天冬氨酸激酶突变体G277K中AK基因的克隆表达及酶学性质表征

通过序列比对和晶体结构分析发现，天冬氨酸激酶（aspartokinase，AK）的G277位点高度保守，并与抑制剂Thr通过氢键相连，对其进行定点突变和酶学性质表征。结果表明：突变体G277K的AK最适反应条件是30 ℃、pH 8.5；动力学实验结果显示突变体AK的正协同效应降低，趋向于米氏酶，参数S0.5、nH、酶比活力、Vmax分别是7.05 mmol/L，1.24、964.3 U/mg、32.143 U/（mg·min）；热稳定性实验显示，其30 ℃半衰期为2.3 h，3 h后酶活力丧失80%左右；Ni2+在低浓度时表现出显著的激活效应；有机溶剂丙三醇、异丙醇和二甲基亚砜的体积分数为1%时，对AK的酶活力有很好的激活作用，表明突变体AK对一些有机溶剂有一定的抗性；低浓度的赖氨酸和蛋氨酸对AK有激活作用。

Gene Cloning,Expression and Enzymatic Characterization of Aspartokinase Mutant G277K

Aspartokinase (AK), a crucial enzyme used in the industrial production of amino acids, is a rate-limiting enzymein the biosynthesis of amino acids of the aspartate family, in which AK is strictly regulated by the feedback of these aminoacids. This study aimed to relieve the metabolic regulation and to create genetically engineered strains that have a highproduction of those amino acids. By using the substrate docking and virtual screening, we found the highly conservedmutation sites of Gly (G) 277 which were bound with Thr501 inhibitory site by hydrogen bonds. The results showed that theoptimal pH, temperature and half-life of the mutant enzyme were 8.5, 30℃ and 2.3 h, respectively. Steady-state kinetic studyshowed that the positive synergistic effect was reduced, and tended to become a Michaelis enzyme. The parameters S0.5, nH,specific activity and Vmax were 7.05 mmol/L, 1.24, 964.3 U/mg and 32.143 U/(mg·min), respectively. Ni2+ showed significantactivation effect at low concentrations. The AK enzyme activity was activated when glycerol, isopropanol or dimethylsulfoxide was present at a level of 1% by volume, suggesting the resistance of the mutant enzyme to these organic solvents.Lysine and methionine at low concentrations were also able to activate the enzyme activity.