• 한국미생물·생명공학회지
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• 제17권4호
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• pp.379-384
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• 1989

E. coli K-12 pgln 6가 지니는 glutamine synthetase와 반응계에 ATP 공급을 위하여 빵효모를 동시에 이용하여 glutamate로부터 glutamine의 생산이 수행되었다. Glutamine synthetase의 효소원으로서 E. coli K-12 pgln 6 생균체를 사용하였을 때, 18시간 배양 후에 11.8g/ι의 glutamine이 생산되었다(기질인 glutamate 기준으로 60%의 수율). 부분정제된 glutamine synthetase를 이용하여, 5시간 후에 19.8g/ι의 glutamine이 생산되었다. 이 양은 glutamate 기준으로 90% 수율에 해당되는 것이다.

• 한국식품영양학회지
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• 제6권3호
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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.

• 한국미생물·생명공학회지
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• 제19권3호
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• pp.259-264
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• 1991

K.pneumoniae의 nif 유전자 발현에 미치는 glnA 산물 (GS)의 영향을 살펴보기 앞서 K.pneumoniae의 GS를 분리.정제하여 그 성질을 조사하였다. K.pneumoniae의 GS는 분자량 약 600,000의 12개 동일 subunit으로 구성되어져 이었으며 최적 반응 pH 및 온도는 각기 pH 7.0, $37^{\circ}C$ 였고 pH 5-8사이에서 활성의 변화가 크지 않았으며 $57^{\circ}C$에서 10분간 열처리 할 때 50의 활성 감소가 나타났다. 또 암모니아 농도의 변화에 따라 adenylylation-deadenylylation 기작에 의한 활성조절기작이 E.coli GS와 동일 했으며 anti-E.coli GS 항체를 이용한 immunodiffusion에서도 K.pneumoniae GS가 E.coli GS와 혈청적으로 매우 유사함이 드러났다.

• Journal of Microbiology
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• 제42권2호
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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.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2005년도 International Meeting of the Microbiological Society of Korea
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• pp.218.1-218
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• 2005

• BMB Reports
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• 제33권6호
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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.

• 한국미생물·생명공학회지
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• 제14권5호
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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.

• 미생물학회지
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• 제26권2호
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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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• 제44권3호
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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%.

• 자연과학논문집
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• 제14권1호
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• pp.7-24
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• 2004

Thiol peroxidase인 E.coli AhpC의 아미노산 서열을 database를 이용해 분석하여, TPx와 상동성이 있는 새로운 형태의 Thiol peroxidase를 찾아내었다. 그 중 병원성을 갖는 박테리아인 Haemophilus Influenzae에서 존재하는 TPx와 유사하고, GRX와 함께 fusion 되어있는 새로운 형태의 단백질의 유전자를 클로닝하여 E.coli에서 과발현시켜 분리정제 하였다. 정제된 TPx-GRX는 환원제로 thiol 성분을 갖는 MCO system(Fe, DTT, Oxygen)에 의하여 Glutamine Synthetase(GS)의 불활성화를 방어하는 티올 특이적 향산화활성을 갖고, peroxides를 제거하는 peroxidase 활성을 갖는 것을 밝혔다. 이 결과들로부터 TPx-GRX는 새로운 형태의 Thiol perosidase임을 알 수 있었다. 더 나아가서 이 결과들은 TPx-GRX가 병원성 박테리아에서 oxidative stress를 막는 생리적으로 중요한 역할을 할 것이라는 것을 시사한다.