• Microbiology and Biotechnology Letters
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• v.17 no.4
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• pp.379-384
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• 1989

Glutamine production from glutamate was carried out using glutamine synthetase from E. coli K-12 pgln 6 and baker's yeast, which supplies ATP into the reaction system through alcohol fermentation, simultaneously. With whole cells of E. coli K-12 pgln 6 as an enzyme source of glutamine synthetase, 11.8 g/ι of glutamine produced after 18-h incubation (60％ yield based on a substrate, glutamate). Using the partially purified glutamine synthetase, 19.8 git of glutamine was produced after 5-h incubation. This amount of glutamine was correspond to 90％ yield, based on substrate, glutamate.

• The Korean Journal of Food And Nutrition
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• v.6 no.3
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• pp.169-177
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• 1993

The conversion of glutamate by glutamine synthetase Is the endergonic reaction that demands ATP as its energy source. In order to supply efficiently ATP that is demanded in the conversion of glutamate to glutamine, the ATP- generating system by acetate kinase partially purified from Escherichia coli K-12 was coupled with glutamine synthetase partially purified 5. coli K-12 Pgln6. The optinum conditions of the coupled reaction were investigated. As the result, the highest conversion of glutamate to glutamine was shown In the reaction mixture containing 100mM glutamate, 100mM NHtCl, 50M acetyl phosphate, 5mM ADP, 40M MgCl2, 300mM potassium phosphate buffer (pH 7.5), 5mM MnCl2, Under this condition, the most effective concentrations of enzyme were 70unit/ml glutamine synthetase and 99unit/ml acetate kinase. Under the optinum conditions, 98% of 100mM glutamate was converted to glutamine within 6 hours.

• Microbiology and Biotechnology Letters
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• v.19 no.3
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• pp.259-264
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• 1991

Glutamine synthetase (GS) of Kkbsiellu pmumonzae was purified and identified it's properties. It was determined to be composed of 12 identical subunits and it's molecular weight was about 600,000. It's optimum pH and temperature were identified as pH 7.0 and $37^{\circ}C$ respectively, and also there was no considerable variation of activity between pH 5 and 8. When GS was incubated at $57^{\circ}C$ for 10 min, it's activity was decreased to half of maximum activity. It was observed that K. pneumoniae has adenylylation-deadenylylation system which regulates activity of GS according to the quality and quantity of nitrogen source like GS of E. coli Also it's GS was very similar to that of E. coli. in structure deduced from the immunodiffuslon experiment using anti-E. coli GS antibody.

• Journal of Microbiology
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• v.42 no.2
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• pp.111-116
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• 2004

It is known that Bacillus subtilis glutamyl-tRNA synthetase (GluRS) mischarges E. coli $tRNA_{1}$$^{Gln}$ with glutamate in vitro. It has also been established that the expression of B. subtilis GluRS in Escherichia coli results in the death of the host cell. To ascertain whether E. coli growth inhibition caused by B. subtilis GluRS synthesis is a consequence of Glu-$tRNA_{1}$$^{Gln}$ formation, we constructed an in vivo test system, in which B. subtilis GluRS gene expression is controlled by IPTG. Such a system permits the investigation of factors affecting E. coli growth. Expression of E. coli glutaminyl-tRNA synthetase (GlnRS) also amelio-rated growth inhibition, presumably by competitively preventing $tRNA_{1}$$^{Gln}$ misacylation. However, when amounts of up to 10 mM L-glutamine, the cognate amino acid for acylation of $tRNA_{1}$$^{Gln}$, were added to the growth medium, cell growth was unaffected. Overexpression of the B. subtilis gatCAB gene encoding Glu-$tRNA^{Gln}$ amidotransferase (Glu-AdT) rescued cells from toxic effects caused by the formation of the mis-charging GluRS. This result indicates that B. subtilis Glu-AdT recognizes the mischarged E. coli Glu-$tRNA_{1}$$^{Gln}$, and converts it to the cognate Gln-$tRNA_{1}$$^{Gln}$ species. B. subtilis GluRS-dependent Glu-$tRNA_{1}$$^{Gln}$ formation may cause growth inhibition in the transformed E. coli strain, possibly due to abnormal protein synthesis.

• Proceedings of the Microbiological Society of Korea Conference
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• 2005.05a
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• pp.218.1-218
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• 2005

• BMB Reports
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• v.33 no.6
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• pp.514-518
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• 2000

Two open reading frames of Haemophilus influenzae, HI0572 and HI0751, showing homology to a yeast thioredoxin peroxidase II (TPx II) and an E. coli thiol peroxidase $P_{20}$, respectively, were cloned and expressed in E. coli, and then the proteins were subsequently purified and characterized. HI0751 protein showed the thioredoxin (Trx)-dependent peroxidase activity, whereas HI0572 protein showed glutathione-dependent peroxidase. The HI0572 is the first peroxiredoxin with glutathione peroxidase activity rather than thioredoxin peroxidase. Purified HI0572 and HI0751 proteins protected specifically the inactivation of glutamine synthetase by metal catalyzed oxidation (MCO) systems composed of $Fe^{3+}$, $O_2$ and mercaptans such as dithiothreitol, ${\beta}-mercaptoethanol$ and glutathione (GSH). Unlike the HI0751 protein, the HI0572 protein was more effective in protecting glutamine synthetase from inactivation by the $GSH/Fe^{3+}/O_2$ system. It seems that these unique properties of the HI0572 protein are due to the structure containing a glutaredoxin domain at it's C-terminal in addition to a peroxiredoxin domain.

