Structural and functional insights of the catalytic GH5 and Calx-β domains from the metagenome-derived endoglucanase CelE2

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dc.contributor.authorPimentel, Agnes C.
dc.contributor.authorLiberato, Marcelo V.
dc.contributor.authorFranco Cairo, João Paulo L.
dc.contributor.authorTomazetto, Geizecler
dc.contributor.authorGandin, César A. [UNESP]
dc.contributor.authorde Oliveira Neto, Mario [UNESP]
dc.contributor.authorAlvarez, Thabata M.
dc.contributor.authorSquina, Fabio M.
dc.contributor.institutionUniversidade Estadual de Campinas (UNICAMP)
dc.contributor.institutionCidade Universitária
dc.contributor.institutionCidade Industrial
dc.contributor.institutionUniversidade Estadual Paulista (UNESP)
dc.date.accessioned2023-07-29T13:40:44Z
dc.date.available2023-07-29T13:40:44Z
dc.date.issued2023-04-01
dc.description.abstractCellulose 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.en
dc.description.affiliationDepartamento de Bioquímica Instituto de Biologia (IB) Universidade Estadual de Campinas (UNICAMP) R. Monteiro Lobato, 255- Cidade Universitária, SP
dc.description.affiliationPrograma de Processos Tecnológicos e Ambientais Universidade de Sorocaba Rodovia Raposo Tavares- km 92,5 Cidade Universitária, SP
dc.description.affiliationUniversidade Positivo Master in Industrial Biotechnology R. Prof. Pedro Viriato Parigot de Souza 5300 Cidade Industrial, PR
dc.description.affiliationDepartamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista (UNESP), Distrito de Rubião Jr. s/n, SP
dc.description.affiliationUnespDepartamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista (UNESP), Distrito de Rubião Jr. s/n, SP
dc.description.sponsorshipFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
dc.description.sponsorshipConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
dc.description.sponsorshipIdFAPESP: 15/23279-6
dc.description.sponsorshipIdFAPESP: 16/09950-0
dc.description.sponsorshipIdFAPESP: 20/05784-3
dc.description.sponsorshipIdFAPESP: 2010/11469-1
dc.description.sponsorshipIdFAPESP: 2014/04105-4
dc.description.sponsorshipIdFAPESP: 2015/50590-4
dc.description.sponsorshipIdFAPESP: 2016/01926-2
dc.description.sponsorshipIdCNPq: 306279/2020-7
dc.description.sponsorshipIdCNPq: 448854/2014-7
dc.identifierhttp://dx.doi.org/10.1016/j.enzmictec.2023.110206
dc.identifier.citationEnzyme and Microbial Technology, v. 165.
dc.identifier.doi10.1016/j.enzmictec.2023.110206
dc.identifier.issn1879-0909
dc.identifier.issn0141-0229
dc.identifier.scopus2-s2.0-85147542807
dc.identifier.urihttp://hdl.handle.net/11449/248321
dc.language.isoeng
dc.relation.ispartofEnzyme and Microbial Technology
dc.sourceScopus
dc.subjectCalx-β
dc.subjectCellulases
dc.subjectCellulose
dc.subjectGH5
dc.subjectMetagenome
dc.titleStructural and functional insights of the catalytic GH5 and Calx-β domains from the metagenome-derived endoglucanase CelE2en
dc.typeArtigo

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