Enhancement of the degradation capacity of Is PETase by acidic amino acids insertion and carbohydrate-binding module fusion.

The biocatalytic degradation of poly(ethylene terephthalate) (PET) through enzymatic methods has garnered considerable attention due to its environmentally friendly and non-polluting nature, as well as its high specificity. While previous efforts in enhancing Is PETase performance have focused on amino acid substitutions in protein engineering, we introduced an amino acid insertion strategy in this work. By inserting a negatively charged acidic amino acid, Glu, at the right-angle bend of Is PETase, the binding capability between the enzyme's active pocket and PET was improved. The resulted mutant Is PETase 9394insE exhibited enhanced hydrolytic activity towards PET at various temperatures ranging from 30 to 45 ℃ compared with the wild-type Is PETase. Notably, a 10.04-fold increase was observed at 45 ℃. To further enhance PET hydrolysis, different carbohydrate-binding modules (CBMs) were incorporated at the C-terminus of Is PETase 9394insE . Among these, the fusion of CBM from Verrucosispora sioxanthis exhibited the highest enhancement, resulting in a 1.82-fold increase in PET hydrolytic activity at 37 ℃ compared with the Is PETase 9394insE . Finally, the engineered variant was successfully employed for the degradation of polyester filter cloth, demonstrating its promising hydrolytic capacity. In conclusion, this research presents an alternative enzyme engineering strategy for modifying PETases and enriches the pool of potential candidates for industrial PET degradation.