6.3.2.2: glutamate-cysteine ligase
This is an abbreviated version!
For detailed information about glutamate-cysteine ligase, go to the full flat file.
Word Map on EC 6.3.2.2
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6.3.2.2
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ganglion
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retinal
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nerve
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heme
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plexiform
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fiber
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sulfoximine
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dismutase
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coherence
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tomography
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buthionine
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s-transferase
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oxygenase-1
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macular
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erythroid
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quinone
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neuroprotective
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2-related
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amacrine
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cytoprotective
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gssg
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nadph:quinone
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glaucoma
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nf-e2-related
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acuity
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transpeptidase
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sd-oct
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peripapillary
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intravitreal
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nrf2-mediated
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nrf2-dependent
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dentate
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spectral-domain
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nrf2-are
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factor-erythroid
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subgranular
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glutathione-related
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gsh-dependent
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phytochelatins
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l-buthionine-s,r-sulfoximine
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tert-butylhydroquinone
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oxidoreductase-1
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ech-associated
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kelch-like
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nrf2-regulated
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fovea
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biotechnology
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medicine
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perimetry
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synthesis
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gsh-depleting
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agriculture
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drug development
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etdrs
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pharmacology
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spectralis
- 6.3.2.2
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ganglion
- retinal
- nerve
- heme
-
plexiform
- fiber
- sulfoximine
- dismutase
-
coherence
-
tomography
-
buthionine
- s-transferase
- oxygenase-1
-
macular
-
erythroid
- quinone
-
neuroprotective
-
2-related
-
amacrine
-
cytoprotective
- gssg
-
nadph:quinone
- glaucoma
-
nf-e2-related
-
acuity
- transpeptidase
-
sd-oct
-
peripapillary
-
intravitreal
-
nrf2-mediated
-
nrf2-dependent
-
dentate
-
spectral-domain
-
nrf2-are
-
factor-erythroid
-
subgranular
-
glutathione-related
-
gsh-dependent
- phytochelatins
-
l-buthionine-s,r-sulfoximine
- tert-butylhydroquinone
-
oxidoreductase-1
-
ech-associated
-
kelch-like
-
nrf2-regulated
-
fovea
- biotechnology
- medicine
-
perimetry
- synthesis
-
gsh-depleting
- agriculture
- drug development
-
etdrs
- pharmacology
-
spectralis
Reaction
Synonyms
Ace-GCL, Asuc_1947, AtGCL, bifunctional glutathione synthetase, bifunctional GSH synthetase, bifunctional L-glutathione synthetase, gamma -GCS, gamma-ECL, Gamma-ECS, gamma-GC, gamma-GCS, gamma-GCS-GS, gamma-glutamate-cysteine ligase-glutathione synthetase, gamma-glutamate-cysteine ligase/glutathione synthetase, gamma-glutaminylcysteine synthetase, gamma-glutamycysteine synthetase, gamma-glutamyl-cysteine ligase, gamma-Glutamyl-L-cysteine synthetase, gamma-glutamylcysteine ligase, gamma-glutamylcysteine synthase, gamma-Glutamylcysteine synthetase, gamma-glutamylcysteine synthetase-glutathione synthetase, gamma-Glutamylcysteinyl-synthetase, gammaGCS, GCL, GCLC, Gclc-X2, GCLM, GCS, GCSGS, ghF, GLCL, GLCLC, GLCLR, glutamate cysteine ligase, glutamate cysteine ligase gene, glutamate-cysteine ligase, glutamate-cysteine-ligase, glutamate–cysteine ligase, glutathione biosynthesis bifunctional protein GshAB, GSH1, GshA, gshAB, GshF, GshFAp, GshFAs, GSHI, I79_022778, L-glutamate L-cysteine ligase, More, PAD2, PhGshA II, PSHAa0937, StGCL-GS, Synthetase, gamma-glutamylcysteine
