Enhancing the catalytic efficiency of non-modular processive endoglucanase EG5C-1 by incorporating an extra carbohydrate binding module

Processive endoglucanase with both endoglucanase and exoglucanase activity exhibits great potential in the degradation of cellulose. Carbohydrate Binding Modules (CBMs) can improve the catalytic efficiency by positioning the substrate closer to the catalytic domain and boosting the affinity between the enzyme and substrate. To investigate the impact of CBMs and linker peptides on the catalytic performance of the processive endoglucanase EG5C-1, various EG5C-1 derivatives with different CBMs and linker peptides were constructed. The results suggested the introduction of CBM1 from Volvariella volvacea and flexible peptide (GGGGS)₃ led to a notable 3.4-fold increase on the Avicel and a 3.7-fold increase on the overall enzymatic activity (FPase). In addition, the kinetic parameters of EG5C-1-F3-VvCBM1 showed higher affinity (K_m) and higher catalytic efficiency (k_cat/K_m) for insoluble substrates. Moreover, inverse kinetic parameters indicated that 99.5% of attack sites were occupied, surpassing the wild type. The processivity enhanced by 60% compared to EG5C-1. A series of cellooligosaccharides were hydrolyzed, with a pattern similar to EG5C-1. These results suggest that the fusing CBMs and linker peptides to the catalytic domain could effectively design a highly efficient processive endoglucanase.