Breeding Of Lactic Acid Bacteria Producing High Yield Ofγ-aminobutyric Acid And Cloning Of The Glutamic Acid Decarboxylase Gene

y-aminobutyric acid is one of non-protein natural amino acids,also known as piperidine acid or GABA. Its chemical name is4-aminobutyric acid. GABA usually exists in both prokaryotes and eukaryotes in a free state, and can be conversion obtained by glutamic acid (Glutamic acid Glu) in the role of glutamic acid decarboxylase (glutamate decarboxylase, GAD). There are many important physiological functions of GABA, such as antianxiety, lower blood pressure, enhance memory, improve sleep and so on. With the on going research of its physiological and physiological functions at this stage, GABA has developed as a new type of functional factor used in food and pharmaceutical industries.Firstly, a strain lactic acid bacteria named3#of high yield of GABA was isolated from kimchi, milk, yogurt and other fermented foods, and GABA content in its fermentation broth can reach8.006g/L. Through analysis of the strain3#’s morphological, physiological, biochemical and16S rDNA sequence, the results showed that:the colonies of bacteria3#were small, rounded and cream, neat edge and opaque; Gram-positive, facultative anaerobic; the strain3#did not produce hydrogen sulfide, and in the sugar fermentation experiments it can produce acid without gas; in the litmus milk experiment it can make litmus red; the result of16S rDNA sequence analysis show that the homology of3#bacteria and Enterococcus faecalis in lactic acid bacteria categories can reach99%,preliminarily identified it as Enterococcus faecalis.The sequence JN990826was applicated in Genbank.Then, through screened GABA-producing strain3#by UV and NTG mutagenesis, Enterococcus faecalis HX-3-6strain with genetic stability can be obtained finally. In the fermentation broth GABA production can reach11.64g/L and the conversion rate increased by55.82%than the original strain. The orthogonal test, the Plackett-Burman (PB), the experimental design of Box-Behnken and response surface analysis methodology were used to optimize the strain of Enterococcus faecalis HX-3-6fermentation conditions and its fermentation medium. The optimized fermentation conditions were as follows:substrate concentration of30g/L, pH5.0, fermentation time of72h, temperature of35℃; The optimized components and contents of the fermentation medium were as follows:glucose12.34g/L,citrate amine0.22g/L, MnSO4. H2O0.10g/L, NaCl0.22g/L,tryptone5g/L, Tween-800.1mL/L, K2HPO40.2g/L and MgSO4.7H2O0.15g/L. In the optimized medium, the yield of y-aminobutyric acid can be increased from the pre-optimization11.006g/L to16.027g/L and conversion rate increased by45.62%than that of the pre-optimization conditions. The paper also explored and optimized the conversion conditions of preparing GABA by lactic acid bacteria cells, and got a set of optimum combinations of conditions as follows:2d of bacterial cells fermentation,0.05mmol/L of PLP, reaction at pH4.8, temperature42℃,180r/min, stirred for40h, and in the transformation process the substrate L-glutamic acid was gradually added to maintain the syetem pH. Finally, concentration of GABA in the converting fluid was35.91g/L and the molar conversion rate can reach83.99%.The last, Enterococcus faecalis HX-3-6strain’s glutamic acid decarboxylase gene was amplified and verified by the means of molecular biology, Compared with glutamic acid decarboxylase gene of the different lactic acid bacteria at the level of gene sequence and amino acid sequence, and found that there was a certain degree of homology among glutamic acid decarboxylase amino acid sequence from different lactic acid bacteria. These results laid some theoretical basis for follow-up experiments.