Structural bioinformatics-based protein engineering of thermo-stable PETase from Ideonella sakaiensis

Poly(ethylene terephthalate) (PET), a widely used plastic around the world, causes various environmental and health problems. Several groups have been extensively conducting research to solve these problems through enzymatic degradation of PET at high temperatures around 70 °C. Recently, Ideonella sakaiensis, a bacterium that degrades PET at mild temperatures, has been newly identified, and further protein engineering studies on the PET degrading enzyme from the organism (IsPETase) have also been conducted to overcome the low thermal stability of the enzyme. In this study, we performed structural bioinformatics-based protein engineering of IsPETase to optimize the substrate binding site of the enzyme and developed two variants, IsPETase^S242T and IsPETase^N246D, with higher enzymatic activity at both 25 and 37 °C compared with IsPETase^WT. We also developed the IsPETase^{S121E/D186H/S242T/N246D} variant by integrating the S242 T and N246D mutations into the previously reported IsPETase^{S121E/D186H/R208A} variant. At the 37 °C incubation, the quadruple variant maintained the PET degradation activity for 20 days, unlike IsPETase^WT that lost its activity within a day. Consequently, this study exhibited 58-fold increase in the activity compared with IsPETase^WT.