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

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.