Abstract: In order to construct mutants with high enzyme activity of 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 mutants M372I,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 are prolonged 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.

Key words: aspartokinase, site\, directed mutagenesis, enzymatic kinetics, enzymatic property