Characterization Of Glutamate Decarboxylase Research Articles

Expression, purification, and characterization of glutamate decarboxylase from human gut-originated Lactococcus garvieae MJF010.

Human gut-originated lactic acid bacteria were cultivated, and high γ-aminobutyric acid (GABA)-producing Lactococcus garvieae MJF010 was identified. To date, despite the importance of GABA, no studies have investigated GABA-producing Lactococcus species, except for Lc. lactis. A recombinant glutamate decarboxylase of the strain MJF010 (rLgGad) was successfully expressed in Escherichia coli BL21(DE3) with a size of 53.9kDa. rLgGad could produce GABA, which was verified using the silylation-derivative fragment ions of GABA. The purified rLgGad showed the highest GABA-producing activity at 35°C and pH 5. rLgGad showed a melting temperature of 43.84°C. At 30°C, more than 80% of the activity was maintained even after 7h; however, it rapidly decreased at 50°C. The kinetic parameters, Km, Vmax, and kcat, of rLgGad were 2.94 mM, 0.023 mM/min, and 12.3min- 1, respectively. The metal reagents of CaCl2, MgCl2, and ZnCl2 significantly had positive effects on rLgGad activity. However, most coenzymes including pyridoxal 5'-phosphate showed no significant effects on enzyme activity. In conclusion, this is the first report of Gad from Lc. garvieae species and provides important enzymatic information related to GABA biosynthesis in the Lactococcus genus.

Molecular cloning, gene overexpression and characterization of glutamate decarboxylase from Enterococcus faecium DO

In most bacteria, Glutamate decarboxylase (GAD) activity significantly reduces at pH > 6. Since l-glutamate is neutral to slightly alkaline, a large amount of hydrochloric acid must be added to the reaction mixture. Acidic reaction conditions greatly increase the corrosion of production equipment and shorten the life of manufacturing equipment. Therefore, this study aims at studying the overexpression of recombinant GAD that is active at the neutral pH and high-level production of γ-aminobutyric acid. A gene sequence encoding GAD of Enterococcus faecium DO was cloned into PET21a (+) and was overexpressed in Escherichia coli BL21 (DE3). The construct was confirmed by colony PCR and double digested with BamHI and XhoI. The result from Western blot analysis showed a signal band at the molecular mass of 54 kDa. The specific activity of the recombinant GAD was 78.17 U mg−1 at 45 °C and pH = 6.6. It was reduced rapidly when pH > 7.8. The Km, Vmax, and kcat values of recombinant GAD were 1.8 mM, 12.9 μmol L−1 min−1, and 0.43 s−1, respectively. Therefore, recombinant GAD is a potent candidate for GABA production in food and pharmaceutical industries, because it increases the efficiency of GABA synthesis, especially in foods with a neutral pH range.

Expression and characterization of glutamate decarboxylase from Lactobacillus brevis HYE1 isolated from kimchi.

A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni-NTA affinity chromatography, and had a molecular mass of 54kDa with optimal hydrolysis activity at 55°C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18°C). Kinetic parameters including Km and Vmax values for GADLbHYE1 were 4.99mmol/L and 0.224mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme.

Purification and characterization of glutamate decarboxylase from Enterococcus raffinosus 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 110kDa, 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 3.45μmolL-1 min-1, respectively.

Characterization of glutamate decarboxylase from Synechocystis sp. PCC6803 and its role in nitrogen metabolism

Glutamate decarboxylase (GAD) (EC 4.1.1.15), an enzyme responsible for the synthesis of γ-aminobutyric acid (GABA), from Synechocystis sp. PCC6803 was cloned and overexpressed in Escherichia coli BL21(DE3). The purified enzyme was expressed as a monomeric protein with a molecular mass of 53 and 55 kDa as determined by SDS-PAGE and gel filtration chromatography, respectively. The enzyme activity was pyridoxal-5′-phosphate dependent with an optimal activity at pH 6.0 and 30 °C. The catalytic properties of this enzyme were, Km = 19.6 mM; kcat = 100.7 s−1; and kcat/Km = 5.1 mM−1 s−1. The transcription levels of genes involved in nitrogen metabolism were up-regulated in the Δgad strain. The mutant showed approximately 4- and 8-fold increases in the transcript levels of kgd and gabdh encoding a novel α-ketoglutarate decarboxylase and γ-aminobutanal dehydrogenase, respectively. Overall results suggested that in Synechocystis lacking a functional GAD, the γ-aminobutanal dehydrogenase might serve as an alternative catalytic pathway for GABA synthesis.

Characterization of Glutamate Decarboxylase (GAD) from Lactobacillus sakei A156 Isolated from Jeot-gal.

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°C 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°C and the activity was dependent on pyridoxal 5'-phosphate. The Km and Vmax of GAD were 0.045 mM and 0.011 mM/min, respectively, when glutamate was used as the substrate.

Characterization of glutamate decarboxylase from Lactobacillus plantarum and its C-terminal function for the pH dependence of activity.

