Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
J Hazard Mater. 2023 Jul 5;453:131386. doi: 10.1016/j.jhazmat.2023.131386. Epub 2023 Apr 7.
Polyethylene terephthalate (PET)-degrading enzymes represent a promising solution to the plastic pollution. However, PET-degrading enzymes, even thermophilic PETase, can effectively degrade low-crystallinity (∼8%) PETs, but exhibit weak depolymerization of more common, high-crystallinity (30-50%) PETs. Here, based on the thermophilic PETase, LCCICCG, we proposed two strategies for rational redesign of LCCICCG using the machine learning tool, Preoptem, combined with evolutionary analysis. Six single-point mutants (S32L, D18T, S98R, T157P, E173Q, N213P) were obtained that exhibit higher catalytic efficiency towards PET powder than wild-type LCCICCG at 75 °C. Additionally, the optimal temperature for degrading 39.07% crystalline PET increased from 65 °C in the wild-type LCCICCG to between 75 and 80 °C in the LCCICCG_I6M mutant that carries all six single-point mutations. Especially, the LCCICCG_I6M mutant has a significantly higher degradation effect on some commonly used bottle-grade plastic powders at 75-80 °C than that of wild type. The enzymatic digestion of ground 31.30% crystalline PET water bottles by LCCICCG_I6M yielded 31.91 ± 0.99 mM soluble products in 24 h, which was 3.64 times that of LCCICCG (8.77 ± 1.52 mM). Overall, this study provides a feasible route for engineering thermostable enzymes that can degrade high-crystallinity PET plastic.
聚对苯二甲酸乙二醇酯（PET）降解酶是解决塑料污染的一种有前途的方法。然而，即使是嗜热的 PETase，也只能有效地降解低结晶度（约 8%）的 PET，但对更常见的高结晶度（30-50%）的 PET 表现出较弱的解聚作用。在这里，基于嗜热的 PETase LCCICCG，我们使用机器学习工具 Preoptem 结合进化分析，提出了两种合理重新设计 LCCICCG 的策略。获得了六个单点突变体（S32L、D18T、S98R、T157P、E173Q、N213P），它们在 75°C 时对 PET 粉末的催化效率比野生型 LCCICCG 更高。此外，在野生型 LCCICCG 中，降解 39.07%结晶 PET 的最佳温度从 65°C 提高到了携带所有六个单点突变的 LCCICCG_I6M 突变体的 75-80°C。特别是，在 75-80°C 下，LCCICCG_I6M 突变体对一些常用的瓶级塑料粉末的降解效果明显高于野生型。LCCICCG_I6M 对研磨的 31.30%结晶 PET 水瓶的酶解在 24 小时内产生了 31.91±0.99mM 的可溶性产物，是 LCCICCG（8.77±1.52mM）的 3.64 倍。总的来说，这项研究为工程化耐热酶提供了一种可行的途径，这些酶可以降解高结晶度的 PET 塑料。
