Enhancing the flexibility of the peripheral regions within the catalytic cavity of a family 5 glycoside hydrolase CelB for improving catalytic efficiency

Glycoside Hydrolase family 5 (GH5) endoglucanases are indispensable biocatalysts in lignocellulosic biorefining and their intrinsic catalytic performances play vital roles in process economics and enable broader industrial applicability. Herein, we engineered the catalytic domain of a bi-modular GH5 endoglucanase CelB from Bacillus sp. Strain N-4 by targeting non-conserved residues around peripheral, solvent-exposed regions. The site-specific mutagenesis identified two single-point mutants Y40G and T177N, which exhibited 1.64- and 1.55-fold increases in specific activity, respectively. Molecular dynamics (MD) simulations further revealed increased fluctuations in the corresponding peripheral regions, suggesting that this altered local backbone dynamics may contribute to the improved catalytic activity. Guided by these observations, iterative saturation mutagenesis was performed at Tyr40 and Thr177, focusing on small or hydrophilic residues to further explore the effects of substitutions at these peripheral, solvent-exposed regions. The final mutant Y40K/T177K exhibited enhanced catalytic efficiency, evidenced by k cat / K m of 11.766 mL mg -1 ·s -1 and 2.719 mL mg -1 ·s -1 towards sodium carboxymethylcellulose and phosphoric acid-swollen cellulose, representing 2.73- and 1.78-fold enhancements over the wild-type enzyme, respectively. MD simulations further showed that Y40K/T177K exhibited enhanced flexibilities across multiple peripheral regions resulted in a more open catalytic cavity, thereby facilitating a greater displacement of cellohexaose ligand from the catalytic center. This behavior is consistent with an increased tendency toward glycan dissociation from the catalytic cavity, which may contribute to the enhanced catalytic turnover. Collectively, these findings underscore the functional importance of peripheral, solvent-exposed regions in modulating the catalytic performance of GH5 endoglucanases.