Gamma-Aminobutyric Acid (GABA) Modulates Nitrate Concentrations and Metabolism in the Leaves of Pakchoi (<Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis> Makino) Treated with a Nitrogen-Rich Solution

Pakchoi plants were grown in 32 mM NO₃^? nutrient solution with or without 2.5 mM γ-aminobutyric acid (GABA) to investigate metabolite changes, gene and protein expression levels, and the activities of key enzymes related to nitrate metabolism in the leaves over a period of 0–12 days. High-nitrogen treatment enhanced plant growth and the NO₃^?, NO₂^?, NH₄⁺, Gln, and Glu contents in the leaves; promoted the gene and protein expression of nitrate reductase (NR) and glutamate decarboxylase (GAD); and increased the activities of NR, nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), and GAD. The endogenous GABA concentration in the leaves was enhanced in parallel with the increase in GAD activity. The GABA-treated leaves displayed the greatest increases in the gene and protein expression levels of NR and GAD and in the activities of NR, NiR, GS, GOGAT, and GAD. In addition, accelerated rates of nitrate reduction and assimilation were detected, and these changes occurred concurrently with the observed increases in gene or protein expression and enzyme activity. As a result, the concentrations of NH₄⁺, Gln, Glu, and endogenous GABA were significantly elevated, and the NO₃^? and NO₂^? contents were significantly decreased, in GABA-treated leaves compared with plants exposed to nitrogen-rich conditions. Our results reveal a potential positive that GABA may act as a nitrogen source to improve the plant growth and the most prominent effect of decreasing nitrate contents by accelerating NO₃^? reduction and assimilation. Exogenous GABA plays an important role in reducing the NO₃^? content of leaves, and thereby improves the ability to harvest leafy vegetables containing higher levels of endogenous GABA.     

Site-directed mutagenesis improves the practical application of L-glutamic acid decarboxylase in Escherichia coli

γ-Aminobutyric acid (GABA) is a kind of non-proteinogenic amino acid which is highly soluble in water and widely used in the food and pharmaceutical industries. Enzymatic conversion is an efficient method to produce GABA, whereby glutamic acid decarboxylase (GAD) is the key enzyme that catalyzes the process. The activity of wild-type GAD is usually limited by temperature, pH or biotin concentration, and hence directional modification is applied to improve its catalytic properties and practical application. GABA was produced using whole cell transformation of the recombinant strains Escherichia coli BL21(DE3)-Gad B, E. coli BL21(DE3)-Gad B-T62S and E. coli BL21(DE3)-Gad B-Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66 g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively. The results showed that Gad B-T62S and Gad B-Q309A are two effective mutation sites. These findings may contribute to ideas for constructing potent recombinant strains for GABA production. Practical Application : Enzymatic properties of the GAD from Escherichia coli and GAD site-specific mutants were examined by analyzing their conserved sequences, substrate contacts, contact between GAD amino acid residues and mutation energy (ΔΔG) of the GAD mutants. The enzyme activity and stability of Gad B-T62S and Gad B-Q309A mutants were improved compared to Gad B. The kinetic parameters K_m and V_max of Gad B, Gad B-T62S, and Gad B-Q309A mutants were 11.3 ± 2.1 mM and 32.1 ± 2.4 U/mg, 7.3 ± 2.5 mM and 76.1 ± 3.1 U/mg, and 7.2 ± 3.8 mM and 87.3 ± 1.1 U/mg, respectively. GABA was produced using whole cell transformation of the recombinant strains E. coli BL21(DE3)-Gad B, E. coli BL21(DE3)-Gad B-T62S, and E. coli BL21(DE3)-Gad B-Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66 g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively.     

Optimization of γ-amino butyric acid production in a newly isolated Lactobacillus brevis

An isolate from kimchi, identified as Lactobacillus brevis, accumulated γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter, in the culture medium. Optimal culture conditions for growth of L. brevis and production of GABA were 6 % (w/v) l-glutamic acid, 4 % (w/v) maltose, 2 % (w/v) yeast extract, 1 % (w/v) NaCl, 1 % (w/v) CaCl₂, 2 g Tween 80/l, and 0.02 mM pyridoxal 5′-phosphate at initial pH 5.25 and 37 °C. GABA reached 44.4 g/l after 72 h cultivation with a conversion rate 99.7 %, based on the amount (6 %) of l-glutamic acid added. GABA was purified using ion exchange column chromatography with 70 % recovery and 97 % purity.     

