6.3.2.3: glutathione synthase
This is an abbreviated version!
For detailed information about glutathione synthase, go to the full flat file.
Word Map on EC 6.3.2.3
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6.3.2.3
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gamma-glutamylcysteine
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dismutase
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gamma-glutamyl
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catalase
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s-transferase
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sulfoximine
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cadmium
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5-oxoprolinuria
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buthionine
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5-oxoproline
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hemolytic
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acidosis
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phytochelatins
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transpeptidase
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gamma-gcs
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school
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school-based
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pyroglutamic
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glutathione-related
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atp-grasp
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bullied
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l-buthionine-s,r-sulfoximine
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juncea
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6.3.2.2
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glutathione-deficient
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homoglutathione
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gsh-dependent
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medicine
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biotechnology
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synthesis
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analysis
- 6.3.2.3
- gamma-glutamylcysteine
- dismutase
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gamma-glutamyl
- catalase
- s-transferase
- sulfoximine
- cadmium
-
5-oxoprolinuria
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buthionine
- 5-oxoproline
-
hemolytic
- acidosis
- phytochelatins
- transpeptidase
- gamma-gcs
-
school
-
school-based
-
pyroglutamic
-
glutathione-related
-
atp-grasp
-
bullied
-
l-buthionine-s,r-sulfoximine
- juncea
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6.3.2.2
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glutathione-deficient
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homoglutathione
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gsh-dependent
- medicine
- biotechnology
- synthesis
- analysis
Reaction
Synonyms
Asuc_1947, bifunctional glutathione synthetase, bifunctional GSH synthetase, bifunctional L-glutathione synthetase, gamma -glutamate-cysteine ligase-glutathione synthetase, gamma-GCS, gamma-GCS-GS, gamma-glutamate-cysteine ligase/glutathione synthetase, gamma-glutamylcysteine synthetase-glutathione synthetase, GCL, GCSGS, ghF, glutamate cysteine ligase, glutathione biosynthesis bifunctional protein GshAB, Glutathione synthase, Glutathione synthetase, Glutathione synthetase (tripeptide), GS, GSH synthase, GSH synthetase, GSH-S, GSH2, gshAB, GSHase, gshB, GshF, GshFAp, GshFAs, GSHII, GSHS, GSHS1, GSS, hGS, L-glutathione synthetase, More, Phytochelatin synthetase, StGCL-GS, Synthetase, glutathione, TAGS1, TaGS2, TbGS, ZmGS
ECTree
6.3.2.3 glutathione synthaseAdvanced search results
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results (Engineering
Engineering on EC 6.3.2.3 - glutathione synthase
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PROTEIN VARIANTS 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
K367N/P368S
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mutant Lys367/Pro368 to Asn/Ser and mutant Gly374 to Val are inactive
E429A
E429Q
G240V
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mutant enzymes replaced with Val at the basal position of the flexible loop (P227V, G240V, and P227V/G240V) are identical with wild-type enzyme in their crystal structures, except the loop region
P227V
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mutant enzymes replaced with Val at the basal position of the flexible loop (P227V, G240V, and P227V/G240V) are identical with wild-type enzyme in their crystal structures, except the loop region
