Information on EC 3.2.1.B1 - extracellular agarase

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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
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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
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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-
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
KCTC23886
UniProt
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
Cellvibrio sp.
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-
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
DSM25967T
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
strains ZP01, ZP02, ZP03, ZP05, ZP06, ZP08, and ZP09
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Manually annotated by BRENDA team
strain F-6
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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
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36.9% neoagarooctaose, 32% neoagarodecaose, 16,7% neoagarododecaose and small amounts of neoagarohexaose and neoagarotetraose
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?
agarose + H2O
neoagarooctaose + neoagarodecaose + neoagarododecaose + neoagarotetradecaose
show the reaction diagram
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in presence of high amount of enzyme, products are predominantly neoagarooctaose plus some neoagarohexaose and neoagarotetraose
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?
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
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-
-
-
?
carrageenan + H2O
?
show the reaction diagram
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-
-
-
?
chitin + H2O
?
show the reaction diagram
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-
-
-
?
neoagarodecaose + H2O
?
show the reaction diagram
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-
-
?
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
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-
?
neoagarododecaose + H2O
?
show the reaction diagram
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-
-
?
neoagarohexaitol + H2O
?
show the reaction diagram
neoagarohexaose + H2O
neoagarotetraose + neoagarobiose
show the reaction diagram
neoagarotetradecaose + H2O
?
show the reaction diagram
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-
-
?
neoagarotetraose + H2O
neoagarobiose
show the reaction diagram
porphyran + H2O
neoagarooctaose
show the reaction diagram
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main product, plus neoagarotetraose and neoagarohexaose
-
?
xylan + H2O
?
show the reaction diagram
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-
-
-
?
additional information
?
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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
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about 30% residual activity at 10% (v/v)
Benzene
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about 70% residual activity at 10% (v/v)
butan-1-ol
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about 30% residual activity at 10% (v/v)
Ca2+
complete inhibition at 5 mM
Chloroform
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about 25% residual activity at 10% (v/v)
Cyclohexane
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about 35% residual activity at 10% (v/v)
Dichloromethane
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about 20% residual activity at 10% (v/v)
DMSO
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about 40% residual activity at 10% (v/v)
ethenol
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about 30% residual activity at 10% (v/v)
heptane
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about 50% residual activity at 10% (v/v)
Isopropanol
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about 40% residual activity at 10% (v/v)
Li+
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about 80% residual activity at 1 mM
methanol
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about 35% residual activity at 10% (v/v)
Na+
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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+
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about 50% residual activity at 1 mM
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Pb2+
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7% residual activity at 1 mM
phenylmethylsulfonyl fluoride
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about 60% residual activity at 1 mM
Sn2+
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30% residual activity at 1 mM
Toluene
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about 40% residual activity at 10% (v/v)
Triton X-100
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58% residual activity at 10% (v/v)
Tween 20
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89.2% residual activity at 10% (v/v)
Tween 80
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60.2% residual activity at 10% (v/v)
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
beta-mercaptoethanol
123% activity at 10 mM
dithiothreitol
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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
25°C, pH 6.0
12.4
neoagarododecaose
25°C, pH 6.0
8.36
neoagarotetradecaose
25°C, pH 6.0
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.19
neoagarodecaose
Pseudoalteromonas sp.
A1A3Y9
25°C, pH 6.0
4.77
neoagarododecaose
Pseudoalteromonas sp.
A1A3Y9
25°C, pH 6.0
3.3 - 5.16
neoagarotetradecaose
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2
Ba2+
Bacillus sp.
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at pH 6.5 and 70°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.04
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culture supernatant, at pH 7.0 and 30°C
0.7
Cellvibrio sp.
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enzyme from culture fluid, at pH 7.0 and 35°C
4.22
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after 103.5fold purification, at pH 7.0 and 30°C
25.26
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crude extract, at pH 9.0 and 40°C
73.87
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crude enzyme, at pH 9.0 and 35°C
84.2
Cellvibrio sp.
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after 120.2fold purification, at pH 7.0 and 35°C
307.4
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agarase AG-b
615.5
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after 8.33fold purification, at pH 9.0 and 35°C
942
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isoform agarase-a, after 107fold purification, at pH 9.0 and 40°C
1338
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isoform agarase-b, after 52fold purification, at pH 9.0 and 40°C
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 9
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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.
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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
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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
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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
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agarase AG-b
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10 - 60
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about 30% activity at 10°C, about 40% activity at 20°C, about 70% activity at 30°C, 100% activity at 40°C, about 50% activity at 50°C, about 20% activity at 60°C
20 - 50
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about 70% activity at 20°C, about 85% activity at 30°C, 100% activity at 40°C, less than 20% activity at 50°C
25 - 55
the enzyme exhibits high activity at temperatures ranging between 25 and 45°C. The enzyme can sustain its enzymatic activity up to 90% of its peak activity at 45°C, while the relative activities rapidly decline to 20 and 3% at 50 and 55°C, respectively
25 - 70
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25°C: about 55% of maximal activity, 70°C: about 65% of maximal activity, agarase AG-b
40 - 50
Cellvibrio sp.
