Structural insights and engineering of deep-sea halophilic PET hydrolytic enzymes

作者：Guoqiang Zhang, Li X, Wei Xia, Chengsong Zhang, Shanmin Zheng, Jieke Du, Ning Wang, Xinzhe Chen, Ge Lv, Yushuo Zhao, Tianheng Wang, Yunjun Pan, Mingzhu Zhang, Jian‐Wen Huang, Chun-Chi Chen, Siqi Huang, Cheng Zeng, Zhengquan Gao, Jianwei Chen, Guangyi Fan, Xingwang Zhang, Hongliang Wang, Yongfu Sun, Jing Wu, Kun Liu, Rey-Ting Guo, Shengying Li · 发表于：bioRxiv (Cold Spring Harbor Laboratory) · 年份：2025 · DOI：10.1101/2025.08.30.673199 · 被引用次数：5 · 研究领域：Microplastics and Plastic Pollution、Graphene and Nanomaterials Applications、Effects and risks of endocrine disrupting chemicals

Abstract Pervasive use of polyethylene terephthalate (PET) poses tremendous challenges for global waste management and environmental sustainability, fueling growing interests in enzymatic degradation as an eco-friendly solution. While PET hydrolases hold significant promise, their industrial deployment is hindered by insufficient performance, particularly under high-salinity conditions raised from high substrate loads. Building on our previous discovery of three deep-sea PET hydrolases (dsPETase01, dsPETase05 and dsPETase06) with exceptional halophilicity and PET-degrading activity, we here present their three-dimensional structures and mechanistic characterization. Structural comparison, site-directed mutagenesis, and domain swapping reveal key structural features and the essential role of C -terminal acidic residues in salt tolerance. Integrating semi-rational protein design, Transformer-based modeling, and disulfide bond engineering, we synergistically enhance the thermostability and catalytic efficiency of dsPETase05. These findings elucidate the unique structural features and salt adaptation mechanisms of deep-sea halophilic PET hydrolases, and inform the future engineering of biocatalysts for application in harsh industrial environments.