Structural and Functional Insights into Catalytic Efficiency and Stability Trade-Offs Induced by CBM Truncation in a GH5 Endoglucanase from Bacillus subtilis DLG.

Cellulose depolymerization into glucose requires the synergistic action of endoglucanase, exoglucanase, and β-glucosidase. In this study, Bacillus subtilis DLG endoglucanase (BsEGL) and its family 3 carbohydrate-binding module (CBM3)-truncated mutant (BsEGLCD) were heterologously expressed in Escherichia coli BL21(DE3) and characterized. Both enzymes displayed optimal activity at 50 °C and pH 4.5, with BsEGLCD exhibiting superior thermal stability and broader pH stability. Both enzymes maintained over 90% relative activity within 0.5-4.5 M NaCl. Notably, BsEGLCD exhibited superior ethanol tolerance compared to BsEGL. Kinetic analysis revealed that, compared to BsEGL, BsEGLCD exhibited a lower substrate affinity but a higher catalytic rate, while BsEGL showed a slightly higher catalytic efficiency. Molecular dynamics simulations revealed that the CBM3 module removal enhanced structural rigidity and reduced salt sensitivity, as evidenced by lower RMSF fluctuations and stable Rg under high ionic strength. Our findings highlighted the trade-off between substrate affinity and stability imparted by the CBM3 module.