Construction Of A Recombinant Escherichia Coli And The Optimization Of Conditions For The Efficient Production Of γ-aminobutyric Acid

Gamma-aminobutyric acid (GABA), serves as a major inhibitory neurotransmitter in mammalian nervous systems. GABA has several positive physiological functions, and it is widely used in food, pharmaceutical, feed and other industries. In order to enhanceγ-aminobutyric acid production from L-glutamate efficiently, the key enzyme glutamate decarboxylase (GAD) encoding gene lpgad from the strain Lactobacillus plantarum GB 01-21 was amplified and overexpressed in E. coli BL21. The results showed that the recombinant E. coli BL21/pET-28a-lpgad produced 8.53 U/mg GAD, which was increased by 3.24 fold compared with the GAD activity in L. plantarum. Then a series of experiments were made to achieve high productivity of GABA. The main contents and results were as follows.Firstly, culture medium and environment conditions were studied in shake flask to improve enzyme activity of GAD . The results were as follows (g/L): glucose 10, soya peptone 12, yeast extract 12, corn steep liquor 15, urea 3, K2HPO4 4.35, KH2PO4 3.4, MgSO4 1.2, L-MSG 3. The optimized environment conditions were temperature 30℃, inoculation amount 1%, initial pH 7.0, 200 r/min, the bacteria was cultured at 37℃for 8 h to achieve later period of logarithmic growth, then it was induced at 30℃for another 8 h, the activity of GAD was 1020.4 U/mL.Secondly, we purified GAD by Ni-NTA affinity chromatography and characterized the enzyme to optimize the conditions of the whole-cell transformation. The optimum pH and temperature of the enzyme were pH 4.8 and 37°C, respectively. At the same time, we found that Ca²⁺ and Mg²⁺ could increase the activity significantly. Based on this,γ-aminobutyric acid transformation in 250 mL shake flask under the optimum transformation conditions was investigated. Accordingly, the mole conversion rate had reached 98.8% from 69.3% at 4 h when the 50 g/L L-glutamate was added, and the production ofγ-aminobutyric acid was improved by 45.2% compared with that under the unoptimized transformation conditions.Thirdly, the effects of pH, stirring speed, ventilation volum and fed-batch of glucose on culture process of E. coli BL21/pET-28a-lpgad in 5 L fermentor were investigated. The best ventilation volum and stirring speed were 1.5 vvm and 300 r/min respectively. When pH was controlled at 7.0, the amount of biomass and activity of GAD were increased by 26.6% and 60.1% respectively. With the initial glucose 10 g/L, the residual glucose solution (total 30 g/L) as fed-batch began feeding steadily after culture for 8 h, the fed-batch was ended after 12 h, then it was induced at 30℃, and the biomass reached the highest value of 10.4 g/L, enzyme activity up to 2205.4 U/mL, after culture for 28 h. The biomass and activity of GAD were increased by 108% and 96.8% compared with batch fermentation conditions.Lastly, the effects of the optimal stirring speed, cell concentration, initial substrate concentration and batch substrate feeding interval of whole cell transformation conditions in 5 L fermentor were investigated. Based on this,γ-aminobutyric acid transformation in 5 L fermentor under the optimum transformation conditions was investigated. Accordingly, the yield ofγ-aminobutyric acid was 278.3 g/L at 21 h when the 800 g L-glutamate was added and the mole conversion rate had reached 99.4%. The production ofγ-aminobutyric acid was improved by 93.9% compared with the original L. plantarum GB 01-21. Compared with the reported highest level, the conversion time was reduced by 15 h and the mole conversion rate was also increased. This paved a way for theγ-aminobutyric acid construction of the industrial applications.