Information on EC 2.3.1.54 - formate C-acetyltransferase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.3.1.54
-
RECOMMENDED NAME
GeneOntology No.
formate C-acetyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
acetyl-CoA + formate = CoA + pyruvate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Acyl group transfer
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Butanoate metabolism
-
-
ethanol fermentation
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-
Metabolic pathways
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mixed acid fermentation
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Propanoate metabolism
-
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pyruvate fermentation to acetate IV
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pyruvate fermentation to ethanol I
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Pyruvate metabolism
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reductive monocarboxylic acid cycle
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superpathway of fermentation (Chlamydomonas reinhardtii)
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SYSTEMATIC NAME
IUBMB Comments
acetyl-CoA:formate C-acetyltransferase
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CAS REGISTRY NUMBER
COMMENTARY hide
9068-08-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
a hyperthermophile, gene pfl2
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-
Manually annotated by BRENDA team
ATCC 14823
-
-
Manually annotated by BRENDA team
DSM552
-
-
Manually annotated by BRENDA team
DSM525
-
-
Manually annotated by BRENDA team
strain JM109
-
-
Manually annotated by BRENDA team
wild-type and mutant strains, anaerobic growth conditions
-
-
Manually annotated by BRENDA team
no activity in Clostridium pasteurianum
-
-
-
Manually annotated by BRENDA team
gene pfl, strain 27405
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-
Manually annotated by BRENDA team
strain MR-1, gene pflB
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-
Manually annotated by BRENDA team
JC2
-
-
Manually annotated by BRENDA team
slightly aerobic and anaerobic growth, and intraperitoneal infection or nasal infection of female MFI mice
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-
Manually annotated by BRENDA team
ATCC 13419
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-
Manually annotated by BRENDA team
DSM20066
-
-
Manually annotated by BRENDA team
DSM20066
-
-
Manually annotated by BRENDA team
strain LMG18311, gene pfl
Q5LYC1
SwissProt
Manually annotated by BRENDA team
strain LMG18311, gene pfl
Q5LYC1
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
physiological function
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enzyme deletion mutant displays pleiotropic effects. In the mutant, no formate is produced, glucose consumption is delayed, and ethanol production is decreased, whereas acetate and lactate production are unaffected. All metabolic alterations can be restored by addition of formate or complementation of the mutant. In compensation reactions, serine and threonine are consumed better by the mutant than by the wild-type. The mutant displays reduced production of formylated peptides compared to the parental strain. Arginine consumption and arc operon transcription are increased in the mutant. Enzyme plays a significant role in the anaerobic layer of a biofilm
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-oxobutyrate + CoA
propionyl-CoA + formate
show the reaction diagram
acetyl-CoA + formate
pyruvate + CoA
show the reaction diagram
CoA + pyruvate
acetyl-CoA + formate
show the reaction diagram
pyruvate + CoA
acetyl-CoA + formate
show the reaction diagram
pyruvate + CoA
formate + acetyl-CoA
show the reaction diagram
pyruvate + dephospho-CoA
dephospho-acetyl-CoA + formate
show the reaction diagram
pyruvate + dithiothreitol
S-acetyl-dithiothreitol + formate
show the reaction diagram
pyruvate + formate
formate + pyruvate
show the reaction diagram
pyruvate + phosphate
acetylphosphate + formate
show the reaction diagram
-
-
-
ir
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
acetyl-CoA + formate
pyruvate + CoA
show the reaction diagram
CoA + pyruvate
acetyl-CoA + formate
show the reaction diagram
pyruvate + CoA
acetyl-CoA + formate
show the reaction diagram
pyruvate + CoA
formate + acetyl-CoA
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
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rapid, reversible inactivation, deactivation is a non-destructive transfer of an H atom equivalent to quench the glycyl radical
Acetylphosphinate
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mechanism-based inactivator
D-Glyceraldehyde-3-phosphate
DEAE-cellulose
dihydroxyacetone phosphate
dioxygen
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-
DTT
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reversible inactivation, deactivation is a non-destructive transfer of an H atom equivalent to quench the glycyl radical
formate
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product inhibition
HPO42-
Hypophosphite
iodoacetate
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-
methacrylate
Oxamate
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isosteric, chemically inert pyruvate analogue
S-adenosyl-L-homocysteine
additional information
-
the putative pyruvate formate-lyase deactivase, as activity of AdhE together with an alcohol dehydrogenase and an acetaldehyde-CoA dehydrogenase activities, is not active on the enzyme PFL, it has no PFL deactivating activity
