Guangdong Provincial Research Center for Environment Pollution Control and Remediation Materials, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
Guangdong Provincial Research Center for Environment Pollution Control and Remediation Materials, College of Life Science and Technology, Jinan University, Guangzhou 510632, China; Guangzhou Customs Technology Center, No. 66 Huacheng Avenue, Tianhe District, Guangzhou, China.
Environ Int. 2023 Aug;178:108054. doi: 10.1016/j.envint.2023.108054. Epub 2023 Jun 19.
Microbial degradation has been confirmed as effective and environmentally friendly approach to remediate phthalates from the environment, and hydrolase is an effective element for contaminant degradation. In the present study, a novel dibutyl phthalate (DBP)-hydrolyzing carboxylesterase (named PS06828) from Pseudomonas sp. PS1 was heterogeneously expressed in E. coli, which was identified as a new member of the lipolytic family VI. Purified PS06828 could efficiently degrade DBP with a wide range of temperature (25-37 °C) and pH (6.5-9.0). Multi-spectroscopy methods combined with molecular docking were employed to study the interaction of PS06828 with DBP. Fluorescence and UV-visible absorption spectra revealed the simultaneous presence of static and dynamic component in the fluorescence quenching of PS06828 by DBP. Synchronous fluorescence and circular dichroism spectra showed inconspicuous alteration in micro-environmental polarity around amino acid residues but obvious increasing of α-helix and reducing of β-sheet and random coil in protein conformation. Based on the information on exact binding sites of DBP on PS06828 provided by molecular docking, the catalytic mechanism mediated by key residues (Ser113, Asp166, and His197) was proposed and subsequently confirmed by site-directed mutagenesis. The results can strengthen our mechanistic understanding of family VI esterase involved in hydrolysis of phthalic acid esters, and provide a solid foundation for further enzymatic modification.
微生物降解已被证实是一种有效且环保的方法，可用于修复环境中的邻苯二甲酸酯，而水解酶是污染物降解的有效元素。本研究从假单胞菌 PS1 中异源表达了一种新型邻苯二甲酸二丁酯（DBP）水解羧酸酯酶（命名为 PS06828），该酶被鉴定为脂解酶家族 VI 的一个新成员。纯化的 PS06828 可以在较宽的温度（25-37°C）和 pH（6.5-9.0）范围内有效降解 DBP。采用多光谱法结合分子对接研究了 PS06828 与 DBP 的相互作用。荧光和紫外-可见吸收光谱表明，DBP 对 PS06828 的荧光猝灭同时存在静态和动态成分。同步荧光和圆二色性光谱表明，氨基酸残基周围微环境极性没有明显变化，但蛋白质构象中α-螺旋增加，β-折叠和无规卷曲减少。基于分子对接提供的 DBP 在 PS06828 上的确切结合位点信息，提出了由关键残基（Ser113、Asp166 和 His197）介导的催化机制，并通过定点突变得到了进一步证实。这些结果可以加强我们对涉及邻苯二甲酸酯水解的家族 VI 酯酶的机制理解，并为进一步的酶修饰提供坚实的基础。
