Qu_2023_Chembiochem_24_e202300373

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(m) values of IsPETase(I168R/S188D) and IsPETase(I168R/S188E) were 7.4 and 8.7 degreesC higher than that of the wild type, respectively. The release of products degraded by IsPETase(I168R/S188E) was 4.3times that of the wild type. Tertiary structure characterization demonstrated that the structure of the variants IsPETase(I168R/S188D) and IsPETase(I168R/S188E) became more compact. Extensive MD simulations verified that a stable salt bridge was formed between the residue R168 and D186 in IsPETase(I168R/S188D) , while in IsPETase(I168R/S188E) 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.