Chaofeng Shao, Ziheng Cui, Yang Zhang, Jianyu Long, Xinyu Tian, Xiaowei Shen, Qian Yang, Hui Zhang, Biqiang Chen, Tianwei Tan
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. inv MM analysis confirmed excellent kinetic properties with the higher catalytic efficiency ( k cat / inv K m ). The variant retained 90% of its initial activity after 250 h incubation at 70 °C and showed a T m 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.