Information on EC 3.2.1.97 - endo-alpha-N-acetylgalactosaminidase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
3.2.1.97
-
RECOMMENDED NAME
GeneOntology No.
endo-alpha-N-acetylgalactosaminidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
beta-D-galactosyl-(1->3)-N-acetyl-alpha-D-galactosaminyl-[glycoprotein]-L-serine/L-threonine + H2O = beta-D-galactosyl-(1->3)-N-acetyl-D-galactosamine + [glycoprotein]-L-serine/L-threonine
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of O-glycosyl bond
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
glycopeptide-D-galactosyl-N-acetyl-alpha-D-galactosamine D-galactosyl-N-acetyl-galactosaminohydrolase
The enzyme catalyses the liberation of Gal-(1->3)-beta-GalNAc alpha-linked to serine or threonine residues of mucin-type glycoproteins. EngBF from the bacterium Bifidobacterium longum specifically acts on core 1-type O-glycan to release the disaccharide Gal-(1->3)-beta-GalNAc. The enzymes from the bacteria Clostridium perfringens, Enterococcus faecalis, Propionibacterium acnes and Alcaligenes faecalis show broader specificity (e.g. they can also release the core 2 trisaccharide Gal-(1->3)-beta-(GlcNAc-(1->6)-beta)-GalNAc or the core 3 disaccharide GlcNAc-(1->3)-beta-GalNAc) [1,2]. The enzyme may play an important role in the degradation and utilization of mucins having core 1 O-glycan.
CAS REGISTRY NUMBER
COMMENTARY hide
59793-96-3
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain F-1906
-
-
Manually annotated by BRENDA team
strain A198
-
-
Manually annotated by BRENDA team
ATCC29521
-
-
Manually annotated by BRENDA team
JCM 7004
-
-
Manually annotated by BRENDA team
JCM 1192
-
-
Manually annotated by BRENDA team
JCM 1192
-
-
Manually annotated by BRENDA team
JCM 1217
SwissProt
Manually annotated by BRENDA team
JCM 7054
-
-
Manually annotated by BRENDA team
strain JCM1217, ATCC29521
-
-
Manually annotated by BRENDA team
strain SS-16
-
-
Manually annotated by BRENDA team
strain SS-16
-
-
Manually annotated by BRENDA team
strain 13
UniProt
Manually annotated by BRENDA team
strain 13
UniProt
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
ATCC BAA-334D
-
-
Manually annotated by BRENDA team
strain OH-11242
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
-
the enzyme is responsible for catalyzing the liberation of galactosyl beta-1,3-N-acetyl-D-galactosamine linked to serine or threonine residues of mucin-type glycoproteins
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2,4-dinitrophenyl beta-D-Gal-(1->3)-alpha-D-GalNAc + H2O
2,4-dinitrophenol + beta-D-Gal-(1->3)-alpha-D-GalNAc
show the reaction diagram
-
-
-
?
2,4-dinitrophenyl-GalNAc-alpha-R + H2O
2,4-dinitrophenol + GalNAc-alpha-R
show the reaction diagram
-
-
-
?
3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosaminyl-L-serine-[protein] + H2O
3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosamine + L-serine-[protein]
show the reaction diagram
3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosaminyl-L-threonine-[protein] + H2O
3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosamine + L-threonine-[protein]
show the reaction diagram
4,6-dimethoxy-1,3,5-triazin-2-yl alpha-N-acetylgalactosaminide + H2O
?
show the reaction diagram
-
-
-
?
4-nitrophenyl 2-(acetylamino)-2-deoxy-alpha-D-galactopyranoside + H2O
4-nitrophenol + N-acetyl-alpha-D-galactosamine
show the reaction diagram
4-nitrophenyl 2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-alpha-D-galactopyranoside + H2O
4-nitrophenol + 3-O-beta-D-galactopyranosyl-2-acetamido-2-deoxy-alpha-D-galactopyranose
show the reaction diagram
-
-
-
-
?
4-nitrophenyl beta-D-Gal-(1->3)-alpha-D-GalNAc + H2O
4-nitrophenol + beta-D-Gal-(1->3)-alpha-D-GalNAc
show the reaction diagram
4-nitrophenyl-N-acetyl-beta-D-galactosaminide + H2O
4-nitrophenol + N-acetyl-beta-D-galactosamine
show the reaction diagram
-
-
-
?
