腈水合酶(nitrile hydratase,NHase,EC 4.2.1.84)是一类将腈类化合物经水合反应生产酰胺类化合物的金属酶,工业上用该酶进行生物法生产烟酰胺。由于水合过程放热,工业用酶对其稳定性提出了更高的要求。实验室前期通过基因挖掘获得了一种来源于嗜热菌温泉热碱芽胞杆菌(Caldalkalibacillus thermarum) TA2.A1的腈水合酶基因,但其催化活性不足,难以满足应用需求。该研究基于底物通道工程,将该酶β亚基的第42位甘氨酸突变为赖氨酸后,其催化3-氰基吡啶的比酶活力提高为野生酶的2.5倍,且仍保留较好的稳定性,经5 L发酵罐培养,细胞酶活力达到5 539.0 U/mL,具有巨大的应用潜力。
Nitrile hydratase (nitrile hydratase, NHase, EC4.2.1.84) is a kind of metalloenzymes that hydrates nitriles to produce amides. It is used to produce nicotinamide through biological methods in the industry. Due to the exothermic process of the hydration reaction, the industry calls for NHase with higher stability. An NHase gene from thermophilic bacteria Caldalkalibacillus thermarum TA2. A1 was obtained by gene mining in our previous study, however, its catalytic activity was relatively low and cannot meet the application requirements in the industry. In this study, substrate access tunnel engineering was applied and after introducing lysine to βGly42, the specific activity of the βG42K mutant toward 3-cyanopyridine was 2.5 times higher than that of its parent enzyme with minimal loss of stability. The activity of the bacterial cells harboring the βG42K mutant reached 5 539.0 U/mL in the 5 L fermentor, which showed great potential for further application of amide production.
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