Amino acid residues with opposite charges were introduced into the flexible region of IsPETase using a computation-based salt bridge design strategy. The mutation sites formed a salt bridge or salt bridge network to improve the thermal stability of IsPETase, and the degradation efficiency of amorphous PET film was enhanced.

Abstract

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 °C higher than that of the wild type, respectively. The release of products degraded by IsPETase^I168R/S188E was 4.3 times 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.