4-nitrophenyl-N-acetyl-beta-D-galactosaminide + L-Ser
?
show the reaction diagram
transglycosylation
-
-
?
asialo kappa-casein + H2O
Galbeta(1-3)GalNAc + ?
show the reaction diagram
asialo-fetuin + H2O
?
show the reaction diagram
-
almost complete removal of O-linked Galbeta1-3GalNAc, the product has a decreased trypsin inhibitory activity
-
?
asialofetuin + H2O
?
show the reaction diagram
asialofetuin + H2O
Galbeta(1-3)GalNAc + ?
show the reaction diagram
asialoglycophorin + H2O
Galbeta(1-3)GalNAc + ?
show the reaction diagram
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-p-nitrophenyl + 1-butanol
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-butyl + p-nitrophenol
show the reaction diagram
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-p-nitrophenyl + 1-heptanol
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-heptyl + p-nitrophenol
show the reaction diagram
-
transfer ratio in presence of sodium cholate is 22.7%
-
?
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-p-nitrophenyl + 1-hexanol
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-hexyl + p-nitrophenol
show the reaction diagram
-
transfer ratio in presence of sodium cholate is 58.8%
-
?
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-p-nitrophenyl + 1-octanol
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-1-octyl + p-nitrophenol
show the reaction diagram
-
transfer ratio in presence of sodium cholate is 14.9%
-
?
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-p-nitrophenyl + 1-pentanol
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-pentyl + p-nitrophenol
show the reaction diagram
-
transfer ratio in presence of sodium cholate is 73.3%
-
?
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-p-nitrophenyl + 1-propanol
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-propyl + p-nitrophenol
show the reaction diagram
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-p-nitrophenyl + 2-benzyloxy-1-pentanol
?
show the reaction diagram
-
transfer ratio in presence of sodium cholate is 56.3%
-
?
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-p-nitrophenyl + 2-benzyloxyethanol
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-(2-benzyloxyethyl) + p-nitrophenol
show the reaction diagram
-
transfer ratio in presence of sodium cholate is 80.8%
-
?
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-p-nitrophenyl + allyl alcohol
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-allyl + p-nitrophenol
show the reaction diagram
-
transfer ratio in presence of sodium cholate is 86.8%
-
?
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-p-nitrophenyl + benzyloxycarbonyl-Leu-Ser-Gln-Val-His-Arg
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-(CBZ-Leu)-Ser-Gln-Val-His-Arg + p-nitrophenol
show the reaction diagram
-
-
-
?
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-p-nitrophenyl + diethylene glycol monomethyl ether
?
show the reaction diagram
-
transfer ratio in presence of sodium cholate is 78.6%
-
?
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-p-nitrophenyl + ethanol
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-ethyl + p-nitrophenol
show the reaction diagram
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-p-nitrophenyl + ethylene glycol monomethyl ether
?
show the reaction diagram
-
transfer ratio in presence of sodium cholate is 79.4%
-
?
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-p-nitrophenyl + methanol
beta-D-Gal-(1-3)-alpha-D-GalNAc-(1-O)-methyl + p-nitrophenol
show the reaction diagram
dansyl-Ser-GalNAc-Gal + H2O
dansyl-Ser + GalNAc-Gal + H2O
show the reaction diagram
-
-
-
?
dansyl-Thr-GalNAc-Gal + H2O
dansyl-Thr + GalNAc-Gal + H2O
show the reaction diagram
-
-
-
?
fetuin + H2O
?
show the reaction diagram
Gal-beta-1,3-GalNAc-alpha-1p-nitrophenol + H2O
p-nitrophenol + Gal-beta-1,3-GalNAc
show the reaction diagram
Galbeta(1,3)GalNAc-asialofetuin + H2O
Galbeta(1,3)GalNAc + asialofetuin
show the reaction diagram
pH 5.0, 37C
-
-
?
Galbeta(1,3)GalNAcalpha-1-p-nitrophenol + H2O
p-nitrophenol + Galbeta(1,3)GalNAc
show the reaction diagram
Galbeta(1-3)GalNAcalpha(1-)OC6H4-o-NO2 + H2O
Galbeta(1-3)GalNAc + o-nitrophenol
show the reaction diagram
-
-
-
-
?
