Site Directed Mutagenesis And Enzymology Properties Characterization Of S381、E382 Mutant Strains From Aspartokinase In Corynebacterium Pekinense

In metabolic pathways microbial fermentation synthesis of amino acids aspartic acid family,Aspartokinase(Aspartokinase,EC 2.7.2.4,AK) catalytic L- aspartic acid and ATP product aspartyl-β- phosphate ADP, AK is a rate limiting enzyme to limit the carbon and nitrogen flow, which is the key yielding aspartic acid family is the key amino acid availability.But aspartate kinase activity feedback inhibition by both metabolites and feedback repression.In this study, bioinformatics and site-directed mutagenesis and high throughput screening toCorynebacterium pekinense(CpAK) S381, E382 sites and site-directed mutagenesis to obtain high activity by high-throughput screening methods AK mutant,AK partially lifted by aspartic acid in the synthesis of aromatic amino acid pathway mutant feedback inhibition and enzymatic properties were characterized lay the foundation for high yield aspartic acid family.1. Basic Local Alignment Search Tool(BLAST) to Beijing Corynebacterium glutamicum AK and AK(Corynebacterium glutamicumAK, CgAK) sequence alignment, sequence similarity between the two was 99%, so we speculate CpAK, CgAK space similar structure and activity of regulatory mechanisms. The use of three-dimensional crystal structure analysis PyMOL CgAK determination S381, E382 active site amino acid residues. By CLUSTALW multiple sequence alignment, is determined Beijing Corynebacterium S381 and E382 site site is conserved sequences and correspond exactly with the site glutamicum S399 and E400 sites. DS 3.5 software to analyze the relationship between the use of AK-D structure, active site inhibitor and between, S381 site lysine and adjacent through van der Waals interactions between amino acid, E382 sites inhibitors and lysine between adjacent amino acids form hydrogen bonds with water molecules to each other via a network connection, in order to remove these covalent role as the goal, to lift inhibitor S381, the interaction of E382.2. S381, E382 saturation point after mutation of plasmid transformed into E. coli(Escherichia coli) BL21 and passed through a high-throughput screening, high activity mutant monomer S381 S381 E, S381 T, S381 F and S381 K, E382 high activity single mutation body is E382 T, E382 H, E382 A. Recombinant strains induced the expression of the resulting bacterial suspension by sonication and non-denaturing nickel column to give a crude enzyme solution and purified liquid. By SDS-PAGE and Western blot protein abundantly expressed further validation purposes.3. The wild type AK and S381 E, S381 T, S381 F, S381 K, E382 T, E382 H, E382 A enzymatic kinetic analysis showed that, E382 A, E382 T, E382 H activity increased 10.49 times, 4.91 times and 3.52 times, indicating that three species mutant enhanced catalytic activity than the wild type, weakened further illustrate the inhibitory effect of inhibitors. S381 E, S381 T, S381 F, S381 K increase compared with WTAK activity multiples are: 17.88,7.13,3.06,15.33. S381 E, S381 T, S381 K activity significant increase in multiples.4. The results showed that: compared with the E382 A WTAK optimum temperature higher than WTAK reached 35 ℃, with the same E382 T WTAK optimum temperature, E382 H WTAK optimum temperature and decreased compared to 25 ℃.Wild-type and mutant were purified AK enzymatic properties characterized.And the wild type AK E382 H same pH optimum, E382 A reduced relative to the optimum pH 7, E382 T and the optimum pH was raised to 8.5 as compared to the wild type. The optimum pH are identical S381 K 8, S381 T optimum pH of 7, S381 E optimum pH was 7.5. For the half-life compared to wild type 4h, E382 A slightly lower thermal stability, thermal stability and E382 T significantly increased half-life extended to 7h, half-E382 H AK also increased, up to 6h. E382 T and E382 H also show good stability. S381 three mutants strains were improved thermal stability. Especially S381 E significantly increase, reaching 8h. Compared to wild type AK, E382 A metal ions on the enzyme activity, affecting mainly metal ions on the E382 T is in addition to Zn2+ and Ni2+ display activation at low concentrations, the majority of metal ions on the enzyme activity inhibition enhanced. E382 H compared to wild type, Ni2+, Mn2+ in 5mmol / L and 10 mmol / L significantly the activation. Compared with wild-type K+, Ca2+, Mn2+, Mg2+, Zn2+, Ni2+ are on the S381 E enzyme activity activation. Compared to wild type AK, E382 A in most organic solvents at a volume fraction of inhibition of the enzyme activity into activation. However, organic solvents E382 T enzyme activity compared with WTAK also vary, but in different concentrations of organic solvents can be detected activity, but did not show significant activation. Compared to wild type, an organic solvent in addition to the ethanol inhibition E382 H activity at high concentrations, the remaining organic solvent AK activity showed activation. Compared to wild type AK S381 E, S381 T, S381 K except glycerine and methanol at different concentrations and inhibited, showed good activation, prove organic solvents S381 mutant enzyme activity greater impact.Substrate inhibitors characterized Showing: WEAK inhibitory activity subject to: Thr + Lys> Thr> Lys, explained mainly by AK threonine and lysine synergistic feedback inhibition. E382 A, S381 E AK activity by activation of: Lys> Thr + Lys> Thr, Lys show feedback inhibition on AK be lifted, and Thr + Lys synergistic feedback inhibition on the AK also weakened after the change of site-directed mutagenesis and substrate inhibitor characterization results are consistent.