Screening, Identification And Optimization Of Fermentation Conditions Of Lactic Acid Bacteria Producing γ-Aminobutyric Acid, And Cloning Of The Glutamic Acid Decarboxylase Gene

γ-Aminobutyric acid (GABA) is a kind of non-protein amino acid in nature. It is an important inhibitory neurotransmitter of the mammals'central nervous system. GABA can participate in cerebral circulation, promote reproduction and activate kidney function. It also influence on depressing blood-pressure, improving growth and treatment of epilepsy. GABA has been widely used in the field of food and pharmaceutical industries. The glutamic acid decarboxylase (GAD) catalyze L-glutamate bya-decarboxylation reaction to generate GABA. It is rate-limiting enzyme for biocatalysis GABA. GAD is the key gene to regulate the production of GABA.In this study, the strain Lb-2 of lactic acid bacteria which could produce higher y-aminobutyric acid was isolated from pickling vegetables. This strain was identified based on morphological, physiological characters and 16S rRNA gene sequence. The culture substrate and fermentation conditions were optimized in order to improve the production of GABA. A full-length gad of the strain Lb-2 was cloned by PCR. The gene was sequenced and analyzed by homology and function.First,21 strains of lactic acid bacteria which could produceγ-aminobutyric acid were isolated from pickling vegetables and yoghourt. The strain Lb-2 with the highest amount of GABA (9.24 g/L) screened from pickling vegetable was determined in MRS with 1% monosodium glutamate (MSG) by improved paper chromatography combined with high-performance liquid chromatography. This strain was identified as Lactobacillus brevis based on morphological, physiological characters and 16 S rRNA gene sequence, and homology analysis was done with the sequence in genebank. Second, the culture substrate and fermentation conditions of the strain Lb-2 were optimized by the method of single factor experiment and L9(33) orthogonal test. Single factors include carbon source, nitrogen source, beginning pH, growing temperature, culture time and etc. which are main factors affecting production of GABA. Based on the single factor experiment, L9(33) orthogonal test was conducted for overall optimization of fermentation conditions with the beginning pH (5.0,5.5,6.0), culture temperature (30℃, 32℃,34℃), and culture time (48 h,60 h,72 h) as the three factors and their GABA production as indicators. The results showed that beginning pH 5.5, culture temperature at 32℃, culture time is 72 h, the higher yield ofγ-Aminobutyric acid could be reached. The content of GABA was 13.45 g/L in the optimum culture conditions.Finally, with the strain Lb-2 as the gad sources, a pair of specific primers was designed based on both ends of Lactobacillus brevis gad sequences region. Then gad gene was amplified by PCR and connected with the pUC18-T Simple Vector. After sequencing, the results showed that A fragment of 1288 bp-length named Lbgad was cloned. Identity analysis of full-length Lbgad sequence shows the sequences of this gene are of the same size with those of Lactobacillus brevis(Lactobacillus brevis) gad in the GenBank database, and homology of the two amounted to 96% at nucleotide level, and there is a certain homology between Lbgad amino acid sequence of glutamate decarboxylase with other micro-organisms.