ECTree
6.3.2.2 glutamate-cysteine ligaseAdvanced search results
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results (Engineering
Engineering on EC 6.3.2.2 - glutamate-cysteine ligase
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PROTEIN VARIANTS 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
C186S
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mutation decreases activity by 20fold and abrogates the response to changes in redox environment
C406S
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mutation decreases activity by 20fold and abrogates the response to changes in redox environment
C356A
the mutant shows reduced inhibition by DTT, but increased inhibition by glutathione
C139S/C267S
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sequential site-directed mutagenesis, exchange of cysteine residues in the modifier subunit, the cysteine residues seem not to be involved in intersubunit disulfide bonding
C213S/C214S/C267S
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sequential site-directed mutagenesis, exchange of cysteine residues in the modifier subunit, which can still associate with the catalytic subunit, but no longer form intersubunit disulfides, the enzyme activity is reduced but still higher than the activity of the catalytic subunit alone, the mutant is more sensitive to inhibition by GSH, human modifier subunit can complement the defect, the mutant strain shows a reduced GSH level
A494G
-
site-specific mutagenesis, 53% increased activity compared to wild-type enzyme
A494L
-
site-specific mutagenesis, 65% increased activity compared to wild-type enzyme
A494V
-
site-specific mutagenesis, 66% increased activity compared to wild-type enzyme
C 164S
-
site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195
C106S
C106S/C164S/C205S/C223S
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site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S
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site-directed mutagenesis, inactive mutant
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/C372S/S395W
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site-directed mutagenesis, no complementation of the gcs yeast mutant strain ABC 1195
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/C372S/S395Y
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site-directed mutagenesis, no complementation of the gcs yeast mutant strain ABC 1195
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/R374Q
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site-directed mutagenesis, no complementation of the gcs yeast mutant strain ABC 1195
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/R374Q/V375F
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site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195, the mutant enzyme lacking cysteine residues shows a decreased in vivo half-life
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372C/S395W
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site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372C/S395Y
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site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372F/C395S
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site-directed mutagenesis, no complementation of the gcs yeast mutant strain ABC 1195
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372F/S395C
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site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/S372W/S395C
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site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195
C106S/C164S/C205S/C223S/C357S/C372S/C395S/C433S/C439S/V375F
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site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195
C106S/C164S/C205S/C223S/C357S/C372S/C433S/C439S
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site-directed mutagenesis, no complementation of the gcs yeast mutant strain ABC 1195
C106S/C164S/C205S/C223S/C357S/C433S/C439S
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site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195
C106S/C164S/C205S/C223S/C433S/C439S
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site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195
C164S
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site-directed mutagenesis, exchange of surface exposed cysteine residue for improved crystallization
C205S
C223S
C433S/C439S
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site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195
S495T
-
site-specific mutagenesis, 62% increased activity compared to wild-type enzyme
C106S
Escherichia coli B / ATCC 11303
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site-directed mutagenesis, exchange of surface exposed cysteine residue for improved crystallization
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C164S
Escherichia coli B / ATCC 11303
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site-directed mutagenesis, exchange of surface exposed cysteine residue for improved crystallization
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C205S
Escherichia coli B / ATCC 11303
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site-directed mutagenesis, exchange of surface exposed cysteine residue for improved crystallization
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C223S
Escherichia coli B / ATCC 11303
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site-directed mutagenesis, exchange of surface exposed cysteine residue for improved crystallization
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C248G
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site-directed mutagenesis in the catalytic subunit, reduced activity of the catalytic subunit, activity of the holoenzyme is similar to the wild-type enzyme
