Information on EC 3.2.1.B1 - extracellular agarase

Word Map on EC 3.2.1.B1
Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Select one or more organisms in this record:
Show additional data
Do not include text mining results
Include (text mining) results (more...)
Include results (AMENDA + additional results, but less precise; more...)

The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.2.1.B1
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
extracellular agarase
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
hydrolysis of 1,3-beta-D-galactosidic linkages in agarose, giving the octamer as the predominant product
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
agarose 8-glycanohydrolase
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
KCTC23886
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Cellvibrio sp.
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
DSM25967T
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strains ZP01, ZP02, ZP03, ZP05, ZP06, ZP08, and ZP09
-
-
Manually annotated by BRENDA team
strain F-6
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
4-nitrophenyl alpha-D-galactopyranoside + H2O
4-nitrophenol + alpha-D-galactopyranose
show the reaction diagram
4-nitrophenyl beta-D-galactopyranoside + H2O
4-nitrophenol + beta-D-galactopyranose
show the reaction diagram
agar + H2O
neoagarobiose
show the reaction diagram
-
-
-
-
?
agar + H2O
neoagarobiose + neoagarotetraose + neoagarohexaose
show the reaction diagram
agarose + H2O
?
show the reaction diagram
agarose + H2O
neoagarobiose + neoagarohexaose + neoagarotetraose
show the reaction diagram
agarose + H2O
neoagarobiose + neoagarotetraose
show the reaction diagram
agarose + H2O
neoagarobiose + neoagarotetraose + neoagarohexaose
show the reaction diagram
agarose + H2O
neoagarohexaose + neoagarotetraose + neoagarobiose
show the reaction diagram
agarose + H2O
neoagarooctaose + neoagarodecaose
show the reaction diagram
agarose + H2O
neoagarooctaose + neoagarodecaose + neoagarododecaose
show the reaction diagram
-
36.9% neoagarooctaose, 32% neoagarodecaose, 16,7% neoagarododecaose and small amounts of neoagarohexaose and neoagarotetraose
-
?
agarose + H2O
neoagarooctaose + neoagarodecaose + neoagarododecaose + neoagarotetradecaose
show the reaction diagram
-
in presence of high amount of enzyme, products are predominantly neoagarooctaose plus some neoagarohexaose and neoagarotetraose
-
?
agarose + H2O
neoagarooctaose + neoagarohexaose
show the reaction diagram
agarose + H2O
neoagarotetraose + neoagarohexaose
show the reaction diagram
agarose + H2O
neoagarotetraose + neoagarohexaose + ?
show the reaction diagram
agarose + H2O
neoagarotetraose + neoagarohexaose + neoagarooctaose
show the reaction diagram
agarose + H2O
neoagarotetraose + neoagarohexaose + neoagarooctaose + neoagarodecaose
show the reaction diagram
alginate + H2O
?
show the reaction diagram
-
-
-
-
?
carboxymethyl cellulose + H2O
?
show the reaction diagram
-
-
-
-
?
carrageenan + H2O
?
show the reaction diagram
-
-
-
-
?
chitin + H2O
?
show the reaction diagram
-
-
-
-
?
neoagarodecaose + H2O
?
show the reaction diagram
-
-
-
?
neoagarodecaose + H2O
neoagarohexaose + neoagarotetraose
show the reaction diagram
enzyme attacks preferentially the sixth linkage and subsequently the eighth linkage from the non-reducing end of neoagarodecaose
-
-
?
neoagarododecaose + H2O
?
show the reaction diagram
-
-
-
?
neoagarohexaitol + H2O
?
show the reaction diagram
neoagarohexaose + H2O
neoagarotetraose + neoagarobiose
show the reaction diagram
neoagarotetradecaose + H2O
?
show the reaction diagram
-
-
-
?
neoagarotetraose + H2O
neoagarobiose
show the reaction diagram
porphyran + H2O
neoagarooctaose
show the reaction diagram
-
main product, plus neoagarotetraose and neoagarohexaose
-
?
xylan + H2O
?
show the reaction diagram
-
-
-
-
?