P227V/G240V
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mutant enzymes replaced with Val at the basal position of the flexible loop (P227V, G240V, and P227V/G240V) are identical with wild-type enzyme in their crystal structures, except the loop region
C294A
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mutant enzymes Cys294Ala and Cys409Ala retain significant residual activity. Substantial decreases in activity are detected with mutant Cys522Ala and Cys-free mutant Cys294/Cys409/Cys422 to Ala294/Ala409/Ala422
C409A
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mutant enzymes Cys294Ala and Cys409Ala retain significant residual activity. Substantial decreases in activity are detected with mutant Cys522Ala and Cys-free mutant Cys294/Cys409/Cys422 to Ala294/Ala409/Ala422
C522A
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mutant enzymes Cys294Ala and Cys409Ala retain significant residual activity. Substantial decreases in activity are detected with mutant Cys522Ala and Cys-free mutant Cys294/Cys409/Cys422 to Ala294/Ala409/Ala422
D219A
naturally occurring missense mutation expressed using a His-tagged, Escherichia coli-based expression system, decreases Vmax to 4% of the wild-type activity
D219G
naturally occurring missense mutation expressed using a His-tagged, Escherichia coli-based expression system, decreases Vmax to 27% of the wild-type activity. Negative cooperativity for L-gamma-glutamyl-L-alpha-aminobutyric acid is changed to positive
D24A
site-directed mutagenesis, the mutant shows a slight increase in catalytic efficiency compared to wild-type enzyme
D458A
site-directed mutagenesis, the mutant shows 10% activity compared to the wild-type enzyme
D458N
site-directed mutagenesis, the mutant shows 15% activity compared to the wild-type enzyme
D458R
site-directed mutagenesis, the mutant shows 7% activity compared to the wild-type enzyme
E144A
site-directed mutagenesis, 0.05% activity compared to the wild-type enzyme, unaltered tertiary structure
E144K
site-directed mutagenesis, inactive mutant, unaltered tertiary structure
G369V
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mutation in G-loop glycine triad, about 0.7% of wild-type activity. Mutation decreases ligand binding and prevent active site closure and protection
G370V
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mutation in G-loop glycine triad, about 0.3% of wild-type activity. Mutation decreases ligand binding and prevent active site closure and protection
K305A
site-directed mutagenesis, 6.5% activity compared to the wild-type enzyme, 7fold increased Km for glycine, loss of negative cooperativity, 105fold increased Km for ATP, unaltered tertiary structure
K305E
site-directed mutagenesis, 5% activity compared to the wild-type enzyme, loss of negative cooperativity, 40fold increased Km for ATP, unaltered tertiary structure
K364A
site-directed mutagenesis, 0.1% activity compared to the wild-type enzyme, unaltered tertiary structure
K364E
site-directed mutagenesis, 0.2% activity compared to the wild-type enzyme, unaltered tertiary structure
L188P
naturally occurring missense mutation expressed using a His-tagged, Escherichia coli-based expression system, decreases Vmax to 9% of the wild-type activity
N146A
site-directed mutagenesis, 0.1% activity compared to the wild-type enzyme, unaltered tertiary structure
N146D
site-directed mutagenesis, 0.05% activity compared to the wild-type enzyme, unaltered tertiary structure
N146K
site-directed mutagenesis, inactive mutant, unaltered tertiary structure
R221A
site-directed mutagenesis, the mutant shows a slight increase in catalytic efficiency compared to wild-type enzyme, the R221A mutation also has a large impact on the intrachain bonding structure
R283C
naturally occurring missense mutation expressed using a His-tagged, Escherichia coli-based expression system, decreases Vmax to 13% of the wild-type activity
S42A
site-directed mutagenesis, the mutant shows a slight increase in catalytic efficiency compared to wild-type enzyme
V44A/V45A
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decrease in melting temperature, initial activity similar to wild-type
Y208C
naturally occurring missense mutation expressed using a His-tagged, Escherichia coli-based expression system, decreases Vmax to 2% of the wild-type activity
Y270H
naturally occurring missense mutation expressed using a His-tagged, Escherichia coli-based expression system, decreases Vmax to 6% of the wild-type activity
A485L/T486P
