Li Xian, Shi Beilei, Huang Jian-Wen, Zeng Ziyin, Yang Yu, Zhang Lilan, Min Jian, Chen Chun-Chi, Guo Rey-Ting
State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Hongshan Laboratory, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, 430062, People's Republic of China.
Bioresour Bioprocess. 2023 Apr 6;10(1):26. doi: 10.1186/s40643-023-00648-1.
Using enzymes to hydrolyze and recycle poly(ethylene terephthalate) (PET) is an attractive eco-friendly approach to manage the ever-increasing PET wastes, while one major challenge to realize the commercial application of enzyme-based PET degradation is to establish large-scale production methods to produce PET hydrolytic enzyme. To achieve this goal, we exploited the industrial strain Pichia pastoris to express a PET hydrolytic enzyme from Caldimonas taiwanensis termed CtPL-DM. In contrast to the protein expressed in Escherichia coli, CtPL-DM expressed in P. pastoris is inactive in PET degradation. Structural analysis indicates that a putative N-glycosylation site N181 could restrain the conformational change of a substrate-binding Trp and hamper the enzyme action. We thus constructed N181A to remove the N-glycosylation and found that the PET hydrolytic activity of this variant was restored. The performance of N181A was further enhanced via molecular engineering. These results are of valuable in terms of PET hydrolytic enzyme production in industrial strains in the future.
利用酶水解和回收聚对苯二甲酸乙二酯（PET）是一种颇具吸引力的环保方法，可用于处理日益增多的PET废弃物，然而，实现基于酶的PET降解商业化应用的一个主要挑战是建立大规模生产方法来生产PET水解酶。为实现这一目标，我们利用工业菌株巴斯德毕赤酵母来表达一种来自台湾嗜热栖热菌的PET水解酶CtPL-DM。与在大肠杆菌中表达的蛋白质不同，在毕赤酵母中表达的CtPL-DM在PET降解中无活性。结构分析表明，一个假定的N-糖基化位点N181可能会抑制底物结合色氨酸的构象变化并阻碍酶的作用。因此，我们构建了N181A以去除N-糖基化，发现该变体的PET水解活性得以恢复。通过分子工程进一步提高了N181A的性能。这些结果对于未来在工业菌株中生产PET水解酶具有重要价值。
