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

Zhang, G.; Li, X.; Xia, W.; Zhang, C.; Zheng, S.; Du, J.; Wang, N.; Chen, X.; Lv, G.; Zhao, Y.; Wang, T.; Pan, Y.; Zhang, M.; Huang, J.-W.; Chen, C.-C.; Huang, S.; Zeng, C.; Gao, Z.; Chen, J.; Fan, G.; Zhang, X.; Wang, H.; Sun, Y.; Wu, J.; Liu, K.; Guo, R.-T.; Li, S.

doi:10.1101/2025.08.30.673199

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bioRxiv · 10.1101/2025.08.30.673199

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

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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.

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Zhang, G., Li, X., Xia, W., Zhang, C., Zheng, S., Du, J., Wang, N., Chen, X., Lv, G., Zhao, Y., Wang, T., Pan, Y., Zhang, M., Huang, J.-W., Chen, C.-C., Huang, S., Zeng, C., Gao, Z., Chen, J., Fan, G., Zhang, X., Wang, H., Sun, Y., Wu, J., Liu, K., Guo, R.-T., Li, S.. 2025-08-30. Structural insights and engineering of deep-sea halophilic PET hydrolytic enzymes. https://doi.org/10.1101/2025.08.30.673199

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