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

Abstract

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 (IsPETase) 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 IsPETase, we selected several polymer-binding domains that can adhere to a hydrophobic polymer surface and fused these to a previously engineered IsPETase^{S121E/D186H/R280A} (IsPETase^EHA) variant. We found that fusing a cellulose-binding domain (CBM) of cellobiohydrolase I from Trichoderma reesei onto the C-terminus of IsPETase^EHA showed a stimulatory effect on enzymatic hydrolysis of PET. Compared to the parental enzyme, IsPETase^EHA_CBM exhibited 71.5 % and 44.5 % higher hydrolytic activity at 30 ℃ and 40 ℃, respectively. The catalytic activity of IsPETase^EHA_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 IsPETase is a promising strategy to stimulate the enzymatic hydrolysis of PET.

Keywords: Biodegradation; Catalytic activity; PET hydrolase; Polyethylene terephthalate (PET); Polymer binding domain.