BRENDA - Enzyme Database show
show all sequences of 3.1.1.84

Subunit stabilization and pegylation of cocaine esterase improves in vivo residence time

Narasimhan, D.; Collins, G.T.; Nance, M.R.; Nichols, J.; Edwald, E.; Chan, J.; Ko, M.C.; Woods, J.H.; Tesmer, J.J.; Sunahara, R.K.; Mol. Pharmacol. 80, 1056-1065 (2011)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
expressed in Escherichia coli BL-21 Gold (DE3) cells
Rhodococcus sp.
Crystallization (Commentary)
Crystallization
Organism
hanging drop vapor diffusion method, using 20% (w/v) PEG 3350, 100 mM 2-(N-morpholino)-ethane sulfonic acid, pH 6.0, and 1 M NaCl
Rhodococcus sp.
Engineering
Amino acid exchange
Commentary
Organism
G173Q/L169K
the mutant has a half-life of 370 min and 2.9 days at 37°C
Rhodococcus sp.
G4C/S10C
the mutant shows about 4fold reduced catalytic efficiency compared to the wild type enzyme. The mutant retains almost all activity after 7 days of 37°C treatment
Rhodococcus sp.
T172R/G173Q
the mutant shows about 4fold reduced catalytic efficiency compared to the wild type enzyme. The mutant remains more than 90% active for longer than 40 days at 37°C, representing a more than 4700fold improvement over wild type. PEGylated mutant enzyme retains full in vitro enzymatic activity
Rhodococcus sp.
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.0057
-
cocaine
wild type enzyme, pH not specified in the publication, 37°C
Rhodococcus sp.
0.019
-
cocaine
pegylated mutant enzyme T172R/G173Q, pH not specified in the publication, 37°C
Rhodococcus sp.
0.021
-
cocaine
mutant enzyme G4C/S10C, pH not specified in the publication, 37°C
Rhodococcus sp.
0.026
-
cocaine
mutant enzyme T172R/G173Q, pH not specified in the publication, 37°C
Rhodococcus sp.
Molecular Weight [Da]
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
127000
-
-
Rhodococcus sp.
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Rhodococcus sp.
-
-
-
Purification (Commentary)
Commentary
Organism
Talon metal chelate column chromatography and Q-Sepharose column chromatography
Rhodococcus sp.
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
cocaine + H2O
-
716281
Rhodococcus sp.
ecgonine methyl ester + benzoate
-
-
-
?
Subunits
Subunits
Commentary
Organism
homodimer
x-ray crystallography
Rhodococcus sp.
Temperature Stability [°C]
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
37
-
the wild type enzyme has a half-life of 12.2 min at 37°C
Rhodococcus sp.
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
40.1
-
cocaine
pegylated mutant enzyme T172R/G173Q, pH not specified in the publication, 37°C
Rhodococcus sp.
49.9
-
cocaine
mutant enzyme G4C/S10C, pH not specified in the publication, 37°C
Rhodococcus sp.
51.4
-
cocaine
wild type enzyme, pH not specified in the publication, 37°C
Rhodococcus sp.
56.6
-
cocaine
mutant enzyme T172R/G173Q, pH not specified in the publication, 37°C
Rhodococcus sp.
Cloned(Commentary) (protein specific)
Commentary
Organism
expressed in Escherichia coli BL-21 Gold (DE3) cells
Rhodococcus sp.
Crystallization (Commentary) (protein specific)
Crystallization
Organism
hanging drop vapor diffusion method, using 20% (w/v) PEG 3350, 100 mM 2-(N-morpholino)-ethane sulfonic acid, pH 6.0, and 1 M NaCl
Rhodococcus sp.
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
G173Q/L169K
the mutant has a half-life of 370 min and 2.9 days at 37°C
Rhodococcus sp.
G4C/S10C
the mutant shows about 4fold reduced catalytic efficiency compared to the wild type enzyme. The mutant retains almost all activity after 7 days of 37°C treatment
Rhodococcus sp.
