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

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.