College of Food Science and Light Industry, Nanjing Tech University, Nanjing, Jiangsu 211816, China.
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, Jiangsu 211816, China.
J Hazard Mater. 2024 Dec 5;480:136389. doi: 10.1016/j.jhazmat.2024.136389. Epub 2024 Nov 1.
Enzymatic degradation offers a promising eco-friendly solution to plastic pollution, especially for polyethylene terephthalate (PET). Current efforts have focused on screening PET-degrading enzymes from microbial and metagenomic sources and obtaining superior candidates with a limited set of templates. More efficient PET hydrolases are required for PET-waste biorefinery. Here, using a structure-guided bioinformatic workflow, we identified a novel PET hydrolase, LSPET4, from Micromonospora sp. HM5-17, by screening the AlphaFold protein structure database. LSPET4 features a unique carbohydrate-binding module (CBM) and a distinctive linear substrate binding conformation. The intrinsic CBM in LSPET4 exhibited superior binding ability on PET surfaces and enhanced PET hydrolysis performance compared to the previously reported most effective CBM3. Through rational protein engineering focused on stabilizing and modifying the linear substrate binding conformation, we developed LSPET4 (D130P, N127F, Y96F, Q209E, A238K, D241S), a variant that achieved a 38.79-fold improvement in activity compared to the wild type, and was comparable to the reported most effective PET hydrolase derived from IsPETase, FAST-PETase at 45 ℃. This variant also demonstrated effectiveness in degrading various commercial PET materials, including PET food sealing films, PET strawberry boxes, and PET tomato boxes used in the food industry. This study not only provides a new template for protein engineering endeavors to create efficient biocatalysts for PET recycling but also offers an effective enzyme discovery approach to uncover enzymes of interest from the AlphaFold protein structure database.
酶降解为塑料污染提供了一种有前途的环保解决方案，特别是对于聚对苯二甲酸乙二醇酯（PET）。目前的研究重点是从微生物和宏基因组来源筛选 PET 降解酶，并利用有限的模板获得具有优异性能的候选酶。需要更有效的 PET 水解酶来实现 PET 废物的生物炼制。在这里，我们通过筛选 AlphaFold 蛋白质结构数据库，使用基于结构的生物信息学工作流程，从 Micromonospora sp. HM5-17 中鉴定出一种新型的 PET 水解酶 LSPET4。LSPET4 具有独特的碳水化合物结合模块（CBM）和独特的线性底物结合构象。LSPET4 中的固有 CBM 表现出对 PET 表面的优异结合能力，并与之前报道的最有效 CBM3 相比，增强了 PET 水解性能。通过聚焦于稳定和修饰线性底物结合构象的合理蛋白质工程，我们开发了 LSPET4（D130P、N127F、Y96F、Q209E、A238K、D241S），与野生型相比，该变体的活性提高了 38.79 倍，与报道的最有效 PET 水解酶 IsPETase 的 FAST-PETase 相当，在 45℃下。该变体在降解各种商业 PET 材料方面也表现出有效性，包括用于食品工业的 PET 食品密封膜、PET 草莓盒和 PET 番茄盒。这项研究不仅为利用蛋白质工程努力创造高效的 PET 回收生物催化剂提供了新的模板，还为从 AlphaFold 蛋白质结构数据库中发现有价值的酶提供了一种有效的酶发现方法。
