Key Laboratory of Industrial Biotechnology (Ministry of Education), School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
Key Laboratory of Industrial Biotechnology (Ministry of Education), School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
Int J Biol Macromol. 2023 Aug 1;245:125531. doi: 10.1016/j.ijbiomac.2023.125531. Epub 2023 Jun 22.
Nitrile hydratase (NHase) has been extensively utilized in industrial acrylamide production. However, the vulnerability to high concentrations of acrylamide limits its further application. Herein, we redesigned the N-terminal loop at the tetramer interface of a thermophilic NHase from Pseudonocardia thermophila JCM3095 (PtNHase), and its catalytic activity, resistance to high acrylamide concentrations, and thermostability were improved. Amino acid residues located in the N-terminal loop of the tetramer interface that are responsible for enhancing the resistance to high acrylamide concentrations were identified via static structural analysis and molecular dynamics simulations. A variant library was used to fine-tune the tetramer interface. Variant αL6T exhibited 3.5-fold greater resistance to 50% (v/v) acrylamide, whereas its activity was 1.2-fold higher than that of the wild-type (WT) enzyme, revealing no activity-stability trade-off. Compared to the use of Escherichia coli harboring the WT enzyme, the use of E. coli harboring αL6T increased the acrylamide concentration from 398.1 g/L to 500 g/L. Crystal structure-guided analysis of αL6T and molecular dynamics simulations revealed that increased enzyme surface hydration and the introduction of positive cross-correlation into the N-terminal loop of the tetramer interface caused the two loop regions to hook to each other, thus improving the resistance to high acrylamide concentrations.
腈水合酶（NHase）已广泛应用于工业丙烯酰胺生产。然而，其对高浓度丙烯酰胺的敏感性限制了其进一步的应用。在此，我们对嗜热假诺卡氏菌 JCM3095 的四聚体界面处的 N 端环进行了重新设计（PtNHase），提高了其催化活性、耐高丙烯酰胺浓度和热稳定性。通过静态结构分析和分子动力学模拟，确定了位于四聚体界面 N 端环中负责提高耐高丙烯酰胺浓度的氨基酸残基。使用变体文库对四聚体界面进行精细调整。变体αL6T 对 50%（v/v）丙烯酰胺的抗性提高了 3.5 倍，而其活性比野生型（WT）酶高 1.2 倍，没有活性-稳定性权衡。与使用携带 WT 酶的大肠杆菌相比，使用携带αL6T 的大肠杆菌将丙烯酰胺浓度从 398.1 g/L 提高到 500 g/L。αL6T 的晶体结构指导分析和分子动力学模拟表明，增加酶表面水合作用并在四聚体界面的 N 端环中引入正交叉相关，使两个环区相互钩住，从而提高了耐高丙烯酰胺浓度的能力。
