Enhanced Adsorption and Enzymatic Hydrolysis of Polyethylene Terephthalate by Cutinase with an N-Terminal Hydrophobic Tether

The environmental challenges presented by plastic waste, particularly polyethylene terephthalate (PET), necessitates innovative biodegradation strategies. The cutinase from Thermobifida cellulosilytica, Thc_Cut1 (Cut), was site-specifically conjugated with alkyl tethers of varying lengths (C3, C6, C9) through 1H-1,2,3-triazole-4-carbaldehyde (TA4C) derivatives. These conjugations were provided to enhance affinity for PET by adjusting the enzyme's hydrophobicity. The enzyme activity and kinetic parameters of both conjugated and unconjugated cutinases revealed that the modifications have minimal impact on catalytic activity. However, a significant improvement in PET hydrolysis efficiency was observed. Specifically, hexyl and nonyl TA4C-containing cutinase display notable increases in terephthalic acid (TPA) release, exceeding the performance of unconjugated cutinase by 65% and 69%, respectively. Scanning electron microscopy and water contact angle measurements confirmed the enhanced erosion and hydrophilicity of the PET surface following enzyme treatment. Increased enzyme adsorption on the PET surface for C6-Cut and C9-Cut was validated by X-ray photoelectron spectroscopy. Moreover, high-speed atomic force microscopy demonstrated faster and more stable adsorption of C6-Cut and C9-Cut on PET surfaces compared to the slower adsorption of unconjugated cutinase. Additionally, molecular dynamics simulations indicate a higher affinity of conjugated cutinase for PET film. These results suggest that conjugating an alkyl tether to the N-terminus strengthens the interaction between cutinase and PET, improving hydrolysis.