Wang_2025_Commun.Biol_8_934

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 beta6-beta7 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 degreesC) 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 degreesC) and Kubu-P(M12)-NM (T(m) = 92.9 degreesC). 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.