Computational loop reconstruction based design of efficient PET hydrolases

Enzymatic PET depolymerization represents a promising approach for establishing a circular economy for PET plastics. Nonetheless, limitations in enzyme activity persist as significant challenges to its industrial application. In this research, the backbone structure of the β6-β7 loop for PET hydrolase Bhr-PETase derived from the thermophilic bacterium HR29 was reconstructed by introducing double mutations (H218N/F222M), resulting in variant Bhr-NMT with high thermal stability (T_m = 92.9 °C) and 87% increase in activity. Moreover, the loop reconstruction mutations are transplanted into the engineered PET hydrolases LCC-ICCG and Kubu-P^M12, resulting variants LCC-ICCG-NM (T_m = 92.4 °C) and Kubu-P^M12-NM (T_m = 92.9 °C). Under high substrate concentration (165 g kg^-1) and an enzyme loading of 0.5 mg_enzyme g_PET^-1, the designed variants Bhr-NMT, LCC-ICCG-NM, and Kubu-P^M12-NM achieve an overall conversion of 93%, 90%, and 94%, respectively, outperforming the benchmark LCC-ICCG (85%). Notably, under reduced enzyme loading (0.3 mg_enzyme g_PET^-1), Kubu-P^M12-NM still reaches an overall conversion of 91%, which is significantly superior to benchmarks Kubu-P^M12 (83%) and LCC-ICCG (71%). Overall, the engineered PET hydrolases demonstrate significant potential for industrial PET waste recycling.