Directed Mutation Of Glutamate Decarboxylase And Its Catalytic Synthesis Of Gamma-aminobutyric Acid

Gamma aminobutyric acid(GABA)is a kind of non-protein amino acid soluble in water,which is widely used in food and pharmaceutical industry and is in great market demand.It can be produced by chemical synthesis,plant enrichment and biological methods.Because of its advantages of short production cycle,high efficiency and low cost,biological method has been attached great importance to by researchers.Glutamate decarboxylase(GAD)is the key factor in the biosynthesis of γ-aminobutyric acid,but there are many limiting factors in natural GAD,which affect the biosynthesis conversion efficiency.In this paper,on the basis of determining the key amino acids affecting the GAD catalytic efficiency of Escherichia coli,the mutant gad B-62 or GAD B-309 was designed,and the target fragments before and after the mutation were transferred into Escherichia coli BL21(DE3)with plasmid pET-30a as the carrier for induction and expression,and the enzymatic characteristics of GAD before and after the mutation were analyzed.Furthermore,the shaking flask fermentation and whole cell transformation production of GABA of the three recombinant strains of GAD before and after the introduction of the mutation were further studied to determine the mutation effect of GAD and the application characteristics of the three recombinant strains.The main research results are as follows:1.Site-directed mutagenesis and construction of Escherichia coli glutamate decarboxylase.Based on homologous sequence alignment and protein structure analysis,the 62 amino acid and 309 amino acid of GAD encoding Escherichia coli MG1655 were mutated,and then the GAD before and after the mutation was introduced into E.coli BL21(DE3)respectively.Sequencing and positive clone validation showed that the target sequence was consistent with expectation,and the plasmid containing the target fragments before and after mutation was successfully introduced into E.coli BL21(DE3),indicating that three recombinant strains of E.coli BL21(DE3)-gad B were successfully constructed.E.coli BL21(DE3)-gad B-T/S,E.coli BL21(DE3)-gad B-Q/A.2.Induction and expression of glutamate decarboxylase and study on its enzymatic properties.After induction with IPTG,the recombinant strain was analyzed by SDS-PAGE,and the GAD enzyme was further extracted.The enzymatic properties before and after the mutation were compared and analyzed.The results showed that there were bands at 53kD before and after mutation.At 37℃ and pH 4.3,the gad B-62 or gad B-309 mutants had 104%or 111%gad B enzyme activity relative to the wild type.At 37 ℃ and pH 6.5,the pH stability of gad B-62 or GAD B-309 mutants relative to wild-type gad B increased from 18%to 24%or 28%.The thermal stability of gad B-62 or gad B-309 mutants relative to wild-type gad B increased from 22%to 41%or 49%at pH 4.3 and temperature 45℃.Km and Vmax of gad B-62 and gad B-309 mutants were 7.3±2.5 mM and 76.1±3.1 U/mg,7.2±3.8 mM and 87.3±1.1 U/mg,respectively.The results showed that all the three recombinant strains could be expressed effectively,and the enzymatic properties of GAD after mutation were improved compared with that before mutation.3.γ-aminobutyric acid was produced by shaking flask fermentation of mutant strain.By shaking flask fermentation,the GABA production capacity of the three recombinant strains was preliminarily verified.The results showed that the initial concentration of substrate L-glutamate in shaking flask medium was 40 g/L,and the fermentation time was about 50 h.Three recombinant strains E.coli BL21(DE3)-gad B,E.coli BL21(DE3)-gad B-62 were found.The accumulated concentrations of GABA in E.coli BL21(DE3)-gad B-309 were 18.14 g/L,19.90 g/L and 22.85 g/L,respectively,and the yields were 65.02%,70.87%and 81.79%,respectively.These results indicated the effectiveness of GABA production by shaking flask fermentation of the three strains,and proved that the GABA production capacity of the corresponding recombinant strains was improved after GAD mutation.4.Study on synthesis of γ-aminobutyric acid by whole cell transformation.In order to furher verify the ability of the three recombinant strains to produce GABA by whole cell transformation before and after mutation,a high-density fermentation technology was proposed to be used.Firstly,the bacteria were enriched,and then the collected bacteria were used for whole cell transformation experiment.The results showed that the total input of substrate L-glutamate was 400 g/L.After 8 h transformation,three recombinant strains E.coli BL21(DE3)-gad B and E.coli BL21(DE3)-gad B-62 were found.The accumulated concentrations of GABA in E.coli BL21(DE3)-gad B-309 were 219.09 g/L,238.42 g/L and 276.66 g/L,respectively,and the conversion rates were 78.02%,85.04%and 98.58%,respectively.These results indicated that whole cell transformation could shorten the production cycle of GABA and significantly increase the production of GABA.