Nakamura_2021_ACS.Catal_11_8550

A thermostable enzyme PET2, found in a metagenome library, has been engineered to improve its hydrolytic activity against polyethylene terephthalate (PET). The PET2 wild-type (WT) showed a melting temperature of 69.0 C and produced water-soluble reaction products at a rate of 0.40 min-1 (2.4 microM products from 0.1 microM enzyme after 60 min reaction) from an amorphous PET film at 60 C. Mutations for surface charge modification, backbone stabilization, and formation of additional disulfide bond were introduced into the PET2 WT, and the best mutant (PET2 7M) showed a melting temperature of 75.7 C and hydrolytic activity of 1.3 min-1 (7.8 micrM products from 0.1 microM enzyme after 60 min reaction at 60 C). X-ray crystal structures of PET2 mutants showed that introduced arginine and lysine residues oriented to the solvent, similar to a PET hydrolase from Ideonella sakaiensis 201-F6. Single-molecule fluorescence imaging revealed that these positively charged surface residues increased binding rate constant of PET2 7M to PET surface 2.7 times, compared with PET2 WT, and resulted in higher activity. Optimal temperature for amorphous PET hydrolysis by PET2 7M (68 C) was 8 C higher than that by PET2 WT (60 C), and hydrolytic activity of PET2 7M at the optimal temperature (2.7 min-1, 16.2 microM products from 0.1 microM enzyme after 60 min reaction) was 6.8 times higher than that of PET2 WT (0.40 min-1). Furthermore, PET2 7M generated reaction products with a constant rate for at least 24 h at 68 C, indicating long-term thermal stability at the optimal temperature.