β-Glucosidases: In Silico Analysis of Physicochemical Properties and Domain Architecture Diversity Revealed by Metagenomic Technology

β-Glucosidases, ubiquitous enzymes with significant contribution to several industries were previously identified as diverse in bacterial metagenomes from Vietnamese native goat rumens, wood humus from Cuc Phuong national forest, and termite gut. In this study, we systematically analyzed their sequence diversity, domain architectures, domain arrangements, physicochemical properties, and producers associated with their structures, conserving catalytic domains. A total of 833 β-glucosidase sequences were categorized into three families: GH1, GH16, and GH3, forming 30 distinct domain architectures with variable isoelectric points, alkaline scores, and melting temperatures across ecological niches. GH1 enzymes exhibited the lowest architectural diversity, whereas GH16 enzymes were frequently associated with carbohydrate-binding module 4 (CBM4) and CBM12 domains. Over 90% of GH3 enzymes contained fibronectin type III (FN3) and accessory domains such as PA14, CBM6, Big_2, or ExoP, with some harboring secondary catalytic domains. Most goat rumen β-glucosidases originated from cellulosome-producing bacteria. A recombinant β-glucosidase GH3-31 expressed in E. coli exhibited optimal activity at 40 °C (lower than the predicted Tm of 49.8 °C), pH5.5 (near the predicted pI of 5.61), Km of 1.37 mM ± 0.08 mM, and Vmax of 43.17 ± 0.6 U/mg. Its activity was enhanced by Tween 20, Tween 80, Triton X-100, and CTAB. These findings provide a comprehensive resource for β-glucosidase engineering and application-oriented screening.