北京棒杆菌天冬氨酸激酶突变体R169H的构建及酶学性质表征

Construction and characterization of R169H mutant of aspartokinase from Corynebacterium pekinense

摘要:

摘要:【目的】提高北京棒杆菌(Corynebacterium pekinense)中天冬氨酸激酶(aspartokinase，AK)活力。【方法】利用定点突变技术对AK基因进行突变，并将突变体转入大肠杆菌(Escherichia coli) BL21中异源表达。重组菌经超声破碎后、利用镍柱对AK进行纯化，并经SDS-PAGE和Western blot验证。通过检测酶活力比较突变体和野生型动力学变化并研究突变体和野生型的部分酶学性质。【结果】成功构建突变体R169H。经验证知，AK分子量为48kDa。突变体R169H的Vmax为226.3U/mg·s－1，较野生型提高2.3倍。最适反应温度为26℃，与野生型经验值相同;最适反应pH为9.0，较野生型经验值8.0有所提高;在最适温度和pH值下的半衰期为5.5 h，比野生型的4h 稳定性要好;代谢产物赖氨酸、苏氨酸和蛋氨酸在低浓度时对AK均具有激活作用。【结论】突变体中R169与E92间氢键消失，能够影响亚基间聚合度，降低酶对底物的亲和力，减弱代谢产物对AK 的反馈抑制作用，从而使R169H中AK的Vmax提高2.3倍。

Abstract:

Abstract:［Objective］Increasing the activity of aspartokinase (AK) from Corynebacterium pekinense.［Methods］The gene of AK was constructed and mutated by site-specific mutagenesis．The mutational recombinant plasmid was heterologously expressed in Escherichia coli BL21．The mutational AK was purified by Ni2 + -NTA column after ultrasonicating of the recombinant bacteria，and then identified by SDS-PAGE and Western blot．We compared the kinetic difference between R169H AK and WT AK by determining the enzymatic activities．Some other characteristics of R169H AK and WTAK were also studied.［Results］The mutant R169H was successfully constructed．The molecular weight of AK was 48kDa．Vmax of R169H AK was 226.3 U/mg·s－1，which was 2.3 times higher than that of WT AK．The optimum reaction temperature of R169H AK was 26℃，the same as that of WT AK．The optimum reaction pH of R169H AK was 9.0，slightly higher than that of WT AK．The half-life period of R169H AK under optimum temperature and pH were 5.5h，much higher than that of WT AK．Lysine，threonine and methionine had an active effect on the activity of R169H AK when they were in low concentration.［Conclusion］ The hydrogen bond between R169 and E92 was broken down in R169H AK，which could affect the degree of polymerization and further lowered the affinity of mutant AK with substrates and then decreased the inhibition inducing by the metabolites. Thus，the Vmax of mutant AK from R169H had increased by 2.3 times compared with that of WT AK.