Gamma-aminobutyrate metabolism in locusts and the effect of exogenous diaminobutyrate

Locust brain homogenate exhibits transaminase activity with γ-aminobutyrate (GABA) as donor and 2-ketoglutarate as acceptor. The Km for GABA is 8–10 mM with 2-ketoglutarate at 20 mM and at pH 8.5. Diaminobutyric acid (DABA) similarly serves as an amino group donor exhibiting both higher affinity (Km = 1.1 mM) together with a third the rate of glutamate formed with GABA as amino group donor. DABA does not inhibit glutamate formation from GABA in a mixed-substrate reaction suggesting that DABA-transaminase may be distinct from GABA-transaminase. Relatively high DABA-transaminase activity is found in the fat body, with pyruvate preferred as acceptor. Haemolymph lacks DABA-transaminase activity. Glutamic acid decarboxylase (GAD), the reciprocal enzyme for GABA synthesis, is present in locust brain homogenate and its activity is not affected by 20 mM DABA.     

Cloning and expression of a full-length glutamate decarboxylase gene from a high-yielding γ-aminobutyric acid yeast strain MJ2

A yeast strain MJ2 that was found to produce a higher amount of γ-aminobutyric acid (GABA) was isolated from the surface of kiwi. Phylogenetic analysis based on the ITS sequence and morphological, biochemical studies indicated that it may belong to Saccharomyces cerevisiae. Under optimum conditions in Czapek’s broth medium with 0.5 % monosodium glutamate, it produced GABA at a concentration of 5.823 g/L after 48 h. A full-length glutamate decarboxylase gene (Scgad) was cloned by PCR amplification. The open reading frame (ORF) of the Scgad gene was composed of 1,755 nucleotides and encoded a protein (585 amino acids) with a predicted molecular weight of 65.897 kDa. The deduced amino acids sequence of Scgad shows 100 %, 65 % and 62 % similarity with S. cerevisiae, Candida glabrata and Kluyveromyces lactis GAD in the polypeptide level, respectively. The Scgad gene was expressed in Escherichia coli BL21 (DE3) cells, and the expression was confirmed by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis. The results suggested that the S. cerevisiae GAD (ScGAD) was successfully encoded in E. coli BL21 (DE3) cells. Furthermore, the enzyme activity of ScGAD encoded in E. coli BL21 (DE3) had been significantly enhanced using artificial neural network linked with genetic algorithm (ANN-GA) method.     

<Emphasis Type="Italic">Lactobacillus futsaii</Emphasis> CS3, a New GABA-Producing Strain Isolated from Thai Fermented Shrimp (<Emphasis Type="Italic">Kung</Emphasis>-<Emphasis Type="Italic">Som</Emphasis>)

Kung-Som is a popular traditional Thai fermented shrimp product. It is rich in glutamic acid, which is the major substrate for the biosynthesis of gamma-aminobutyric acid (GABA) by lactic acid bacteria (LAB). In the present study, LAB from Kung-Som were isolated, screened for GABA formation, and the two isolates that transform glutamic acid most efficiently into GABA were identified. Based on the API-CHL50 fermentation profile and a phylogenetic tree of 16S rDNA sequences, strain CS3 and CS5 were identified as Lactobacillus futsaii, which was for the first time shown to be a promising GABA producer. L. futsaii CS3 was the most efficient microorganism for the conversion of 25 mg/mL monosodium glutamate (MSG) to GABA, with a maximum yield of more than 99% conversion rate within 72 h. The open reading frame (ORF) of the glutamate decarboxylase (gad) gene was identified by PCR. It consists of 1410 bp encoding a polypeptide of 469 amino acids with a predicted molecular weight of 53.64 kDa and an isoelectric point (pI) of 5.56. Moreover, a good quality of the constructed model of L. futsaii CS3 was also estimated. Our results indicate that L. futsaii CS3 could be of interest for the production of GABA-enriched foods by fermentation and for other value-added products.     

