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Information on EC 3.2.1.81 - beta-agarase and Organism(s) Zobellia galactanivorans and UniProt Accession G0L322

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IUBMB Comments
Also acts on porphyran, but more slowly . This enzyme cleaves the beta-(1->4) linkages of agarose in a random manner with retention of the anomeric-bond configuration, producing beta-anomers that give rise progressively to alpha-anomers when mutarotation takes place . The end products of hydrolysis are neoagarotetraose and neoagarohexaose in the case of AgaA from the marine bacterium Zobellia galactanivorans, and neoagarotetraose and neoagarobiose in the case of AgaB .
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Zobellia galactanivorans
UNIPROT: G0L322
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Word Map
The taxonomic range for the selected organisms is: Zobellia galactanivorans
The expected taxonomic range for this enzyme is: Bacteria, Archaea
Synonyms
agarase, beta-agarase, aga50d, lsl-1, endo-type beta-agarase, agaac, agarase 0107, beta-agarase a, beta-agarase c, agab34, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
agarase
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-
-
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Agarase 0107
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-
-
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Hydrolysis of (1->4)-beta-D-galactosidic linkages in agarose, giving the tetramer as the predominant product
show the reaction diagram
mechanism
Hydrolysis of (1->4)-beta-D-galactosidic linkages in agarose, giving the tetramer as the predominant product
show the reaction diagram
enzyme might be able to unwind the double-helical structure of substrate prior to the catalytic cleavage
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of O-glycosyl bond
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PATHWAY SOURCE
PATHWAYS
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-, -
SYSTEMATIC NAME
IUBMB Comments
agarose 4-glycanohydrolase
Also acts on porphyran, but more slowly [1]. This enzyme cleaves the beta-(1->4) linkages of agarose in a random manner with retention of the anomeric-bond configuration, producing beta-anomers that give rise progressively to alpha-anomers when mutarotation takes place [6]. The end products of hydrolysis are neoagarotetraose and neoagarohexaose in the case of AgaA from the marine bacterium Zobellia galactanivorans, and neoagarotetraose and neoagarobiose in the case of AgaB [6].
CAS REGISTRY NUMBER
COMMENTARY hide
37288-57-6
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
agarose + H2O
neoagarotetraose + neoagarohexaose
show the reaction diagram
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-
-
?
agarose + H2O
?
show the reaction diagram
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-
-
-
?
agarose + H2O
neoagarotetraose
show the reaction diagram
agarose + H2O
neoagarotetraose + neoagarohexaose
show the reaction diagram
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-
-
?
liquid-phase agarose + H2O
?
show the reaction diagram
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-
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
agarose + H2O
neoagarotetraose
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
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the activity and stability of the wild type enzyme at 45°C with 1 mM CaCl2 are double those of the enzyme at 40°C without CaCl2. Activities of both the wild type and mutant E99K/T307I enzymes increase in 1 mM CaCl2. The E99K/T307I mutant enzyme is stable at 55°C with 1 mM CaCl2, reaching 260% of the activity the wild type enzyme held at 40°C without CaCl2. No further increases in activity are observed in 10 mMCaCl2
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1 - 2
liquid-phase agarose
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.1
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AgaB, 1 ml of 0.125% melted agarose, 44°C, pH 7.5
0.16
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AgaAc, 1 ml of 0.125% melted agarose, 44°C, pH 7.5
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60000
recombinant enzyme
13400
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AgaAc, calculated by sequence
31000
recombinant enzyme
32150
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AgaAc, ESI MS
353000
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AgaB, calculated by sequence
36000
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SDS-PAGE
40010
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AgaB, ESI MS
539000
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AgaA, calculated by sequence
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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AgaB, gel filtration and ESI MS
monomer
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AgaA, gel filtration and ESI MS
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
E147S mutant in complex with agaro-octaose, 1.7 A resolution, structure determination by molecular replacment, hanging drop vapor duffusion method, 20.5°C, 30% PEG 4000, 200 mM ammonium acetate, 100 mM sodium acetate
hanging drop vapor diffusion method, using 28-34% (w/v) PEG 8000 and 0.1 M imidazole pH 8.0, at 19°C
inactive mutant A-E174S, in complex with agaro-octaose
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recombinant protein expressed in Escherichia coli
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E147S
inactive mutant
E99K
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the mutant shows 140% relative activity compared to the wild type enzyme
E99K/T307I
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the mutant shows 200% relative activity compared to the wild type enzyme
T307I
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the mutant shows 190% relative activity compared to the wild type enzyme
additional information
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inactive mutant A-E147S, crystallization data
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50 - 60
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mutant enzymes E99K, T307I and E99K/T307I are stable up to 50°C, the melting temperature of E99K/T307I is increased by 5.2°C over that of the wild type enzyme (54.6°C). The E99K/T307I mutant enzyme is stable at 55°C with 1 mM CaCl2, reaching 260% of the activity the wild type enzyme held at 40°C without CaCl2
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Ni-NTa affinity chromatography and exclusion chromatography with Sephacryl S100
ammonium sulfate precipitation, Sepharose CL6B, anion-exchange Mono Q HR5, Ni-NTA column, AgaB further gel filtration with Sephacryl S200
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chitin bead column chromatography
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Ni affinity column chromatography and Sephacryl S-200 gel filtration
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli DH5alpha cells
expression in Escherichia coli XL21 Blue and ORIGAMI (DE3) pLysS cells
AgaA, AgaB and truncated form of AgaA referred to as AgaAc with His tag, overexpression in Escherichia coli
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expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli DH5alpha cells
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Allouch, J.; Jam, M.; Helbert, W.; Barbeyron, T.; Kloareg, B.; Henrissat, B.; Czjzek, M.
