对 EKylated PETase 对 PET 降解的增强性能的分子见解,ACS Catalysis

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对 EKylated PETase 对 PET 降解的增强性能的分子见解
ACS Catalysis ( IF 11.3 ) Pub Date : 2021-06-07 , DOI: 10.1021/acscatal.1c01062
Kun Chen ₁ , Yang Hu ₁ , Xiaoyan Dong _{1,

2} , Yan Sun _{1,

2}

Affiliation

聚对苯二甲酸乙二醇酯（PET）在环境中的积累给全球生态系统带来了巨大威胁。尽管最近报道的 PET 水解酶 (PETase) 显示出对 PET 的有效分解，但其低活性和热稳定性限制了其实际应用。在此，我们通过将由带交替电荷的谷氨酸 (E) 和赖氨酸 (K) 残基组成的两性离子多肽 (5–30 kDa) 融合到 PETase 的 C 端来引入生物修饰策略，并发现增加融合肽长度会导致以提高催化性能。与 PETase 相比，PETase-EK30 降解高度结晶的 PET 薄膜（结晶度为 45.2%）时的产物释放提高了 11 倍以上。通过结构分析、底物结合和分子模拟研究了增强催化性能的分子机制。二级和三级结构的表征证实了 EKylated PETase 的结构稳定性增强。同步荧光光谱表明 EKylation 后更开放的底物结合口袋。酶-底物复合物的 MD 模拟支持 EKylation 诱导底物结合口袋中疏水性氨基酸（W185、I208 和 W159）的暴露和 Y87 苯环的旋转，这促进了底物结合动力学。这导致底物亲和力增强，具体表现为结合常数增加和结合自由能降低。此外，从 MD 模拟中观察到缩短的催化距离，这也可能有助于增强对 PET 降解的催化活性。对增强的酶性能的分子见解将有助于扩展 EKylation 策略在各种酶中的应用。

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Molecular Insights into the Enhanced Performance of EKylated PETase Toward PET Degradation

The accumulation of polyethylene terephthalate (PET) in the environment has brought an enormous threat to the global ecosystem. Although the recently reported PET hydrolase (PETase) displays an efficient decomposition to PET, the low activity and thermostability limit its practical applications. Herein, we introduce a biomodification strategy by fusing a zwitterionic polypeptide (5–30 kDa) consisting of alternating-charged glutamic acid (E) and lysine (K) residues to the C-terminus of PETase and find that increasing the fusion peptide length leads to the improved catalytic performance. The product release in the degradation of highly crystallized PET films (45.2% in crystallinity) by PETase-EK30 is promoted by over 11 times as compared with PETase. The molecular mechanism of the enhanced catalytic performance is investigated via structural analysis, substrate binding, and molecular simulations. Characterizations of the secondary and tertiary structures verify a strengthened structural stability of the EKylated PETases. Synchronous fluorescence spectra indicate a more open substrate-binding pocket after EKylation. MD simulations of enzyme–substrate complexes support that the EKylation induces the exposure of hydrophobic amino acids (W185, I208, and W159) in the substrate-binding pocket and the rotation of the benzene ring of Y87, which promote the substrate binding kinetics. This leads to the enhanced substrate affinity, exactly represented by the increased association constant and the decreased binding free energy. Besides, a shortened catalytic distance is observed from the MD simulations, which might also contribute to the enhanced catalytic activity toward PET degradation. The molecular insights into the enhanced enzyme performance would benefit in extending the application of the EKylation strategy in various enzymes.

更新日期：2021-06-18

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