An isobutyronitrile-induced bienzymatic system of Alcaligenes sp. MTCC 10674 and its application in the synthesis of α-hydroxyisobutyric acid

Alcaligenes sp. MTCC 10674 was isolated as acetone cyanohydrin hydrolyzing bacterium from soil of orchid gardens of Himachal Pradesh. Acetone cyanohydrin hydrolyzing activity of this organism comprised nitrile hydratase and amidase activities. It exhibited higher substrate specificity towards aliphatic hydroxynitrile (acetone cyanohydrin) in comparison to arylaliphatic hydroxynitrile. Isobutyronitrile (40 mM) acted as a carbon source as well as inducer for growth of Alcaligenes sp. MTCC 10674 and expression of acetone cyanohydrin hydrolyzing activity. Optimization of culture condition using response surface methodology increased acetone cyanohydrin hydrolyzing activity by 1.3-fold, while inducer mediation approach increased the activity by 1.2-fold. The half life of this enzyme was 25 h at 15 °C. V _max and K _m value for acetone cyanohydrin hydrolyzing enzyme was 0.71 μmol mg^?1 min^?1 and 14.3 mM, when acetone cyanohydrin was used as substrate. Acetone cyanohydrin hydrolyzing enzyme encountered product inhibition and IC50 and K _i value were calculated to be 28 and 10.2 mM, respectively, when product α-hydroxyisobutyric acid was added in the reaction. Under optimized reaction conditions at 40 ml fed batch scale, 3 mg dcw ml ^? resting cells of Alcaligenes sp. MTCC 10674 fully converted 0.33 M acetone cyanohydrin into α-hydroxyisobutyric acid (1.02 g) in 6 h 40 min. The characterization of acetone cyanohydrins hydrolyzing activity revealed that it comprises bienzymatic nitrile hydrolyzing system, i.e. nitrile hydratase and amidase for the production of α-hydroxyisobutyric acid from acetone cyanohydrin and maximum 70 % yield is being reported for the first time.     

Induction of larval settlement and metamorphosis in the donkey-ear abalone,Haliotis asinina Linnaeus,by chemical cues

The effects of the chemical inducers, gamma-aminobutyric acid (GABA) and potassium chloride (KCl), on the larval settlement and metamorphosis of the donkey-ear abalone, Haliotis asinina, was investigated. H. asinina larvae (5–6 h post-hatch) were exposed to a range of GABA (0.125–2.00 μM) and KCl (1.00–12.00 mM) concentrations for 72 h. Results of the dose response experiments showed that settlement and metamorphosis vary according to the dose levels of the inducer compounds. Under controlled laboratory conditions, 0.45–0.50 μM and 6.0 mM seemed to be the optima for GABA and KCl, respectively, as these concentrations elicited the greatest number of postlarvae that metamorphosed, settled or survived. However, GABA generally promoted better attachment and metamorphic response as well as survival than KCl in H. asinina postlarvae.     

Expression,characterization and mutagenesis of a novel glutamate decarboxylase from <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Objectives

To search for a novel glutamate decarboxylase (GAD) with an optimum pH towards near-neutrality in order to improve production of gamma-aminobutyric acid (GABA) in recombinant hosts.

Results

A novel glutamate decarboxylase, BmGAD, from Bacillus megaterium was overexpressed and purified. BmGAD was approximately 53 kDa by SDS-PAGE analysis. Its optimum activity was at pH 5 and 50 °C. BmGAD had a specific activity of 59 ± 5.2 U mg^?1 at pH 6, which is the highest value reported so far. The apparent Km and V_max values of BmGAD were 8 ± 0.5 mM and 150 ± 4.7 U mg^?1, respectively. Through site-directed mutagenesis, two BmGAD mutants (E294R and H467A) showed higher V_max values than that of wild-type, with the values of 210 ± 6.9 and 180 ± 4.1 U mg^?1 at pH 5 and 50 °C, respectively.

Conclusions

The unusual high activity of BmGAD at pH 6 makes it an attractive GABA-producing candidate in industrial application.

     

Glutamate decarboxylase of the parasitic arthropods Ctenocephalides felis and Rhipicephalus microplus: Gene identification,cloning, expression,assay development,identification of inhibitors by high throughput screening and comparison with the orthologs from Drosophila melanogaster and mouse

Glutamate decarboxylase (l-glutamate 1-carboxylyase, E.C. 4.1.1.15, GAD) is the rate-limiting enzyme for the production of γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in vertebrates and invertebrates. We report the identification, isolation and characterization of cDNAs encoding GAD from the parasitic arthropods Ctenocephalides felis (cat flea) and Rhipicephalus microplus (cattle tick). Expression of the parasite GAD genes and the corresponding Drosophila melanogaster (fruit fly) GAD1 as well as the mouse GAD₆₅ and GAD₆₇ genes in Escherichia coli as maltose binding protein fusions resulted in functional enzymes in quantities compatible with the needs of high throughput inhibitor screening (HTS). A novel continuous coupled spectrophotometric assay for GAD activity based on the detection cascade GABA transaminase/succinic semialdehyde dehydrogenase was developed, adapted to HTS, and a corresponding screen was performed with cat flea, cattle tick and fruit fly GAD. Counter-screening of the selected 38 hit substances on mouse GAD₆₅ and GAD₆₇ resulted in the identification of non-specific compounds as well as inhibitors with preferences for arthropod GAD, insect GAD, tick GAD and the two mouse GAD forms. Half of the identified hits most likely belong to known classes of GAD inhibitors, but several substances have not been described previously as GAD inhibitors and may represent lead optimization entry points for the design of arthropod-specific parasiticidal compounds.     