The three-dimensional structures of two beta-agarases
J. Biol. Chem.
278
47171-47180
2003
Zobellia galactanivorans (G0L322), Zobellia galactanivorans (Q9RGX8), Zobellia galactanivorans
Manually annotated by BRENDA team
Allouch, J.; Helbert, W.; Henrissat, B.; Czjzek, M.
Parallel substrate binding sites in a beta-agarase suggest a novel mode of action on double-helical agarose. [Erratum to document cited in CA141:067260]
Structure
12
905
2004
Zobellia galactanivorans
-
Manually annotated by BRENDA team
Jam, M.; Flament, D.; Allouch, J.; Potin, P.; Thion, L.; Kloareg, B.; Czjzek, M.; Helbert, W.; Michel, G.; Barbeyron, T.
The endo-beta-agarases AgaA and AgaB from the marine bacterium Zobellia galactanivorans: two paralogue enzymes with different molecular organizations and catalytic behaviours
Biochem. J.
385
703-713
2005
Zobellia galactanivorans
Manually annotated by BRENDA team
Allouch, J.; Helbert, W.; Henrissat, B.; Czjzek, M.
Parallel substrate binding sites in a beta-agarase suggest a novel mode of action on double-helical agarose
Structure
12
623-632
2004
Zobellia galactanivorans (G0L322), Zobellia galactanivorans, Zobellia galactanivorans Dsij (G0L322)
Manually annotated by BRENDA team
Hehemann, J.H.; Michel, G.; Barbeyron, T.; Czjzek, M.
Expression, purification and preliminary X-ray diffraction analysis of the catalytic module of a beta-agarase from the flavobacterium Zobellia galactanivorans
Acta Crystallogr. Sect. F
66
413-417
2010
Zobellia galactanivorans (Q9RGX8), Zobellia galactanivorans
Manually annotated by BRENDA team
Jang, M.K.; Lee, S.W.; Lee, D.G.; Kim, N.Y.; Yu, K.H.; Jang, H.J.; Kim, S.; Kim, A.; Lee, S.H.
Enhancement of the thermostability of a recombinant beta-agarase, AgaB, from Zobellia galactanivorans by random mutagenesis
Biotechnol. Lett.
32
943-949
2010
Zobellia galactanivorans, Zobellia galactanivorans KCTC 12921
Manually annotated by BRENDA team
Fu, X.T.; Kim, S.M.
Agarase: review of major sources, categories, purification method, enzyme characteristics and applications
Mar. drugs
8
200-218
2010
Agarivorans albus (A8W969), Agarivorans albus YKW-34 (A8W969), Agarivorans sp., Agarivorans sp. (A1E2A6), Agarivorans sp. (C9WIW7), Agarivorans sp. JA-1 (A1E2A6), Alteromonas sp., Alteromonas sp. C-1, Alteromonas sp. SY37-12, Bacillus sp. MK03, Cytophaga sp., Flavobacterium flevense, Microbulbifer thermotolerans (Q6F4N4), Microbulbifer thermotolerans JAMB-A94 (Q6F4N4), Pseudoalteromonas antarctica, Pseudoalteromonas antarctica N-1, Pseudomonas sp., Pseudomonas sp. (Q9KK55), Pseudomonas sp. SK38, Pseudomonas sp. W7 (Q9KK55), Saccharophagus degradans (Q21HB4), Saccharophagus degradans (Q21HC5), Saccharophagus degradans (Q21LJ2), Saccharophagus degradans 2-40 (Q21HB9), Saccharophagus degradans 2-40 (Q21LJ1), Saccharophagus degradans 2-40 / ATCC 43961 (Q21HB4), Saccharophagus degradans 2-40 / ATCC 43961 (Q21HC5), Saccharophagus degradans 2-40 / ATCC 43961 (Q21LJ2), Vibrio sp., Vibrio sp. (A1E2B6), Vibrio sp. JT0107, Vibrio sp. PO-303 (A5A6R7), Vibrio sp. V134 (A1E2B6), Zobellia galactanivorans (G0L322), Zobellia galactanivorans (Q9RGX8)
Manually annotated by BRENDA team