Galbeta(1-3)GalNAcalpha(1-)OC6H4-p-NO2 + H2O
Galbeta(1-3)GalNAc + p-nitrophenol
show the reaction diagram
-
-
-
-
?
Galbeta(1-3)GalNAcalpha(1-)OC6H5 + H2O
Galbeta(1-3)GalNAc + phenol
show the reaction diagram
-
-
-
-
?
Galbeta(1-3)GalNAcalpha(1-)p-nitrophenyl + galactose
?
show the reaction diagram
-
transglycosylation
-
-
?
Galbeta(1-3)GalNAcalpha(1-)p-nitrophenyl + glucose
?
show the reaction diagram
-
transglycosylation
-
-
?
Galbeta(1-3)GalNAcalpha(1-)p-nitrophenyl + H2O
Galbeta(1-3)GalNAc + p-nitrophenol
show the reaction diagram
-
-
-
-
?
Galbeta(1-3)GalNAcalpha(1-)p-nitrophenyl + maltose
?
show the reaction diagram
-
transglycosylation
-
-
?
Galbeta(1-3)GalNAcalpha(1-)p-nitrophenyl + mannitol
?
show the reaction diagram
-
transglycosylation
-
-
?
Galbeta(1-3)GalNAcalpha(1-)p-nitrophenyl + mannose
?
show the reaction diagram
-
transglycosylation
-
-
?
Galbeta(1-3)GalNAcalpha(1-)p-nitrophenyl + sorbitol
?
show the reaction diagram
-
transglycosylation
-
-
?
Galbeta(1-3)GalNAcalpha(1-)p-nitrophenyl + sucrose
?
show the reaction diagram
-
transglycosylation
-
-
?
Galbeta(1-3)GalNAcalpha1-p-nitrophenol + H2O
Galbeta(1-3)GalNAc + p-nitrophenol
show the reaction diagram
-
-
-
?
Galbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-pNP + H2O
?
show the reaction diagram
Galbeta1-3GalNAcalpha-pNP + H2O
Galbeta1-3GalNAc + p-nitrophenol
show the reaction diagram
Galbeta1-3GalNAcalpha1-pNP + H2O
?
show the reaction diagram
Galbeta1-3[Galbeta1-3GlcNAcbeta1-6]GalNAcalpha-pNP + H2O
?
show the reaction diagram
Galbeta1-3[GlcNAcbeta1-6]GalNAcalpha1-pNP + H2O
?
show the reaction diagram
GalNAcbeta1-3GalNAcalpha1-pNP + H2O
?
show the reaction diagram
gastric mucin + H2O
disaccharide + ?
show the reaction diagram
Glc-NAcbeta1-3GalNAcalpha1-pNP + H2O
?
show the reaction diagram
-
-
-
?
Glcbeta1-3GalNAcalpha1-pNP + H2O
?
show the reaction diagram
GlcNAcbeta1-3GalNAcalpha1-pNP + H2O
?
show the reaction diagram
glycopeptide + H2O
Galbeta(1-3)GalNAc + ?
show the reaction diagram
glycoprotein + H2O
Galbeta(1-3)GalNAc + ?
show the reaction diagram
mucin + H2O
Galbeta1-3GalNAc + ?
show the reaction diagram
Tn antigen + H2O
?
show the reaction diagram
i.e. N-acetyl-beta-D-galactosaminyl-L-Ser/Thr
-
-
?
asialofetuin + H2O
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
3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosaminyl-L-serine-[protein] + H2O
3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosamine + L-serine-[protein]
show the reaction diagram
3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosaminyl-L-threonine-[protein] + H2O
3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosamine + L-threonine-[protein]
show the reaction diagram
Tn antigen + H2O
?