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ferredoxin
NADPH
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more effective for activation than NADPH, 0.02 mM NADPH activates over 80% inactive enzyme in cell-free extracts
Oxamate
pyruvate
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obligatory component of the activation reaction
S-adenosylmethionine
Y06I
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autonomous glycyl radical cofactor, reconstituting the catalytic center of oxygen-fragmented enzyme
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additional information
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conversion into the active form is carried out by an activating system containing a flavodoxin system and an activating enzyme (pyruvate format lyase-activase, EC 1.97.1.4)
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.051 - 0.26
acetyl-CoA
0.0068 - 0.012
CoA
10 - 24.5
formate
0.6 - 3.6
pyruvate
additional information
additional information
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steady-state kinetics of wild-type and mutant enzymes
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3.8 - 4.33
formate
11 - 12.8
pyruvate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.42
methacrylate
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pH 7.4, 30C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 7.5
7.6
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assay at
7.8 - 8.4
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7.8 - 8.5
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forward direction
8.5
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8.5
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6.5 - 8.5
activity assay at, wild-type and mutant protein
6.5 - 8.3
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40% of activity maximum at pH 6.5, between pH 7.0-8.3 80% of maximum activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
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assay at room temperature
30
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assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 50
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22 - 35
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activity is about 78 times of that at 22C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
formate accumulation in the medium of anaerobically adapted Chlamydomonas reinhardtii CC-277 cells measured
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
80000 - 85000
80000
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identification of pfl gene product
85120
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amino acid sequence calculation
140000
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gel filtration, sedimentation velocity
150000
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gel filtration
151000
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sedimentation equilibrium
170000 - 180000
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gel filtration
180000
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TOF-MS flight mass spectrometry
297000
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dynamic light scattering, analytical ultracentrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
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4 * 95000, structure analysis, crystal packing, solution X-ray scattering, and ultracentrifugation
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
PFL-activating enzyme
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified enzyme, sitting drop vapour diffusion method, mixing 0.001 ml of 20 mg/ml protein solution with 0.001 ml precipitant solution containing 16% w/v PEG 8000, 8% v/v isopropanol, 80 mM Hepes, pH 7.5, 160 mM ammonium sulfate and 1 mM DTT, plate-like crystals, X-ray diffraction structure determination and analysis at 2.9 A resolution
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space group P4(1)2(1)2
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space group P4(3)2(1)2 a = b = 159 A, c = 160 A
space group P4(3)2(1)2, cell parameters a = b = 158.36 A, c = 159.30 A
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structure of the pyruvate formate-lyase monomer in complex with pyruvate and CoA, structure of active site, small model computational studies of the pyruvate formate-lyase substrate transformation
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tetragonal crystals in complex with pyruvate, monoclinic cocrystals with CoA and either pyruvate or oxamate obtained by hanging drop method, space group C222_1, a = 54.90 A, b = 153.05 A, c = 205.95
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
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activity decreases to less than 10% of the maximal activity
487234
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0 - 30
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stable in anaerobic buffers for days at 0C, at 30C for several hours
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
PFL2 appears to be stabilized by several factors including an increased number of ion pairs, differences in buried charges, a truncated N terminus, anchoring of loops and N terminus via salt-bridges,changes in the oligomeric interface and perhaps also the higher oligomerization state of the protein
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stable for 2 h at 35C in untreated cell-free extracts under strictly anaerobic conditions. In dialyzed cell-free extracts, activity decreases gradually.
