Redesigning N-glycosylation sites in a GH3 β-xylosidase improves the enzymatic efficiency

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Autor(es): dc.contributorUniversidade Estadual de Campinas (UNICAMP)-
Autor(es): dc.contributorUniversidade Estadual Paulista (Unesp)-
Autor(es): dc.contributorUniversidade de São Paulo (USP)-
Autor(es): dc.creatorRubio, Marcelo Ventura-
Autor(es): dc.creatorTerrasan, César Rafael Fanchini-
Autor(es): dc.creatorContesini, Fabiano Jares-
Autor(es): dc.creatorZubieta, Mariane Paludetti-
Autor(es): dc.creatorGerhardt, Jaqueline Aline-
Autor(es): dc.creatorOliveira, Leandro Cristante [UNESP]-
Autor(es): dc.creatorDe Souza Schmidt Gonçalves, Any Elisa-
Autor(es): dc.creatorAlmeida, Fausto-
Autor(es): dc.creatorSmith, Bradley Joseph-
Autor(es): dc.creatorDe Souza, Gustavo Henrique Martins Ferreira-
Autor(es): dc.creatorDias, Artur Hermano Sampaio-
Autor(es): dc.creatorSkaf, Munir-
Autor(es): dc.creatorDamasio, André-
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Descrição: dc.descriptionBackground: β-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. Aspergillus nidulans is an established genetic model and good source of carbohydrate-active enzymes (CAZymes). Most fungal enzymes are N-glycosylated, which influences their secretion, stability, activity, signalization, and protease protection. A greater understanding of the N-glycosylation process would contribute to better address the current bottlenecks in obtaining high secretion yields of fungal proteins for industrial applications. Results: In this study, BxlB-a highly secreted GH3 β-xylosidase from A. nidulans, presenting high activity and several N-glycosylation sites-was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of N-glycans on BxlB secretion and function. The non-glycosylated mutant (BxlBnon-glyc) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlBwt), while a partially glycosylated mutant (BxlBN1;5;7) exhibited increased activity. Additionally, there was no enzyme secretion in the mutant in which the N-glycosylation context was changed by the introduction of four new N-glycosylation sites (BxlBCC), despite the high transcript levels. BxlBwt, BxlBnon-glyc, and BxlBN1;5;7 formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlBN1;5;7, to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlBN1;5 and BxlBN5;7) showed improved catalytic efficiency, whereas the other four mutants' catalytic efficiencies were reduced. The N-glycosylation site N5 is important for improved BxlB catalytic efficiency, but needs to be complemented by N1 and/or N7. Molecular dynamics simulations of BxlBnon-glyc and BxlBN1;5 reveals that the mobility pattern of structural elements in the vicinity of the catalytic pocket changes upon N1 and N5 N-glycosylation sites, enhancing substrate binding properties which may underlie the observed differences in catalytic efficiency between BxlBnon-glyc and BxlBN1;5. Conclusions: This study demonstrates the influence of N-glycosylation on A. nidulans BxlB production and function, reinforcing that protein glycoengineering is a promising tool for enhancing thermal stability, secretion, and enzymatic activity. Our report may also support biotechnological applications for N-glycosylation modification of other CAZymes.-
Descrição: dc.descriptionDepartment of Biochemistry and Tissue Biology Institute of Biology University of Campinas (UNICAMP), Rua Monteiro Lobato, 255 Cidade Universitária Zeferino Vaz-
Descrição: dc.descriptionDepartment of Physics Institute of Biosciences Humanities and Exact Sciences São Paulo State University (UNESP)-
Descrição: dc.descriptionDepartment of Medical Science Faculty of Medicine University of Campinas (UNICAMP)-
Descrição: dc.descriptionDepartment of Biochemistry and Immunology Ribeirão Preto Medical School University of São Paulo (USP)-
Descrição: dc.descriptionInstitute of Chemistry and Center for Computing in Engineering and Sciences University of Campinas (UNICAMP)-
Descrição: dc.descriptionDepartment of Physics Institute of Biosciences Humanities and Exact Sciences São Paulo State University (UNESP)-
Idioma: dc.languageen-
Relação: dc.relationBiotechnology for Biofuels-
???dc.source???: dc.sourceScopus-
Palavras-chave: dc.subjectAspergillus nidulans-
Palavras-chave: dc.subjectCAZyme-
Palavras-chave: dc.subjectEnzyme secretion-
Palavras-chave: dc.subjectGlycomutants-
Palavras-chave: dc.subjectGlycoside hydrolase family 3-
Palavras-chave: dc.subjectN-glycosylation-
Palavras-chave: dc.subjectβ-Xylosidase-
Título: dc.titleRedesigning N-glycosylation sites in a GH3 β-xylosidase improves the enzymatic efficiency-
Tipo de arquivo: dc.typelivro digital-
Aparece nas coleções:Repositório Institucional - Unesp

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