Integrated Enzyme Engineering and One-Step Pretreatment Enable Scalable PET Waste Depolymerization

Polyethylene terephthalate (PET), the most abundant polyester plastic, poses significant environmental and health risks. Biotechnological recycling has emerged as a promising solution, yet efficient PET biodepolymerization at industrial scale require robust PET hydrolases and compatible pretreatment process. Here, we report a two-stage engineering strategy combining rational optimization of substrate-binding residues and computational thermostability design on the PET hydrolase ThcCut1-AICCG. The resulting variant, NI-M7, exhibited a balanced enhancement in catalytic activity, thermostability, and depolymerization efficiency. invMM analysis confirmed excellent kinetic properties with the higher catalytic efficiency (kcat/invKm). The variant retained 90% of its initial activity after 250 h incubation at 70 °C and showed a Tm of 91.8 °C. The exceptional thermal durability also enabled efficient heat-based purification. Furthermore, the scale-up bioreactor trials validated its industrial scalability. We combined the variant with a one-step high-speed milling pretreatment that simultaneously induced amorphization and micronization of postconsumer PET. In a 3 L bioreactor, the integrated enzymatic depolymerization system yielded >90% conversion within 24 h, with 100% of the released product being TPA. By integrating enzyme engineering with a one-step pretreatment, this work establishes a practical workflow for PET biorecycling with reduced process complexity and offers a viable blueprint for closed-loop plastic recycling.