A thermostable CBM3-containing endo-β-1,4-glucanase from Paenibacillus sp. XP01 efficiently produces prebiotic...

Konjac glucomannan (KGM), a natural high-molecular-weight polysaccharide, is widely utilized as a functional thickener, gelling agent, and dietary fiber in food and health products. However, its large molecular size and high viscosity restrict its efficacy in intestinal and industrial applications, prompting the development of enzymatic strategies to produce KGM-derived oligosaccharides. In this study, a novel endo-β-1,4-glucanase, EglGH5A, belonging to glycoside hydrolase subfamily GH5_1, was identified from Paenibacillus sp. XP01. EglGH5A exhibited maximum activity at 70 °C, remained stable over a broad pH range (4.0-9.0), and maintained high residual activity after incubation at 60 °C for 12 h, which was further enhanced by its C-terminal carbohydrate-binding module (CBM3). The enzyme displayed high hydrolytic activity toward KGM and barley β-glucan (BG). Site-directed mutagenesis revealed that E118, E164, Y245, E287, and W320 are critical catalytic residues. Enzymatic hydrolysis of KGM by EglGH5A yielded KGM oligosaccharides (KGM-OS) with a degree of polymerization ranging from 1 to 8. In vitro fermentation demonstrated that KGM-OS stimulated the growth of beneficial bacteria (such as Bifidobacterium and Megamonas), suppressed pathogenic bacteria (e.g., Streptococcus), and increased the production of SCFA. Furthermore, untargeted metabolomics showed that KGM-OS was associated with alterations across a wider range of metabolic pathways than KGM, particularly involving amino acid, carbohydrate, and sphingolipid metabolism. These findings highlight EglGH5A is particularly valuable for applications requiring high thermostability and specific oligosaccharide production with potential applications in functional food development.