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unstable even when stored anaerobically
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virtually stable at 0C and pH 8.0 if kept in media which display a redoxpotential of 0.2 V or below
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
extreme sensitivity towards inactivation by oxygen, admittance of oxygen inactivates completely within a few seconds
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487220, 487221
immediately inactivated by exposure to the air
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487222
obligate anaerobic enzyme, enzymatic conversion by a unique homolytic mechanism that involves a free radical harbored in the protein structure, protein based organic free radical is essential for catalysis, oxygen destruction of the protein radical
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487225, 487226, 487236
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 10 mM MOPS-KOH, pH 7.0, 50 mM KCl, thiols, EDTA in 50% ethyleneglycol, proved stable for months
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-20C, stable for at least several months
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4C, 50 mM potassium phosphate buffer, containing 1 mM dithiothreitol, storage of the purified enzyme in an anaerobic glove box for 2 weeks reduces activity by 50%
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
centrifugation, washing in 50 mM potassium phosphate buffer, pH 7.5, resuspension, French press disruption, centrifugation of crude extract and of resulting supernatant
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gel filtration, Western Blot, heterologously expressed protein
native enzyme at pH 6.4, by anion exchange and hydrophobic interaction chromatography, and gel filtration, to homogeneity
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native enzyme from strain BL21(DE3) by gel filtration and anion exchange chromatography, recombinant His6-tagged enzyme from strain BL21(DE3) by nickel affinity chromatography as single enzyme or as AdhE-Pfl
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partially
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recombinant enzyme
recombinant His-tagged wild-type and mutant enzymes from strain BL21(DE3) by nickel affinity chromatography to homogeneity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Escherichia coli SP 264 contains plasmid p29with pfl gene, identification of pfl gene product as a 80kDa polypeptide
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expressed in Escherichia coli BL21(DE3), pET9a expression vector, transformation into pyruvate formate-lyase-deficient Escherichia coli strain BL21, capacity for formate excretion shown by in vitro tests
gene deletion is transduced via Bacteriophage P1
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gene pfl from strain JM109, DNA and amino acid sequence determination and analysis, expression of His-tagged wild-type and mutant enzymes in strain BL21(DE3)
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gene pfl, DNA and amino acid sequence determination and analysis, detailed phylogenetic analysis, overview
gene pfl, DNA and amino acid sequence determination and analysis, sequence comparison
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gene pflB, expression analysis
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overexpression in Escherichia coli
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overexpression of the His6-tagged enzyme as single enzyme or as AdhE-Pfl in strain BL21(DE3)
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overproducing of the enzyme in Escherichia coli 234M1 transformed with expression vector p153E1
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PCR-amplificatioin, mariner plasmid
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pfl gene cloned and expressed in Escherichia coli HB101
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pfl gene expressed under control of different constitutive promoters, PFL-deficient strain CRM40 complemented
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recombinant PFL expressed in Escherichia coli BL21 (DE3)
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recombinant pfl in Escherichia coli RM220
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recombinant pfl in Escherichia coli RM221
structural gene pfl cloned and sequenced, homologous expression and overproduction in Escherichia coli K12
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
9fold increase in SPD0420 mutants in galactose compared to glucose-grown bacteria in anaerobic conditions, in aerobiosis, 2.4fold upregulation of SpD0420 mutant, 3.5fold in SPD1774 mutant in galactose compared to glucose-grown bacteria
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A273C
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site-directed mutagenesis, the mutant is less thermostable than the wild-type enzyme
E336C
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site-directed mutagenesis, the mutant is more thermostable than the wild-type enzyme, Tm is increased 3.7fold, half-life 1.8fold
E400I
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site-directed mutagenesis, the mutant is more thermostable than the wild-type enzyme, Tm is increased 2.2fold, half-life 2.21fold
A273C
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site-directed mutagenesis, the mutant is less thermostable than the wild-type enzyme
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E336C
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site-directed mutagenesis, the mutant is more thermostable than the wild-type enzyme, Tm is increased 3.7fold, half-life 1.8fold
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E400I
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site-directed mutagenesis, the mutant is more thermostable than the wild-type enzyme, Tm is increased 2.2fold, half-life 2.21fold
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additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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D-lactate production for the use in biopolymer production as biodegradable alternative for oil-derived plastics
nutrition
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plays a significant role in industrial milk fermentation
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