Rational Protein Engineering of Thermo-Stable PETase from Ideonella sakaiensis 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.