Enhancing PET hydrolytic enzyme activity by fusion of the cellulose–binding domain of cellobiohydrolase I from Trichoderma reesei.

• Various polymer-binding domains were fused to a PET hydrolase variant Is PETaseEHA. • Is PETaseEHA_CBM fused with a cellulose-binding domain showed enhanced PET hydrolysis. • Dose-dependent effects of Is PETaseEHA_CBM on PET hydrolysis were observed. The large amounts of polyethylene terephthalate (PET) that enter and accumulate in the environment have posed a serious threat to global ecosystems and human health. A PET hydrolase from PET-assimilating bacterium Ideonella sakaiensis (Is PETase) that exhibits superior PET hydrolytic activity at mild conditions is attracting enormous attention in development of plastic biodegrading strategies. In order to enhance the PET hydrolysis capacity of Is PETase, we selected several polymer-binding domains that can adhere to a hydrophobic polymer surface and fused these to a previously engineered Is PETaseS121E/D186H/R280A (Is PETaseEHA) variant. We found that fusing a cellulose–binding domain (CBM) of cellobiohydrolase I from Trichoderma reesei onto the C-terminus of Is PETaseEHA showed a stimulatory effect on enzymatic hydrolysis of PET. Compared to the parental enzyme, Is PETaseEHA_CBM exhibited 71.5 % and 44.5 % higher hydrolytic activity at 30 ℃ and 40 ℃, respectively. The catalytic activity of Is PETaseEHA_CBM was increased by 86 % when the protein concentration was increased from 2.5 μg/mL to 20 μg/mL. These findings suggest that the fusion of polymer-binding module to Is PETase is a promising strategy to stimulate the enzymatic hydrolysis of PET. [ABSTRACT FROM AUTHOR]