Identifying and characterising a plant GH1 β-glucosidase that exhibits hydrolytic activity on N-linked glucopyranoside

β-Glucosidases (Bgls) catalyse the hydrolysis of β-D-glycosidic bonds and are widely exploited as industrial biocatalysts. While their activity on O- and S-linked glycopyranosides is well established, hydrolysis of N-linked glycopyranosides by Bgls has not previously been demonstrated. Here, we screened a eukaryotic Bgl library for activity on methyl anthranilate-N-β-D-glucopyranoside (MANT-N-glucose), a novel N-glucopyranoside we recently produced biocatalytically. We identify ZmGlu1 as capable of hydrolysing this substrate, albeit with substantially reduced catalytic efficiency relative to native O-glucopyranoside substrates. Structural modelling of the enzyme–substrate complex reveals interactions likely underlying this reduced activity. These findings establish N-glycopyranosides as Bgl substrates and lay the groundwork for engineering Bgls with broader glycosidic bond specificity.