BRENDA - Enzyme Database show
show all sequences of 1.14.12.3

Subtle difference between benzene and toluene dioxygenases of Pseudomonas putida

Bagneris, C.; Cammack, R.; Mason, J.R.; Appl. Environ. Microbiol. 71, 1570-1580 (2005)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
expressed in Escherichia coli JM109; expression of wild-type and mutant enzymes in Escherichia coli strains JM109 and CJ236
Pseudomonas putida
Engineering
Amino acid exchange
Commentary
Organism
A291S
reduced activity with ethylbenzene
Pseudomonas putida
E444D
no effect on activity
Pseudomonas putida
G404D
reduced activity with ethylbenzene
Pseudomonas putida
I301V
the mutation in the C-terminal part of subunit alpha enhances the substrate specificity for ethylbenzene, the mutant shows altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains
Pseudomonas putida
I301V/T305S/I307L/L309V
increased activity with ethylbenzene; the mutations in the C-terminal part of subunit alpha enhance the substrate specificity for ethylbenzene, the quadruple mutant also shows a high uncoupled rate of electron transfer without product formation
Pseudomonas putida
I307L
the mutation in the C-terminal part of subunit alpha enhances the substrate specificity for ethylbenzene, the mutant shows altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains
Pseudomonas putida
I412V
reduced activity with ethylbenzene
Pseudomonas putida
K436R
reduced activity with ethylbenzene
Pseudomonas putida
L285W
reduced activity with ethylbenzene
Pseudomonas putida
L285W/A291S/G404D
slightly reduced activity with ethylbenzene
Pseudomonas putida
L28W/A291S
reduced activity with ethylbenzene
Pseudomonas putida
L309V
the mutation in the C-terminal part of subunit alpha enhances the substrate specificity for ethylbenzene, the mutant shows altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains
Pseudomonas putida
additional information
construction of chimeric proteins and mutants of the benzene dioxygenase alpha subunit, the chimera are formed by benzene and toluene dioxygenases, the amino acid sequences of the alpha subunits of both enzymes differ at only 33 of 450 amino acids, these residues are primarily responsible for the change in specificity, the chimeric protein containing toluene dioxygenase C-terminal region residues 281 to 363 shows greater substrate preference for alkyl benzenes, identification of four amino acid substitutions in this region, I301V, T305S, I307L, and L309V, that particularly enhance the preference for ethylbenzene, structure modeling, overview
Pseudomonas putida
T305S
the mutation in the C-terminal part of subunit alpha enhances the substrate specificity for ethylbenzene, the mutant shows altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains
Pseudomonas putida
V324I/I327V
reduced activity with ethylbenzene
Pseudomonas putida
Metals/Ions
Metals/Ions
Commentary
Organism
Structure
Fe2+
the alpha subunit of the enzyme contains a catalytic [2Fe-2S] cluster involved in electron transfer, the catalytic nonheme iron center is also responsible for substrate specificity, overview
Pseudomonas putida
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
benzene + NADH + O2
Pseudomonas putida
bioremediation of aromatic environmental pollutants, initial step of benzene degradation
(1R,2S)-cis-cyclohexa-3,5-diene-1,2-diol + NAD+
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Pseudomonas putida
-
-
-
Reaction
Reaction
Commentary
Organism
benzene + NADH + H+ + O2 = cis-cyclohexa-3,5-diene-1,2-diol + NAD+
the catalytic iron-sulfur proteins of the enzyme consist of two dissimilar alpha and beta subunits, the alpha subunit contains a [2Fe-2S] cluster involved in electron transfer, the catalytic nonheme iron center, and is also responsible for substrate specificity
Pseudomonas putida
Specific Activity [micromol/min/mg]
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
0.025
-
with ethylbenzene as substrate
Pseudomonas putida
0.071
-
with toluene as substrate
Pseudomonas putida
0.115
-
with benzene as substrate
Pseudomonas putida
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
benzene + NADH + O2
-
657604
Pseudomonas putida
(1R,2S)-cis-cyclohexa-3,5-diene-1,2-diol + NAD+
-
-
-
?
benzene + NADH + O2
bioremediation of aromatic environmental pollutants, initial step of benzene degradation
657604
Pseudomonas putida
(1R,2S)-cis-cyclohexa-3,5-diene-1,2-diol + NAD+
-
-
-
?
benzene + O2 + NADH
-
657604
Pseudomonas putida
cis-cyclohexa-3,5-diene-1,2-diol + NAD+
-
-
-
?
ethylbenzene + NADH + O2
-
657604
Pseudomonas putida
cis-2,3-dihydroxy-1-ethyl-cyclohexa-4,6-diene + 1-phenethyl alcohol + NAD+
-
-
-
?
ethylbenzene + [reduced NADPH-hemoprotein reductase] + O2
-
657604
Pseudomonas putida
cis-2,3-dihydroxy-1-ethyl-cyclohexa-4,6-diene + [oxidized NADPH-hemoprotein reductase] + H2O
-
-
-
?
toluene + NADH + O2
-
657604
Pseudomonas putida
cis-2,3-dihydroxy-1-methyl-cyclohexa-4,6-diene + NAD+
-
-
-
?