site-directed mutagenesis, the mutant shows 70% reduced activity compared to the wild-type activity, and a shift of substrate specificity with increased affinity of GSH2 for Ser as a substrate, while affinity to Gly is preserved. This provides another biosynthetic pathway for hydroxymethyl glutathione, which is known to be synthesized from glutathione and Ser in a reaction catalysed by carboxypeptidase Y
I471M/C472M/A485L/T486P
site-directed mutagenesis, the mutant shows 38% reduced activity compared to the wild-type activity, and a shift of substrate specificity with 1.2fold increased affinity of GSH2 for beta-Ala and lowered affinity for Gly, which is a characteristic of the enzyme homoglutathione synthetase found in plants, EC 6.3.2.23
I471M/C472V
site-directed mutagenesis, the mutant shows showed much lower affinity towards Gly and 78% reduced activity compared to the wild-type activity, but no other differences
additional information
additional information
construction of gsh2 insertion mutants, the mutants have a seedling lethal phenotype in contrast to the embryo lethal phenotype of gamma-glutamate cysteine ligase, gsh1, EC 6.3.2.2, null mutants, overview. The mutants show hyperaccumulation of gamma-glutamylcysteine to levels 5000-fold that in the wild-type and 200fold wild-type levels of GSH, phenotype, overview. Complementation of gsh2 mutants with the cytosol-specific GSH2 give rise to phenotypically wild-type transgenic plants
additional information
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construction of transgenic plants by overexpression of the Escherichia coli enzyme, encoded by gene gshII, in the cytosol of transgenic plants, the plants show 3fold increased ability in the shoot compared to the wild-type to accumulate and tolerate heavy metals, e.g. cadmium
additional information
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creation of targeted X chromosomal deficiency lines. Relative arsenite sensitivity arises when the dose of the glutathione synthetase gene expression is reduced by half. Knockdown of GS expression by RNAi in cultured S2 cells leads to enhanced arsenite sensitivity, while GS RNAi applied to intact organisms dramatically reduces the concentration of food-borne arsenite compatible with successful growth and development, phenotypes, overview
additional information
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deletion mutant of the loop and mutant with a nicked multifunctional loop. Cleavage of the loop results in a drastic increase in activity, which is similar to the results for the loop deletion. High concentrations of ATP inhibit the wild-type enzyme, while both nicked and loopless enzyme are not inhibited
additional information
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the mutant enzymes Arg86 and Asn283 are altered in their kinetic parameters, especially the Michaelis constant of gamma-Glu-Cys
additional information
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the crystal structure of the loopless mutant, in which the loop is replaced by 3 Gly residues, is identical with that of the wild-type enzyme. Replacement of the loop increases the Km-values, especially for glycine, and a 930fold decrease in turnover number. The loopless mutant shows gamma-Glu-L-Cys-dependent ATP hydrolase activity to almost the same extent as its glutathione synthetase activity
additional information
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construction of transgenic plants by overexpression of the enzyme in the cytosol of Brassica juncea, i.e. Indian mustard, transgenic plants show 3fold increased ability in the shoot compared to the wild-type to accumulate and tolerate heavy metals, e.g. cadmium, for use in detoxification
additional information
Escherichia coli B / ATCC 11303
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the mutant enzymes Arg86 and Asn283 are altered in their kinetic parameters, especially the Michaelis constant of gamma-Glu-Cys
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additional information
Escherichia coli B / ATCC 11303
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the crystal structure of the loopless mutant, in which the loop is replaced by 3 Gly residues, is identical with that of the wild-type enzyme. Replacement of the loop increases the Km-values, especially for glycine, and a 930fold decrease in turnover number. The loopless mutant shows gamma-Glu-L-Cys-dependent ATP hydrolase activity to almost the same extent as its glutathione synthetase activity
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additional information
all Asp458 mutants display a change in cooperativity from negative cooperativity to non-cooperative. All mutants show similar stability as compared to wild-type enzyme, differential scanning calorimetry
additional information
