Dataset Information

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

ABSTRACT:

Background

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 (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^CC), 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.

SUBMITTER: Rubio MV

PROVIDER: S-EPMC6854716 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

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

Rubio Marcelo Ventura MV Terrasan César Rafael Fanchini CRF Contesini Fabiano Jares FJ Zubieta Mariane Paludetti MP Gerhardt Jaqueline Aline JA Oliveira Leandro Cristante LC de Souza Schmidt Gonçalves Any Elisa AE Almeida Fausto F Smith Bradley Joseph BJ de Souza Gustavo Henrique Martins Ferreira GHMF Dias Artur Hermano Sampaio AHS Skaf Munir M Damasio André A

Biotechnology for biofuels 20191114

<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 t ...[more]

PMID: 31754374

Similar Datasets

Project description:BackgroundGlycosides are compounds displaying crucial biological roles and plenty of applications. Traditionally, these molecules have been chemically obtained, but its efficient production is limited by the lack of regio- and stereo-selectivity of the chemical synthesis. As an interesting alternative, glycosidases are able to catalyze the formation of glycosides in a process considered green and highly selective. In this study, we report the expression and characterization of a fungal β-xylosidase in Pichia pastoris. The transglycosylation potential of the enzyme was evaluated and its applicability in the synthesis of a selective anti-proliferative compound demonstrated.ResultsThe β-xylosidase BxTW1 from the ascomycete fungus Talaromyces amestolkiae was cloned and expressed in Pichia pastoris GS115. The yeast secreted 8 U/mL of β-xylosidase that was purified by a single step of cation-exchange chromatography. rBxTW1 in its active form is an N-glycosylated dimer of about 200 kDa. The enzyme was biochemically characterized displaying a K m and k cat against p-nitrophenyl-β-D-xylopyranoside of 0.20 mM and 69.3 s-1 respectively, and its maximal activity was achieved at pH 3 and 60 °C. The glycan component of rBxTW1 was also analyzed in order to interpret the observed loss of stability and maximum velocity when compared with the native enzyme. A rapid screening of aglycone specificity was performed, revealing a remarkable high number of potential transxylosylation acceptors for rBxTW1. Based on this analysis, the enzyme was successfully tested in the synthesis of 2-(6-hydroxynaphthyl) β-D-xylopyranoside, a well-known selective anti-proliferative compound, enzymatically obtained for the first time. The application of response surface methodology, following a Box-Behnken design, enhanced this production by eightfold, fitting the reaction conditions into a multiparametric model. The naphthyl derivative was purified and its identity confirmed by NMR.ConclusionsA β-xylosidase from T. amestolkiae was produced in P. pastoris and purified. The final yields were much higher than those attained for the native protein, although some loss of stability and maximum velocity was observed. rBxTW1 displayed remarkable acceptor versatility in transxylosylation, catalyzing the synthesis of a selective antiproliferative compound, 2-(6-hydroxynaphthyl) β-D-xylopyranoside. These results evidence the interest of rBxTW1 for transxylosylation of relevant products with biotechnological interest.

Dataset Information

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

Background

Results

Conclusions

Publications

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

Similar Datasets

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