Structural and functional insights of the catalytic GH5 and Calx-β domains from the metagenome-derived endoglucanase CelE2
Autor(a) principal: | |
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Data de Publicação: | 2023 |
Outros Autores: | , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1016/j.enzmictec.2023.110206 http://hdl.handle.net/11449/248321 |
Resumo: | Cellulose is the most abundant natural polymer on Earth, representing an attractive feedstock for bioproducts and biofuel production. Cellulases promote the depolymerization of cellulose, generating short oligosaccharides and glucose, which are useful in biotechnological applications. Among the classical cellulases, those from glycoside hydrolase family 5 (GH5) are one of the most abundant in Nature, displaying several modular architectures with other accessory domains attached to its catalytic core, such as carbohydrate-binding modules (CBMs), Ig-like, FN3-like, and Calx-β domains, which can influence the enzyme activity. The metagenome-derived endoglucanase CelE2 has in its modular architecture an N-terminal domain belonging to the GH5 family and a C-terminal domain with a high identity to the Calx-β domain. In this study, the GH5 and the Calx-β domains were subcloned and heterologously expressed in E. coli, to evaluate the structural and functional properties of the individualized domains of CelE2. Thermostability analysis by circular dichroism (CD) revealed a decrease in the denaturation temperature values around 4.6 °C for the catalytic domain (CelE21–381) compared to CelE2 full-length. The CD analyses revealed that the Calx-β domain (CelE2382–477) was unfolded, suggesting that this domain requires to be attached to the catalytic core to become structurally stable. The three-dimensional structure of the catalytic domain CelE21–381 was determined at 2.1 Å resolution, showing a typical (α/β)8-barrel fold and a narrow active site compared to other cellulases from the same family. The biochemical characterization showed that the deletion of the Calx-β domain increased more than 3-fold the activity of the catalytic domain CelE21–381 towards the insoluble substrate Avicel. The main functional properties of CelE2, such as substrate specificity, optimal pH and temperature, thermal stability, and activation by CaCl2, were not altered after the deletion of the accessory domain. Furthermore, the Small Angle X-ray Scattering (SAXS) analyses showed that the addition of CaCl2 was beneficial CelE21–381 protein solvency. This work contributed to fundamental concepts about the structure and function of cellulases, which are useful in applications involving lignocellulosic materials degradation into food and feedstuffs and biofuel production. |
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Structural and functional insights of the catalytic GH5 and Calx-β domains from the metagenome-derived endoglucanase CelE2Calx-βCellulasesCelluloseGH5MetagenomeCellulose is the most abundant natural polymer on Earth, representing an attractive feedstock for bioproducts and biofuel production. Cellulases promote the depolymerization of cellulose, generating short oligosaccharides and glucose, which are useful in biotechnological applications. Among the classical cellulases, those from glycoside hydrolase family 5 (GH5) are one of the most abundant in Nature, displaying several modular architectures with other accessory domains attached to its catalytic core, such as carbohydrate-binding modules (CBMs), Ig-like, FN3-like, and Calx-β domains, which can influence the enzyme activity. The metagenome-derived endoglucanase CelE2 has in its modular architecture an N-terminal domain belonging to the GH5 family and a C-terminal domain with a high identity to the Calx-β domain. In this study, the GH5 and the Calx-β domains were subcloned and heterologously expressed in E. coli, to evaluate the structural and functional properties of the individualized domains of CelE2. Thermostability analysis by circular dichroism (CD) revealed a decrease in the denaturation temperature values around 4.6 °C for the catalytic domain (CelE21–381) compared to