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all Asp458 mutants display a change in cooperativity from negative cooperativity to non-cooperative. All mutants show similar stability as compared to wild-type enzyme, differential scanning calorimetry
additional information
structure and stability comparisons of wild-type enzyme and mutant enzymes, overview
additional information
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structure and stability comparisons of wild-type enzyme and mutant enzymes, overview
additional information
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ApoE-deficient mice show reduced enzyme expression and activity
additional information
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decreased hepatic GSH levels occur, which correlated with a fall in GS activity, in Tat transgenic mice. GCLC and GS are coordinately regulated but GCLM is unchanged in liver-specific retinoid X receptor alpha knockout mice
additional information
deletion of genes GSH1 and GSH2 (encoding glutathione synthetase) using the CRISPR-Cas9 nuclease system
additional information
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deletion of genes GSH1 and GSH2 (encoding glutathione synthetase) using the CRISPR-Cas9 nuclease system
additional information
generation of an GSH2 enzyme deletion mutant using the CRISPR-Cas9 nuclease system
additional information
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generation of an GSH2 enzyme deletion mutant using the CRISPR-Cas9 nuclease system
additional information
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deletion of genes GSH1 and GSH2 (encoding glutathione synthetase) using the CRISPR-Cas9 nuclease system
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additional information
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generation of an GSH2 enzyme deletion mutant using the CRISPR-Cas9 nuclease system
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additional information
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elimination of the protease cleavage site in the 56 kDa subunit by site-directed mutagenesis results in expression of a stable and functional enzyme
additional information
enzymatic production of glutathione by recombinant cell-free bifunctional gamma-glutamylcysteine synthetase/glutathione synthetase (gamma-GCS-GS or GshF) coupled with in vitro acetate kinase-based ATP generation in Escherichia coli strain Rosetta (DE3), method optimization. The recombinant enzyme comprises both the activities of gamma-glutamylcysteine synthetase (gamma-GCS or GSHI, EC 6.3.2.2) and GSH synthetase (GS or GSHII, EC 6.3.2.3). The gshF from Streptomyces thermophilus shows poor expression levels compared to gshF from Streptomyces agalactiae. GSH production resulting from a combination of recombinant Escherichia coli BL21(DE3) expressing gshF from Streptomyces agalactiae with recombinant Escherichia coli BL21(DE3) expressing acetate kinase from Lactobacillus sanfranciscensis is 2.5 times higher than that of gshF from Streptomyces thermophilus
additional information
Streptococcus agalactiae serogroup V ATCC BAA-611 / 2603 V/R
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enzymatic production of glutathione by recombinant cell-free bifunctional gamma-glutamylcysteine synthetase/glutathione synthetase (gamma-GCS-GS or GshF) coupled with in vitro acetate kinase-based ATP generation in Escherichia coli strain Rosetta (DE3), method optimization. The recombinant enzyme comprises both the activities of gamma-glutamylcysteine synthetase (gamma-GCS or GSHI, EC 6.3.2.2) and GSH synthetase (GS or GSHII, EC 6.3.2.3). The gshF from Streptomyces thermophilus shows poor expression levels compared to gshF from Streptomyces agalactiae. GSH production resulting from a combination of recombinant Escherichia coli BL21(DE3) expressing gshF from Streptomyces agalactiae with recombinant Escherichia coli BL21(DE3) expressing acetate kinase from Lactobacillus sanfranciscensis is 2.5 times higher than that of gshF from Streptomyces thermophilus
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additional information
a recombinant Escherichia coli strain expressing gshF encoding the bifunctional glutathione synthetase of Streptococcus thermophilus is constructed for efficient GSH production. The cultivation process is optimized by controlling dissolved oxygen, amino acid addition, and glucose feeding. 36.8 mM (11.3 g/l) GSH are formed at a productivity of 2.06 mM/h when the amino acid precursors (75 mM each) are added and glucose is supplied as the sole carbon and energy source. The fed-batch fermentations are performed in a 5-l bioreactor containing 2.5 l medium for fed-batch culture inoculated with 140 ml secondary seed culture. The temperature and pH are controlled at 37°C and 7.0, respectively. The GSH production is extremely limited by the precursors of GSH, and the GSH productivity is only 0.18 mM/h. Method evaluation, overview
