Rational Selection of XyGH74 over XyGH5 in <i>Paenibacillus</i> sp. XP01 for High-Yield Production of Prebiotic Xyloglucan Oligosaccharides

Xyloglucan oligosaccharides (XyGOs) are emerging prebiotics with limited sustainable production strategies. Here, two endoxyloglucanases, XyGH5 and XyGH74, from the bacterium <i>Paenibacillus</i> sp. XP01 were identified. Genetic knockout revealed their synergistic role in bacterial growth on xyloglucan. Biochemical characterization showed that XyGH74 exhibited superior activity (<i>V</i>_max = 76.25 U/mg) and thermal stability (optimum at 70 °C) compared to XyGH5, with its carbohydrate-binding modules (CBMs) crucially enhancing catalytic efficiency and substrate affinity. We further employed the highly efficient XyGH74 to produce tamarind xyloglucan oligosaccharides (TXOS). In vitro fermentation with human gut microbiota demonstrated that TXOS significantly modulated microbial composition, enriching beneficial genera such as <i>Bacteroides</i> and <i>Parabacteroides</i>, and notably enhanced the production of short-chain fatty acids, particularly butyrate, compared to the native polysaccharide. This study provides direct genetic evidence for synergistic glycoside hydrolase function in vivo and establishes XyGH74 as a robust enzymatic tool for scalable, high-value prebiotic production with enhanced health benefits.