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 thermophilus HB8 Aspartate Aminotransferase in Escherichia coli

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1 h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K_m^KG为7.559mmol/L，V_max^KG为0.086mmol/(L·min)，K_m^Asp为2.031mmol/L，V_max^Asp为0.024mmol/(L·min)。Ca²⁺、Fe³⁺、Mn²⁺等金属离子对酶活性有微弱抑制作用。

Abstract:

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. K_m^KG was 7.559mmol/L, V_max^KGwas 0.086 mmol/(L·min)， Ksub>m^Asp was 2.031mmol/L， V_max^Asp was 0.024mmol/(L·min). Ca²⁺，Fe³⁺，Mn²⁺ inhibited enzyme activity softly.