Wang_2025_Biochem.Eng.J_219_109708

The extensive application of polyethylene terephthalate (PET) has led to severe environmental pollution. Enzymatic degradation provides a sustainable solution for PET recycling, but the limited hydrolytic activity of current PET hydrolases hinders its practical application. Here, we designed an Energy-Guided Accumulated Mutation Strategy (EGAMS) for the rational design of PET enzyme PHL7. By applying multiple rounds of evolution to enhance the degradation activity and thermostability of PHL7, we achieved the excellent PET degradation enzyme FlashPETase (PHL7E148K/T158P/S184E/H185Y) with 238.8 % PET degradation activity of PHL7 and 82.93 degreesC melting temperature. FlashPETase completely degrades PET at 72 degreesC with an enzyme loading of 1.5 mgenzyme gPET1, without any substrate pretreatment or additional processing. As for PET substrates with high crystallinity, FlashPETase still performs well. Furthermore, molecular simulations revealed the structural mechanisms of FlashPETase's enhanced activity and stability. In summary, the EGAMS protocol and the variants discussed in this work advance the fields of PET degradation and enzyme engineering, offering valuable insights for future research.