C249G
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site-directed mutagenesis in the catalytic subunit, reduced activity of the catalytic subunit, reduced activity of the holoenzyme compared to the wild-type enzyme
C295G
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site-directed mutagenesis in the catalytic subunit, reduced activity of the catalytic subunit, activity of the holoenzyme is similar to the wild-type enzyme
C491G
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site-directed mutagenesis in the catalytic subunit, reduced activity of the catalytic subunit, activity of the holoenzyme is similar to the wild-type enzyme
C501G
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site-directed mutagenesis in the catalytic subunit, reduced activity of the catalytic subunit, activity of the holoenzyme is similar to the wild-type enzyme
C52G
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site-directed mutagenesis in the catalytic subunit, reduced activity of the catalytic subunit, activity of the holoenzyme is similar to the wild-type enzyme
C553G
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site-directed mutagenesis in the catalytic subunit, slightly reduced activity of the catalytic subunit, about 3.5fold reduced activity of the holoenzyme compared to the wild-type enzyme
C605G
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site-directed mutagenesis in the catalytic subunit, reduced activity of the catalytic subunit, activity of the holoenzyme is similar to the wild-type enzyme
H370L
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clinically relevant mutation in catalytic subunit GCLC. Significantly lower levels of glutathione relative to that of the wild type. Compromised enzymatic activity can largely be rescued by the addition of GCLM
P158L
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clinically relevant mutation in catalytic subunit GCLC. Significantly lower levels of glutathione relative to that of the wild type, kinetic constants comparable to those of wild-type GCLC
P414L
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clinically relevant mutation in catalytic subunit GCLC. Significantly lower levels of glutathione relative to that of the wild type, most compromised mutant among those studied. Compromised enzymatic activity can largely be rescued by the addition of GCLM
P462S
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non-synonymous polymorphism in the gene encoding the catalytic subunit of glutamate-cysteine ligase. The polymorphism is present only in individuals of African descent and encodes an enzyme with significantly decreased in vitro activity when expressed by either a bacterial or mammalian cell expression system. Overexpression of the P462 wild-type GCLC enzyme results in higher intracellular glutathione concentrations than overexpression of the P462S isoform. Apoptotically stimulated mammalian cells overexpressing the P462S enzyme have increased caspase activation and increased DNA laddering compared to cells overexpressing the wild-type enzyme. The P462S polymorphism is in Hardy-Weinberg disequilibrium, with no individuals homozygous for the P462S polymorphism identified
R127C
C248A/C249A
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mutant enzyme shows the same strength of binding to regulatory subunit (GCLM) as does wild-type GCLC, yet the catalytic activity is dramatically decreased
E103A
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transduction of Hepa-1c1c7 cells with a catalytically inactive GCL catalytic subunit E103A mutant decreases cellular GCL activity in a dose-dependent manner
P158L
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mutant enzyme shows the same strength of binding to regulatory subunit (GCLM) as does wild-type GCLC, yet the catalytic activity is dramatically decreased
K38A
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mutant enzyme Lys38Arg: small changes in the catalytic properties, mutant enzyme K38N and K38E show marked decrease in enzymatic activity and about 2fold increase in Km for Glu
K38N
site-directed mutagenesis, 50% reduced activity and 2 to 3fold increased Km for L-Glu compared to the wild-type
K38Q
site-directed mutagenesis, 50% reduced activity and 2 to 3fold increased Km for L-Glu compared to the wild-type
D520stop
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KM-value for L-Glu is 1.3fold higher than wild-type value, KM-values for L-Cys and ATP are nearly identical to wild-type value, Ki-value for GSH is 2.2fold higher than wild-type value, Ki-value for gamma-glutamylcysteine is 1.3fold higher than wild-type value
E494stop
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KM-value for L-Glu, L-Cys and ATP are nearly identical to wild-type value, Ki-value for GSH is 7.7fold lower than wild-type value, Ki-value for gamma-glutamylcysteine is 2.4fold higher than wild-type value
G441stop
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KM-value for L-Glu is 3.5fold higher than wild-type value, KM-values for L-Cys and ATP are nearly identical to wild-type value
H144A
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KM-value for L-Glu is 10fold higher than wild-type value, KM-value for L-Cys is 6.4fold higher than wild-type value, KM-value for ATP is nearly identical to wild-type value
K526A
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KM-value for L-Glu, L-Cys and ATP are nearly identical to wild-type value
R508stop
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KM-value for L-Glu is 1.6fold higher than wild-type value, KM-value for L-Cys is 1.7fold higher than wild-type value, KM-value for ATP is nearly identical to wild-type value, Ki-value for GSH is 2fold higher than wild-type value, Ki-value for gamma-glutamylcysteine is 3.3fold higher than wild-type value
