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<p>Enzymatic PET depolymerization represents a promising approach for establishing a circular economy for PET plastics. Nonetheless, limitations in enzyme activity persist as significant challenges to its industrial application. In this research, the backbone structure of the β6-β7 loop for PET hydrolase Bhr-PETase derived from the <i>thermophilic bacterium HR29</i> was reconstructed by introducing double mutations (H218N/F222M), resulting in variant Bhr-NMT with high thermal stability (<i>T</i><sub>m</sub> = 92.9 °C) and 87% increase in activity. Moreover, the loop reconstruction mutations are transplanted into the engineered PET hydrolases LCC-ICCG and Kubu-P<sup>M12</sup>, resulting variants LCC-ICCG-NM (<i>T</i><sub>m</sub> = 92.4 °C) and Kubu-P<sup>M12</sup>-NM (<i>T</i><sub>m</sub> = 92.9 °C). Under high substrate concentration (165 g kg<sup>−1</sup>) and an enzyme loading of 0.5 mg<sub>enzyme</sub> g<sub>PET</sub><sup>−1</sup>, the designed variants Bhr-NMT, LCC-ICCG-NM, and Kubu-P<sup>M12</sup>-NM achieve an overall conversion of 93%, 90%, and 94%, respectively, outperforming the benchmark LCC-ICCG (85%). Notably, under reduced enzyme loading (0.3 mg<sub>enzyme</sub> g<sub>PET</sub><sup>−1</sup>), Kubu-P<sup>M12</sup>-NM still reaches an overall conversion of 91%, which is significantly superior to benchmarks Kubu-P<sup>M12</sup> (83%) and LCC-ICCG (71%). Overall, the engineered PET hydrolases demonstrate significant potential for industrial PET waste recycling.</p>

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Computational loop reconstruction based design of efficient PET hydrolases

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年6月17日

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  • Hongzhao Wang,
  • Yuntao Cun,
  • Minxuan Wang,
  • Xiaoyu Du,
  • Zhenwu Yang,
  • Hao Wang,
  • Jun Zhang,
  • Pengyu Wang,
  • Yue Feng,
  • Yushan Zhu

摘要

Enzymatic PET depolymerization represents a promising approach for establishing a circular economy for PET plastics. Nonetheless, limitations in enzyme activity persist as significant challenges to its industrial application. In this research, the backbone structure of the β6-β7 loop for PET hydrolase Bhr-PETase derived from the thermophilic bacterium HR29 was reconstructed by introducing double mutations (H218N/F222M), resulting in variant Bhr-NMT with high thermal stability (Tm = 92.9 °C) and 87% increase in activity. Moreover, the loop reconstruction mutations are transplanted into the engineered PET hydrolases LCC-ICCG and Kubu-PM12, resulting variants LCC-ICCG-NM (Tm = 92.4 °C) and Kubu-PM12-NM (Tm = 92.9 °C). Under high substrate concentration (165 g kg−1) and an enzyme loading of 0.5 mgenzyme gPET−1, the designed variants Bhr-NMT, LCC-ICCG-NM, and Kubu-PM12-NM achieve an overall conversion of 93%, 90%, and 94%, respectively, outperforming the benchmark LCC-ICCG (85%). Notably, under reduced enzyme loading (0.3 mgenzyme gPET−1), Kubu-PM12-NM still reaches an overall conversion of 91%, which is significantly superior to benchmarks Kubu-PM12 (83%) and LCC-ICCG (71%). Overall, the engineered PET hydrolases demonstrate significant potential for industrial PET waste recycling.