• Applied Biological Chemistry
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• v.51 no.4
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• pp.344-345
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• 2008

• Microbiology and Biotechnology Letters
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• v.47 no.1
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• pp.139-147
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• 2019

Glutamate decarboxylase catalyzes the conversion of glutamate to gamma-aminobutyric acid (GABA), contributing to pH homeostasis through proton consumption. The reaction is the first step toward the GABA shunt. To date, the enzymes involved in the glutamate metabolism of Photobacterium damselae subsp. piscicida have not been elucidated. In this study, an open reading frame of P. damselae subsp. piscicida, showing homology to the glutamate decarboxylase or putative pyridoxal-dependent aspartate 1-decarboxylase genes, was isolated and cloned into an expression vector to produce the recombinant enzyme. Preliminary gas chromatography-mass spectrometry characterization of the purified recombinant enzyme revealed that it catalyzed not only the decarboxylation of glutamate but also the transamination of GABA. This enzyme of P. damselae subsp. piscicida could be bifunctional, combining decarboxylase and transaminase activities in a single polypeptide chain.

• BMB Reports
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• v.31 no.3
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• pp.233-239
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• 1998

A GABA synthesizing enzyme, glutamate decarboxylase, has been purified from bovine brain by several chromatographic procedures. The preparation appeared homogeneous on SDS-PAGE. The enzyme is a homodimeric protein with a molecular mass of 120 kDa. The activation of glutamate decarboxylase by ginesenosides from Panax ginseng C.A. Meyer has been studied. Preincubation of the enzyme with total ginsenoside, $Rb_2$ and Rc ginsenosides, increased glutamate decarboxylase activities in a dose-dependent manner. There was a reproducible decrease in $K_m$, in addition to a increase in $V_{max}$, in response to increasing concentrations of the Rc ginsenoside fraction. Upon addition of the ginsenoside to the enzyme, a decrease in flurorescence intensity was discernible, together with an increase in emission anisotropy. Judging from the anisotropy values, the ginsenoside is rapidly trapped by the protein matrix. Total ginsenoside was administered to rats and the rat brains were removed for the measurement of the changes of GABA shunt regulating enzyme activities. Among the GABA shunt regulating enzymes, only the glutamate decarboxylase activities were increased after ginsenoside treatment. Therefore, it is suggested that the ginsenosides may elevate the GABA level in brain by activation of glutamate decarboxylase and the enzymatic activation might be due to the conformational change induced by binding of ginsenoside to the enzyme.

• KSBB Journal
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• v.29 no.6
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• pp.426-431
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• 2014

pH controlled batch reactor and bubble column reactors have been developed in this research. They were used to produce high concentration of GABA and to determine optimal pH for GABA production. Glutamate decarboxylase (GAD) was isolated from recombinant E. coli and used for GABA production from monosodium glutamate (MSG). pH control was inevitable because the pH increased with MSG consumption. GAD showed highest activity at acidic conditions at pH 5.5 but the optimal pH for GABA production was pH 6.0. When 1.5 mole of MSG was used as reactant, the 1.05 mole of GABA was produced after 10 hrs batch reaction. Using bubble column reactors, 80 % of MSG was converted to GABA for 6 hrs reaction and 1.2 mole of GABA was produced.

• Journal of Microbiology and Biotechnology
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• v.27 no.9
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• pp.1664-1669
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• 2017

Gamma-aminobutyric acid is a precursor of nylon-4, which is a promising heat-resistant biopolymer. GABA can be produced from the decarboxylation of glutamate by glutamate decarboxylase. In this study, a synthetic scaffold complex strategy was employed involving the Neurospora crassa glutamate decarboxylase (GadB) and Escherichia coli GABA antiporter (GadC) to improve GABA production. To construct the complex, the SH3 domain was attached to the N. crassa GadB, and the SH3 ligand was attached to the N-terminus, middle, and C-terminus of E. coli GadC. In the C-terminus model, 5.8 g/l of GABA concentration was obtained from 10 g/l glutamate. When a competing pathway engineered strain was used, the final GABA concentration was further increased to 5.94 g/l, which corresponds to 97.5% of GABA yield. With the introduction of the scaffold complex, the GABA productivity increased by 2.9 folds during the initial culture period.

• Microbiology and Biotechnology Letters
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• v.43 no.3
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• pp.300-305
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• 2015

The glutamate decarboxylase gene (gadB) from Lactobacillus plantarum WCFS1 was cloned and expressed as an N-terminal hexa-histidine-tagged fusion protein in Escherichia coli BL21 (DE3) as the host strain. Purified glutamate decarboxylase (GAD) was immobilized onto porous silica beads by covalent coupling. The pH dependence of activity and stability of the immobilized GAD was significantly altered, when compared to those of the free enzyme. Immobilized GAD was stable in the range of pH 3.5 to 6.0. The resulting packed-bed reactor produced 41.7 g of γ-aminobutyric acid/l·h at 45℃.

