With the continuous growing demand of poly (ethylene terephthalate) (PET), the consequent environmental pollution has captured attention of researchers. The PET hydrolase from Ideonalla sakaiensis (IsPETase) exhibited highest PET degradation activity under ambient temperature, while poor thermostability limited its practical application. Herein, we applied an electrostatic interaction-based strategy by introducing ion-pair to stabilize flexible areas W156-T161 and E245-R251 of IsPETase, and generated variants with enhanced thermostability. The IsPETaseS92K/R251A variant and IsPETaseI139R variant showed satisfactory Tm value (53.0 °C and 56.4 °C, respectively) and PET degradation activity over wild type. Further MD simulation demonstrated that the stable electrostatic interaction introduced strengthened the connection between flexible loop and β-sheet structure and stabilized IsPETase globally, leading to a more robust thermostability. Meanwhile, not only the IsPETaseS92K/D157E/R251A variant exhibited increased thermostability, but also its hydrolysis product was solely terephthalic acid (TPA), without MHET detected, which realized PET depolymerization to complete monomer TPA by IsPETase for the first time. The turnover rate (kcat) of IsPETaseS92K/D157E/R251A variant towards MHET substrate was 1.74-fold higher than that of wild type, which informed the enhanced MHETase activity. Molecular docking and MD simulation results indicated that the increased substrate affinity and closer catalytic distance of IsPETaseS92K/D157E/R251A towards MHET substrate contributed to the enhanced MHETase activity. Overall, based on the electrostatic interaction strategy, we constructed variants with enhanced PET degradation activity and enhanced MHETase activity which contribute to complete depolymerization of PET to TPA by one IsPETase variant, without extra demand of MHETase. Therefore, the strategy, together with the derived PET hydrolyses variants, may contribute to the bio-recycle of PET, reducing the severe global burden of PET waste.

Structure-based engineering of PET hydrolase from Ideonella sakaiensis

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