Subunits
Subunits
Commentary
Organism
dimer
alpha, beta, iron sulfur protein component; the enzyme consist of two dissimilar alpha and beta subunits, alpha subunit structure modeling
Pseudomonas putida
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
22
-
assay at room temperature
Pseudomonas putida
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.2
-
assay at
Pseudomonas putida
Cofactor
Cofactor
Commentary
Organism
Structure
Ferredoxin
-
Pseudomonas putida
iron-sulfur centre
[2Fe-2S] cluster
Pseudomonas putida
NADH
-
Pseudomonas putida
Cloned(Commentary) (protein specific)
Commentary
Organism
expressed in Escherichia coli JM109; expression of wild-type and mutant enzymes in Escherichia coli strains JM109 and CJ236
Pseudomonas putida
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
Ferredoxin
-
Pseudomonas putida
iron-sulfur centre
[2Fe-2S] cluster
Pseudomonas putida
NADH
-
Pseudomonas putida
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
A291S
reduced activity with ethylbenzene
Pseudomonas putida
E444D
no effect on activity
Pseudomonas putida
G404D
reduced activity with ethylbenzene
Pseudomonas putida
I301V
the mutation in the C-terminal part of subunit alpha enhances the substrate specificity for ethylbenzene, the mutant shows altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains
Pseudomonas putida
I301V/T305S/I307L/L309V
increased activity with ethylbenzene; the mutations in the C-terminal part of subunit alpha enhance the substrate specificity for ethylbenzene, the quadruple mutant also shows a high uncoupled rate of electron transfer without product formation
Pseudomonas putida
I307L
the mutation in the C-terminal part of subunit alpha enhances the substrate specificity for ethylbenzene, the mutant shows altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains
Pseudomonas putida
I412V
reduced activity with ethylbenzene
Pseudomonas putida
K436R
reduced activity with ethylbenzene
Pseudomonas putida
L285W
reduced activity with ethylbenzene
Pseudomonas putida
L285W/A291S/G404D
slightly reduced activity with ethylbenzene
Pseudomonas putida
L28W/A291S
reduced activity with ethylbenzene
Pseudomonas putida
L309V
the mutation in the C-terminal part of subunit alpha enhances the substrate specificity for ethylbenzene, the mutant shows altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains
Pseudomonas putida
additional information
construction of chimeric proteins and mutants of the benzene dioxygenase alpha subunit, the chimera are formed by benzene and toluene dioxygenases, the amino acid sequences of the alpha subunits of both enzymes differ at only 33 of 450 amino acids, these residues are primarily responsible for the change in specificity, the chimeric protein containing toluene dioxygenase C-terminal region residues 281 to 363 shows greater substrate preference for alkyl benzenes, identification of four amino acid substitutions in this region, I301V, T305S, I307L, and L309V, that particularly enhance the preference for ethylbenzene, structure modeling, overview
Pseudomonas putida
T305S
the mutation in the C-terminal part of subunit alpha enhances the substrate specificity for ethylbenzene, the mutant shows altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains
Pseudomonas putida
V324I/I327V
reduced activity with ethylbenzene
Pseudomonas putida
Metals/Ions (protein specific)
Metals/Ions
Commentary
Organism
Structure
Fe2+
the alpha subunit of the enzyme contains a catalytic [2Fe-2S] cluster involved in electron transfer, the catalytic nonheme iron center is also responsible for substrate specificity, overview
Pseudomonas putida
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
benzene + NADH + O2
Pseudomonas putida
bioremediation of aromatic environmental pollutants, initial step of benzene degradation
(1R,2S)-cis-cyclohexa-3,5-diene-1,2-diol + NAD+
-
-
?
Specific Activity [micromol/min/mg] (protein specific)
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
0.025
-
with ethylbenzene as substrate
Pseudomonas putida
0.071
-
with toluene as substrate
Pseudomonas putida
0.115
-
with benzene as substrate
Pseudomonas putida
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
benzene + NADH + O2
-
657604
Pseudomonas putida
(1R,2S)-cis-cyclohexa-3,5-diene-1,2-diol + NAD+
-
-
-
?
benzene + NADH + O2
bioremediation of aromatic environmental pollutants, initial step of benzene degradation
657604
Pseudomonas putida
(1R,2S)-cis-cyclohexa-3,5-diene-1,2-diol + NAD+
-
-
-
?
benzene + O2 + NADH
-
657604
Pseudomonas putida
cis-cyclohexa-3,5-diene-1,2-diol + NAD+
-
-
-
?
ethylbenzene + NADH + O2
-
657604
Pseudomonas putida
cis-2,3-dihydroxy-1-ethyl-cyclohexa-4,6-diene + 1-phenethyl alcohol + NAD+
-
-
-
?
