• Applied Biological Chemistry
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• v.40 no.6
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• pp.478-483
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• 1997

${\gamma}-Glutamylcysteine$ synthetase was purified from E. coli K-12 strain and its properties related to the in vitro synthesis of glutathione by enzymatic method were investigated. The activity of purified ${\gamma}-glutamylcysteine$ synthetase was increased with increasing concentration of L-glutamate up to 60 mM, while it was decreased by about 50% and 40% under 60 mM of L-cysteine and 45 mM of glycine, respectively. The enzyme activity was reduced not only by ADP, one of the reaction products, but also by the reduced form of glutathione. Therefore, because the reduced glutathione as well as glycine which is the substrate for glutathione synthetase inhibit the activity of ${\gamma}-glutamylcysteine$ synthetase, it is recommended to design a bioreactor system with two separate reactions for glutathione synthesis : one with ${\gamma}-glutamylcysteine$ synthetase reaction and the other glutathione synthetase reaction. In addition since ADP, resulted from these reactions, reduces the activity of ${\gamma}-glutamylcysteine$ synthetase, it is necessary to introduce an ATP regeneration system for glutathione synthesis.

• Korean Journal of Microbiology
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• v.29 no.5
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• pp.278-283
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• 1991

Two amino acid residues ($Ala^{494}$ and $Ser^{495}$ of E. coli .gamma.-glutamylcysteine synthetase have been investigated whether they are the site of feedback inhibition by site specific mutagenesis. Single substitution of $serine^{495}$ (S495F), and double substitutions of alanine$^{494}$ and $serine^{495}$ (A494G-S495F) resulted in the inactivation of the .gamma.-glutamylcysteine synthetase activity. Substitution of $alanine^{494}$ with $glycine^{494}$ resulted in a higher level of feedback inhibition. These results suggest that $serine^{495}$ in .gamma.-glutamylcysteine synthetase is required for its catalytic acitvity and $alanine^{494}$ is presumably related to the feeback inhibition site.

• BMB Reports
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• v.31 no.3
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• pp.254-257
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• 1998

The gshI gene from the Escherichia coli K-12 strain codes for ${\gamma}-glutamylcysteine$ synthetase which mediates the rate-limiting step of glutathione biosynthesis. The isolated gshI gene from E. coli K-12 has an unusual translation initiation codon, UUG. The 494th amino acid is Ala rather than Gly which was found in a mutant strain E. coli B. In order to improve the translational rate of the gshI gene of E. coli K-12, the initiation codon, UUG, was changed to the usual AUG codon by the site-specific mutagenesis. This change has resulted in a 53% increase of ${\gamma}-glutamylcysteine$ synthetase activity. The enzyme activity was also improved by replacing $Ala^{494}$ with Val (A494V) or Leu (A494L). The replacement of $Ser^{495}$ with Thr (S495T) also resulted in a 62% increase of the enzyme activity. Therefore, the specific activity of ${\gamma}-glutamylcysteine$ synthetase was increased with the increasing chain length of the aliphathic amino acid at the site of the 494th amino acid (Ala<$Val{\leq}Leu$).

• Journal of Life Science
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• v.7 no.4
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• pp.253-261
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• 1997

In order to enhance glutathione production, various recombinant plasmids containing gshI and/or gshII genes isolated from E. coli K-12 were constructed and introduced into E. coli. Some plasmids contained one to three copies of gshI genes in pBR325 and others contained both gshI and genes for glutathione biosynthesis. $\gamma$-Glutamylcysteine synthetase activities of E, coli strains amplified tandem repeated gshI genes were dependent on the number of inserted gshI genes. The glutathione productivity of E. coli strains harboring various plasmids was investigated using an E. coli acetate kinase reaction as an ATP regenerating system. The glutathione productivity of E. coli strains harboring tandem repeated gshI genes was increased in proportion to the number of inserted gshI genes. By the introduction of gshII gene, the glutathione productivity of the E. coli was increased by two-fold compared with E. coli strain amplified gshI gene only. The enzymatic production of glytathione in E. coli was mainly affected by the increase of $\gamma$-glutamylcysteine synthetase activity. The highest glutathione productivity was obtained in E. coli strains harboring pGH-501 plasmid containing two copies of gshI and copy of gshII genes in pUC8 vector.

• KSBB Journal
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• v.10 no.3
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• pp.249-256
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• 1995

${\gamma}$-Glutamylcysteine production by the immobilized microbial system of recombinant Escherichia coli and yeast was investigated. ${\gamma}$-Glutamylcysteine was synthesized from L-glutamic acid and L-cysteine in the presence of ATP by the reaction catalyzed by ${\gamma}$-glutamylcysteine synthetase. An immobilized microbial cell system was developed for the efficient ${\gamma}$-glutamylcysteine production. Recombinant Escherichia coli and yeast were immobilized by alginate. Production of ${\gamma}$-glutamylcysteine was better with the recombinant Escherichia coli for both the synthesis of ${\gamma}$-glutamylcysteine and the ATP regeneration than the mixed system of recombinant Escherichia coli and yeast. The proper radio of recombinant Escherichia coli to yeast was experimentary observed to be 1:4 in the mixed system. Although the immobi1ized system had the slower reaction rate, its reaction stability was increased by 10%.