CelE2 full-length. The CD analyses revealed that the Calx-β domain (CelE2382–477) was unfolded, suggesting that this domain requires to be attached to the catalytic core to become structurally stable. The three-dimensional structure of the catalytic domain CelE21–381 was determined at 2.1 Å resolution, showing a typical (α/β)8-barrel fold and a narrow active site compared to other cellulases from the same family. The biochemical characterization showed that the deletion of the Calx-β domain increased more than 3-fold the activity of the catalytic domain CelE21–381 towards the insoluble substrate Avicel. The main functional properties of CelE2, such as substrate specificity, optimal pH and temperature, thermal stability, and activation by CaCl2, were not altered after the deletion of the accessory domain. Furthermore, the Small Angle X-ray Scattering (SAXS) analyses showed that the addition of CaCl2 was beneficial CelE21–381 protein solvency. This work contributed to fundamental concepts about the structure and function of cellulases, which are useful in applications involving lignocellulosic materials degradation into food and feedstuffs and biofuel production.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Departamento de Bioquímica Instituto de Biologia (IB) Universidade Estadual de Campinas (UNICAMP) R. Monteiro Lobato, 255- Cidade Universitária, SPPrograma de Processos Tecnológicos e Ambientais Universidade de Sorocaba Rodovia Raposo Tavares- km 92,5 Cidade Universitária, SPUniversidade Positivo Master in Industrial Biotechnology R. Prof. Pedro Viriato Parigot de Souza 5300 Cidade Industrial, PRDepartamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista (UNESP), Distrito de Rubião Jr. s/n, SPDepartamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista (UNESP), Distrito de Rubião Jr. s/n, SPFAPESP: 15/23279-6FAPESP: 16/09950-0FAPESP: 20/05784-3FAPESP: 2010/11469-1FAPESP: 2014/04105-4FAPESP: 2015/50590-4FAPESP: 2016/01926-2CNPq: 306279/2020-7CNPq: 448854/2014-7Universidade Estadual de Campinas (UNICAMP)Cidade UniversitáriaCidade IndustrialUniversidade Estadual Paulista (UNESP)Pimentel, Agnes C.Liberato, Marcelo V.Franco Cairo, João Paulo L.Tomazetto, GeizeclerGandin, César A. [UNESP]de Oliveira Neto, Mario [UNESP]Alvarez, Thabata M.Squina, Fabio M.2023-07-29T13:40:44Z2023-07-29T13:40:44Z2023-04-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlehttp://dx.doi.org/10.1016/j.enzmictec.2023.110206Enzyme and Microbial Technology, v. 165.1879-09090141-0229http://hdl.handle.net/11449/24832110.1016/j.enzmictec.2023.1102062-s2.0-85147542807Scopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengEnzyme and Microbial Technologyinfo:eu-repo/semantics/openAccess2023-07-29T13:40:44Zoai:repositorio.unesp.br:11449/248321Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-08-05T21:16:36.173850Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Structural and functional insights of the catalytic GH5 and Calx-β domains from the metagenome-derived endoglucanase CelE2 |
title |
Structural and functional insights of the catalytic GH5 and Calx-β domains from the metagenome-derived endoglucanase CelE2 |
spellingShingle |
Structural and functional insights of the catalytic GH5 and Calx-β domains from the metagenome-derived endoglucanase CelE2 Pimentel, Agnes C. Calx-β Cellulases Cellulose GH5 Metagenome |
title_short |
Structural and functional insights of the catalytic GH5 and Calx-β domains from the metagenome-derived endoglucanase CelE2 |
title_full |
Structural and functional insights of the catalytic GH5 and Calx-β domains from the metagenome-derived endoglucanase CelE2 |
title_fullStr |
Structural and functional insights of the catalytic GH5 and Calx-β domains from the metagenome-derived endoglucanase CelE2 |
title_full_unstemmed |
Structural and functional insights of the catalytic GH5 and Calx-β domains from the metagenome-derived endoglucanase CelE2 |
title_sort |
Structural and functional insights of the catalytic GH5 and Calx-β domains from the metagenome-derived endoglucanase CelE2 |
author |
Pimentel, Agnes C. |
author_facet |