Y464stop
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KM-values for L-Glu, L-Cys and ATP are nearly identical to wild-type value, Ki-value for GSH is 11fold lower than wild-type value, Ki-value for gamma-glutamylcysteine is 1.3fold lower than wild-type value
H121A
site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme
H121Q
site-directed mutagenesis, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
R167K
site-directed mutagenesis, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
R248K
site-directed mutagenesis, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
T117S
site-directed mutagenesis, the mutant shows altered kinetics and reduced activity compared to the wild-type enzyme
C319A
E100A
-
site-directed mutagenesis, n1 metal binding site mutant, inactive mutant
E489A
-
site-directed mutagenesis, n2 metal binding site mutant, reduced activity
E53A
-
site-directed mutagenesis, n2 metal binding site mutant, reduced activity
E55A
-
site-directed mutagenesis, n1 metal binding site mutant, inactive mutant
E93A
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site-directed mutagenesis, n1 metal binding site mutant, only capable of catalyzing L-Glu-dependent ATP hydrolysis and not the ligation between L-Glu and L-alpha-aminobutyrate
Q321A
-
site-directed mutagenesis, n2 metal binding site mutant, reduced activity
R179A
-
site-directed PCR-based mutagenesis, conserved residue, gamma-aminobutyrate-binding determinant, increase of Km for both L-Cys and L-Glu
R366A
-
site-directed PCR-based mutagenesis, mutant is active with gamma-aminobutyrate, increase of dissociation constant for L-Glu by 160fold, elimination of positive cooperativity of binding of L-Glu and ATP, interacts with the alpha-carboxylate of L-Glu, 220fold increase in Ki for GSH
R474A
-
site-directed PCR-based mutagenesis, R474 is an ATP binding determinant, increase of Km for ATP by 20-100fold
R487A
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site-directed PCR-based mutagenesis, R487 is an ATP binding determinant, increase of Km for ATP by 20-100fold
R491A
-
site-directed PCR-based mutagenesis, decrease of kcat for ATP hydrolysis
T323A
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site-directed PCR-based mutagenesis, T323 is an alpha-phosphate of ATP binding determinant, increase of Km for ATP by 20-100fold
additional information
C106S
-
site-directed mutagenesis, exchange of surface exposed cysteine residue for improved crystallization
C106S
-
site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195
C205S
-
site-directed mutagenesis, exchange of surface exposed cysteine residue for improved crystallization
C205S
-
site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195
C223S
-
site-directed mutagenesis, exchange of surface exposed cysteine residue for improved crystallization
C223S
-
site-directed mutagenesis, complementation of the gcs yeast mutant strain ABC 1195
R127C
-
naturally occuring mutation in patients with hemolytic anemia resulting from low erythrocyte enzyme levels, fibroblast cells from these patients bearing the mutation show 95% reduced enzyme activity and lowered Km for glutamate and aminobutyrate, as well as an increased Ki for GSH
R127C
-
clinically relevant mutation in catalytic subunit GCLC. Significantly lower levels of glutathione relative to that of the wild type. Compromised enzymatic activity can largely be rescued by the addition of GCLM
C319A
-
mutant is insensitive to cystamine, but the catalytic efficiency, the kinetic mechanism, or the substrate affinities remain unaltered
C319A
-
site-directed mutagenesis, loss of sensitivity against cysteamine inactivation
additional information
-
mutation of Cys102, Cys251, Cys349, or Cys364 does not alter the response to redox environment
additional information
G3HIY9; G3IG96
overexpression of subunit Gclc-X2 leads to upregulation of the proteins Abcf1, Fkbp4, and Eif3h, as well as to downregulation of protein Lamb1. There is no significant difference in growth rate during exponential phase at 32C between Gclm+ or Gclc-X2+ populations and the wild-type population. A higher cell proliferation observed in the Gclc overexpressing population
additional information
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overexpression of subunit Gclc-X2 leads to upregulation of the proteins Abcf1, Fkbp4, and Eif3h, as well as to downregulation of protein Lamb1. There is no significant difference in growth rate during exponential phase at 32C between Gclm+ or Gclc-X2+ populations and the wild-type population. A higher cell proliferation observed in the Gclc overexpressing population
additional information
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construction of a quadruple mutant of the enzyme termed gamma-GCS4CS
additional information
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natural K-12 mutant B possesses a Gly at position 494 compared to Ala for the K-12 wild-type enzyme, the initiation codon exchange mutant shows increased activity
additional information
overexpression of Escherichia coli gamma-glutamylcysteine synthetase in the cytosol of Populus tremula x Populus alba produces higher glutathione (GSH) concentrations in leaves, thereby indicating the potential for cadmium (Cd) phytoremediation. Analysis of net Cd2+ influx in association with H+/Ca2+, Cd tolerance, and the underlying molecular and physiological mechanisms, overview. Transgenic plants have higher Cd2+ uptake rates and elevated transcript levels of several genes involved in Cd2+ transport and detoxification compared with wild-type poplar plants. Transgenic plants exhibit greater Cd2+ accumulation in the aerial parts than wild-type plants in response to Cd2+ exposure. Transgenic poplars show lower concentrations of superoxide anions and H2O2, higher concentrations of total thiols, GSH and oxidized GSH in roots and/or leaves, and stimulated foliar GSH reductase activity compared with wild-type plants. The transgenic plants are more tolerant of 0.1 mM Cd2+ than wild-type plants, probably due to the GSH-mediated induction of the transcription of genes involved in Cd2+ transport and detoxification