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more than 90% activity between 40°C and 50°C
40 - 60
40 - 50
optimal temperature is at 40°C, while activity is dramatically dropped to 28% at 45°C and completely lost at 50°C
50
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at 50°C, isoform agarase-a has 35.8% of its optimal activity
50 - 80
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the enzyme activity consistently increases from 55°C to 70°C with optimum activity at 70°C, and an obvious decrease is observed when the agarase is incubated at temperatures above 80°C
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
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x * 34500, agarase AG-b, SDS-PAGE
35000
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x * 35000, SDS-PAGE
40000
x * 40000, SDS-PAGE
46900
x * 46900, calculated from amino acid sequence
50000
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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
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x * 54000, SDS-PAGE
56000
-
x * 56000, native PAGE
58000
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x * 58000, SDS-PAGE
58700
x * 58700, calculated from amino acid sequence
66000
-
x * 66000, SDS-PAGE
70000
Cellvibrio sp.
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x * 70000, SDS-PAGE
75000
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x * 75000, SDS-PAGE
87613
x * 87613, calculated from amino acid sequence
90000
x * 90000, SDS-PAGE
107000
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x * 107000, isoform agarase-b, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
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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
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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 4.0–9.0 for 12 h at 4°C
732434
4 - 10
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4°C, 6 h, stable in the pH range of 4.0–10.0, agarase AG-b
699362
4 - 8
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677674
5.7 - 10.6
-
-
681563
12 - 13
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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
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the activity of agarase is stable at a low temperature and retains more than 90% of its activity up to a temperature of 40°C, after 30 min incubation
20 - 50
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isoform garase-a is stable at 30°C, while agarase-b is stable at 50°C. Agarase-a shows residual activity remaining above 70% after 60 min at 20–30°C. After incubation at 40°C for 60 min, the remaining activity decreases to 28%. Agarase-b shows more than 75% of the maximal activity at 50°C
20 - 80
100% activity between 20 and40°C, about 50% activity at 50°C, about 10% activity between 60-80°C (after incubation for 1 h)
30 - 40
the residual activity is above 79% at 30 and 35°C. The half-life is 56 min at 40°C
40 - 45
the enzyme is stable up to 40°C (100%), and retains more than 70% of its initial activity at 45°C after heat treatment for 30 min. The enzyme stability dramatically declines at 50°C to 19%
40
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half-life of mutant S2 is 350 min, which was 18.4fold longer than that of wild-type AgaB
45
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30 min, about 10% loss of activity, agarase AG-b
49
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melting temperature of wild-type enzyme is 49.2°C
50 - 80
the enzyme retains more than 80% agarolytic activity after being kept in 50°C for 1 h, 13% residual activity after incubation in 80°C for 1 h, and 12% residual activity if boiled for 5 min
50 - 70
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the agarase retains 62% of its activity after incubating at 50°C for 30 min. Further, 45% of the agarase activity is still retained at 60°C. The agarase activity is completely abolished at 70°C
54
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melting temperature of mutant enzyme S2 is 53.8°C
55
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30 min, about 20% loss of activity, agarase AG-b
65
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30 min, about 50% loss of activity, agarase AG-b
70
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30 min, about 90% loss of activity, agarase AG-b
80
-
more than 50% activity is retained at 80°C 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
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, pH 7.5 (phosphate buffer), 6 months, agarase AG-b loses 5% activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation and dialysis
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ammonium sulfate precipitation, DEAE column chromatography, and Q-Sepharose column chromatography
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ammonium sulfate precipitation, DEAE Sepharose column chromatography, and DEAE Sephadex A-50 gel filtration
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ammonium sulfate precipitation, Q-Sepharose column chromatography and Sephacryl S-200 gel filtration
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ammonium sulfate precipitation, Resource Q column chromatography, and Superdex 200 gel filtration
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ammonium sulfate precipitation, Toyopearl QAE-550C column chromatography, Toyopearl HW-55F column chromatography, and MonoQ column chromatography
Cellvibrio sp.
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glutathione Sepharose column chromatography, and gel filtration
Ni-NTA column chromatography
Ni-NTA column chromatography, and gel filtration
recombinant protein, purification from inclusion bodies
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ultrafiltration, DEAE-Sepharose column chromatography, and Sephacryl S-300HR gel filtration
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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
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expression in Escherichia coli
expression in Escherichia coli with C-terminal His-tag
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the mature agarase is highly expressed extracellularly in Escherichia coli BL21 (DE3) cells
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
L122Q
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mutant enzyme shows similar thermostability with wild-type AgaB. Specific activity 1.3fold higher than that of wild-type enzyme
N446I
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mutant enzyme has enhanced thermostability. 10-20% decrease of the specific activity compared to wild-type enzyme
N446L
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half-life of mutant enzyme at 40°C is 12.9fold longer than that of wild-type AgaB
N446V
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half-life of mutant enzyme at 40°C is 18.2fold longer than that of wild-type AgaB
additional information
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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 4.6°C higher than that of wild-type AgaB, and the half-life of S2 is 350 min at 40°C, 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 4°C
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
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use of enzyme for preparation of neoagarooctaose and neoagarodecaose and separation of neoagaro-oligosaccharides by consecutive column chromatography