Accumulation of gamma-aminobutyric acid in the halotolerant cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis> under salt and acid stress

γ-Aminobutyric acid (GABA) is known as an inhibitory neurotransmitter in human, while in plants, GABA is an intermediate for amino acid metabolism and also is accumulated in response to a wide range of environmental stress. In the present study, GABA accumulation in Aphanothece halophytica was increased 2-fold in mid-log phase cells grown under salt stress (2.0 M NaCl). When mid-log phase cells were subjected to changes in NaCl concentrations and pH for 4 h, the highest GABA accumulation was observed in cells adapted in medium that contained 2.0 M NaCl and that was adjusted to pH 4.0, respectively. The increase of GABA accumulation was accompanied by an increased glutamate decarboxylase activity. Addition of glutamate to growth medium stimulated GABA accumulation under acid stress but had no effect under salt stress. However, the highest GABA accumulation was detected in cells exposed to both high salt and acid stresses combined with the 5 mM glutamate supplementation with an approximately 3-fold increase as compared to the control. The unicellular A. halophytica showed a similarly high content of GABA to that of a filamentous Arthrospira platensis suggesting the possibility of genetic manipulation of the genes of A. halophytica involved in GABA synthesis to increase GABA yield.     

Antioxidant Lactobacilli Could Protect Gingival Fibroblasts Against Hydrogen Peroxide: A Preliminary In Vitro Study

Oxidative stress and tissue destruction are at the heart of periodontal diseases. The dental research area is geared toward the prevention of free radicals by nutrient antioxidants. Lactic acid bacteria (LAB) have recently attracted attention in alternative dental therapies. We aimed at highlighting the antioxidative property of Lactobacilli and Bifidobacterium strains and at determining their protective effect on gingival fibroblasts (GFs). Two Lactobacilli and 2 Bifidobacterium strains were screened for their exopolysaccharide (EPSs) production. Antioxidative assays were conducted by spectrophotometer analysis. Resistance to different concentrations of hydrogen peroxide (H₂O₂) was determined by the serial dilution technique. The protective effect of strains on GFs on hydrogen peroxide exposure was also examined by a new trypan blue exclusion assay method. Bifidobacterium breve A28 showed the highest EPS production (122 mg/l) and remarkable antioxidant activity, which were demonstrated by its ability to scavenge 72 % α,α-diphenyl-1-picrylhydrazyl free radical and chelate 88 % of iron ion, respectively. Inhibition of lipid peroxidation was determined as 71 % for the A28 strain. We suggest that LAB with antioxidative activity could be a good natural therapy agent for periodontal disorders.     

Purification and characterization of glutamate decarboxylase from <Emphasis Type="Italic">Enterococcus raffinosus</Emphasis> TCCC11660

Glutamate decarboxylase (GAD) is the sole enzyme that synthesizes γ-aminobutyric acid through the irreversible decarboxylation of l-glutamate. In this study, the purification and characterization of an unreported GAD from a novel strain of Enterococcus raffinosus TCCC11660 were investigated. The native GAD from E. raffinosus TCCC11660 was purified 32.3-fold with a recovery rate of 8.3%, using ultrafiltration and ammonium sulfate precipitation, followed by ion-exchange and size-exclusion chromatography. The apparent molecular weight of purified GAD, as determined by SDS-PAGE and size-exclusion chromatography was 55 and 110 kDa, respectively, suggesting that GAD exists as a dimer of identical subunits in solution. In the best sodium citrate buffer, metal ions of Mo⁶⁺ and Mg²⁺ had positive effects, while Cu²⁺, Fe²⁺, Zn²⁺ and Co²⁺ showed significant adverse effects on enzyme activity. The optimum pH and temperature of GAD were determined to be 4.6 and 45 °C, while the K _m and V _max values for the sole l-glutamate substrate were 5.26 and 3.45 μmol L^?1 min^?1, respectively.     