Pimentel, Agnes C. Liberato, Marcelo V. Franco Cairo, João Paulo L. Tomazetto, Geizecler Gandin, César A. [UNESP] de Oliveira Neto, Mario [UNESP] Alvarez, Thabata M. Squina, Fabio M. |
author_role |
author |
author2 |
Liberato, Marcelo V. Franco Cairo, João Paulo L. Tomazetto, Geizecler Gandin, César A. [UNESP] de Oliveira Neto, Mario [UNESP] Alvarez, Thabata M. Squina, Fabio M. |
author2_role |
author author author author author author author |
dc.contributor.none.fl_str_mv |
Universidade Estadual de Campinas (UNICAMP) Cidade Universitária Cidade Industrial Universidade Estadual Paulista (UNESP) |
dc.contributor.author.fl_str_mv |
Pimentel, Agnes C. Liberato, Marcelo V. Franco Cairo, João Paulo L. Tomazetto, Geizecler Gandin, César A. [UNESP] de Oliveira Neto, Mario [UNESP] Alvarez, Thabata M. Squina, Fabio M. |
dc.subject.por.fl_str_mv |
Calx-β Cellulases Cellulose GH5 Metagenome |
topic |
Calx-β Cellulases Cellulose GH5 Metagenome |
description |
Cellulose is the most abundant natural polymer on Earth, representing an attractive feedstock for bioproducts and biofuel production. Cellulases promote the depolymerization of cellulose, generating short oligosaccharides and glucose, which are useful in biotechnological applications. Among the classical cellulases, those from glycoside hydrolase family 5 (GH5) are one of the most abundant in Nature, displaying several modular architectures with other accessory domains attached to its catalytic core, such as carbohydrate-binding modules (CBMs), Ig-like, FN3-like, and Calx-β domains, which can influence the enzyme activity. The metagenome-derived endoglucanase CelE2 has in its modular architecture an N-terminal domain belonging to the GH5 family and a C-terminal domain with a high identity to the Calx-β domain. In this study, the GH5 and the Calx-β domains were subcloned and heterologously expressed in E. coli, to evaluate the structural and functional properties of the individualized domains of CelE2. Thermostability analysis by circular dichroism (CD) revealed a decrease in the denaturation temperature values around 4.6 °C for the catalytic domain (CelE21–381) compared to CelE2 full-length. The CD analyses revealed that the Calx-β domain (CelE2382–477) was unfolded, suggesting that this domain requires to be attached to the catalytic core to become structurally stable. The three-dimensional structure of the catalytic domain CelE21–381 was determined at 2.1 Å resolution, showing a typical (α/β)8-barrel fold and a narrow active site compared to other cellulases from the same family. The biochemical characterization showed that the deletion of the Calx-β domain increased more than 3-fold the activity of the catalytic domain CelE21–381 towards the insoluble substrate Avicel. The main functional properties of CelE2, such as substrate specificity, optimal pH and temperature, thermal stability, and activation by CaCl2, were not altered after the deletion of the accessory domain. Furthermore, the Small Angle X-ray Scattering (SAXS) analyses showed that the addition of CaCl2 was beneficial CelE21–381 protein solvency. This work contributed to fundamental concepts about the structure and function of cellulases, which are useful in applications involving lignocellulosic materials degradation into food and feedstuffs and biofuel production. |
publishDate |
2023 |
dc.date.none.fl_str_mv |
2023-07-29T13:40:44Z 2023-07-29T13:40:44Z 2023-04-01 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1016/j.enzmictec.2023.110206 Enzyme and Microbial Technology, v. 165. 1879-0909 0141-0229 http://hdl.handle.net/11449/248321 10.1016/j.enzmictec.2023.110206 2-s2.0-85147542807 |
url |
http://dx.doi.org/10.1016/j.enzmictec.2023.110206 http://hdl.handle.net/11449/248321 |
identifier_str_mv |
Enzyme and Microbial Technology, v. 165. 1879-0909 0141-0229 10.1016/j.enzmictec.2023.110206 2-s2.0-85147542807 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Enzyme and Microbial Technology |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.source.none.fl_str_mv |
Scopus reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
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1808129303959830528 |