Characterization of a novel cold-active endo-β-1,3-1,4-glucanase from Paenibacillus sp. XP01 and prebiotic potential...

Barley β-glucan (BG), a natural polysaccharide with demonstrated health benefits, faces application limitations due to its high molecular weight and viscosity, creating a demand for specific β-glucan-degrading enzymes. In this study, we report a novel endo-β-1,3-1,4-glucanase, XPGH16, from Paenibacillus sp. XP01, which belongs to the GH16_3 subfamily. XPGH16 exhibited optimal activity at 40 °C and pH 6.0, retained over 20% activity at 0 °C, and was stable from pH 5.0 to 9.0. These properties are critically governed by its unique multi-domain architecture, which comprises S-layer homology (SLH) domains and carbohydrate-binding modules (CBM4 and CBM6). Systematic truncation analysis revealed that these auxiliary domains synergistically govern enzymatic stability, structural integrity, and catalytic efficiency, with CBM4b identified as the primary module for BG binding. Site-directed mutagenesis confirmed E582 and E587 as essential catalytic residues. The enzyme specifically hydrolyzes BG to produce oligosaccharide mixture, designated BGOS, which is predominantly composed of tri- and tetrasaccharides. In vitro fermentation demonstrated that BGOS, compared to native BG, more effectively modulated the gut microbiota by promoting beneficial bacteria (e.g., Clostridium butyricum and Megamonas sp.), suppressing potential pathogens, and significantly enhancing the production of short-chain fatty acids, particularly acetate and butyrate. Metabolomic analysis further revealed that BGOS distinctively influenced microbial carbohydrate and lipid metabolism pathways. This study elucidates the structure-function relationship of a multi-domain β-glucanase and highlights the potential of XPGH16 as an efficient biocatalyst for the tailored production of prebiotic BGOS for functional food and health applications.