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
agar + H2O
neoagarobiose
show the reaction diagram
-
-
-
-
?
agar + H2O
neoagarobiose + neoagarotetraose + neoagarohexaose
show the reaction diagram
agarose + H2O
?
show the reaction diagram
agarose + H2O
neoagarobiose + neoagarohexaose + neoagarotetraose
show the reaction diagram
agarose + H2O
neoagarobiose + neoagarotetraose
show the reaction diagram
agarose + H2O
neoagarobiose + neoagarotetraose + neoagarohexaose
show the reaction diagram
agarose + H2O
neoagarohexaose + neoagarotetraose + neoagarobiose
show the reaction diagram
agarose + H2O
neoagarotetraose + neoagarohexaose + ?
show the reaction diagram
agarose + H2O
neoagarotetraose + neoagarohexaose + neoagarooctaose
show the reaction diagram
agarose + H2O
neoagarotetraose + neoagarohexaose + neoagarooctaose + neoagarodecaose
show the reaction diagram
neoagarotetraose + H2O
neoagarobiose
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
125% activity at 1 mM
Cu2+
-
1 mM, increases activity of agarase AG-b 1.2fold
Sr2+
-
400% activity at 2 mM
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5,5'-dithiobis-(2-nitrobenzoic acid)
69% residual activity at 1 mM
acetone
-
about 30% residual activity at 10% (v/v)
Benzene
-
about 70% residual activity at 10% (v/v)
butan-1-ol
-
about 30% residual activity at 10% (v/v)
Ca2+
complete inhibition at 5 mM
Chloroform
-
about 25% residual activity at 10% (v/v)
Cyclohexane
-
about 35% residual activity at 10% (v/v)
Dichloromethane
-
about 20% residual activity at 10% (v/v)
DMSO
-
about 40% residual activity at 10% (v/v)
ethenol
-
about 30% residual activity at 10% (v/v)
heptane
-
about 50% residual activity at 10% (v/v)
Isopropanol
-
about 40% residual activity at 10% (v/v)
Li+
-
about 80% residual activity at 1 mM
methanol
-
about 35% residual activity at 10% (v/v)
Na+
-
60% residual activity at 1 mM, 72% residual activity at 10 mM, 91% residual activity at 100 mM, 83% residual activity at 1 M, complete inhibition at 3 M
Ni2+
1% residual activity at 10 mM
Pb+
-
about 50% residual activity at 1 mM
-
Pb2+
-
7% residual activity at 1 mM
phenylmethylsulfonyl fluoride
-
about 60% residual activity at 1 mM
Sn2+
-
30% residual activity at 1 mM
Toluene
-
about 40% residual activity at 10% (v/v)
Triton X-100
-
58% residual activity at 10% (v/v)
Tween 20
-
89.2% residual activity at 10% (v/v)
Tween 80
-
60.2% residual activity at 10% (v/v)
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
beta-mercaptoethanol
123% activity at 10 mM
dithiothreitol
-
at 2 mM, isoforms agarase-a and agarase-b show 117.47% and 113.44% activity, respectively
DL-dithiothreitol
121% activity at 10 mM
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
12.8
neoagarodecaose
25C, pH 6.0
12.4
neoagarododecaose
25C, pH 6.0
8.36
neoagarotetradecaose
25C, pH 6.0
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.19
neoagarodecaose
Pseudoalteromonas sp.
A1A3Y9
25C, pH 6.0
4.77
neoagarododecaose
Pseudoalteromonas sp.
A1A3Y9
25C, pH 6.0
3.3 - 5.16
neoagarotetradecaose
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2
Ba2+
Bacillus sp.
-
at pH 6.5 and 70C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.04
-
culture supernatant, at pH 7.0 and 30C
0.7
Cellvibrio sp.
-
enzyme from culture fluid, at pH 7.0 and 35C
4.22
-
after 103.5fold purification, at pH 7.0 and 30C
25.26
-
crude extract, at pH 9.0 and 40C
73.87
-
crude enzyme, at pH 9.0 and 35C
84.2
Cellvibrio sp.
-
after 120.2fold purification, at pH 7.0 and 35C
307.4
-
agarase AG-b
615.5
-
after 8.33fold purification, at pH 9.0 and 35C
942
-
isoform agarase-a, after 107fold purification, at pH 9.0 and 40C
1338
-
isoform agarase-b, after 52fold purification, at pH 9.0 and 40C
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 9
-
about 15% activity at pH 4.0, about 30% activity at pH 5.0, 100% activity at pH 6.0, about 60% activity at pH 7.0, about 45% activity at pH 8.0, about 40% activity at pH 9.0
5.5 - 7.5
Cellvibrio sp.
