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The conversion of lignocellulosic feedstocks by cellulases to glucose is a critical step in biofuel production. β-glucosidases catalyze the final step in cellulose breakdown, producing glucose, and is often the rate-limiting step in biomass hydrolysis. Rationally engineering previously characterized enzymes may be one strategy to increase catalytic activity and the efficiency of cellulose hydrolysis. The specific activity of most natural and engineered β-glucosidase is higher on the artificial substrate p-Nitrophenyl β-D-glucopyranoside (pNPGlc) than on the natural substrate, cellobiose. Based on our hypothesis of increasing catalytic activity by reducing the interaction of residues present near the active site tunnel entrance with glucose without disturbing any existing interactions with cellobiose, we report an engineered β-glucosidase (Q319A H0HC94) with a 1.8-fold specific activity increase (366.3 ± 36 μmol/min/mg), an almost 1.5-fold increase in kcat (340.8 ± 27 s-1), and a 3-fold increase in Q319A H0HC94 cellobiose specificity (236.65 mM-1 s-1) over HOHC94. Molecular dynamic simulations and protein structure network analysis indicate that Q319A significantly increased the dynamically stable communities and hub residues, leading to a change in enzyme conformation and higher enzymatic activity. This study shows the impact of rational engineering of non-conserved residue to increase β-glucosidase substrate accessibility and enzyme specificity.

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