Structural elucidation and distribution pattern of glucuronic acid in β-glucan from Hypsizygus marmoreus using glycosidase hydrolysis

A ∼ 20 kDa heteroglucan composed of glucose (Glc) and glucuronic acid (GlcA) was isolated from Hypsizygus marmoreus . Preliminary analysis through methylation analysis and nuclear magnetic resonance (NMR) showed the backbone consisted of β-1,6-linked d -glucopyranosyl (Glc p ) residues, with branches at O-3 positions containing t-β-D-Glc p residues. The linkages of GlcA was carried out using partial acid and glycosidase hydrolysis. HILIC-FLR/ESI-MS n , methylation and NMR of the partial acid and glycosidase hydrolysis products revealed Glc p A was in the forms of t-β-D-Glc p A and β-1,4-D-Glc p A attached to the C-3 position of part of the Glc in the main chain. Furthermore, the β-glucan exhibited structural heterogeneity, comprising three distinct domains: (a) unsubstituted β-1,6-glucan; (b) β-1,6-heteroglucan substituted with GlcA (1–2 sugar residues); and (c) β-1,6-heteroglucan substituted with Glc (single sugar residue). The proposed heteropolysaccharide structure provides critical insights into the structural diversity of mushroom-derived β-1,6-glucans, thereby facilitating future investigations into their structure-activity relationships.