-
more than 90% activity between pH 5.5 and pH 7.6
5.7 - 10.6
more than 95% of maximum acitivity
6 - 9
the enzyme retains more than 42% of its activity at pH 8.0. The enzyme exhibits low activity at both acidic (pH 6.0) and basic (pH 9.0) conditions
6 - 10
8 - 10
-
above 90% activity is obtained at pH 8.0 and 9.0. Less than 60% of the maximum activity is detected at pH 10.0
10 - 11
-
the enzyme shows residual activities of about 85.2 and 78.2% at pH 10 and 11.0
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55
-
agarase AG-b
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10 - 60
-
about 30% activity at 10C, about 40% activity at 20C, about 70% activity at 30C, 100% activity at 40C, about 50% activity at 50C, about 20% activity at 60C
20 - 50
-
about 70% activity at 20C, about 85% activity at 30C, 100% activity at 40C, less than 20% activity at 50C
25 - 55
the enzyme exhibits high activity at temperatures ranging between 25 and 45C. The enzyme can sustain its enzymatic activity up to 90% of its peak activity at 45C, while the relative activities rapidly decline to 20 and 3% at 50 and 55C, respectively
25 - 70
-
25C: about 55% of maximal activity, 70C: about 65% of maximal activity, agarase AG-b
40 - 50
Cellvibrio sp.
-
more than 90% activity between 40C and 50C
40 - 60
40 - 50
optimal temperature is at 40C, while activity is dramatically dropped to 28% at 45C and completely lost at 50C
50
-
at 50C, isoform agarase-a has 35.8% of its optimal activity
50 - 80
-
the enzyme activity consistently increases from 55C to 70C with optimum activity at 70C, and an obvious decrease is observed when the agarase is incubated at temperatures above 80C
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.6
calculated from amino acid sequence
5.1
calculated from amino acid sequence
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
34500
-
x * 34500, agarase AG-b, SDS-PAGE
35000
-
x * 35000, SDS-PAGE
40000
x * 40000, SDS-PAGE
46900
x * 46900, calculated from amino acid sequence
50000
-
x * 50000, isoform agarase-a, SDS-PAGE
50400
x * 47200, calculated, x * 50400, SDS-PAGE
51200
x * 51200, calculated from amino acid sequence
53000
-
x * 53000, SDS-PAGE
54000
-
x * 54000, SDS-PAGE
56000
-
x * 56000, native PAGE
58000
-
x * 58000, SDS-PAGE
58700
x * 58700, calculated from amino acid sequence
66000
-
x * 66000, SDS-PAGE
70000
Cellvibrio sp.
-
x * 70000, SDS-PAGE
75000
-
x * 75000, SDS-PAGE
87613
x * 87613, calculated from amino acid sequence
90000
x * 90000, SDS-PAGE
107000
-
x * 107000, isoform agarase-b, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
-
the enzyme shows about 15% N-linked glycosylation
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3 - 11
the enzyme still retains more than 95% activity after incubation at pH 3.0-11.0 for 1 h
705482
4 - 10
-
isoform agarase-a is stable over a wide pH range of 4.0-10.0 with more than 60% activity. Isoform agarase-b retains more than 60% activity over a pH range of 5.0-10.0
732848
4 - 9
the enzyme retains more than 80% of activity after incubation at a wide range of 4.09.0 for 12 h at 4C
732434
4 - 10
-
4C, 6 h, stable in the pH range of 4.010.0, agarase AG-b
699362
4 - 8
-
677674
5.7 - 10.6
-
-
681563
12 - 13
-
the enzyme retains about 62% of its activity at pH 12.0 and almost 50% at pH 13.0 after a pre-incubation of 1 h
731420
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10 - 40
-
the activity of agarase is stable at a low temperature and retains more than 90% of its activity up to a temperature of 40C, after 30 min incubation
20 - 50
-
isoform garase-a is stable at 30C, while agarase-b is stable at 50C. Agarase-a shows residual activity remaining above 70% after 60 min at 2030C. After incubation at 40C for 60 min, the remaining activity decreases to 28%. Agarase-b shows more than 75% of the maximal activity at 50C
20 - 80
100% activity between 20 and40C, about 50% activity at 50C, about 10% activity between 60-80C (after incubation for 1 h)
30 - 40
the residual activity is above 79% at 30 and 35C. The half-life is 56 min at 40C
40 - 45
the enzyme is stable up to 40C (100%), and retains more than 70% of its initial activity at 45C after heat treatment for 30 min. The enzyme stability dramatically declines at 50C to 19%
