[2] l-Glutamate decarboxylase from bacteria

Fonda, Margaret L.

doi:10.1016/s0076-6879(85)13005-3

Cited by 58 publications

(44 citation statements)

References 35 publications

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“…The properties of L. lactis GAD were similar to those of E. coli GAD (Fonda, 1985 The role of bacterial amino acid decarboxylases has been proposed to be in the maintenance of acidic pH by the consumption of hydrogen ions as decarboxylation proceeds (Gale, 1946). Three amino-acid-dependent acid-resistance systems have been described for enterobacteria (Bearson et al, 1997).…”

Section: Discussionmentioning

confidence: 88%

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Lactococcus lactis contains only one glutamate decarboxylase gene

et al. 1999

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Glutamate decarboxy~ase, which is associated with a glutamate-dependent acid-resistance mechanism, was purified from Lactococcus lactis subsp. lactis by a three-step procedure. The specific activity was increased about 114-fold with a yield of 16%. The N-terminal amino acid sequence of the enzyme was determined. The gene encoding this enzyme was cloned in kcherichia coli, and its nucleotide sequence was determined. The deduced amino acid sequence suggests that the enzyme is produced as a mature form (466 amino acid residues), not as a precursor protein. The subunit molecular mass of L. lactis glutamate decarboxylase was calculated to be 53926 Da. The enzyme was maximally active a t pH 4 7 and reacted only with L-glutamate among 20 a-amino acids. The apparent Km value was calculated to be 0.51 mM. The activity was stable at acidic pH values; there was no activity in the neutral pH range. A t pH 4 1 the enzyme activity was retained at temperatures up to 70 "C in 10 min incubations. L. lactis glutamate decarboxylase behaved as a single protein when the enzyme was purified. A single band corresponding to the glutamate decarboxylase gene was detected on Southern blot analysis. These data suggest that there is one glutamate decarboxylase gene in L. lactis.

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Section: Discussionmentioning

confidence: 88%

“…GAD has been isolated from a wide variety of sources and its biochemical properties have been characterized. Escherichia coli GAD (Fonda, 1985) is a homohexameric enzyme with a subunit size of 50 kDa. Each subunit contains a pyridoxal phosphate bound to a lysyl residue.…”

Section: Introductionmentioning

confidence: 99%

Lactococcus lactis contains only one glutamate decarboxylase gene

et al. 1999

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“…of the mycelia of A. oryzae. After the final step of the purification, the specific activity increased to 48.2 U/mg, which is twice that of GAD from squash (Matsumoto et al, 1986) and 6 times that of GAD from L. brevis, but lower than that of GAD from E. coli (Fonda, 1985) (Table2).…”

Section: Resultsmentioning

confidence: 96%

“…In higher plants, GAD has been purified from squash (Matsumoto et al, 1986). In bacteria, the enzymatic properties of GAD have been reported in both Escherichia coli (Shikuya & Schwert, 1960;Fonda, 1985) and Lactobacillus brevis . Although GAD obtained from A. oryzae has not been purified, the gene was recently cloned and the nucleotide sequence was determined (Iwai et al, 2001).…”

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confidence: 99%

Purification and Characterization of Glutamate Decarboxylase from Aspergillus oryzae

Tsuchiya

Nishimura

Iwahara

2003

FSTR

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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 g 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 K m 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.Keywords: glutamate decarboxylase, Aspergillus oryzae, purification, ␥-amino-butyric acid Glutamate decarboxylase (GAD; EC4.1.1.15) produces ␥-amino-butyric acid (GABA) from glutamate. GABA has several physiological effects on the human body, including neurotransmitting, hypotensive and diuretic effects. Several attempts to enrich GABA in functional foods have been reported: GABArich green tea produced by anaerobic treatment of green tea (Ohmori et al., 1987), GABA accumulation in red mold rice (Tsuji et al., 1992), GABA accumulation in rice germ by soaking in water (Saikusa et al., 1994), GABA enrichment in brown rice by high-pressure treatment (Kinefuchi et al., 1999) and GABA production by lactic acid bacteria .We have recently found high GAD activity in Aspergillus oryzae cultured in a liquid medium, and it was specially rich in A. oryzae used for miso and sake brewing (Tsuchiya & Nishimura, 2002a). Furthermore, we also reported that the batch reactor using pellet form mycelium of A. oryzae (for miso production), which was cultured in liquid medium achieved an extremely high GABA production rate (8.5 mmol GABA/g Aspergillus mycelium/h) (Tsuchiya et al., 2002b).GAD is widely distributed in animate nature. In mammals, GAD has been purified from human brain (Blindermann et al., 1978) and mouse brain (Wu et al., 1973). In higher plants, GAD has been purified from squash (Matsumoto et al., 1986). In bacteria, the enzymatic properties of GAD have been reported in both Escherichia coli (Shikuya & Schwert, 1960;Fonda, 1985) and Lactobacillus brevis . Although GAD obtained from A. oryzae has not been purified, the gene was recently cloned and the nucleotide sequence was determined (Iwai et al., 2001). The detailed biochemical nature of the enzyme is unclear, which we believe important in promoting the GABA production in food processing by effective utilization of the GAD from A. oryzae. In this study, we purified GAD from the mycelium of A. oryzae and examined some biochemical characteristics of the enzyme. Materials and MethodsMicroorganism and the culture conditions Aspergillus oryzae used in this study was isolated from tane-koji SR-108...

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“…The amino acid decarboxylases are widely distributed in procaryotes and eucaryotes (see Table). Therefore it is surprising to see that glutamate decarboxylases, though common in eucaryotes, appear to be uncommon among procaryotes (7,14). Not only are gadBC lacking in enterobacteriaceae other than Shigella species and E. coil, they are also absent from other gram negative bacteria assayed such as Yersinia enterocolitica, Pseudomonas aeruginosa.…”

Section: Introductionmentioning

confidence: 99%

Shigella and Escherichia coli Strategies for Survival at Low pH

Small¹

1998

JJMSB

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[2] l-Glutamate decarboxylase from bacteria

Cited by 58 publications

References 35 publications

Lactococcus lactis contains only one glutamate decarboxylase gene

Lactococcus lactis contains only one glutamate decarboxylase gene

Purification and Characterization of Glutamate Decarboxylase from Aspergillus oryzae

Shigella and Escherichia coli Strategies for Survival at Low pH

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