Cutinase fused with C-terminal residues of α-synuclein improves polyethylene terephthalate degradation by enhancing the substrate binding

Abstract

Environmental-friendly treatment of poly (ethylene terephthalate) (PET) is important for the circular plastic economy, and enzymatic degradation provided a promising approach. However, enzyme binding toward the PET surface is a rate-limiting factor in PET hydrolysis. Thus, a 16-residue peptide (αSP) from the C-terminal sequence of α-synuclein was used to fuse to the C-terminal of a variant (ICCG) of the leaf-branch compost cutinase, and supposed to serve as an anchor peptide for PET degradation. The fusion enzyme (ICCG-αSP) was characterized to keep the same secondary and tertiary structures as ICCG, but enhanced enzyme binding onto bis(2-hydroxyethyl) terephthalate and PET, respectively. Moreover, αSP-fusion enhanced enzyme expression by over 50%, and increased the catalytic efficiency of p-nitrophenyl butyrate by 20% without compromising the enzyme stability. For PET degradation, ICCG-αSP was 3.89 times more active than ICCG by 3-h reaction at 50 °C, and exhibited 1.05–1.45 times higher degradation activity than ICCG in 4-d reaction at 50–70 °C, and pH 7.0 to pH 9.0, respectively. Furthermore, αSP-fusion lowered the optimal long-term degradation temperature from 65 °C to 50 °C at pH 8.0. Therefore, αSP-fusion was effective to increase the hydrolytic efficiency of PET hydrolases by increasing the degradation efficiency and decreasing the degradation temperature.