Abstract
Exo-β-1,3-glucanase EXG1 (ScEXG1) from Saccharomyces cerevisiae hydrolyzes β-1,3-glucans and exhibits transglycosylation activity, enabling biocatalytic synthesis of prebiotic oligosaccharides. In this study, recombinant ScEXG1-6 × His was tested with laminaribiose as both the donor and acceptor. Under low-enzyme dosage, transglycosylation products were detected across a range of substrate concentrations (4.38-32.85 mM), with 21.9 mM laminaribiose producing 270.70 ± 10.32 nmol/mL of the main trisaccharide product-DP3 (DP3) after 3 h. Structural elucidation by HPLC-ESI-MS/MS identified DP3 as β-Glc-(1→6)-β-Glc-(1→3)-Glc based on glycosidic and cross-ring fragmentation patterns. Two additional tetrasaccharides were also characterized as β-Glc-(1→3)-β-Glc-(1→6)-β-Glc-(1→3)-Glc (DP4-1) and β-Glc-(1→6)-β-Glc-(1→3)-β-Glc-(1→3)-Glc (DP4-2). The findings highlight ScEXG1's ability to produce structurally diverse β-glucan oligosaccharides under mild conditions, thereby expanding enzymatic strategies for prebiotic oligosaccharide production.