A Mixed-Linkage-Selective β-1,4-Glucanase from Pleurotus tuber-regium: Functional Characterization and Mechanistic Insights into Substrate Cleavage Specificity

Macrofungal β-glucanases hold promise for the targeted bioconversion of dietary polysaccharides, yet their functional diversity and mechanistic specificity remain limited. In this study, a β-1,4-glucanase (PTRBGL4) was identified from Pleurotus tuber-regium genome via CAZy and dbCAN3-guided mining and heterologously expressed in Pichia pastoris. Biochemical assays revealed that PTRBGL4 exhibits low activity toward typical β-1,4-linked substrates, but demonstrates high specificity for mixed-linked β-1,3-1,4-glucans, including oat and barley β-glucans. Enzyme activity was enhanced by Fe²⁺ and Co²⁺, but inhibited by Cu²⁺ and Mn²⁺. HPAEC-PAD, MALDI-TOF-MS, and NMR analyses revealed that PTRBGL4 preferentially cleaves β-1,4-linkages adjacent to β-1,3-bonds near the nonreducing end, yielding defined oligosaccharides (e.g., DP3, DP4, DP7, DP10). Structurally, PTRBGL4 features a TIM-barrel (β/α)₈ fold and a C-terminal fungal-type CBM1 that likely aids substrate binding. In addition to the catalytic residues E235 and E347, key residues such as H191 and A313 also play significant roles in substrate binding. These findings clarify the cleavage specificity of PTRBGL4 and support its potential in the enzymatic production of structurally defined β-glucooligosaccharides for application in food, pharmaceutical, and biotechnological industries.