Rational Protein Engineering of Thermo-Stable PETase from <i>Ideonella sakaiensis</i> for Highly Efficient PET Degradation

Widespread utilization of polyethylene terephthalate (PET) has caused a variety of environmental and health problems; thus, the enzymatic degradation of PET can be a promising solution. Although PETase from Ideonalla sakaiensis (IsPETase) has been reported to have the highest PET degradation activity under mild conditions of all PET-degrading enzymes reported to date, its low thermal stability limits its ability for efficient and practical enzymatic degradation of PET. Using the structural information on IsPETase, we developed a rational protein engineering strategy using several IsPETase variants that were screened for high thermal stability to improve PET degradation activity. In particular, the IsPETaseS121E/D186H/R280A variant, which was designed to have a stabilized β6-β7 connecting loop and extended subsite IIc, had a Tm value that was increased by 8.81 °C and PET degradation activity was enhanced by 14-fold at 40 °C in comparison with IsPETaseWT. The designed structural modifications were further verified through structure determination of the variants, and high thermal stability was further confirmed by a heat-inactivation experiment. The proposed strategy and developed variants represent an important advancement for achieving the complete biodegradation of PET under mild conditions.