Rational design of IsPETase variant toward efficient poly(ethylene terephthalate) ambient degradation

The accumulation of poly(ethylene terephthalate) (PET) waste in the environment poses a severe ecological threat. While extensive research has focused on high-performance PET degradation by thermophilic enzymes, PET hydrolases are efficient under lower-temperature conditions, which would better align with green and energy-saving demands the energy-efficient centralized treatment of PET waste remains underexplored. Herein, based on our previously engineered mesophilic IsPETase^S121P/D186A, we performed rational design to improve its PET degradation activity at relatively low temperature. Through rational design methods including salt bridge construction and hydrophobic engineering, we obtained effective variant PADFL (IsPETase^{S121P/D186A/N246D/Y87F/N233L}), demonstrating an 8.37-fold activity of IsPETase^S121P/D186A in PET degradation efficiency (56.52-fold of IsPETase). Molecular dynamics (MD) simulations further revealed stronger PET binding affinity, enhanced hydrogen bonding network, and reduced acylation energy barrier. Overall, this work enhances the degradation activity of the PET hydrolase through energy-based rational design and obtained optimized variant PADFL, offering a promising candidate for future efficient PET degradation under mild temperature conditions.