腈水合酶(nitrile hydratase,NHase,EC 4.2.1.84)是一种催化腈类化合物生成酰胺类化合物的金属酶,工业上用于生物法生产丙烯酰胺和烟酰胺。由于腈类的水合反应是放热反应,工业上对腈水合酶的热稳定性较为关注。该实验室此前通过基因挖掘获得了具有高热稳定性的温泉热碱芽孢杆菌(Caldalkalibacillus thermarum TA2.A1)来源的腈水合酶,但其活性与工业生产要求有较大的差距,因此,提高该来源腈水合酶的酶活力具有重要意义。该研究采用分子动力学模拟的方法,通过对β亚基上位于腈水合酶底物通道入口的N47和N181这2个位点进行代表性氨基酸突变,破坏氢键,提高底物通道入口柔性,增强通道入口构象可变性,继而提高酶促反应催化效率。研究得到了最优突变体βN47F,与野生型酶相比,催化丙烯腈的比酶活力提高了2.57倍,且保持了良好的热稳定性,为工业化应用奠定了坚实的基础。
Nitrile hydratase (nitrile hydratase, NHase, EC 4.2.1.84) is one type of metalloenzyme that catalyzes the formation of amides from nitriles. It is used in the biological production of acrylamide and nicotinamide in industry. Since the hydration of nitriles is an exothermic reaction, the thermal stability of nitrile hydratase attracted increasing attention. Previously, a novel nitrile hydratase derived from Caldalkalibacillus thermarum TA2.A1 was obtained through gene mining. Cal.t NHase possesses excellent thermal stability, while the enzyme activity is far behind the requirements of industrial production. Therefore, improving catalytic efficiency is important for its further applications. This study adopted molecular dynamics simulation methods and found two key residues, N47 and N181, located at the entrance of the substrate channel on the b subunit. Making representative residues mutations at these sites breaks the hydrogen bond, increases the flexibility of the substrate channel entrance, and then improves the enzyme activity. The optimal mutant, βN47F, compared with the wild-type enzyme, shows a 2.57-fold increase in acrylonitrile catalytic activity, and maintain good thermal stability at the same time. This variant enables the industrial application of Cal.t NHase in the future.
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