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. 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 , presenting high activity and several N-glycosylation sites-was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of -glycans on BxlB secretion and function. The non-glycosylated mutant (BxlB) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlB), while a partially glycosylated mutant (BxlB) 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), despite the high transcript levels. BxlB, BxlB, and BxlB formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlB, to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlB and BxlB) 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 and BxlB 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 and BxlB.

Conclusions: This study demonstrates the influence of N-glycosylation on 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.