Gamma-amino butyric acid (GABA) is a kind of pharmacological and biological component and its application is wide and useful in Korea specially, becoming aging society in the near feature. GABA is request special dose for the purposed biological effect but the production of concentrated GABA is very difficult due to low concentration of glutamic acid existed in the fermentation broth. To increase GABA concentrate using fermentation technology, high content of glutamic acid is required. For this reason, various strains which have the glutamic acid decarboxylase (GAD) and can convert glutamic acid to GABA, were isolated from various fermented foods. Most of GABA producing strains are lactic acid bacteria isolated from kimchi, especially added monosodium glutamate (MSG) as a taste enhancer. Optimizing the formulation of culture media and the culture condition, GABA conversion yield and amounts were increased. Finally GABA concentration of fermentation broth in batch or fed batch fermentation reached 660 mM or 1000 mM, respectively. Furthermore formulation of culture media for GABA production developed commercially. Many studies about GABA-rich product have been continued, so GABA-rich kimchi, cheese, yogurt, black raspberry juice and tomato juices has been also developed. In Korea many biological effects of GABA are evaluated recently and GABA will be expected to be used in multipurpose.

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Production of GABA (gamma amino butyric acid) by Lactic Acid Bacteria

In previous work, our research group isolated and bred a γ-aminobutyric acid (GABA)-producing microorganism, Lactobacillus brevis CGMCC 1306, from fresh unpasteurized milk and found that the strain possessed high glutamate decarboxylase (GAD) activity. However, the cells of the strain grew poorly during submerged fermentation, with the highest dry cell weight only 2.78 g/L. To enhance the efficiency of GABA biosynthesis and achieve GAD production in high cell-density fermentations, the gad gene from L. brevis CGMCC 1306 was cloned using degenerate PCR. Nucleotide sequencing analysis showed that the cloned gene comprised 1407 bp and encoded a 468-amino acid protein. The gad gene with a hexa-His tag was inserted into pET-28a(+) and expressed in Escherichia coli BL21. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the recombinant protein product had a molecular weight of approximately 53 kDa, which corresponded to the predicted size of the deduced protein (53.47 kDa). High-performance liquid chromatography analysis showed that the purified recombinant protein was capable of catalyzing α-decarboxylation of L-sodium glutamate into GABA, which confirmed that the protein was derived from a gad gene. GAD activity was the highest at pH 4.8 and 48°C. Using Lineweaver–Burk plots, the K
 m
 and V
 max
 of the recombinant enzyme were 10.26 mM and 8.86 U/mg, respectively. Its activity was not dependent on the addition of pyridoxal 5'-phosphate. These results strongly suggest that cloning of the gad gene is not only of interest to researchers in terms of affecting GAD production by protein engineering strategies, but that it may increase GAD production by high cell-density fermentation and enhance the efficiency of GABA biosynthesis.

Cloning, sequencing and expression of a glutamate decarboxylase gene from the GABA-producing strain Lactobacillus brevis CGMCC 1306

γ-aminobutyric acid (GABA) is an important bioactive regulator, and its biosynthesis is primarily through the α-decarboxylation of glutamate by glutamate decarboxylase (GAD). The procedures to obtain GABA by bioconvertion with high activity recombinant Escherichia coli GAD have been seldom understood. In this study, Escherichia coli GAD (gadA) was highly expressed (about 70–75% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-gadA, which was induced by 0.4 mM IPTG in LB medium, and maximal GABA-forming activity of the recombinant GAD was 40 U/mL at a concentration (0.15 mM) of pyridoxal phosphate (PLP) and a concentration (0.6 mM) of Ca2+ at optimal pH of 3.8. The optimal concentration (7.5 mM) of Mn2+ can also improve the activity of recombinant enzyme, but the co-effect of Ca2+ and Mn2+ exhibited antagonism effect when added simultaneously. LB and 0.1% (w/v) lactose were selected as culture medium and inducer, respectively. The relative activity was markedly higher activated by Ca2+ (174%), Mn2+ (164%) than that by other seven bivalent cations. Finally, the yield of GABA was high of 94 g/L detected by paper chromatography or HPLC in 1 L reaction system with 30 mL crude GAD (12 U/mL). By entrapping Escherichia coli glutamate decarboxylase into sodium alginate and carrageenan gel beads, the activity of immobilized GAD (IGAD) remained 85% during the initial five batches and the activity still remained 50% at the tenth batch, these results indicated that the recombinant Escherichia coli GAD was feasible for the future industrial production of GABA.

Enhanced γ-aminobutyric acid-forming activity of recombinant glutamate decarboxylase (gadA) from Escherichia coli

An aspartate aminotransferase (AATase) gene from Lactobacillus brevis CGMCC 1306 was cloned, which contains a 1182-bp open reading frame coding for 393 amino acids (41.43 kDa). When expressed in Escherichia coli BL21 (DE3), the recombinant AATase was purified and subsequently characterized. The recombinant AATase can catalyse the conversion of L-Asp to L-Glu, and the kcat/Km was determined to be 25.5 (mmol/L)−1 s−1 for L-Asp and 207.8 m(mol/L)−1 s−1 for α-ketoglutarate. With optimum temperature as 25 ˚C, the AATase may be a novel and special psychrophilic enzyme which exhibited a good thermal stability below 55 ˚C. The conserved active site residue of AATase was identified as Lys237 by phylogenetic analysis. Secondary structure of the enzyme includes α-helix (39.2%), β-sheet (5.5%), β-turn (8.8%), and random coil (36.5%) by circular dichroism spectral analysis. Phase diagram for the fluorescence data analysis showed that guanidinium chloride-induced unfolding of AATase involved at least one interm...

Cloning, expression and characterization of an aspartate aminotransferase gene from Lactobacillus brevis CGMCC 1306

OBJECTIVE To establish a method for the content of glutamic acid and γ-aminobutyric acid in hippocampus of rat by O-phthalaldehyde(OPA) pre-column derivatization high performance liquid chromatograph-fluorescence (HPLC-FLU). METHODS The separation and determination were carried out by linear gradient elution under following conditions: Alltima C18(250 mm×4.6 mm, 5 μm)column, 50 mmol·L-1 acetic acid solution (pH 6.8), methanol and trifluorofuran as mobile phase, flow-rate was 1.0 mL·min-1. The fluorescence excitation and emission wavelengths were set at λEx 338 and λEm 425 nm, respectively. RESULTS The linear range of the method was 1.03-20.6 μg·mL-1(r=0.999 2) for glutamic acid(Glu) and 1.13-22.6 μg·mL-1(r=0.999 7) for r-aminobutyric acid(GABA). The detection limits of GLU and GABA were 5.27×10-4 μg·mL-1,and 7.35×10-4 μg·mL-1, respectively, when the ratio of signal to noise was 3. The inter-assay precisions were 3.4% and 3.5%, respectively. The recoveries were in the range of 90%-96% and 85%-91%. CONCLUSION The method is simple, sensitive, reliable and has strong specificity and good repetition. It is suitable for the content determination of GLU, GABA in hippocampus of rat by HPLC.

Study on the Determination of Glutamic Acid and γ-Aminobutyric Acid in Hippocampus of Rat by HPLC

[Determination of gamma-aminobutyric acid and glutamic acid in human cerebrospinal fluid by high performance liquid chromatography].

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[Determination of gamma-aminobutyric acid and glutamic acid in human cerebrospinal fluid by high performance liquid chromatography].