Rational Design And Recombinant Expression Of Alanine Dehydrogenase From Bacillus Subtilis 168

L-Alanine is a non-essential amino acid also the highest content of amino acids in human blood.It has been used in chemical synthesis,food industry,medical care,daily chemical products,and feed industry.Alanine dehydrogenase is an amino acid dehydrogenase.The effect of this enzyme is dependent on the coenzymes NADH and NAD⁺.Positive reaction catalyzes oxidative deamination of alanine to pyruvate,and reverse catalytic reaction amination of pyruvate produces alanine.The aim of this study was to increase the activity of alanine dehydrogenase amination by protein site-directed mutagenesis,express the modified enzyme in lactic acid bacteria,and increase L-alanine production by lactic acid bacteria fermentation.In this study,the alanine dehydrogenase was from Bacillus subtilis 168.After PCR amplification of coding gene ald,the E.coli expression vector pET-28a?+?was ligated into E.coli BL21?DE3?strain,expressed protein,isolated and purified by Nickel ion affinity chromatography to obtain recombinant protein.The recombinant protein concentration was1.33 mg/mL,the total deamination reaction activity was 2.82×10⁴U/L,the specific activity was 21.21 U/mg,the substrate L-alanine K_m was 10.12 mM,the k_catat was 12.23 s^-11 The total activity of the amination reaction was 1.23×10⁶U/L,and the specific activity was 9.28×10²U/mg.The substrate pyruvate K_m was 2.40 mM,the k_catat was 23.21 s^-1.Mutants of Y93F and N299Q were obtained by homology modeling,molecular docking and molecular dynamics simulation and change the hydrophilicity of the active pocket and the size of the amino acid side chains.The concentration of Y93F mutant protein was 1.15 mg/mL,the total deaminationg reaction activity was 3.44×10⁴ U/L,and the specific activity was 29.9 U/mg.The substrate L-alanine K_m is 4.51 mM,the k_catat is 10.02s^-1;the total activity of the amination reaction is 1.32×10⁶ U/L,the specific activity is 1.15×10³ U/mg,and the substrate pyruvate K_m is 3.09 mM,the k_catat was 31.05 s^-1.The N299Q mutant protein concentration was 1.48 mg/mL,the total deamination reaction activity was 1.47×10⁴ U/L,and the specific activity was 9.91U/mg.The substrate L-alanine K_m was 2.69 mM,the k_catat was 1.01s^-1.The total activity of the amination reaction was 1.09×10⁴ U/L,and the specific activity was 7.35 U/mg.The substrate pyruvate K_m was 3.68 mM,the k_catat was 19.08 s^-1.We use lactic acid bacteria expression vector pNZ 8148 to construct pNZ 8148-ald,pNZ8148-y93f recombinant plasmid,transfer to L.lactis NZ9000 wild-type and knockout strains for shake flask fermentation.The flasks were fermented for 72 h,ALD produced L-alanine at 71.56?g/m L;Y93F mutant produced L-alanine at 245.72?g/mL.ALD?L.lactis?ldh?produced L-alanine 242.65?g/mL;Y93F?L.lactis?ldh?produced L-alanine253.48?g/mL.The results showed that the modified Y93F mutant effectively increased the fermentation yield of L-alanine,and the deletion of the ldh gene increased the amount of L-alanine produced by L.lactis fermentation.