极端嗜热菌Thermus thermophilus HB8中天冬氨酸转氨酶在大肠杆菌中的表达、纯化及酶学性质研究  

Expression,Purification and Enzymatic Characterization of Thermus thermophilu s HB8 Aspartate Aminotransferase in Escherichia coli

作　　者：周华[1] 洪媛[1] 严明[1] 许琳[1] 

机构地区：[1]南京工业大学制药与生命科学学院,南京210009

出　　处：《生物工程学报》2007年第2期278-283,共6页Chinese Journal of Biotechnology

基　　金：国家973项目(No.2003CB716004);国家自然科学重点基金项目(No.20336010)资助~~

摘　　要：为获得具有热稳定性的天冬氨酸转氨酶,从极端嗜热细菌Thermus thermophilus HB8中克隆得到天冬氨酸转氨酶基因aspC,并在大肠杆菌BL21(DE3)和Rosetta(DE3)中进行表达,发现在Rosetta(DE3)中具有较高的表达量。重组酶的最适反应pH是7.0,37℃下在pH8～10的缓冲液中保温1h酶活几乎不改变。重组酶反应的最适温度为75℃,酶活稳定的温度范围为25～55℃。重组酶在65℃时半衰期为3.5h,75℃时为2.5h。重组酶的KmKG为7.559mmol/L,VmaxKG为0.086mmol/(L·min),KmAsp为2.031mmol/L,VmaxAsp为0·024mmol/(L·min)。Ca2+、Fe3+、Mn2+等金属离子对酶活性有微弱抑制作用。To obtain thermostable aspartate aminotransferase, the gene aspC from an extremely thermophilic bacterium, Thermus thermophilus HB8 was cloned, and its product was overexpressed in Escherichia coli BL21（DE3） and Rosetta（DE3）. The expression in Rosetta（DE3） was more efficient. The optimum reactive pH was 7, and the recombinant enzyme activity changed little when incubated in the buffer of pH8 - 10 on 37℃ for 1 h. The optimum reactive temprature was 75℃, and the recombinant enzyme was more stable on the temperature of 25 - 55℃. The half life of recombinant enzyme on 65℃ was 3.5 h, on 75℃ was 2.5h. KmKG was 7.559mmol/L, Vmax^KC was 0.086 mmol/（L· min）, Km^Aap was 2.031mmol/L, Vmax^Asp was 0. 024mmol/（L· min）. Ca^2＋, Fe^3＋, Mn^2 ＋ inhibited enzyme activity softly.

关 键 词：天冬氨酸转氨酶 THERMUS THERMOPHILUS HB8 表达 纯化 酶学性质 

分 类 号：Q78[生物学—分子生物学]