{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Rubio MV"],"funding":["Conselho Nacional de Desenvolvimento Científico e Tecnológico","Fundação de Amparo à Pesquisa do Estado de São Paulo"],"pagination":["269"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC6854716"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["12"],"pubmed_abstract":["<h4>Background</h4>β-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. <i>Aspergillus nidulans</i> 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.<h4>Results</h4>In this study, BxlB-a highly secreted GH3 β-xylosidase from <i>A. nidulans</i>, presenting high activity and several N-glycosylation sites-was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of <i>N</i>-glycans on BxlB secretion and function. The non-glycosylated mutant (BxlB<sup>non-glyc</sup>) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlB<sup>wt</sup>), while a partially glycosylated mutant (BxlB<sup>N1;5;7</sup>) 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<sup>CC</sup>), despite the high transcript levels. BxlB<sup>wt</sup>, BxlB<sup>non-glyc</sup>, and BxlB<sup>N1;5;7</sup> formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlB<sup>N1;5;7</sup>, to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlB<sup>N1;5</sup> and BxlB<sup>N5;7</sup>) 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<sup>non-glyc</sup> and BxlB<sup>N1;5</sup> 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<sup>non-glyc</sup> and BxlB<sup>N1;5</sup>.<h4>Conclusions</h4>This study demonstrates the influence of N-glycosylation on <i>A. nidulans</i> 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."],"journal":["Biotechnology for biofuels"],"pubmed_title":["Redesigning N-glycosylation sites in a GH3 β-xylosidase improves the enzymatic efficiency."],"pmcid":["PMC6854716"],"funding_grant_id":["442352/2014-0","441912/2014-1","2014/10068-4","2017/22669-0","2019/00098-7","2012/20549-4","304816/2017-5","2013/08293-7"],"pubmed_authors":["Contesini FJ","Rubio MV","Dias AHS","Smith BJ","Zubieta MP","Terrasan CRF","Gerhardt JA","Skaf M","Oliveira LC","de Souza GHMF","Damasio A","de Souza Schmidt Goncalves AE","Almeida F"],"additional_accession":[]},"is_claimable":false,"name":"Redesigning N-glycosylation sites in a GH3 β-xylosidase improves the enzymatic efficiency.","description":"<h4>Background</h4>β-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. <i>Aspergillus nidulans</i> 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.<h4>Results</h4>In this study, BxlB-a highly secreted GH3 β-xylosidase from <i>A. nidulans</i>, presenting high activity and several N-glycosylation sites-was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of <i>N</i>-glycans on BxlB secretion and function. The non-glycosylated mutant (BxlB<sup>non-glyc</sup>) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlB<sup>wt</sup>), while a partially glycosylated mutant (BxlB<sup>N1;5;7</sup>) 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<sup>CC</sup>), despite the high transcript levels. BxlB<sup>wt</sup>, BxlB<sup>non-glyc</sup>, and BxlB<sup>N1;5;7</sup> formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlB<sup>N1;5;7</sup>, to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlB<sup>N1;5</sup> and BxlB<sup>N5;7</sup>) 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<sup>non-glyc</sup> and BxlB<sup>N1;5</sup> 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<sup>non-glyc</sup> and BxlB<sup>N1;5</sup>.<h4>Conclusions</h4>This study demonstrates the influence of N-glycosylation on <i>A. nidulans</i> 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.","dates":{"release":"2019-01-01T00:00:00Z","publication":"2019","modification":"2024-11-19T17:36:46.188Z","creation":"2020-05-21T19:57:09Z"},"accession":"S-EPMC6854716","cross_references":{"pubmed":["31754374"],"doi":["10.1186/s13068-019-1609-2"]}}