Abstract: Aspartate kinase (AK) is the first key allosteric enzyme involved in the metabolic pathway of the aspartate family of amino acids, which is subject to synergistic feedback inhibition by lysine and threonine. This study aimed to increase AK activity and alleviate the feedback inhibition by site-directed mutation. The monomer aspartate kinase from Corynebacterium pekinense was obtained and its structure was analyzed. On this basis, the key residue sites Tyr198 and Asp201 around ATP were selected for site-directed mutation. The double mutant Y198N/D201M was successfully obtained by high-throughput screening, whose enzymatic activity was improved by 18.26 folds compared to that of the wild type (WT) AK. The Km value of the mutant was reduced (2.37 versus 3.58 mmol/L), and the substrate affinity was enhanced compared to the WT enzyme. The n value was reduced (1.58 versus 1.91), and the positive synergy was weakened. The enzymatic properties showed that optimum temperature, pH and half-life of the mutant were 28 ℃, 7.5 and 5.32 h as opposed to 25 ℃, 8.0 and 4.66 h for the WT enzyme. The mutant was less inhibited by lysine and threonine at all tested concentrations than the WT enzyme, and was even activated by Lys + Met, Thr + Met and Lys + Thr + Met at 5 and 10 mmol/L concentrations.

Key words: Corynebacterium pekinense; aspartate kinase; double mutant; enzymatic activity; enzymatic properties