• Applied Biological Chemistry
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• v.43 no.4
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• pp.231-235
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• 2000

The changes of calmodulin levels, calmodulin-binding proteins, and $Ca^{2+}$/calmodulin-dependent glutamate decarboxylase during the growth of tobacco suspension cells were investigated. Tobacco cells exhibited a typical growth curve, including an exponential growth phase between 3 and 5 days after inoculation, and an apparent stationary phase occurring after 5 day. Although slight changes were observed from sample to sample, calmodulin protein levels remained similar during the phases of culture growth. Several $Ca^{2+}-dependent$ calmodulin-binding proteins including 56, 46, 36, and 32-kDa proteins were detected in tobacco cell extracts. The 56-kDa protein was identified as glutamate decarboxylase by Western-blot analysis using an anti-GAD monoclonal antibody. The levels of GAD protein and the specific activity of GAD enzyme were highest during the middle exponential phase of the culture growth cycle. These data suggest that $Ca^{2+}$/calmodulin-dependent glutamate decarboxylase is modulated during the growth of tobacco suspension cells.

• Journal of Applied Biological Chemistry
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• v.49 no.3
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• pp.106-109
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• 2006

Improvement of L-asparate ${\beta}-decarboxylase$ production from Pseudomonas dacunhae ATCC 21192 was attempted by optimizing fermentation conditions. Optimum carbon and nitrogen sources for cell growth and enzyme production were determined. L-Glutamate (2%) was the most suitable carbon source, and D-glucose, D-glycerol and fumarate repressed enzyme production. Yeast extract (2%) was the most effective as nitrogen source. A slight change of pH to 6.5 from medium pH resulted in a meaningful increase in the production of enzyme. The production of the enzyme was highly improved by using 2% yeast extract and 2% L-glutamate in culture media. Maximum L-asparate ${\beta}-decarboxylase$ activity reached up to over 24 U/mL-broth by 15 h flask fermentation.

• KSBB Journal
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• v.16 no.1
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• pp.36-40
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• 2001

In order to study the molecular mechanism of $\gamma$-aminobutyric acid (GABA) production in plants, we cloned and sequenced a partial glutamate decarboxylase (GAD) cDNA from the Arabidopsis thaliana cDNA library, using primers targeted at highly conserved sequences of the petunia GAD gene. The cDNA fragment was inserted into TA cloning vector with T7 promoter and the recombinant plasmid obtained was used to transform E. coli. The plasmid DNA purified from the transformed E. coli was digested with EcoRI and the presence of the insert was confirmed. Nucleotide sequence analysis showed that the fragment is a partial Arabidopsis thaliana GAD gene and that the sequence showed 98% and 78% identity to the region of the putative Arabidopsis thaliana GAD sequences deposited in GenBank, Accession nos: U46665 and U10034, respectively. The amino acid sequence deduced from the partial Arabidopsis thaliana GAD gene showed 99% and 91% identities to the GAD sequences deduced from the genes of the U46665 and U10034, respectively. The partial cDNA sequence determined may facilitate the study of the molecular mechanism of GABA metabolism in plants.

• Journal of Microbiology and Biotechnology
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• v.25 no.5
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• pp.696-703
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• 2015

A gamma-aminobutyric acid (GABA)-producing microorganism was isolated from jeot-gal (anchovy), a Korean fermented seafood. The isolate, A156, produced GABA profusely when incubated in MRS broth with monosodium glutamate (3% (w/v)) at 37℃ for 48 h. A156 was identified as Lactobacillus sakei by 16S rRNA gene sequencing. The GABA conversion yield was 86% as determined by GABase enzyme assay. The gadB gene encoding glutamate decarboxylase (GAD) was cloned by PCR. gadC encoding a glutamate/GABA antiporter was located immediately upstream of gadB. The operon structure of gadCB was confirmed by RT-PCR. gadB was overexpressed in Escherichia coli BL21(DE3) and recombinant GAD was purified. The purified GAD was 54.4 kDa in size by SDS-PAGE. Maximum GAD activity was observed at pH 5.0 and 55℃ and the activity was dependent on pyridoxal 5'-phosphate. The K_m and V_max of GAD were 0.045 mM and 0.011 mM/min, respectively, when glutamate was used as the substrate.