极端嗜热菌Thermus thermophilus HB27中天冬氨酸激酶在大肠杆菌中表达、纯化及酶学性质研究  

EXPRESSION IN ESCHERICHIA COLI,PRELIMINARY PURIFICATION AND CHARACTERIZATION OF THE ASPARTOKINASE FROM THERMUS THERMOPHILUS HB27

作　　者：李敬坡[1] 韩宏岩[2] 夏勇 孟凡国 蔡平[1] 许维岸[2] 

机构地区：[1]苏州大学金螳螂城市建设学院,苏州215123 [2]苏州大学医学部基础医学与生物科学学院,苏州215123 [3]浙江清华长三角研究院,嘉兴314050

出　　处：《山东农业大学学报（自然科学版）》2009年第4期479-483,488,共6页Journal of Shandong Agricultural University：Natural Science Edition

摘　　要：为获得具有热稳定性的天冬氨酸激酶,从极端嗜热菌Thermus therm ophilus HB27基因组中扩增出天冬氨酸激酶(AK)基因,构建重组质粒,并在Escherichia coliBL21(DE3)中进行表达,得到了较高的表达量。经热处理纯化后测定重组天冬氨酸激酶(tAK)的最适pH为7.5,最适反应温度为80℃,80℃半衰期是18 h。酶动力学分析表明tAK的KmATP为0.23 mmol/L,Vm axATP为840.34 nmol/(L.m in),KmAsp为0.95 mmol/L,Vm axAsp为190.76 nmol/(L.m in)。赖氨酸和甲硫氨酸对tAK活性没有抑制作用,当苏氨酸浓度大于0.5 mmol/L时,对tAK活性有明显抑制作用,并且抑制作用与苏氨酸呈现浓度依赖性。To obtain thermostable aspartokinase （AK）, the gene ak from an extremely thermophilic bacterium, Thermus thermophilus HB27 was cloned , and was introduced into Escherichia coli BL21 （DE3） . The gene ak can be functionally expressed in Escherichia coll. The recombina aspartokinase（tAK） were partially purified from E. coli cellular extracts by the heated treatment and their kinetic characteristics were studied. The optimum reactive pH was 7.5 , the optimum reactive temperature was 80 ℃, and the half life of tAK under 80 ℃ was 18 h. tAK has Km values of 0.23 mmol/L for ATP and 0.95 mmo]/L for aspartate, it has Vmax values of 840.34 nmol/（ L · min）for ATP and 190.76 nmol/（L · min）for aspartate, tAK was not inhibited by L- Lysine and L - Methionine , but was inhibited by L - Threonine at a concentration more than 0.5 mmoL/ L in concentration dependent manner.

关 键 词：天冬氨酸激酶 THERMUS THERMOPHILUS HB27 表达 纯化 酶学性质 

分 类 号：Q814[生物学—生物工程]