Catalytic tunnel engineering of thermostable endoglucanase of GH7 family (W356C) from Aspergillus fumigatus gains catalytic rate

Tunnel engineering targets the access tunnels in enzymes, which is crucial for substrate binding and product release. Modifying the tunnels can lead to better biomass-degrading abilities of the lignocellulolytic enzymes. In this report, we have engineered the thermostable GH7 family endoglucanase from Aspergillus fumigatus (AfEgl7). The residues in the open tunnel having the highest bottleneck radius are mutated. Mutations are created (T229F, W356C) in the non-conserved region. The mutant W356C showed a 2-fold increase in product release rate (V_max = 375.8 µM/min) and 2.5-fold higher catalytic activity (K_cat = 75.1 min⁻¹) compared to wild-type (V_max= 232 µM/min; K_cat = 30.9 min⁻¹) using CM cellulose as substrate. The mutant T229F lost both catalytic activity and thermostability. Molecular dynamic simulations and docking studies of W356C revealed a change in structure near the product exit region, which may facilitate faster product release and account for the increased catalytic efficiency of the mutant. This study showed how redesigning the access pathways can be a promising strategy for protein engineering and de novo protein design by tailoring the open tunnel geometry to a ligand-specific manner.