北京棒杆菌天冬氨酸激酶M372I-T379S双突变体的构建及其酶学性质

为构建高酶活力天冬氨酸激酶(aspartokinase, AK), 并削弱或解除Lys(lysine)反馈抑制作用突变体, 通过定点突变和高通量筛选技术构建突变体M372I,T379S和M372I-T379S, 对野生型（WT）和突变体分别进行诱导表达、 纯化及酶学性质表征. 结果表明： 突变体M372I,T379S和M372I-T379S AK与WTAK相比, V_max分别提高了13.77,15.02,15.60倍, K_m和n值均降低； 最适pH值分别升高为8.0,8.5,8.5, 且半衰期分别延长了1.0,0.9,2.3 h； M372I-T379S AK最适温度为30 ℃, 比WT AK高2 ℃； 当浓度为1~10 mmol/L时, 突变体均削弱或部分解除了抑制剂Lys的反馈抑制作用.

Construction and Enzymatic Properties of Double Mutant M372I-T379S of Aspartokinase from Corynebacterium pekinense#br#

In order to construct mutants with high enzyme activityof aspartokinase (AK) and weaken or relieve the feedback inhibition of Lys (lysine),mutants M372I,T379S and M372I-T379S were constructed by site\|directed mutagenesis and high-throughput screening techniques. The wild type (WT) and mutants were induced, expressed and characterized by enzymatic properties. The results show that compared with the WTAK, the V_max of mutantsM372I,T379S and M372I-T379S AK increases 13.77,15.02 and 15.60 times, respectively, and the values of K_m and n are lower than that of WTAK,the optimum pH values of mutants M372I,T379S and M372I-T379S AK are increased slightly, which are 8.0,8.5,8.5, and the half\|life period of mutants areprolonged by 1.0,0.9 and 2.3 h, respectively. The optimal temperature of M372I-T379S AK is 30 ℃, which is higher than WT AK 2 ℃. When the concentration is 1—10 mmol/L, the mutants weaken or partially relieve the feedback inhibition of Lys inhibitor.