show the reaction diagram
G5ELM1
i.e. N-acetyl-beta-D-galactosaminyl-L-Ser/Thr
-
-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
at a level twice the EDTA concentration completely restores enzyme activity
Co2+
-
activates at 5 mM
Cu2+
7% residual activity at 5 mM
NaCl
effects on kinetics, overview
NaN3
effects on kinetics, overview
Ni2+
-
activates at 5 mM
Sodium formate
effects on kinetics, overview
Zn2+
30% residual activity at 5 mM
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ag+
-
5 mM, 35% inhibition
C2H5HgCl
-
5 mM, 23% inhibition
CH3HgCl
-
5 mM, 18% inhibition
Co2+
-
5 mM, 18% inhibition
Cr3+
-
5 mM, 58% inhibition
DFP
-
1 mM, 48% inhibition
Fe2+
-
1 mM
Fe3+
-
5 mM, 15% inhibition
Galactonolactone
-
5 mM, 11% inhibition
glucose
-
5 mM, 11% inhibition
iodoacetate
-
5 mM, 17% inhibition
L-Cys
-
2 mM, 12% inhibition
N-bromosuccinimide
-
1 mM, complete inhibition
p-chloromercuribenzenesulfonate
-
1 mM, 60% inhibition
p-Nitrophenyl-2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-beta-D-galactopyranoside
-
-
PCMB
-
1 mM, 26% inhibition
SDS
-
strong inhibition
Sodium deoxycholate
-
slightly effective
Triton X-100
-
slightly effective
Tween 20
-
slightly effective
Tween 80
-
slightly effective
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0012 - 0.048
2,4-dinitrophenyl beta-D-Gal-(1->3)-alpha-D-GalNAc
2.1 - 2.4
2,4-dinitrophenyl-GalNAc-alpha-R
17.1
4,6-dimethoxy-1,3,5-triazin-2-yl alpha-N-acetylgalactosaminide
at pH 5.0 and 37C
-
0.021 - 0.212
4-nitrophenyl beta-D-Gal-(1->3)-alpha-D-GalNAc
3.2
asialo kappa-casein
-
-
-
0.78
asialo-kappa-casein
-
-
-
0.52 - 3.7
asialofetuin
0.32
asialoglycophorin
-
-
-
0.17
dansyl-Ser-GalNAc-Gal
-
-
1.43
dansyl-Thr-GalNAc-Gal
-
-
0.0218 - 0.165
Galbeta(1,3)GalNAcalpha-1-p-nitrophenol
0.23
Galbeta(1-3)GalNAcalpha(1-)OC6H4-o-NO2
-
-
0.25
Galbeta(1-3)GalNAcalpha(1-)OC6H4-p-NO2
-
-
0.0218 - 0.107
Galbeta(1-3)GalNAcalpha1-p-nitrophenol
0.0596
Galbeta1-3GalNAcalpha-pNP
-
0.051
Galbeta1-3GalNAcalpha1-pNP
-
2.2
Tn antigen
at pH 5.0 and 37C
-
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.76 - 748
2,4-dinitrophenyl beta-D-Gal-(1->3)-alpha-D-GalNAc
1.6 - 5
2,4-dinitrophenyl-GalNAc-alpha-R
16.7
4,6-dimethoxy-1,3,5-triazin-2-yl alpha-N-acetylgalactosaminide
Bifidobacterium bifidum
G5ELM1
at pH 5.0 and 37C
-
1 - 579
4-nitrophenyl beta-D-Gal-(1->3)-alpha-D-GalNAc
0.23 - 37
Galbeta(1,3)GalNAcalpha-1-p-nitrophenol
8.17 - 17.8
Galbeta(1-3)GalNAcalpha1-p-nitrophenol
49.9
Galbeta1-3GalNAcalpha-pNP
Enterococcus faecalis
Q833X5
-
110
Galbeta1-3GalNAcalpha1-pNP
Clostridium perfringens
Q8XMJ5
-
47.6
Tn antigen
Bifidobacterium bifidum
G5ELM1
at pH 5.0 and 37C
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.023 - 15.9
2,4-dinitrophenyl beta-D-Gal-(1->3)-alpha-D-GalNAc
0.0006 - 0.002
2,4-dinitrophenyl-GalNAc-alpha-R
0.0099 - 9.3
4-nitrophenyl beta-D-Gal-(1->3)-alpha-D-GalNAc
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30.1
-
-
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 5
-
-
4.5 - 6
-
hydrolysis of dansyl-Thr-GalNAc-Gal
4.5 - 7.5
-
hydrolysis of dansyl-Ser-GalNAc-Gal
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 9
-
about 50% of maximal activity at pH 4.0 and at pH 9.0
4 - 6
68% activity at pH 4.0, 73% activity at pH 6.0
5.5 - 7
-
about 50% of maximal activity at pH 5.5 and at pH 7.0
6 - 9
retains more than 80% activity
6 - 8.8
-
pH 6.0: about 40% of maximal activity, pH 8.8: about 60% of maximal activity
additional information
-
-
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bifidobacterium longum subsp. longum (strain ATCC 15707 / DSM 20219 / JCM 1217 / NCTC 11818 / E194b)
Streptococcus pneumoniae (strain ATCC BAA-255 / R6)
Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334 / TIGR4)
Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334 / TIGR4)
Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334 / TIGR4)
Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334 / TIGR4)
Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334 / TIGR4)
Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334 / TIGR4)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
105000
-
gel filtration
110000
-
gel filtration
160000
200000
gel filtration
210000
210300
calculated from amino acid sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant His-tagged enzyme, X-ray diffraction structure analysis at 2.0-2.5 A resolution
-