The gadB gene encoding glutamate decarboxylase (GAD) from Lactobacillus plantarum was cloned and expressed in Escherichia coli. The recombinant enzyme exhibited maximal activity at 40 °C and pH 5.0. The 3D model structure of L. plantarum GAD proposed that its C-terminal region (Ile454-Thr468) may play an important role in the pH dependence of catalysis. Accordingly, C-terminally truncated (Δ3 and Δ11 residues) mutants were generated and their enzyme activities compared with that of the wild-type enzyme at different pH values. Unlike the wild-type GAD, the mutants showed pronounced catalytic activity in a broad pH range of 4.0-8.0, suggesting that the C-terminal region is involved in the pH dependence of GAD activity. Therefore, this study may provide effective target regions for engineering pH dependence of GAD activity, thereby meeting industrial demands for the production of γ-aminobutyrate in a broad range of pH values.

Characterization of Glutamate Decarboxylase from a High γ-Aminobutyric Acid (GABA)-Producer,Lactobacillus paracasei

Gamma-aminobutyric acid (GABA) has several physiological functions in humans. We have reported that Lactobacillus paracasei NFRI 7415 produces high levels of GABA. To gain insight into the higher GABA-producing ability of this strain, we analyzed glutamate decarboxylase (GAD), which catalyzes the decarboxylation of L-glutamate to GABA. The molecular weight of the purified GAD was estimated to be 57 kDa by SDS-PAGE and 110 kDa by gel filtration, suggesting that GAD forms the dimer under native conditions. GAD activity was optimal at pH 5.0 at 50 degrees C. The Km value for the catalysis of glutamate was 5.0 mM, and the maximum rate of catalysis was 7.5 micromol min(-1) mg(-1). The N-terminal amino acid sequence of GAD was determined, and the gene encoding GAD from genomic DNA was cloned. The findings suggest that the ability of Lb. paracasei to produce high levels of GABA results from two characteristics of GAD, viz., a low Km value and activity at low pH.

Purification and characterization of glutamate decarboxylase from rice germ

Glutamate decarboxylase (EC 4.1.1.15, GAD) is a pyridoxal 5′-phosphate (PLP) dependent enzyme, which catalyses the irreversible α-decarboxylation of l-glutamic acid to γ-aminobutyric acid. GAD was purified 186-fold from rice germ using a combination of ammonium sulfate fractionation, DEAE-Sepharose FF ion exchange chromatography, Superdex-200 gel filtration chromatography, and Glu-Sepharose CL 4B affinity chromatography. The purified preparation showed a single peak on SE-HPLC with an approximate molecular mass of 78 kDa and a single band on SDS–PAGE with a subunit Mr of 40 kDa. This indicated that the GAD from rice germ existed as a dimer of homological subunits. Rice germ GAD has an optimum pH range between 5.5 and 5.8, and an optimum temperature at 40 °C. K m values for glutamic acid and PLP were determined at 32.3 mM and 1.7 μM, respectively. Chemicals reagents such as HgCl 2, KI and AgNO 3 decreased the enzyme activity by 68.5%, 44.9% and 32.4%, respectively, but 500 μM of CaCl 2 at the optimum pH could increase the activity by 145%.

Purification and Characterization of Glutamate Decarboxylase from Aspergillus oryzae

We purified glutamate decarboxylase (GAD) [EC4.1.1.15] from Aspergillus oryzae and characterized its biochemical and kinetic properties. GAD was purified by ammonium sulfate at 30–70% saturation and chromatographies on Sephacryl S-300, DEAE-FF and CM-FF. The purification of GAD from the crude enzyme solution was 40-fold and the recovery rate was 4.9%. About 230 μg of purified enzyme was obtained from 20 g of the mycelia of A. oryzae. The purified preparation of the enzyme showed a single protein band on SDS-PAGE. The molecular weight of purified GAD by SDS-PAGE and gel filtration was estimated to be 48 kDa and 300 kDa, respectively, suggesting that purified GAD had a hexameric structure. The Km value for L-glutamic acid, a substrate of the enzyme, was estimated to be 13 mM. The optimum pH and temperature of GAD were 5.5 and 60°C, respectively. The GAD activity was stable up to 40°C.

Purification and characterization of glutamate decarboxylase from cowpea

Glutamate decarboxylase (GAD) was purified 300-fold from green cowpea ( Vigna unguiculata L.) pods using a combination of PEG precipitation, DEAE cellulose chromatography, hydroxylapatite chromatography, and Q-resin chromatography. The partially purified preparation demonstrated 2 primary bands in SDS-polyacrylamide gel electrophoresis with up to 3 additional minor bands. Cowpea GAD has a pH optimum at between pH 5.5–6.0, and an apparent K M for glutamate of 3.2 mM at pH 5.8. Both crude GAD preparations and preparations partially purified through the hydroxylapatite step can be stimulated by Ca 2+ and calmodulin when assayed at pH 5.8. However, the purified enzyme does not show activation by Ca 2+ and/or calmodulin at pH 5.8 or at pH 7.0. © 1997 Elsevier Science Ltd. All rights reserved

Purification and characterization of glutamate decarboxylase from Lactobacillus brevis IFO 12005.