• Bulletin of Food Technology
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• v.17 no.4
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• pp.98-106
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• 2004

Two mutant enzymes of $\gamma$-glutamylcysteine synthetase ($\gamma$-GCS) which catalyzed the synthesis of $\gamma$-glutamylcysteine from L-glutamic acid and L-cysteine in the presence of ATP, were prepared bypoint mutation of $\gamma$-GCS gene with site-directed mutagensis in E. coli. Conformational structuresand catalytic reaction kinetics of mutant enzymes were compared with wild type $\gamma$-GCS afterpurification. The S495F mutant enzyme (serine at 495 residue was substituted with phenylalanine),which had no catalytic activity for $\gamma$-glutamylcysteine synthesis, rarely folded even in neutral pH.However, the mutant A494V (alanine of 494 residue was replaced by valnine) which showed 50 %increase of activity, had a high folding structure. The folding structure of A494V also more stable athigh temperature and extreme pH compared to wild type and S495F. Reaction kinetics of wild typeand A494V were also investigated, Km value of A494V was smaller than that of wild type, while itshowed a little difference at Vmax values. This result evolved that alanine at 494 may be involved inbinding site of substrate rather than catalytic site. In addition, change of catalytic activity by onepoint mutation was highly correlated with the folding structure of enzyme.

• Microbiology and Biotechnology Letters
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• v.26 no.3
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• pp.213-220
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• 1998

An ATP regeneration system was used for the production of glutathione which was synthesized by a sequential action of ${\gamma}$-glutamyl-cysteine synthetase and glutathione synthetase. The synthetases above were produced in the recombinant E. coli (TG1/pDG7) with the highest specific production yield of 31 mg glutathione/g wet cell. Bakers yeast was considered to have economically a better ATP regeneration system although the glutathione production yield was lower than that of acetate kinase. It was also observed that the ATP regeneration system of bakers yeast was superior to that of Saccharomyces cerevisiae ATCC24858. The yield of glutathione production with bakers yeast was 36% with the ATP concentration of 5 mM. To avoid the cysteine limitation during the early phase of glutatione production, an extra cysteine was added at 2 hours after reaction and the production yield increased 1.91 times. The effectiveness of bakers yeast as an ATP regeneration system was proved by several sets of extra feeding experiments. The product inhibition by glutathione above 14 mM was also observed.

• Korean Journal of Crystallography
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• v.4 no.2
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• pp.100-104
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• 1993

Reduced glutathione (GSH) plays a vital role in the metabolism of all cells. Glutathions, a tripeptide cowfosed of glutamic acid, cysteane, and gtycina is synthesized by two synthesized reutions. The first is catalyzed by Y-glutamylcysteine synthetase (GSH-I) and the second by glutathione synthetase (GSH-ll). The glutathione biosynthetic pathway of E. coziis mainly controlled by nonallosteric feedback inhibition of GHS-I by GSH. Determination of the three-dimensional structure of GSH-I by X-ray crystallography is necessary in order to understand the structure-function relationship at the molecular level. As the (irst step toward its structure determination, crystallization of 5. coli V-glutamylcystfine synthetase (GSH-I) has been achived using the hanging drop vapor diffusion method and capillaw method. Crystals of GSH-I have been grown from ammonium sulfate solution. The crystals grew at room temperature within 10 days to dimensions of 0.2 m x 0.2 m x 0.2 ml by hanging drop vapor diffusion method and diffracted to about 4 A resolution using synchrotron X-rays. Another crystal, grown by the capillary method to dimensions of 0.25 mm x 0.25 mm x 0.3 mm within 40 days, diffracted to about 4 A resolution using X-rays from a rotating anode.

• Microbiology and Biotechnology Letters
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• v.19 no.6
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• pp.575-582
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• 1991

To increase the production of glutathione by the expression of recombinant gsh plasmids, two genes responsible for the biosynthesis of glutathione were isolated and cloned. To clone a gshI gene, the GS903 mutant strain, which is deficient in $\gamma$-glutamylcysteine synthetase activity, has been raised. A gshI gene was cloned using pBR322 plasmid as a 3.6 Kb PstI DNA fragment isolated from E. coli K-12 chromosomal DNA. Also a gshIl gene was cloned using pUC13 plasmid as a 2.2 Kb PstI-BamHI DNA fragment. To study the effects of plasmid copy number and passenger DNA size on the expression levels of the gsh genes, various recombinant plasmids containing different sets of genes were constructed. The expression levels of the gsh genes were increased approximately twice higher in pUC series plasmids than that in pBR322 plasmid. But the sizes of the passenger DNA containing the gsh genes in the vector plasmid did not affect on the expression levels of the gsh genes.