School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, China.
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, China; Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, PR China.
Environ Res. 2022 Sep;212(Pt D):113472. doi: 10.1016/j.envres.2022.113472. Epub 2022 May 14.
Ideonella sakaiensis PET hydrolase (IsPETase) is a well-characterized enzyme for effective PET biodegradation. However, the low soluble expression level of the enzyme hampers its practical implementation in the biodegradation of PET. Herein, the expression of IsPETase, one of the most active mutants of IsPETase obtained so far, was systematically explored in E. coli by adopting a series of strategies. A notable improvement of soluble IsPETase was observed by using chaperon co-expression and fusion expression systems. Under the optimized conditions, GroEL/ES co-expression system yielded 75 ± 3.4 mg·L purified soluble IsPETase (GroEL/ES), and NusA fusion expression system yielded 80 ± 3.7 mg·L purified soluble NusA-IsPETase, which are 12.5- and 4.6-fold, respectively, higher than its commonly expression in E. coli. The two purified enzymes were further characterized. The results showed that IsPETase (GroEL/ES) displayed the same catalytic behavior as IsPETase, while the fusion of NusA conferred new enzymatic properties to IsPETase. Although NusA-IsPETase displayed a lower initial hydrolysis capacity than IsPETase, it showed a 1.4-fold higher adsorption constant toward PET. Moreover, the product inhibition effect of terephthalic acid (TPA) on IsPETase was reduced with NusA-IsPETase. Taken together, the latter two catalytic properties of NusA-IsPETase are more likely to contribute to the enhanced product release by NusA-IsPETase PET degradation for two weeks.
解淀粉欧文氏菌 PET 水解酶（IsPETase）是一种经过充分研究的有效 PET 生物降解酶。然而，由于该酶的可溶性表达水平较低，限制了其在 PET 生物降解中的实际应用。本研究通过采用一系列策略，在大肠杆菌中对该酶的一个最活跃突变体 IsPETase 的表达进行了系统探索。通过伴侣蛋白共表达和融合表达系统，可显著提高可溶形式的 IsPETase 表达水平。在优化条件下，GroEL/ES 共表达系统可得到 75±3.4mg·L 的纯化可溶 IsPETase（GroEL/ES），NusA 融合表达系统可得到 80±3.7mg·L 的纯化可溶 NusA-IsPETase，分别比其在大肠杆菌中的常规表达提高了 12.5 倍和 4.6 倍。进一步对这两种纯化酶进行了表征。结果表明，IsPETase（GroEL/ES）表现出与 IsPETase 相同的催化行为，而 NusA 的融合赋予了 IsPETase 新的酶学特性。尽管 NusA-IsPETase 的初始水解能力比 IsPETase 低，但它对 PET 的吸附常数提高了 1.4 倍。此外，NusA-IsPETase 对邻苯二甲酸（TPA）的产物抑制作用也有所降低。综上所述，NusA-IsPETase 的后两种催化特性可能有助于其在两周的 PET 降解过程中提高产物释放效率。