T172R/G173Q
the mutant shows about 4fold reduced catalytic efficiency compared to the wild type enzyme. The mutant remains more than 90% active for longer than 40 days at 37°C, representing a more than 4700fold improvement over wild type. PEGylated mutant enzyme retains full in vitro enzymatic activity
Rhodococcus sp.
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.0057
-
cocaine
wild type enzyme, pH not specified in the publication, 37°C
Rhodococcus sp.
0.019
-
cocaine
pegylated mutant enzyme T172R/G173Q, pH not specified in the publication, 37°C
Rhodococcus sp.
0.021
-
cocaine
mutant enzyme G4C/S10C, pH not specified in the publication, 37°C
Rhodococcus sp.
0.026
-
cocaine
mutant enzyme T172R/G173Q, pH not specified in the publication, 37°C
Rhodococcus sp.
Molecular Weight [Da] (protein specific)
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
127000
-
-
Rhodococcus sp.
Purification (Commentary) (protein specific)
Commentary
Organism
Talon metal chelate column chromatography and Q-Sepharose column chromatography
Rhodococcus sp.
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
cocaine + H2O
-
716281
Rhodococcus sp.
ecgonine methyl ester + benzoate
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
homodimer
x-ray crystallography
Rhodococcus sp.
Temperature Stability [°C] (protein specific)
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
37
-
the wild type enzyme has a half-life of 12.2 min at 37°C
Rhodococcus sp.
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
40.1
-
cocaine
pegylated mutant enzyme T172R/G173Q, pH not specified in the publication, 37°C
Rhodococcus sp.
49.9
-
cocaine
mutant enzyme G4C/S10C, pH not specified in the publication, 37°C
Rhodococcus sp.
51.4
-
cocaine
wild type enzyme, pH not specified in the publication, 37°C
Rhodococcus sp.
56.6
-
cocaine
mutant enzyme T172R/G173Q, pH not specified in the publication, 37°C
Rhodococcus sp.
KCat/KM [mM/s]
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
2110
-
cocaine
pegylated mutant enzyme T172R/G173Q, pH not specified in the publication, 37°C
Rhodococcus sp.
2120
-
cocaine
mutant enzyme T172R/G173Q, pH not specified in the publication, 37°C
Rhodococcus sp.
2380
-
cocaine
mutant enzyme G4C/S10C, pH not specified in the publication, 37°C
Rhodococcus sp.
8990
-
cocaine
wild type enzyme, pH not specified in the publication, 37°C
Rhodococcus sp.
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
2110
-
cocaine
pegylated mutant enzyme T172R/G173Q, pH not specified in the publication, 37°C
Rhodococcus sp.
2120
-
cocaine
mutant enzyme T172R/G173Q, pH not specified in the publication, 37°C
Rhodococcus sp.
2380
-
cocaine
mutant enzyme G4C/S10C, pH not specified in the publication, 37°C
Rhodococcus sp.
8990
-
cocaine
wild type enzyme, pH not specified in the publication, 37°C
Rhodococcus sp.
Other publictions for EC 3.1.1.84
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)
750291
Kim
Kinetic characterization of c ...
Homo sapiens
Chem. Biol. Interact.
293
107-114
2018
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1
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3
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3
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3
1
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1
3
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3
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5
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1
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3
1
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1
1
-
3
3
728856
Fang
Rational design, preparation, ...
Rhodococcus sp., Rhodococcus sp. MB1 Bresler
ACS Chem. Biol.
9
1764-1772
2014
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1
1
3
-
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3
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2
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3
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1
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2
1
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3
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1
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1
3
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3
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2
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1
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2
1
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3
-
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-
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-
3
3
730250
Brim
The fate of bacterial cocaine ...
Rhodococcus sp.