• Microbiology and Biotechnology Letters
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• v.14 no.5
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• pp.427-432
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• 1986

The regulation of three ammonia assimilatory enzymes, GDH (glutamate dehydrogenase), GS (glutamine synthetase) and GOGAT (glutamate synthase), has been examined in C. glutamicum. Three kinds of arginine auxotrophs blocked in each step of arginine biosynthetic pathway from glutamate were selected as arg 5, arg 6, arg 8. Histidine and tryptophan auxotrophs were also selected because histidine and tryptophan repressed GS biosynthesis in E. coli. These strains were cultured on the media containing nitrogen-excess and limited conditions, to compare the specific activities of ${\alpha}$-ketoglutarate dehydrogenase(${\alpha}-KGDH$), GDH, GS, GOGAT from the cell-free extracts. These results showed that enzyme levels of ${\alpha}-KGDH$ and GDH from 3 kinds of arginine auxotrophs, histidine and tryptophan auxotrophs in nitrogen-excess condition and those of GS and GOGAT in nitrogen limited condition were increased compared with opposite condition. The tryptophan and histidine auxotrophs showed higher level of glutamate and glutamine than parental strains and other mutants. it is assumed that the higher levels of ${\alpha-KGDH}$ and GDH from mutants in nitrogen-excess condition promoted the accumulation of glutamate and glutamine in fermentation broth. The inhibition of GS activities by ADP suggested that GS is regulated by energy charge in C. glutamicum. The results with histidine, tryptophan, glycine, alanine, serine and GMP implied that a system of feedback inhibition were effective. The GDH, GS and GOGAT biosynthesis in culture broth was markedly repressed by the nature and kinds of available nitrogen sources such as tryptophan, proline, glycine, alanine, serine and tyrosine.

• Korean Journal of Microbiology
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• v.26 no.2
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• pp.93-100
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• 1988

In order to understand the regulation of glutamate dehydrogenase(GDH) synthesis in Brevibacterium flavum, we have isolated a mutant lacking NADP-linked GDH activity by ethlmethane sulfonate treatment. The $gdh^-$ mutant was grown on the minimal plate with 1mM ammonium chloride and not that with 300mM ammonium chloride. The cell-free extracts from $gdh^-$ mutant and prototroph were also examined with glutamine synthetase(GS) and glutamate synthase (GOGAT) production by niteogen sources. The growth of $gdh^-$ mutant in presence of 20mM ammonium chloride means that GOGAT synthesis is sufficient to allow growth in this condition. GS production of $gdh^-$ mutant as well as parental strain was induced by 1mM urea and ammonium tartrate, but it was repressed by higher concentration of ammonia, and also induced by 20mM to 50mM glutamate as a substrate. It was special attention that GOGAT synthesis from $gdh^-$ strain was more repressed by higher concentration of ammonia than prototroph as described in E. coli system.

• Journal of Microbiology
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• v.44 no.3
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• pp.301-310
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• 2006

Two pathways of ammonium assimilation and glutamate biosynthesis have been identified in microorganisms. One pathway involves the NADP-linked glutamate dehydrogenase, which catalyzes the amination of 2-oxoglutarate to form glutamate. An alternative pathway involves the combined activities of glutamine synthetase, which aminates glutamate to form glutamine, and glutamate synthase, which transfers the amide group of glutamine to 2-oxoglutarate to yield two molecules of glutamate. We have cloned the large subunit of the glutamate synthase (GOGAT) from Salmonella typhimurium by screening the expression of GOGAT and complementing the gene in E. coli GOGAT large subunit-deficient mutants. Three positive clones (named pUC19C12, pUC19C13 and pUC19C15) contained identical Sau3AI fragments, as determined by restriction mapping and Southern hybridization, and expressed GOGAT efficiently and constitutively using its own promoter in the heterologous host. The coding region expressed in Escherichia coli was about 170 kDa on SDS-PAGE. This gene spans 4,732 bases, contains an open reading frame of 4,458 nucleotides, and encodes a mature protein of 1,486 amino acid residues (Mr =166,208). The EMN-binding domain of GOGAT contains 12 glycine residues, and the 3Fe-4S cluster has 3 cysteine residues. The comparison of the translated amino acid sequence of the Salmonella GOGAT with sequences from other bacteria such as Escherichia coli, Salmonella enterica, Shigella flexneri, Yersinia pestis, Vibrio vulnificus and Pseudomonas aeruginosa shows sequence identity between 87 and 95%.

• The Journal of Natural Sciences
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• v.14 no.1
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• pp.7-24
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• 2004

We found new type of thiol peroxidase, fused with GRX.(TPx-GRX) The TPx-GRX exists in pathogenic bacteria including -. This protein was homogeneously purified from the E.coli recombinant overexpressing TPx-GRX. In the presence of a thiol-containing electron donor such as DTT, the purified TPx-GRX has potent the antioxidant to prevent the inactivation of GS by the MCO system, which is comprised of DTT, $Fe^{3+}$, and $O^2$. The antioxidant activity is much higher that other thiol peroxidase. The investigate the peroxidase activity of TPx-GRX, we directly measured the peroxidase activity of TPx-GRX toward peroxides in terms of the removal of peroxides in the presence of GSH. This result demonstrates that the peroxidase activity of TPx-GRX. These taken together results suggest that TPx-GRX is a new member of thiol peroxidase. These observations also suggest that in the pathogenic bacteria, TPx-GRX plays an important antioxidative role as a multiple array defence mechanism against oxidative stress.