Computation-Based Design of Salt Bridges in PETase for Enhanced Thermostability and Performance for PET Degradation.

Polyethylene terephthalate (PET) is one of the most widely used plastics, and the accumulation of PET poses a great threat to the environment. IsPETase can degrade PET rapidly at moderate temperatures, but its application is greatly limited by the low stability. Herein, molecular dynamics (MD) simulations combined with a sequence alignment strategy were adopted to introduce salt bridges into the flexible region of IsPETase to improve its thermal stability. In the designed variants, the T values of IsPETase and IsPETase were 7.4 and 8.7 °C higher than that of the wild type, respectively. The release of products degraded by IsPETase was 4.3 times that of the wild type. Tertiary structure characterization demonstrated that the structure of the variants IsPETase and IsPETase became more compact. Extensive MD simulations verified that a stable salt bridge was formed between the residue R168 and D186 in IsPETase , while in IsPETase an R168-D186-E188 salt bridge network was observed. These results confirmed that the proposed computation-based salt bridge design strategy could efficiently generate variants with enhanced thermal stability for the long-term degradation of PET, which would be helpful for the design of enzymes with improved stability.