additional information
Escherichia coli B / ATCC 11303
-
construction of a quadruple mutant of the enzyme termed gamma-GCS4CS
-
additional information
mutational analysis of the 5'-flanking sequence of the heavy subunit, site-directed mutagenesis
additional information
mutational analysis of the 5'-flanking sequence of the heavy subunit, site-directed mutagenesis
additional information
-
mutational analysis of the 5'-flanking sequence of the heavy subunit, site-directed mutagenesis
additional information
-
a GAG-repeat polymorphism in the 5'-UTR of the gene coding for the catalytic subunit, GCLC, is associated with altered GSH levels in vitro and in vivo
additional information
-
GCLC and GCLM polymorphisms increase disease susceptibility in humans, overview
additional information
-
GCLC polymorphisms are associated with lower lung function levels causing lung disease, especially in association with oxidative stress due to smoking
additional information
-
the presence of at least one T allele in the -129 C/T polymorphism of the GCL catalytic subunit gene is independently associated with non-alcoholic steatohepatitis
additional information
-
deletion analysis indicats that most regions, except a portion of the C-terminal region of catalytic subunit (GCLC) and a portion of the N-terminal region of regulatory subunit (GCLM), are required for the interaction to occur
additional information
-
construction of Gclc and Gclm transgenic mice designed to conditionally overexpress GCL in the liver, conditional Gcl transgene expression in these mice promotes resistance to acetaminophen-induced liver injury
additional information
-
transgenic mice that conditionally overexpress GCL subunits are protected from acetaminophen induced liver injury. Gclm null mice exhibit low GSH levels and enhanced sensitivity to acetaminophen. When Gclm expression and GCL activity are restored in Gclm conditional transgenic X Gclm null mice, they become resistant to APAP-induced liver damage. Construction of transgenic mouse models of inducible GCL subunit expression in the liver, overview
additional information
-
a protein transduction approach whereby recombinant GCL protein can be rapidly and directly transferred into cells when coupled to the HIV TAT protein transduction domain. The TAT-GCL fusion proteins are capable of heterodimerization and formation of functional GCL holoenzyme in vitro. Exposure of Hepa-1c1c7 cells to the TAT-GCL fusion proteins results in the time- and dose-dependent transduction of both GCL subunits and increased cellular GCL activity and glutathione levels. A heterodimerization-competent, enzymatically deficient GCLC-TAT mutant was also generated in an attempt to create a dominant-negative suppressor of GCL
additional information
-
construction of transgenic poplar plants expressing the bacterial enzyme in the cytosol and chloroplasts, transgenic plants show increased enzyme expression and induction, as well as increased detoxification activity and less phytootxic effects by the herbcides, overview
additional information
deletion of genes GSH1 and GSH2 (encoding glutathione synthetase) using the CRISPR-Cas9 nuclease system
additional information
-
deletion of genes GSH1 and GSH2 (encoding glutathione synthetase) using the CRISPR-Cas9 nuclease system
additional information
generation of an GSH1 enzyme deletion mutant using the CRISPR-Cas9 nuclease system
additional information
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generation of an GSH1 enzyme deletion mutant using the CRISPR-Cas9 nuclease system
additional information
-
deletion of genes GSH1 and GSH2 (encoding glutathione synthetase) using the CRISPR-Cas9 nuclease system
-
additional information
-
generation of an GSH1 enzyme deletion mutant using the CRISPR-Cas9 nuclease system
-
additional information
-
mutants with a deletion of GSH1 cannot grow, a high level expression from a plasmid of the enzyme can compensate for adeletion of gene GSH2 encoding glutathione synthatase, the enzyme catalyzing the second step of glutathione biosynthesis
additional information
mutation of yAP-1 consensus sequence inhibits binding of yAP-1 protein, rendering the GSH1 promotor nonresponsive to exogenously expressed yAP-1
additional information
-
wild-type enzyme is nearly uninhibited by GSH, shorter gamma-glutamylcysteine synthetase domain constructs are strongly inhibited. Chimeras of Streptococcus agalactiae gamma-glutamylcysteine synthetase-glutathione synthetase are made containing gamma-glutamylcysteine synthetase domain flexible loop sequences from Enterococcus faecalis and Pasteurella multocida gamma-glutamylcysteine synthetase-glutathione synthetase isoforms that are inhibited by GSH. Inhibition remains Streptococcus agalactiae-like (very weak)
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
-
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
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
-
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
-
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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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
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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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