Glutamate decarboxylase (GAD) [EC 4.1.1.15] was purified from a cell-free extract of Lactobacillus brevis IFO 12005 by chromatographies on Sephadex G-100, DEAE-Sepharose CL-6B, and Mono Q. About 9 mg of purified GAD was obtained from 90.2 g of wet cells. The purified preparation showed a single protein band on SDS-PAGE. The molecular weights of purified GAD by SDS-PAGE and gel filtration on Superdex 200 were 60,000 and 120,000, respectively, indicating that GAD from L. brevis exists as a dimer. The N-terminal amino acid sequence of the purified GAD was NH2-Met-Asn-Lys-Asn-Asp-Gln-Glu-Gln-Thr-. The optimum pH and temperature of GAD were at pH 4.2 and at 30 degrees C. The GAD activity was increased by the addition of sulfate ions in a dose-dependent manner. The order of effects was as follows: ammonium sulfate > sodium sulfate > magnesium sulfate, indicating that the increase of hydrophobic interaction between subunits causes the increase of GAD activity. The purified GAD reacted only with L-glutamic acid as a substrate and the K(m), kcat, and kcat/K(m) values were 9.3 mM, 6.5 S-1, and 7 x 10(2) M-1 S-1, respectively.

Purification and characterization of glutamate decarboxylase from Neurospora crassa conidia.

L-Glutamate decarboxylase, an enzyme under the control of the asexual developmental cycle of Neurospora crassa, was purified to homogeneity from conidia. The purification procedure included ammonium sulfate fractionation and DEAE-Sephadex and cellulose phosphate column chromatography. The final preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gels with a molecular weight of 33,200 +/- 200. A single band coincident with enzyme activity was found on native 7.5% polyacrylamide gels. The molecular weight of glutamate decarboxylase was 30,500 as determined by gel permeation column chromatography at pH 6.0. The enzyme had an acidic pH optimum and showed hyperbolic kinetics at pH 5.5 with a Km for glutamic acid of 2.2 mM and a Km for pyridoxal-5'-phosphate of 0.04 microM.

Purification and characterization of glutamate decarboxylase from Solanum tuberosum.

Glutamate decarboxylase has been purified from potato tubers. The final preparation was homogeneous as judged from native and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Gel filtration on Sephadex G-200 gave a relative molecular mass Mr, of 91 000 for the native enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a subunit Mr of 43 000. Thus the enzyme appears to be a dimer of identical subunits. It has 2 mol pyridoxal 5'-phosphate/mol protein, which could not be removed by exhaustive dialysis or gel filtration on Sephadex G-25. The enzyme has an absorption maximum at 370 nm in sodium phosphate buffer, pH 5.8. Reduction of the enzyme with sodium borohydride abolished the absorption maximum at 370 nm with attendant loss of catalytic activity. The enzyme exhibited pH-dependent spectral changes. The enzyme was specific for L-glutamate and could not decarboxylate other amino acids tested. The enzyme was maximally active at pH 5.8 and a temperature of 37 degrees C. Isoelectric focussing gave a pI of 4.7 Km values for L-glutamate and pyridoxal 5'-phosphate were 5.6 mM and 2 microM respectively. Thiol-directed reagents and heavy metal ions inhibited the enzyme, indicating that an -SH group is required for activity. The nature of the functional groups at the active site of the enzyme was inferred from competitive inhibition studies. L-Glutamate promoted inactivation of the enzyme caused by decarboxylation-dependent transamination was demonstrated. The characteristics of potato enzyme were compared with enzyme from other sources.

Purification and Characterization of Glutamate Decarboxylase from Mouse Brain

Abstract Glutamate decarboxylase catalyzes the formation of γ-aminobutyric acid, an important inhibitory transmitter in invertebrate and vertebrate nervous systems. Approximately 700-fold purification of the enzyme from mouse brain has been achieved by a combination of ammonium sulfate fractionation, gel filtration, calcium phosphate gel, and DEAE-Sephadex chromatography. The most highly purified preparation appeared to be monodisperse on high speed sedimentation equilibrium analysis and was found to have a partial specific volume, v, of 0.732 and a molecular weight of 85,000 ± 2,000. A single major protein band coincident with the enzyme activity was found with polyacrylamide gel. In addition, a faint band also was present. In tests with 20 naturally occurring amino acids and α-ketoglutarate the enzyme showed α-decarboxylation only with glutamate to a great extent; but it also catalyzed α-decarboxylation of aspartate to a slight extent. A sharp pH optimum at pH 7.0 has been observed. Km values of 0.7 mm and 0.05 µm were found for glutamate and pyridoxal phosphate, respectively. The enzyme was dissociated into two physically indistinguishable subunits with a molecular weight of 44,000 ± 2,000 in 6 m guanidine HCl containing 0.1 m β-mercaptoethanol.