ethylbenzene + [reduced NADPH-hemoprotein reductase] + O2
-
657604
Pseudomonas putida
cis-2,3-dihydroxy-1-ethyl-cyclohexa-4,6-diene + [oxidized NADPH-hemoprotein reductase] + H2O
-
-
-
?
toluene + NADH + O2
-
657604
Pseudomonas putida
cis-2,3-dihydroxy-1-methyl-cyclohexa-4,6-diene + NAD+
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
dimer
alpha, beta, iron sulfur protein component; the enzyme consist of two dissimilar alpha and beta subunits, alpha subunit structure modeling
Pseudomonas putida
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
22
-
assay at room temperature
Pseudomonas putida
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.2
-
assay at
Pseudomonas putida
Other publictions for EC 1.14.12.3
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
711842
Di Gennaro
Development of microbial engin ...
Pseudomonas putida, Pseudomonas putida MST
Ecotoxicol. Environ. Saf.
74
542-549
2010
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1
1
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1
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4
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1
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1
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2
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1
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1
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657604
Bagneris
Subtle difference between benz ...
Pseudomonas putida
Appl. Environ. Microbiol.
71
1570-1580
2005
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-
1
-
15
-
-
-
-
1
-
1
-
2
-
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-
1
-
-
3
-
6
1
1
-
-
-
1
-
-
3
-
-
-
-
-
1
3
-
15
-
-
-
-
-
-
1
-
1
-
-
-
-
-
-
3
-
6
1
1
-
-
-
1
-
-
-
-
-
-
-
-
-
674962
Na
Isolation and characterization ...
Rhodococcus opacus
J. Biosci. Bioeng.
99
378-382
2005
-
-
-
-
-
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-
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4
-
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1
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1
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1
-
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-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
439072
Swift
Expression of benzene dioxygen ...
Pseudomonas putida, Pseudomonas putida ML2
Appl. Microbiol. Biotechnol.
55
721-726
2001
-
-
1
-
-
-
-
-
-
1
-
8
-
15
-
-
-
-
-
-
5
-
13
-
1
-
-
-
1
-
-
1
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
1
-
8
-
-
-
-
-
-
5
-
13
-
1
-
-
-
1
-
-
-
-
-
-
-
-
-
439070
Mason
Structural studies on the cata ...
Pseudomonas putida, Pseudomonas putida ML2
Biochem. Soc. Trans.
25
90-95
1997
-
-
1
-
6
-
-
-
-
-
1
2
-
12
-
-
-
-
-
-
-
-
4
1
-
-
-
-
-
-
-
1
-
-
-
-
-
1
1
-
6
-
-
-
-
-
-
-
1
2
-
-
-
-
-
-
-
-
4
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
439071
Shergill
EPR, ENDOR and ESEEM studies o ...
Pseudomonas putida, Pseudomonas putida ML2
Biochem. Soc. Trans.
22
288S
1994
-
-
1
-
-
-
-
-
-
1
-
-
-
11
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
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-
1
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
439069
Wackett
Benzene dioxygenase from Pseud ...
Pseudomonas putida, Pseudomonas putida ML2
Methods Enzymol.
188
52-60
1990
-
-
-
-
-
-
-
1
-
2
3
2
-
12
-
-
1
-
-
-
-
1
6
2
-
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-
3
-
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-
3
-
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-
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-
1
-
2
3
2
-
-
-
1
-
-
-
1
6
2
-
-
-
-
-
-
-
-
-
-
-
-
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-
439068
Morrice
Primary structure of protein B ...
Pseudomonas putida, Pseudomonas putida ML2
FEBS Lett.
231
336-340
1988
-
-
-
-
-
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-
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1
1
2
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13
-
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4
-
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1
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1
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1
1
2
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4
-
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439064
Zamanian
Benzene dioxygenase in Pseudom ...
Pseudomonas putida, Pseudomonas putida ML2
Biochem. J.
244
611-616
1987
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3
2
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13
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4
1
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1
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1
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3
2
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4
1
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439066
Geary
An investigation of the iron-s ...
Pseudomonas putida
Biochem. J.
217
667-673
1984
-
-
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2
1
1
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2
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2
1
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2
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2
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2
1
1
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2
1
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439065
Geary
Moessbauer spectroscopic studi ...
Pseudomonas putida
Biochem. J.
195
199-203
1981
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1
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1
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2
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2
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1
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1
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1
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1
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2
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439063
Crutcher
Properties of the iron--sulphu ...
Pseudomonas putida
Biochem. J.
177
393-400
1979
-
-
-
-
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-
1
3
1
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2
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1
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2
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1
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1
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1
3
1
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1
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2
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-
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439067
Axcell
Purification and some properti ...
Pseudomonas sp.
Biochem. J.
146
173-183
1975
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1
1
2
1
1
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1
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1
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1
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3
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2
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2
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1
1
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1
1
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1
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1
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3
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