Glutamic acid decarboxylase gene disruption reveals signalling pathway(s) governing complex morphogenic and metabolic events in Trichoderma atroviride

Glutamate decarboxylase (GAD) catalyses decarboxylation of glutamate to gamma-aminobutyrate (GABA) in a metabolic pathway connected to citrate cycle and known as GABA shunt. The gene (gad) was disrupted in Trichoderma atroviride CCM F-534 and viable mutants were characterized. Two of them were found to arise by homologous recombination and were devoid of both GAD activity and GABA. Mutants grew slower as compared to the wild type (F534). In the submerged culture, mutants developed less CO₂ and consumed less O₂ than the F534 without changing their respiratory quotients. Hyphae of mutants were more ramified than those of F534. Their ramification, in contrast to F534, was not increased by cyclosporin A, a drug causing hyphae ramification of several fungi and which is a calcineurin/cyclophilin inhibitor, or by FK506. Rapamycin, which is a cyclophilin but not calcineurin inhibitor, had a different effect on hyphae ramification in F534 and mutants. To examine the presence of GABA receptors in the fungus the effect of mammalian GABA-receptor modulators, such as bicuculline, gabapentin or carbamazepine on fungal morphology were investigated. Conidia of mutants germinated in a multipolar manner more frequently (up to 80 %) than those of F534. This trait was modified with cyclosporine A, FK506 and GABA receptor modulators in a different manner. Transport of chlorides, an intimate feature of GABA-regulated receptors/channels in animal cells, was measured in vegetative mycelia by means ³⁶Cl^? uptake. It was significantly reduced in gad mutants. The results suggest that T. atroviride possesses a signalling pathway that involves GABA, putative GABA receptor(s), calcineurin, target of rapamycin and chloride transporter(s) to regulate physiological functions.     

2-Ketogluconic Acid Secretion by Incorporation of Pseudomonas putida KT 2440 Gluconate Dehydrogenase (gad) Operon in Enterobacter asburiae PSI3 Improves Mineral Phosphate Solubilization

Enterobacter asburiae PSI3 is known to efficiently solubilize rock phosphate by secretion of approximately 50 mM gluconic acid in Tris-buffered medium in the presence of 75 mM glucose and in a mixture of seven aldosugars each at 15 mM concentration, mimicking alkaline vertisol soils. Efficacy of this bacterium in the rhizosphere requires P release in the presence of low amount of sugars. To achieve this, E. asburiae PSI3 has been manipulated to express gluconate dehydrogenase (gad) operon of Pseudomonas putida KT 2440 to produce 2-ketogluconic acid. E. asburiae PSI3 harboring gad operon had 438 U of GAD activity, secreted 11.63 mM 2-ketogluconic and 21.65 mM gluconic acids in Tris-rock phosphate-buffered medium containing 45 mM glucose. E. asburiae PSI3 gad transformant solubilized 0.84 mM P from rock phosphate in TRP-buffered liquid medium. In the presence of a mixture of seven sugars each at 12 mM, the transformant brought about a drop in pH to 4.1 and released 0.53 mM P.     

Effects of two sodium salts on fatty acid and essential oil composition of basil (Ocimum basilicum L.) leaves

The effects of two sodium salts on growth, fatty acids, and essential oil compositions were investigated in a medicinal and aromatic plant, Ocimum basilicum cultivated in hydroponic medium. Plants were subjected to an equimolar concentration of Na₂SO₄ (25 mM) and NaCl (50 mM) for 15 days. Our results showed that leaf growth rate was more depressed by 25 mM Na₂SO₄ than by 50 mM NaCl. The total fatty acid contents did not show any change in plants. α-Linolenic, palmitic, and linoleic acids were the major fatty acids. The identification of basil leaf fatty acids has not been previously studied and this work revealed the predominance of polyunsaturated fatty acids. Under both salts, leaf fatty acid composition remained unchanged. Regarding the essential oil yield, it decreased significantly by 28 % under 25 mM Na₂SO₄ and showed an increase by 27 % under 50 mM NaCl. The major volatile compound in leaves was linalool with 34.3 % of total essential oil constituents, followed by eugenol (19.8 %), 1.8-cineole (14.4 %) and methyl eugenol (5.2 %). Further, levels of eugenol and methyl eugenol were most modulated by salt, and the negative correlation between these two compounds reflects the stimulation of O-methyltransferase activity under both salts.