Fusion of Hydrophobic Anchor Peptides Promotes the Hydrolytic Activity of PETase but not the Extent of PET Depolymerization

Abstract

Enzymatic recycling of polyethylene terephthalate (PET) has attracted significant attention in recent years. While the fusion of anchor peptides to PET hydrolases is believed to enhance PET hydrolytic activity, a quantitative analysis is yet lacking. Here, we construct four fusion enzymes by fusing anchor peptides (including hydrophobic LCI, LCIM1 and TA2, and hydrophilic EK4) to the C terminus of HotPETase, one of the most active PET hydrolases for high-crystallinity PET (HC-PET). Single-molecule force spectroscopy (SMFS) demonstrates that hydrophobic anchor peptides promote adhesive interactions between the fusion enzymes and the PET surface. This is also validated by the adsorption kinetics and isotherms, and the saturated adsorption capacity remains unaltered compared to HotPETase. At low substrate loadings, the apparent hydrolytic activity of these fusion enzymes is positively related to the hydrophobicity of the anchor peptides. Among them, HotPETase-LCI stands out as the most effective enzyme for HC-PET degradation, demonstrating a 1.5-fold increase in hydrolytic activity. At high substrate loadings, the advantages of fusion with anchor peptides diminish. We conclude that fusion enzymes only facilitate the hydrolytic rates of reactions for HC-PET but have little effect on the final conversion extent.