Li Xian, Huang Jian-Wen, Ning Zhiyuan, Huang Siqi, Zeng Cheng, Zeng Ziyin, Ji Rui, Peng Rouming, Liu Xin, Min Jian, Chen Chun-Chi, Guo Rey-Ting
Zhejiang Key Laboratory of Medical Epigenetics, Hubei Hongshan Laboratory, Department of Immunology and Pathogen Biology, School of Basic Medical Sciences, Hangzhou Normal University, 311121 Hangzhou, China; State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan 430062, China.
Zhejiang Key Laboratory of Medical Epigenetics, Hubei Hongshan Laboratory, Department of Immunology and Pathogen Biology, School of Basic Medical Sciences, Hangzhou Normal University, 311121 Hangzhou, China.
Int J Biol Macromol. 2025 Aug;320(Pt 2):145862. doi: 10.1016/j.ijbiomac.2025.145862. Epub 2025 Jul 11.
Enzymatic degradation of polyethylene terephthalate (PET) provides a sustainable and promising strategy for the recycling of plastic waste. Herein, we employed site-directed mutagenesis and machine learning methods to further enhance the performance of an efficient mutant of IsPETase, FAST-PETase-N212A, and resulting in seven variants with enhanced activity. We also found that the α3-β5 loop containing the T140D mutation plays a significant role in both type I and type II cutinases. Subsequently, we determined the complex structures of two activity-elevated mutants with the PET analogue mono(2-hydroxyethyl)terephthalic acid, revealing a different binding mode. Finally, to facilitate the industrial application of PET hydrolases, we exploited the industrial strain Pichia pastoris to express the activity-enhanced mutants. Compared with E. coli-produced proteins, these mutants expressed by P. pastoris exhibited higher activity and thermal stability.
聚对苯二甲酸乙二酯（PET）的酶促降解为塑料废物的回收利用提供了一种可持续且有前景的策略。在此，我们采用定点诱变和机器学习方法进一步提高高效突变体IsPETase、FAST-PETase-N212A的性能，从而得到了七个活性增强的变体。我们还发现，含有T140D突变的α3-β5环在I型和II型角质酶中都起着重要作用。随后，我们确定了两个活性提高的突变体与PET类似物单（2-羟乙基）对苯二甲酸的复合物结构，揭示了不同的结合模式。最后，为促进PET水解酶的工业应用，我们利用工业菌株巴斯德毕赤酵母来表达活性增强的突变体。与大肠杆菌产生的蛋白质相比，这些由巴斯德毕赤酵母表达的突变体表现出更高的活性和热稳定性。
