CAJ | 학술논문

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NHaseM-TH-SBM（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NHaseM-TH（TH）、盐桥突变酶NHaseM-TH-SB（SB）和盐桥耦合定点突变的重组酶NHaseM-TH-SBM （SBM），研究了3种酶的催化反应动力学和失活动力学。结果表明，SBM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Kcat比TH提高了20%。SBM的表观失活常数KD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。SBM是活性和稳定性同时提高的重组腈水合酶。
쌍아기정수합매시최화병희정수합생산병희선알적중요공업매。통과Discovery Studio 2.5연건대C-말단염교우합정점돌변개조적내열형기인중조정수합매NHaseM-TH-SBM（S344K-S346K-L347E-N362S-435DT436(+)）진행염교망락분석，발현기전국염교총수유소하강，단쌍아기계면간염교수량제고도7개。재중조대장간균중표체료원정수합매NHaseM-TH（TH）、염교돌변매NHaseM-TH-SB（SB）화염교우합정점돌변적중조매NHaseM-TH-SBM （SBM），연구료3충매적최화반응동역학화실활동역학。결과표명，SBM적정수합매활성위543.9 U·mg-1，비TH제고료31.0%；기미씨방정최화속솔상수Kcat비TH제고료20%。SBM적표관실활상수KD재25～42℃범위내균명현저우대조TH，재42℃시위TH적68.0%，현시료량호적열은정성。SBM시활성화은정성동시제고적중조정수합매。
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, NHaseM-TH-SBM (SBM), 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 SBM decreases but the number inα-β interface increases to 7 pairs. Using both the original NHase TH and SB mutant as controls, SBM is successfully expressed in recombinantE. 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 constantKcat of Michaelis-Menten equation is increased by 20% and 60% compared to TH and SB, respectively. The apparent inactivation constantKD is 68.0% of TH at 42℃, indicating that SBM also improves thermal stability.