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.

/pdf/production-of-gaba-gamma-amino-butyric-acid-by-lactic-acid-50eu2yraul.pdf

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].

在线固相萃取-超高效液相色谱-三重四极杆质谱法测定水源水和饮用水中107种典型农药及代谢产物

Disinfection of drinking water is critical to prevent waterborne diseases. An unexpected consequence of water disinfection is the formation of disinfection by-products by the interaction of disinfectants with organic matter (natural or anthropogenic) and halides, which present significant toxicological effects and carcinogenic risks. As an emerging disinfection by-product, halobenzoquinones (HBQs) have attracted increasing attention owing to their severe toxicity and high detection rates. The credible determination of HBQs is essential for further studies on their occurrence, toxicity, and control measures; however, HBQs are usually detected in drinking water at trace levels. Therefore, accurate and efficient analytical techniques are critical for HBQ determination and quantitation. In this study, a method based on solid phase extraction (SPE) combined with ultra performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS) was developed to determine 13 HBQs, including six chlorobenzoquinones, six bromobenzoquinones, and one iodobenzoquinone, in drinking water. One-liter water samples were added with 2.5 mL of formic acid, and 500 mL of each sample was collected for further enrichment. Pretreatment optimization mainly focused on the SPE column, washing solvent, and nitrogen blowing temperature. After extraction using Plexa SPE columns (200 mg/6 mL), the samples were washed with ultrapure water containing 0.25% formic acid combined with 30% methanol aqueous solution containing 0.25% formic acid, eluted with 6 mL of methanol containing 0.25% formic acid, and then nitrogen blown at 30 ℃. The UPLC-MS/MS parameters were optimized by comparing the results of two reversed-phase columns (BEH C18 and HSS T3) and various concentrations of formic acid in the mobile phase, as well as by establishing the best instrumental conditions. The separation of 13 HBQs was performed using an HSS T3 column (100 mm×2.1 mm, 1.8 μm) via gradient elution with a mixture of 0.1% formic acid aqueous solution and methanol as the mobile phase for 16 min. The 13 HBQs were detected using a triple quadrupole mass spectrometer equipped with a negative electrospray ionization source (ESI-) in multiple reaction monitoring (MRM) mode. Matrix-matched calibration curves were used to quantify the HBQs owing to intense matrix inhibitory effects. The results reflected the good linear relationships of the 13 HBQs and yielded correlation coefficients (r) greater than 0.999. The method detection limits (MDLs, S/N=3) were 0.2-10.0 ng/L, while the method quantification limits (MQLs, S/N=10) were 0.6-33.0 ng/L. The recoveries of the 13 HBQs were 56%-88% at three spiked levels (10, 20, 50 ng/L), and the relative standard deviations (RSDs, n=6) were less than or equal to 9.2%. The optimization method was applied to analyze HBQs in five drinking water samples. Four HBQs, namely, 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ), 2,5-dibromo-1,4-benzoquinone (2,5-DBBQ), 2,6-dibromo-1,4-benzoquinone (2,6-DBBQ), and 2,6-dibromo-3,5-dimethyl-1,4-benzoquinone (2,6-DBDMBQ), were detected in the samples with detection rates of 100%, 20%, 80%, and 20%, respectively. The most frequently detected HBQ, 2,6-DCBQ, also exhibited the highest content (15.0-56.2 ng/L). The method showed high sensitivity, stability, accuracy, and efficiency, rendering it suitable for the analysis of 13 HBQs in drinking water. Compared with previous methods that mainly focused on 2,6-DCBQ and 2,6-DBBQ, the developed method achieved higher throughput and enabled the simultaneous analysis of 13 HBQs. The method presented in this study provides an opportunity to explore different types and concentrations of HBQs in drinking water, offers a deeper understanding of the occurrence of HBQs, and facilitates further studies on the health risks and control measures of these compounds.

[Determination of 13 halobenzoquinone disinfection by-products in drinking water using solid phase extraction-ultra performance liquid chromatography-triple quadrupole mass spectrometry].

In order to monitor the risk of pesticide pollutants in drinking water, an analytical method based on online-solid phase extraction coupled with ultra performance liquid chromatography-triple quadrupole mass spectrometry (online-SPE-UPLC-MS/MS) was established for the simultaneous rapid screening and determination of 107 pesticides and metabolites (organophosphorus, organic nitrogen, organic heterocycle, carbamate, amide, benzoyl urea, neonicotinoid, etc.) in raw water and drinking water. Different injection volumes (5, 10, and 15 mL) were compared. The detection response increased with an increase in the injection volume, but the matrix effect also became more pronounced. Under the premise of ensuring the sensitivity of the method and meeting the detection requirements, the injection volume was selected as 5 mL. Accordingly, the samples were filtered through a 0.22-μm hydrophilic polytetrafluoroethylene filter, and then, 5 mL samples were injected into the online-SPE system by the automatic sampler. After adsorption on an X Bridge C18 online-SPE column, the samples were washed with pure water and eluted by gradient elution using acetonitrile and 0.1% formic acid aqueous solution as the mobile phases, with separation on an ACQUITY HSS T3 column. The samples were detected by multiple reaction monitoring with electrospray ionization in positive and negative ion modes, and quantified by an external standard method. Using raw water and drinking water as the sample matrices, the accuracy and precision of the method were verified. The 107 pesticides and metabolites showed good linear relationships in different ranges with correlation coefficients (r2)>0.995. The limits of detection (LODs, S/N=3) of the method were 0.03-1.5 ng/L, and the limits of quantification (LOQs, S/N=10) were 0.1-5.0 ng/L. The target pesticides were spiked at concentration levels of 1, 20, and 50 ng/L. The spiked recoveries of the 107 targets in raw water and drinking water samples were 60.6%-119.8% and 61.2%-119.0%, respectively. The corresponding relative standard deviations (RSDs, n=6) were 0.3%-18.6% and 0.4%-17.1%. The pesticide residues in raw water and drinking water were determined by this method. Amide herbicides, triazine herbicides, triazole insecticides, carbamate insecticides, and neonicotinoid insecticides had high detection rates. The detected concentrations ranged from 0.1 to 97.1 ng/L in raw water and from 0.1 to 93.6 ng/L in drinking water. The sample consumption of online-SPE method was lower than that in the traditional off-line SPE methods, which greatly improved the convenience of sample collection, storage, and transportation. The samples only need to be filtered before injection and analysis. The method is simple to operate and shows good reproducibility. With this online-SPE method, only 23 min were required from online enrichment to detection completion. The developed method has the advantages of high analytical speed and high sensitivity. The method is suitable for the trace analysis and determination of 107 typical pesticides in raw water and drinking water, which effectively improves the detection efficiency of pesticides in water and has high potential for practical application. It can extend technical support for the pollution-level analysis of typical pesticides and metabolites in drinking water and provide an objective basis for human health risk assessment.

[Determination of 107 typical pesticides and metabolites in raw water and drinking water by online-solid phase extraction coupled with ultra performance liquid chromatography-triple quadrupole mass spectrometry].

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

在线固相萃取-超高效液相色谱-三重四极杆质谱法测定水源水和饮用水中107种典型农药及代谢产物

[Determination of 13 halobenzoquinone disinfection by-products in drinking water using solid phase extraction-ultra performance liquid chromatography-triple quadrupole mass spectrometry].

[Determination of 107 typical pesticides and metabolites in raw water and drinking water by online-solid phase extraction coupled with ultra performance liquid chromatography-triple quadrupole mass spectrometry].