Synergistic engineering PETase reveals loop-region mutations for enhanced catalytic activity and thermal stability
2026
Yuxin Han | Shilong Xing | Mingzhu Ding | Ying Wang | Wenhai Xiao | Yixun Jiang | Mingdong Yao
Biodegradation of Polyethylene Terephthalate (PET) offer a strategic avenue for addressing the global plastic pollution crisis. However, the degradation performance of PET hydrolases remains a technological bottleneck for the industrial realization of plastic biodegradation. In this study, we employed an integrated strategy combining semi-rational design and directed evolution to discover new mutation sites (N114, N205, N233 and S269) that enhance the catalytic activity and thermostability of Ideonella sakaiensis PETase (IsPETase). Subsequently, through the combined design of newly discovered mutation sites, we screened the novel quadruple mutant (N114I/N205K/N233K/S269V, named QM-PETase-2), which exhibited a 4.9-fold increase in catalytic efficiency and a ΔTm of +12.4 °C. Interestingly, the four newly discovered mutation sites are all located in the loop region of the enzyme structure, which might play a crucial role on the structural stability of enzyme. Also, molecular dynamics simulations revealed that the QM-PETase-2 exhibited a more stable structure and an expanded substrate-binding cleft, which would facilitate the binding of the polymer PET substrate. Especially, the newly quadruple mutation sites were introduced into three reported high-performance PETase mutants FAST-PETase, PA-PETase, and DepoPETase. The obtained combined mutants, named QMFAST-PETase, QMPA-PETase, and QM-DepoPETase, demonstrated higher activity and thermal stability, indicating that the newly discovered mutations are universal for improving the performance of PETase. This research would be helpful in guiding the optimization and development of PETase.
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