40
-
half-life of mutant S2 is 350 min, which was 18.4fold longer than that of wild-type AgaB
45
-
30 min, about 10% loss of activity, agarase AG-b
49
-
melting temperature of wild-type enzyme is 49.2C
50 - 80
the enzyme retains more than 80% agarolytic activity after being kept in 50C for 1 h, 13% residual activity after incubation in 80C for 1 h, and 12% residual activity if boiled for 5 min
50 - 70
-
the agarase retains 62% of its activity after incubating at 50C for 30 min. Further, 45% of the agarase activity is still retained at 60C. The agarase activity is completely abolished at 70C
54
-
melting temperature of mutant enzyme S2 is 53.8C
55
-
30 min, about 20% loss of activity, agarase AG-b
65
-
30 min, about 50% loss of activity, agarase AG-b
70
-
30 min, about 90% loss of activity, agarase AG-b
80
-
more than 50% activity is retained at 80C for 15 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the enzyme remains stable in the presence of 50-200 mM NaCl (more than 80% activity)
the enzyme remains stable in the presence of up to 4 M NaCl
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, pH 7.5 (phosphate buffer), 6 months, agarase AG-b loses 5% activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation and dialysis
-
ammonium sulfate precipitation, DEAE column chromatography, and Q-Sepharose column chromatography
-
ammonium sulfate precipitation, DEAE Sepharose column chromatography, and DEAE Sephadex A-50 gel filtration
-
ammonium sulfate precipitation, Q-Sepharose column chromatography and Sephacryl S-200 gel filtration
-
ammonium sulfate precipitation, Resource Q column chromatography, and Superdex 200 gel filtration
-
ammonium sulfate precipitation, Toyopearl QAE-550C column chromatography, Toyopearl HW-55F column chromatography, and MonoQ column chromatography
Cellvibrio sp.
-
glutathione Sepharose column chromatography, and gel filtration
Ni-NTA column chromatography
Ni-NTA column chromatography, and gel filtration
recombinant protein, purification from inclusion bodies
-
ultrafiltration, DEAE-Sepharose column chromatography, and Sephacryl S-300HR gel filtration
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21 (DE3) pLysS cells
expressed in Escherichia coli BL21(DE3) cells
expressed in Pichia pastoris strain GS115
-
expression in Escherichia coli
expression in Escherichia coli with C-terminal His-tag
-
the mature agarase is highly expressed extracellularly in Escherichia coli BL21 (DE3) cells
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
L122Q
-
mutant enzyme shows similar thermostability with wild-type AgaB. Specific activity 1.3fold higher than that of wild-type enzyme
N446I
-
mutant enzyme has enhanced thermostability. 10-20% decrease of the specific activity compared to wild-type enzyme
N446L
-
half-life of mutant enzyme at 40C is 12.9fold longer than that of wild-type AgaB
N446V
-
half-life of mutant enzyme at 40C is 18.2fold longer than that of wild-type AgaB
additional information
-
to increase the thermostability of beta-agarase AgaB by directed evolution, the mutant gene libraries are generated by error-prone polymerase chain reaction and deoxyribonucleic acid shuffling. A mutant S2 is obtained through two rounds of error-prone polymerase chain reaction and a single round of DNA shuffling and selection. It has higher thermostability and slightly increased catalytic activity than wild-type AgaB. Melting temperature (Tm) of S2, as determined by circular dichroism, is 4.6C higher than that of wild-type AgaB, and the half-life of S2 is 350 min at 40C, which is 18.4-fold longer than that of the wild-type enzyme. Saturation mutagenesis and hydrophobic cluster analysis indicate that hydrophobic interaction might be the key factor that enhances the enzyme stability
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
refolding of enzyme from aggregates in inclusion bodies is optimal in buffer containing 20 mM phosphate, 1 M urea, 500 mM L-arginine, and 10% glycerol, pH 7.0, at 4C
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
-
use of enzyme for preparation of neoagarooctaose and neoagarodecaose and separation of neoagaro-oligosaccharides by consecutive column chromatography