Thermostability and Activity Improvements of PETase from <i>Ideonella sakaiensis</i>
Jansen Stevensen, Rifqi Zahroh Janatunaim, Aisy Humaira Ratnaputri, Safero Hedi Aldafa, Resmila Rachelisa Rudjito, Dwi Hadi Saputro, Sony Suhandono, Rindia M. Putri, Reza Aditama, Azzania Fibriani
Abstract
High Resolution Image Download MS PowerPoint Slide Polyethylene terephthalate (PET), a widely used plastic, is a significant environmental pollutant due to its persistence. While the PET-degrading enzyme PETase from Ideonella sakaiensis offers promising solutions, its limited activity at higher temperatures hinders its practical application. This study aimed to enhance the PETase performance through protein engineering. We introduced multiple amino acid substitutions to the wild-type I. sakaiensis PETase to improve its thermostability, substrate binding, and catalytic activity. Several potential mutant IsPETases were generated using computational design and evaluated in silico . The selected mutant was then produced in E. coli BL21(DE3). Finally, the catalytic activity of the purified mutant IsPETase was examined in vitro using p -nitrophenyl butyrate and PET substrates. Is PETase MT has been confirmed to be catalytically active and more thermostable with a maximum temperature reaching 60 °C and the T m value increasing up to 15.3 °C compared to the wild-type PETase, Is PETase WT . Is PETase MT also showed better degradation toward the PET plastic film in comparison to Is PETase WT . Thus, these findings demonstrate successful protein engineering to create a more robust PETase for potential plastic waste management applications.