Rational Engineering\nof a β‑Glucosidase\n(H0HC94) from Glycosyl Hydrolase Family I (GH1) to Improve Catalytic\nPerformance on Cellobiose

The conversion of lignocellulosic feedstocks by cellulases\nto glucose\nis a critical step in biofuel production. β-Glucosidases catalyze\nthe final step in cellulose breakdown, producing glucose, and are\noften the rate-limiting step in biomass hydrolysis. The specific activity\nof most natural and engineered β-glucosidase is higher on the\nartificial substrate <i>p</i>-nitrophenyl β-d-glucopyranoside (<i>p</i>NPGlc) than on the natural substrate,\ncellobiose. We report an engineered β-glucosidase (Q319A H0HC94)\nwith a 1.8-fold higher specific activity (366.3 ± 36 μmol/min/mg),\na 1.5-fold increase in <i>k</i><sub><i>cat</i></sub> (340.8 ± 27 s<sup>–1</sup>), and a 3-fold increase\nin catalytic efficiency (236.65 mM<sup>–1</sup> s<sup>–1</sup>) over H0HC94 (WT) on cellobiose. Molecular dynamic simulations and\nprotein structure network analysis indicate that the Q319A H0HC94\nactive site pocket is significantly remodeled compared to the WT,\nleading to changes in enzyme conformation, better accessibility of\ncellobiose inside the active site pocket, and higher enzymatic activity.\nThis study shows the impact of rational engineering of a nonconserved\nresidue to increase β-glucosidase substrate accessibility and\ncatalytic efficiency by reducing crowding interaction between cellobiose\nand active site pocket residues near the gatekeeper region and increasing\npocket volume and surface area. Thus, rational engineering of previously\ncharacterized enzymes could be an excellent strategy to improve cellulose\nhydrolysis.