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	耦合末端盐桥与定点突变的重组腈水合酶催化动力学
	陈杰 ; 贾旭 ; 于慧敏 ; 罗晖 ; 沈忠耀 ; CHEN Jie ; JIA Xu ; YU Huimin ; LUO Hui ; SHEN Zhongyao
	2016-03-30 ; 2016-03-30
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 nitrile hydratase terminal salt bridge point-mutation reaction kinetics Michaelis-Menten equation deactivation kinetics Q814
其他题名	Catalytic kinetics of recombinant nitrile hydratase coupling terminal salt bridge and point-mutation
中文摘要	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。; 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 in ?-? interface increases to 7 pairs. Using both the original NHase TH and SB mutant as controls, SBM 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 Kcat of Michaelis-Menten equation is increased by 20% and 60% compared to TH and SB, respectively. The apparent inactivation constant KD is 68.0% of TH at 42℃, indicating that SBM also improves thermal stability.
语种	中文 ; 中文
内容类型	期刊论文
源URL	[http://ir.lib.tsinghua.edu.cn/ir/item.do?handle=123456789/144054]
专题	清华大学
推荐引用方式 GB/T 7714	陈杰,贾旭,于慧敏,等. 耦合末端盐桥与定点突变的重组腈水合酶催化动力学[J],2016, 2016.
APA	陈杰.,贾旭.,于慧敏.,罗晖.,沈忠耀.,...&SHEN Zhongyao.(2016).耦合末端盐桥与定点突变的重组腈水合酶催化动力学..
MLA	陈杰,et al."耦合末端盐桥与定点突变的重组腈水合酶催化动力学".(2016).

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