J. Pharmacol. Exp. Ther.
340
83-95
2012
-
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1
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-
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1
1
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-
-
-
-
-
-
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-
730421
Lee
Cell permeable cocaine esteras ...
Rhodococcus sp., Rhodococcus sp. MB1
Mol. Pharm.
9
1361-1373
2012
-
-
1
-
5
-
-
6
-
-
1
2
-
6
-
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1
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2
1
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1
6
-
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1
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5
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6
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1
2
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1
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2
1
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1
6
-
-
-
-
-
-
-
-
6
6
716278
Xue
Design, preparation, and chara ...
Homo sapiens
Mol. Pharmacol.
79
290-297
2011
-
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1
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3
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4
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4
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1
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3
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4
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1
-
1
-
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1
-
-
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4
-
-
-
-
-
-
-
-
4
4
716281
Narasimhan
Subunit stabilization and pegy ...
Rhodococcus sp.
Mol. Pharmacol.
80
1056-1065
2011
-
-
1
1
3
-
-
4
-
-
1
-
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3
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1
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1
1
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1
4
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1
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3
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4
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1
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1
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1
1
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1
4
-
-
-
-
-
-
-
-
4
4
714226
Zheng
Design of high-activity mutant ...
Homo sapiens
Biochemistry
49
9113-9119
2010
-
-
1
1
10
-
-
-
-
-
-
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-
1
-
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1
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1
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1
10
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1
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-
9
9
714702
Yang
Characterization of a high-act ...
Homo sapiens
Chem. Biol. Interact.
187
148-152
2010
-
-
-
-
1
-
-
2
-
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1
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1
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2
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1
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2
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-
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2
2
716276
Brim
A thermally stable form of bac ...
Rhodococcus sp., Rhodococcus sp. MB1
Mol. Pharmacol.
77
593-600
2010
-
1
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1
1
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-
4
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7
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1
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2
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1
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716824
Narasimhan
Structural analysis of thermos ...
Rhodococcus sp., Rhodococcus sp. MB1
Protein Eng.
23
537-547
2010
-
1
1
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5
-
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4
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6
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1
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2
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1
5
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5
5
692028
Ko
Effects of cocaine esterase fo ...
Rhodococcus sp.
Drug Alcohol Depend.
101
202-209
2009
-
1
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3
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694267
Gao
Thermostable variants of cocai ...
Rhodococcus sp.
Mol. Pharmacol.
75
318-323
2009
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1
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4
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3
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1
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1
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1
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1
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699760
Collins
Zhan. C.G.; Woods. J.H.: Cocai ...
Rhodococcus sp.
J. Pharmacol. Exp. Ther.
331
445-455
2009
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1
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4
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698483
Liu
Fundamental reaction mechanism ...
Rhodococcus sp.
J. Am. Chem. Soc.
131
11964-11975
2003
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650108
Turner
Biochemical characterization a ...
Rhodococcus sp.
Biochemistry
41
12297-12307
2002
-
1
1
1
11
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10
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11
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9
1
1
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653322
Larsen
Crystal structure of a bacteri ...
Rhodococcus sp.
Nat. Struct. Biol.
9
17-21
2002
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695729
Bresler
Gene cloning and nucleotide se ...
Rhodococcus sp., Rhodococcus sp. MB1
Appl. Environ. Microbiol.
66
904-908
2000
-
-
1
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1
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2
2
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11
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1
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1
1
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6
1
1
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1
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2
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1
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6
1
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1
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-
171019
Pindel
Purification and cloning of a ...
Homo sapiens
J. Biol. Chem.
272
14769-14775
1997
-
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1
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1
4
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2
1
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1
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1
1
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1
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1
1
4
-
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2
1
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1
1
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1
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5
1
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1
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-
-
698540
Britt
Identification of a cocaine es ...
Stenotrophomonas maltophilia, Stenotrophomonas maltophilia MB11L
J. Bacteriol.
174
2087-2094
1992
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