High Yield Gamma-aminobutyric Acid Lactic Acid Bacteria Breeding And Expression Of GadB

?-aminobutyric acid (GABA) is a kind of non-protein amino acid, which is synthesized by a-decarboxylation of glutamate acid with glutamate decarboxylase. It widely exists in plants, animals and microorganisms. As an inhibitory neurotransmitter, GABA has many physiological functions, such as depressing blood-pressure, antianxiety, diabetes treatment, promoting the metabolism of alcohol and deodorant, promoting reproductive and so on. It widely uses in widely uses in food, medicine and animal husbandry.Firstly, one lactic acid bacteria with which the production of GABA is 2.461 g/L in 72 h fermentation was isolated. Combined calcium carbonate-bromocresol green plates with high performance liquid chromatography (HPLC), a high-yield GABA strain FJNU-GA01 was screened from aging pickle juice, it was identified as Lactococcus lactis based on morphological characteristics, physiological and biochemical indicators and 16SrDNA sequence analysis.Secondly, the cultural system of L. lactis FJNU-GA01 was optimized with single-factor experiments, factorial design and central composite design. With glucose 12.46 g/L, sodium glutamate 15.80 g/L, K2HPO40.49 g/L, tryptone 4 g/L, peptone 16 g/L, MgSO41.6 g/L (initial pH:7.5) as medium composite, according to 9% inoculum size, rotate speed 60 r/min, temperature 30?, the yield of GABA was increased from 2.461 g/L to 7.539 g/L in 72 h fermentation.Thirdly, the process of L. lactis FJNU-GA01 whole-cell catalysis to product GABA was studied. With 5.81 g/L glucose,12.04 g/L corn steep powder,5 g/L peptone, 1.5 g/L K2HPO4,0.5 g/L MgSO4,10.67 g/L sodium glutamate as medium composite to culture bacteria 12 hours, on the condition of acetic acid-sodium acetate buffer (pH 4.2, 0.3 mol/L, including 20 g/L sodium glutamate), cell concentration 25 g/L, temperature 50? and rotate speed 150 r/min, GABA production was improved from 3.080 g/L to 9.360 g/L in 4 h reaction time. It was increased 203.9%. Applied 10t fermentor into the culturing bacteria cells by the method of three-stage cultivation, and 2t fermentor to transformation, the yield of GABA can reach 12 g/L.Finally, E.coli engineering bacteria which can transfer glutamate acid to GABA efficiently was constructed. According to the sequence of gadB on the web of NCBI, the primer was designed and Lactococcus lactis FJNU-GA01 gadB gene was cloned, ligation gadB and pET-28a(+), transformed into E.coli BL21(DE3), the engineering bacteria that could producte GAB A was achieved. On the condition of lmmol/L IPTG, induction time 12 hours and induction temperature 30?, GABA production can reach 327.06 g/L with L-glutamate acid as substrate.