Abstract:

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.

Key words: aspartokinase (AK), cloning and expression, site-directed mutation, enzymatic properties