A New GH3 β-Glucosidase from <i>Chryseobacterium</i> sp. with Applications in Cellulosic Ethanol Production and Agri-Biotechnological Processes

β-Glucosidases catalyze the hydrolysis of β-glycosidic bonds and play key roles in biomass conversion and glycoside processing. We report the identification and characterization of Cr_B1, a GH3 β-glucosidase from Chryseobacterium sp. containing a predicted signal peptide. Cr_B1 hydrolyzed pNPG, cellobiose, salicin, and daidzin, showing optimal activity at pH 5.0−5.5 and 45−55 °C. The enzyme retained over 90% activity after 190 days at 4°C and 25 °C and above 80% activity after 24 h at 50 °C, indicating remarkable long-term and thermal stability. Cr_B1 exhibited high glucose tolerance (IC50: 1.5−1.8 M) and substrate-dependent kinetics. In synergy with Celluclast, it increased glucose release from CMC by 69%, demonstrating its potential to enhance enzymatic saccharification. These properties highlight Cr_B1 as a promising biocatalyst for improving saccharification, enhancing isoflavone bioavailability, and reducing bitterness in food and feed applications.