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(Referência obtida automaticamente do Web of Science, por meio da informação sobre o financiamento pela FAPESP e o número do processo correspondente, incluída na publicação pelos autores.)

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

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Autor(es):
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Rubio, Marcelo Ventura [1] ; Fanchini Terrasan, Cesar Rafael [1] ; Contesini, Fabiano Jares [1] ; Zubieta, Mariane Paludetti [1] ; Gerhardt, Jaqueline Aline [1] ; Oliveira, Leandro Cristante [2] ; de Souza Schmidt Goncalves, Any Elisa [3] ; Almeida, Fausto [4] ; Smith, Bradley Joseph [1] ; Martins Ferreira de Souza, Gustavo Henrique [1] ; Sampaio Dias, Artur Hermano [5] ; Skaf, Munir [5] ; Damasio, Andre [1]
Número total de Autores: 13
Afiliação do(s) autor(es):
[1] Univ Estadual Campinas, Dept Biochem & Tissue Biol, UNICAMP, Inst Biol, Rua Monteiro Lobato 255, Cidade Univ, BR-13083862 Campinas, SP - Brazil
[2] Sao Paulo State Univ, Inst Biosci Humanities & Exact Sci, Dept Phys, UNESP, BR-15054000 Sao Jose Do Rio Preto, SP - Brazil
[3] Univ Estadual Campinas, Fac Med, Dept Med Sci, UNICAMP, BR-13083862 Campinas, SP - Brazil
[4] Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Biochem & Immunol, BR-14049900 Ribeirao Preto, SP - Brazil
[5] Univ Estadual Campinas, Inst Chem & Ctr Comp Engn & Sci, UNICAMP, BR-13084862 Campinas, SP - Brazil
Número total de Afiliações: 5
Tipo de documento: Artigo Científico
Fonte: BIOTECHNOLOGY FOR BIOFUELS; v. 12, n. 1 NOV 14 2019.
Citações Web of Science: 0
Resumo

Background beta-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 beta-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 (BxlB(non-glyc)) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlB(wt)), while a partially glycosylated mutant (BxlB(N1;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 (BxlB (c)), despite the high transcript levels. BxlB(wt), BxlB(non-glyc), and BxlB(N1;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 BxlB(N1;5;7), to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlB(N1;5) and BxlB(N5;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 BxlB(non-glyc) and BxlB(N1;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 BxlB(non-glyc) and BxlB(N1;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. (AU)

Processo FAPESP: 12/20549-4 - Secreção de glicoproteínas heterólogas em Aspergillus: efeito do padrão de glicosilação em parâmetros funcionais de glicosil hidrolases
Beneficiário:André Ricardo de Lima Damasio
Linha de fomento: Auxílio à Pesquisa - Programa BIOEN - Apoio a Jovens Pesquisadores
Processo FAPESP: 13/08293-7 - CECC - Centro de Engenharia e Ciências Computacionais
Beneficiário:Munir Salomao Skaf
Linha de fomento: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
Processo FAPESP: 16/16306-0 - Análise funcional e biológica de LPMOs (Lytic Polysaccharide Monooxygenases) e proteínas acessórias (não-CAZymes) frente a degradação de palha de cana-de-açúcar por fungos filamentosos
Beneficiário:César Rafael Fanchini Terrasan
Linha de fomento: Bolsas no Brasil - Pós-Doutorado
Processo FAPESP: 17/22669-0 - N-glicosilação e secreção de enzimas em fungos filamentosos
Beneficiário:André Ricardo de Lima Damasio
Linha de fomento: Auxílio à Pesquisa - Programa BIOEN - Regular
Processo FAPESP: 17/10083-1 - Engenheiramento genético de uma plataforma fúngica para secreção de enzimas lignocelulolíticas visando a produção de oligossacarídeos
Beneficiário:Fabiano Jares Contesini
Linha de fomento: Bolsas no Brasil - Pós-Doutorado
Processo FAPESP: 19/00098-7 - EMU concedido no processo 2017/25588-1: cromatógrafo Acquity UPLC I-Class
Beneficiário:Daniel Martins-de-Souza
Linha de fomento: Auxílio à Pesquisa - Programa Equipamentos Multiusuários
Processo FAPESP: 14/15403-6 - Aspergillus nidulans como modelo para manipulação de genes envolvidos no processo de "Unfolded Protein Response"
Beneficiário:Mariane Paludetti Zubieta
Linha de fomento: Bolsas no Brasil - Doutorado
Processo FAPESP: 13/24988-5 - Secreção de glicoproteínas heterólogas em Aspergillus: efeito do padrão de glicosilação em parâmetros funcionais de glicosil hidrolases
Beneficiário:Marcelo Ventura Rubio
Linha de fomento: Bolsas no Brasil - Doutorado Direto