Abstract: Nitrile hydratase (NHase) is a double-subunit enzyme widely used for industrial production of acrylamide from acrylonitrile. By Discovery Studio 2.5, salt-bridge network simulation toward different recombinant NHases was carried out. A new mutant, NHase^M-TH-SB^M (SB^M), was obtained, which couples the salt-bridge mutation in C-terminal (SB, S344K-S346K-L347E-435DT436(+)) with the point-mutation of N362S. Simulation results show that the total number of salt-bridges in SB^M decreases but the number in a-b interface increases to 7 pairs. Using both the original NHase TH and SB mutant as controls, SB^M is successfully expressed in recombinant E. coli. The activity is 543.9 U·mg^-1, increased by 31.0% with respect to TH. The reaction and inactivation kinetics was also investigated. The catalytic rate constant K_cat of Michaelis-Menten equation is increased by 20% and 60% compared to TH and SB, respectively. The apparent inactivation constant K_D is 68.0% of TH at 42℃, indicating that SB^M also improves thermal stability.

Key words: nitrile hydratase, terminal salt bridge, point-mutation, reaction kinetics, Michaelis-Menten equation, deactivation kinetics

摘要： 双亚基腈水合酶是催化丙烯腈水合生产丙烯酰胺的重要工业酶。通过Discovery Studio 2.5软件对C-末端盐桥耦合定点突变改造的耐热型基因重组腈水合酶NHase^M-TH-SB^M（S344K-S346K-L347E-N362S-435DT436（+））进行盐桥网络分析，发现其全局盐桥总数有所下降，但双亚基界面间盐桥数量提高到7个。在重组大肠杆菌中表达了原腈水合酶NHase^M-TH（TH）、盐桥突变酶NHase^M-TH-SB（SB）和盐桥耦合定点突变的重组酶NHase^M-TH-SB^M（SB^M），研究了3种酶的催化反应动力学和失活动力学。结果表明，SB^M的腈水合酶活性为543.9 U·mg^-1，比TH提高了31.0%；其米氏方程催化速率常数K_cat比TH提高了20%。SB^M的表观失活常数K_D在25～42℃范围内均明显低于对照TH，在42℃时为TH的68.0%，显示了良好的热稳定性。SB^M是活性和稳定性同时提高的重组腈水合酶。

关键词: 腈水合酶, 末端盐桥, 定点突变, 反应动力学, 米氏方程, 失活动力学