School of Biological Science, Jining Medical University, Jining, China.
College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
Ecotoxicol Environ Saf. 2024 Jul 15;280:116540. doi: 10.1016/j.ecoenv.2024.116540. Epub 2024 Jun 3.
The widespread utilization of polyethylene terephthalate (PET) has caused a variety of environmental and health problems. Compared with traditional thermomechanical or chemical PET cycling, the biodegradation of PET may offer a more feasible solution. Though the PETase from Ideonalla sakaiensis (IsPETase) displays interesting PET degrading performance under mild conditions; the relatively low thermal stability of IsPETase limits its practical application. In this study, enzyme-catalysed PET degradation was investigated with the promising IsPETase mutant HotPETase (HP). On this basis, a carbohydrate-binding module from Bacillus anthracis (BaCBM) was fused to the C-terminus of HP to construct the PETase mutant (HLCB) for increased PET degradation. Furthermore, to effectively improve PET accessibility and PET-degrading activity, the truncated outer membrane hybrid protein (FadL) was used to expose PETase and BaCBM on the surface of E. coli (BL21with) to develop regenerable whole-cell biocatalysts (D-HLCB). Results showed that, among the tested small-molecular weight ester compounds (p-nitrophenyl phosphate (pNPP), p-Nitrophenyl acetate (pNPA), 4-Nitrophenyl butyrate (pNPB)), PETase displayed the highest hydrolysing activity against pNPP. HP displayed the highest catalytic activity (1.94 μM(p-NP)/min) at 50 °C and increased longevity at 40 °C. The fused BaCBM could clearly improve the catalytic performance of PETase by increasing the optimal reaction temperature and improving the thermostability. When HLCB was used for PET degradation, the yield of monomeric products (255.7 μM) was ∼25.5 % greater than that obtained after 50 h of HP-catalysed PET degradation. Moreover, the highest yield of monomeric products from the D-HLCB-mediated system reached 1.03 mM. The whole-cell catalyst D-HLCB displayed good reusability and stability and could maintain more than 54.6 % of its initial activity for nine cycles. Finally, molecular docking simulations were utilized to investigate the binding mechanism and the reaction mechanism of HLCB, which may provide theoretical evidence to further increase the PET-degrading activities of PETases through rational design. The proposed strategy and developed variants show potential for achieving complete biodegradation of PET under mild conditions.
聚对苯二甲酸乙二醇酯（PET）的广泛应用导致了各种环境和健康问题。与传统的热机械或化学 PET 循环相比，PET 的生物降解可能提供了更可行的解决方案。尽管来自 Ideonella sakaiensis 的 PETase（IsPETase）在温和条件下显示出有趣的 PET 降解性能；但 IsPETase 的相对较低的热稳定性限制了其实际应用。在这项研究中，使用有前途的 IsPETase 突变体 HotPETase（HP）研究了酶催化的 PET 降解。在此基础上，将来自炭疽杆菌的碳水化合物结合模块（BaCBM）融合到 HP 的 C 末端，构建了用于增加 PET 降解的 PETase 突变体（HLCB）。此外，为了有效提高 PET 的可及性和 PET 降解活性，使用截短的外膜杂合蛋白（FadL）将 PETase 和 BaCBM 暴露在大肠杆菌（BL21with）表面，以开发可再生的全细胞生物催化剂（D-HLCB）。结果表明，在所测试的小分子酯化合物（对硝基苯酚磷酸盐（pNPP）、对硝基苯乙酸酯（pNPA）、4-硝基丁酸酯（pNPB））中，PETase 对 pNPP 表现出最高的水解活性。HP 在 50°C 时表现出最高的催化活性（1.94μM(p-NP)/min），并在 40°C 时延长了寿命。融合的 BaCBM 可以通过提高最佳反应温度和改善热稳定性，明显提高 PETase 的催化性能。当 HLCB 用于 PET 降解时，单体产物的产率（255.7μM）比 HP 催化 PET 降解 50 小时后的产率高约 25.5%。此外，D-HLCB 介导的系统中单体产物的最高产率达到 1.03mM。全细胞催化剂 D-HLCB 表现出良好的可重复使用性和稳定性，可在九轮循环中保持初始活性的 54.6%以上。最后，利用分子对接模拟研究了 HLCB 的结合机制和反应机制，这可能为通过合理设计进一步提高 PETase 的 PET 降解活性提供理论依据。所提出的策略和开发的变体有望在温和条件下实现 PET 的完全生物降解。
