Muchut, Robertino José; Garay, Alberto Sergio; Arias, Diego Gustavo; Iglesias, Alberto Alvaro; et al.; Functional and structural characterization of an endo-β-1,3-glucanase from Euglena gracilis; Elsevier France-Editions Scientifiques Medicales Elsevier; Biochimie; 208; 5-2023; 117-128
0300-9084
CONICET Digital
CONICET
url http://hdl.handle.net/11336/212055
identifier_str_mv Calloni, Rodrigo Daniel; Muchut, Robertino José; Garay, Alberto Sergio; Arias, Diego Gustavo; Iglesias, Alberto Alvaro; et al.; Functional and structural characterization of an endo-β-1,3-glucanase from Euglena gracilis; Elsevier France-Editions Scientifiques Medicales Elsevier; Biochimie; 208; 5-2023; 117-128
0300-9084
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://linkinghub.elsevier.com/retrieve/pii/S0300908422003418
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.biochi.2022.12.016
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 France-Editions Scientifiques Medicales Elsevier
publisher.none.fl_str_mv Elsevier France-Editions Scientifiques Medicales Elsevier
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 13.011256

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