Characterization and Functional Mechanism Analysis of Two Glycoside Hydrolase Family-39 β-Xylosidases Identified from <i>Caldicellulosiruptor saccharolyticus</i>

β-Xylosidase, crucial for xylan degradation, catalyzes xylose release from xylo-oligosaccharides. However, enzymatic characters of β-xylosidases possess certain limitations, including poor thermal stability, and inhibitors are unclear. This study identified two GH39 family β-xylosidases, <i>Cs</i>Xyl39A and <i>Cs</i>Xyl39B, from the thermophilic bacterium <i>Caldicellulosiruptor saccharolyticus</i>. Both enzymes exhibited exceptional thermostability and compatibility with organic solvents, making them promising for industrial applications. <i>Cs</i>Xyl39A specifically cleaved d-xylopyranosyl groups from substrates such as notoginsenoside R1 and <i>p</i>-nitrophenyl-β-d-xylopyranoside. The catalytic efficiency in organic-solvent/water-miscible (O/Wm) systems was enhanced, and the physical immobilization on macroporous polystyrene strengthened its resistance to organic solvents in high-ratio O/Wm solutions. <i>Cs</i>Xyl39B demonstrated high specific activity and thermal stability at 70 °C, maintaining a 26 h half-life while producing xylose and glucose, indicating its suitability for lignocellulose biodegradation. Together, these enzymes address key industrial challenges, offering enhanced stability and solvent adaptability for sustainable bioprocessing applications.