Construction of the Five-point Mutant in Novel Aspartokinase and Its Enzymatic Characterization from Corynebacterium pekinense

Purpose: To improve the activity of the first key allosteric enzyme-aspartate kinase (AK) in the aspartate amino acid synthesis pathway, and to weaken or remove the terminal products lysine (Lys) and threonine (Thr) synergistic feed back inhibition of AK. Methods: On this basis of the four-point mutant T379N/A380C/G171I/Y198N AK(NCIN AK) of Corynebacterium pekinense, it was found that the highly conserved mutation sites of Gly295 was bound with inhibitor Thr by hydrogen bonds. The site of Gly295 were selected for site-directed mutation. The plasmid was extracted and transformed into E. coli competent BL21 to induce expression. High-throughput screening was used to obtain mutant strains with significantly improved enzyme activity. Enzyme kinetic study and characterization of enzyme properties were performed on the wild type(WT) and the five-point mutant T379N/A380C/G171I/Y198N/G295L AK(NCINL AK). Results: The five-point mutant NCINL AK was successfully constructed, and its maximum reaction rate V_max was 259 U/(mg·min). The K_m value of the Michaelis constant of the mutant reduced from 3.44 mmol/L to 0.93 mmol/L, and the substrate affinity was enhanced compared with the wild type enzyme. The hill coefficient n value was reduced from 2.73 to 1.21, and the positive synergy was decreased. The optimum temperature of NCINL AK increased from 25 ℃ to 28 ℃, the optimum pH value increased from 8.0 to 8.5, and the half-life was extended from 4.7 h to 5.2 h. Compared with the wild type WT AK, the five-point mutant NCINL AK had different inhibitory effects at different substrate inhibitors with concentration of 0.2, 1.0, 5.0, 10.0 mmol/L and even the inhibitory effect of Lys was completely lifted, and it had an activation effect under the condition of 10 mmol/L Thr. Conclusion: In this experiment, the five-point mutant NCINL AK with enzyme activity increased by 87.20 times was obtained, and the enzymatic properties were also significantly improved. Within a certain inhibitor concentration range, the feedback-inhibition effect of Lys on AK was basically lifted, and to a certain extent releasing Thr's feedback inhibition provided the possibility for the accumulation of large amounts of aspartate amino acids and provided reference for constructing high-yield aspartate amino acid strains. It was possible for us to get the pollution-free and high-efficiency amino acids such as methionine (Met).