additional information
enzymatic production of glutathione by recombinant cell-free bifunctional gamma-glutamylcysteine synthetase/glutathione synthetase (gamma-GCS-GS or GshF) coupled with in vitro acetate kinase-based ATP generation in Escherichia coli strain Rosetta (DE3), method optimization. The recombinant enzyme comprises both the activities of gamma-glutamylcysteine synthetase (gamma-GCS or GSHI, EC 6.3.2.2) and GSH synthetase (GS or GSHII, EC 6.3.2.3). The gshF from Streptomyces thermophilus shows poor expression levels compared to gshF from Streptomyces agalactiae. GSH production resulting from a combination of recombinant Escherichia coli BL21(DE3) expressing gshF from Streptomyces agalactiae with recombinant Escherichia coli BL21(DE3) expressing acetate kinase from Lactobacillus sanfranciscensis is 2.5 times higher than that of gshF from Streptomyces thermophilus with acetate kinase from Escherichia coli
additional information
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enzymatic production of glutathione by recombinant cell-free bifunctional gamma-glutamylcysteine synthetase/glutathione synthetase (gamma-GCS-GS or GshF) coupled with in vitro acetate kinase-based ATP generation in Escherichia coli strain Rosetta (DE3), method optimization. The recombinant enzyme comprises both the activities of gamma-glutamylcysteine synthetase (gamma-GCS or GSHI, EC 6.3.2.2) and GSH synthetase (GS or GSHII, EC 6.3.2.3). The gshF from Streptomyces thermophilus shows poor expression levels compared to gshF from Streptomyces agalactiae. GSH production resulting from a combination of recombinant Escherichia coli BL21(DE3) expressing gshF from Streptomyces agalactiae with recombinant Escherichia coli BL21(DE3) expressing acetate kinase from Lactobacillus sanfranciscensis is 2.5 times higher than that of gshF from Streptomyces thermophilus with acetate kinase from Escherichia coli
additional information
to convert feather hydrolysates into GSH with high values, the bifunctional glutathione synthetase, that comprises the activities of EC 6.3.2.2 and EC 6.3.2.3, encoded by gcsgs from Streptococcus thermophilus is surface displayed on Saccharomyces cerevisiae strain EBY100, the potential in glutathione (GSH) production from feather hydrolysates is analyzed. The surface-displayed GCSGS can be used to convert feather hydrolysates into GSH. 10 g/l of feather are converted into 321.8 mg/l GSH by the Trichoderma atroviride F6, with feather degrading ability, and surface-displayed GCSGS. Method optimization, overview
additional information
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enzymatic production of glutathione by recombinant cell-free bifunctional gamma-glutamylcysteine synthetase/glutathione synthetase (gamma-GCS-GS or GshF) coupled with in vitro acetate kinase-based ATP generation in Escherichia coli strain Rosetta (DE3), method optimization. The recombinant enzyme comprises both the activities of gamma-glutamylcysteine synthetase (gamma-GCS or GSHI, EC 6.3.2.2) and GSH synthetase (GS or GSHII, EC 6.3.2.3). The gshF from Streptomyces thermophilus shows poor expression levels compared to gshF from Streptomyces agalactiae. GSH production resulting from a combination of recombinant Escherichia coli BL21(DE3) expressing gshF from Streptomyces agalactiae with recombinant Escherichia coli BL21(DE3) expressing acetate kinase from Lactobacillus sanfranciscensis is 2.5 times higher than that of gshF from Streptomyces thermophilus with acetate kinase from Escherichia coli
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additional information
-
a recombinant Escherichia coli strain expressing gshF encoding the bifunctional glutathione synthetase of Streptococcus thermophilus is constructed for efficient GSH production. The cultivation process is optimized by controlling dissolved oxygen, amino acid addition, and glucose feeding. 36.8 mM (11.3 g/l) GSH are formed at a productivity of 2.06 mM/h when the amino acid precursors (75 mM each) are added and glucose is supplied as the sole carbon and energy source. The fed-batch fermentations are performed in a 5-l bioreactor containing 2.5 l medium for fed-batch culture inoculated with 140 ml secondary seed culture. The temperature and pH are controlled at 37°C and 7.0, respectively. The GSH production is extremely limited by the precursors of GSH, and the GSH productivity is only 0.18 mM/h. Method evaluation, overview
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