purified recombinant isolated catalytic domain, hanging drop vapour diffusion method at 19C, 1 ml of 15 mg/ml protein in 20 mM Tris-HCl, pH 8.0, is mixed with 1 ml 25% PEG 1500. Removal of the His6-tag is unnecessary for crystallization. Crystals are cryoprotected in 1 ml 33% PEG 1500 supplemented with 6% MPD, X-ray diffraction structure determination and analysis at 2.0 A resolution
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3 - 11
the enzyme is stable over the pH range of 3.0-11.0
732082
3.5 - 9
-
37C, 1 h, stable
208866
4 - 10
-
37C, 30 min, stable
208869
4.5 - 6.5
-
30C, 1 h, stable
208859
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35
-
10 min, stable below
70
-
1 h, complete inactivation
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 2 months, stable
-
-20C, stable for 3 months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by gel filtration
HisTrap column chromatography and Superdex 200 gel filtration
partial
recombinant enzyme
recombinant His-tagged EngBF from Escherichia coli strain BL21(DE3) by affinity chromatography and gel filtration
-
recombinant His-tagged EngBF truncation and point mutants from Escherichia coli strain BL21(DE3) by nickel affinity and anion exchange chromatography, and gel filtration
-
recombinant isolated catalytic domain from Escherichia coli strain BL21 Star (DE3)
-
recombinant wild-type and mutant enzymes from Escherichia coli strain AD202 by anion exchange chromatography, ultrafiltration, gel filtration, and hydrophobic interaction chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
EngCP expressed in Escherichia coli
expressed in Escherichia coli BL21(DE3)DELTAlacZ cells
expression in Escherichia coli
expression in Escherichia coli BL21; expression in Escherichia coli, wild-type and mutants D601N, D605N, D612N, D655N, D665N, E679Q, D682N, D682A, D700N, E723Q, D762N, D762A, D789N, D789A, E822Q, E822A, E828Q, D839N, D864N, D898N, D1058N, D1106N, D1248N, E1276Q, E1302Q, E1350Q
expression of His-tagged EngBF in Escherichia coli strain BL21(DE3)
-
expression of His-tagged truncated EngBF mutants, comprising residues 340-1528 and 340-1694, respectively, and point mutants in Escherichia coli strain BL21(DE3)
-
expression of the isolated catalytic domain in Escherichia coli strain BL21 Star (DE3)
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expression of wild-type and mutant enzymes in Escherichia coli strain AD202
into vector pET23a and expressed with a histidine-tag in Escherichia coli BL21(DE3)
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D1058N
activity in cell-free extracts of Escherichia coli almost comparable to wild-type
D1106N
10-30% activity compared to wild-type; Escherichia coli cells expressing the mutant enzyme exhibit 10-30% of the wild-type endo-alpha-GalNAc activity
D1248N
10-30% activity compared to wild-type
D1295A
-
site-directed mutagenesis, the mutant shows highly reduced activity compared to the truncated wild-type enzyme
D601N
activity in cell-free extracts of Escherichia coli almost comparable to wild-type; Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type
D605N
10-30% activity compared to wild-type; Escherichia coli cells expressing the mutant enzyme exhibit 10-30% of the wild-type endo-alpha-GalNAc activity
D612N
activity in cell-free extracts of Escherichia coli almost comparable to wild-type; Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type
D655N
10-30% activity compared to wild-type; Escherichia coli cells expressing the mutant enzyme exhibit 10-30% of the wild-type endo-alpha-GalNAc activity
D665N
activity in cell-free extracts of Escherichia coli almost comparable to wild-type; Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type
D682N
Escherichia coli cells expressing the mutant enzyme exhibit less than 2% of the wild-type endo-alpha-GalNAc activity; significant decrease in activity (below 2%)
D700N
activity in cell-free extracts of Escherichia coli almost comparable to wild-type; Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type
D762A
activity almost comparable to wild-type; kcat/KM is 3.9fold lower than wild-type value
D762N
Escherichia coli cells expressing the mutant enzyme exhibit less than 2% of the wild-type endo-alpha-GalNAc activity; significant decrease in activity (below 2%)
D789N
Escherichia coli cells expressing the mutant enzyme exhibit less than 2% of the wild-type endo-alpha-GalNAc activity; significant decrease in activity (below 2%)
D839N
activity in cell-free extracts of Escherichia coli almost comparable to wild-type; Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type
D864N
activity in cell-free extracts of Escherichia coli almost comparable to wild-type; Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type
D898N
activity in cell-free extracts of Escherichia coli almost comparable to wild-type; Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type
E1276Q
10-30% activity compared to wild-type; Escherichia coli cells expressing the mutant enzyme exhibit 10-30% of the wild-type endo-alpha-GalNAc activity
E1302Q
activity in cell-free extracts of Escherichia coli almost comparable to wild-type; Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type
E1350Q
activity in cell-free extracts of Escherichia coli almost comparable to wild-type; Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type
E679Q
activity in cell-free extracts of Escherichia coli almost comparable to wild-type; Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type
E723Q
10-30% activity compared to wild-type; Escherichia coli cells expressing the mutant enzyme exhibit 10-30% of the wild-type endo-alpha-GalNAc activity
E822Q
significant decrease in activity (below 2%)
E828Q
10-30% activity compared to wild-type; Escherichia coli cells expressing the mutant enzyme exhibit 10-30% of the wild-type endo-alpha-GalNAc activity
E882Q
Escherichia coli cells expressing the mutant enzyme exhibit less than 2% of the wild-type endo-alpha-GalNAc activity
K1199A
-
site-directed mutagenesis, the mutant shows increased activity compared to the truncated wild-type enzyme
N720A
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site-directed mutagenesis, the mutant shows increased activity compared to the truncated wild-type enzyme
Q894A
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site-directed mutagenesis, the mutant shows increased activity compared to the truncated wild-type enzyme
S1248N
Escherichia coli cells expressing the mutant enzyme exhibit 10-30% of the wild-type endo-alpha-GalNAc activity
W748A
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site-directed mutagenesis, the mutant shows highly reduced activity compared to the truncated wild-type enzyme
W748F
-
site-directed mutagenesis, the mutant shows highly reduced activity compared to the truncated wild-type enzyme
W748Y
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site-directed mutagenesis, the mutant shows highly reduced activity compared to the truncated wild-type enzyme
W750A
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site-directed mutagenesis, the mutant shows highly reduced activity compared to the truncated wild-type enzyme
W750F
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site-directed mutagenesis, the mutant shows reduced activity compared to the truncated wild-type enzyme
W750Y
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site-directed mutagenesis, the mutant shows reduced activity compared to the truncated wild-type enzyme
Y787F
-
site-directed mutagenesis, the mutant shows highly reduced activity compared to the truncated wild-type enzyme
D601N
-
activity in cell-free extracts of Escherichia coli almost comparable to wild-type; Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type
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D612N
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activity in cell-free extracts of Escherichia coli almost comparable to wild-type
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D762N
-
Escherichia coli cells expressing the mutant enzyme exhibit less than 2% of the wild-type endo-alpha-GalNAc activity; significant decrease in activity (below 2%)
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D658A
site-directed mutagenesis, the substrate specificity and activity is altered compared to the wild-type enzyme
D764A
site-directed mutagenesis, the substrate specificity and activity is altered compared to the wild-type enzyme
D764F
site-directed mutagenesis, the substrate specificity and activity is altered compared to the wild-type enzyme
E796A
site-directed mutagenesis, the substrate specificity and activity is altered compared to the wild-type enzyme
E796Q
site-directed mutagenesis, the substrate specificity and activity is altered compared to the wild-type enzyme
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
synthesis
Show AA Sequence (140 entries)
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