Enhancement of proteolytic activity of a thermostable papain-like protease by structure-based rational design
Dutta, S.; Dattagupta, J.K.; Biswas, S.; PLoS ONE 8, e62619 (2013) 
Data extracted from this reference:
Activating Compound
Activating Compound
Commentary
Organism
Structure
L-cysteine
20 mM used in assay conditions
Tabernaemontana divaricata
Cloned(Commentary)
Cloned (Commentary)
Organism
expressed in Escherichia coli BL21(DE3) cells
Tabernaemontana divaricata
Engineering
Protein Variants
Commentary
Organism
A67Y
the mutation enhances the catalytic efficiency of the enzyme about 8fold while the thermostability of the mutant enzyme remains unchanged. The specific activity against azo-casein is almost 2.4 times higher than wild type
Tabernaemontana divaricata
S32T
the mutation enhances the catalytic efficiency of the enzyme about 8fold while the thermostability of the mutant enzyme remains unchanged
Tabernaemontana divaricata
S32T/A67Y
the mutations enhance the catalytic efficiency of the enzyme about 8fold while the thermostability of the mutant enzyme remains unchanged
Tabernaemontana divaricata
Inhibitors
Inhibitors
Commentary
Organism
Structure
E-64
-
Tabernaemontana divaricata
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.09687
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
mutant enzyme S32T/A67Y, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
0.1273
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
wild type enzyme, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
0.1499
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
mutant enzyme S32T, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
0.1769
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
mutant enzyme A67Y, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
Molecular Weight [Da]
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
40000
-
x * 40000, SDS-PAGE
Tabernaemontana divaricata
Organism
Organism
UniProt
Commentary
Textmining
Tabernaemontana divaricata
P83654
-
-
Purification (Commentary)
Purification (Commentary)
Organism
Ni-NTA column chromatography
Tabernaemontana divaricata
Renatured (Commentary)
Renatured (Commentary)
Organism
dialysis
Tabernaemontana divaricata
Specific Activity [micromol/min/mg]
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
30.78
-
wild type enzyme, with azocasein as substrate, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
63.11
-
mutant enzyme S32T/A67Y, with azocasein as substrate, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
73.63
-
mutant enzyme S32T, with azocasein as substrate, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
86.95
-
mutant enzyme A67Y, with azocasein as substrate, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
Substrate Product ID
azocasein + H2O
-
732725
Tabernaemontana divaricata
?
-
-
-
?
Gelatin + H2O
-
732725
Tabernaemontana divaricata
?
-
-
-
?
N-benzoyl-Phe-Val-Arg-4-nitroanilide + H2O
-
732725
Tabernaemontana divaricata
N-benzoyl-Phe-Val-Arg + 4-nitroaniline
-
-
-
?
Subunits
Subunits
Commentary
Organism
?
x * 40000, SDS-PAGE
Tabernaemontana divaricata
Synonyms
Synonyms
Commentary
Organism
Erv-C
-
Tabernaemontana divaricata
ervatamin-C
-
Tabernaemontana divaricata
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
70
-
-
Tabernaemontana divaricata
Temperature Stability [°C]
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
40
80
after 10 incubation, the enzyme shows 100% activity between 40 and 55°C, about 97% activity at 60°C, about 65% activity at 70°C, 50% activity at 72°C, about 25% activity at 75°C, and no activity at 80°C
Tabernaemontana divaricata
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.23
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
wild type enzyme, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
1.43
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
mutant enzyme S32T/A67Y, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
2.23
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
mutant enzyme S32T, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
2.65
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
mutant enzyme A67Y, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
6.5
7.5
-
Tabernaemontana divaricata
pH Range
pH Minimum
pH Maximum
Commentary
Organism
4.5
8
more than 50% activity between pH 4.5 and 8.0
Tabernaemontana divaricata
IC50 Value
IC50 Value
IC50 Value Maximum
Commentary
Organism
Inhibitor
Structure
0.0001712
-
mutant enzyme S32T/A67Y, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
E-64
0.0002137
-
mutant enzyme S32T, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
E-64
0.0002507
-
mutant enzyme A67Y, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
E-64
0.000349
-
wild type enzyme, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
E-64
Activating Compound (protein specific)
Activating Compound
Commentary
Organism
Structure
L-cysteine
20 mM used in assay conditions
Tabernaemontana divaricata
Cloned(Commentary) (protein specific)
Commentary
Organism
expressed in Escherichia coli BL21(DE3) cells
Tabernaemontana divaricata
Engineering (protein specific)
Protein Variants
Commentary
Organism
A67Y
the mutation enhances the catalytic efficiency of the enzyme about 8fold while the thermostability of the mutant enzyme remains unchanged. The specific activity against azo-casein is almost 2.4 times higher than wild type
Tabernaemontana divaricata
S32T
the mutation enhances the catalytic efficiency of the enzyme about 8fold while the thermostability of the mutant enzyme remains unchanged
Tabernaemontana divaricata
S32T/A67Y
the mutations enhance the catalytic efficiency of the enzyme about 8fold while the thermostability of the mutant enzyme remains unchanged
Tabernaemontana divaricata
IC50 Value (protein specific)
IC50 Value
IC50 Value Maximum
Commentary
Organism
Inhibitor
Structure
0.0001712
-
mutant enzyme S32T/A67Y, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
E-64
0.0002137
-
mutant enzyme S32T, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
E-64
0.0002507
-
mutant enzyme A67Y, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
E-64
0.000349
-
wild type enzyme, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
E-64
Inhibitors (protein specific)
Inhibitors
Commentary
Organism
Structure
E-64
-
Tabernaemontana divaricata
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.09687
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
mutant enzyme S32T/A67Y, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
0.1273
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
wild type enzyme, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
0.1499
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
mutant enzyme S32T, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
0.1769
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
mutant enzyme A67Y, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
Molecular Weight [Da] (protein specific)
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
40000
-
x * 40000, SDS-PAGE
Tabernaemontana divaricata
Purification (Commentary) (protein specific)
Commentary
Organism
Ni-NTA column chromatography
Tabernaemontana divaricata
Renatured (Commentary) (protein specific)
Commentary
Organism
dialysis
Tabernaemontana divaricata
Specific Activity [micromol/min/mg] (protein specific)
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
30.78
-
wild type enzyme, with azocasein as substrate, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
63.11
-
mutant enzyme S32T/A67Y, with azocasein as substrate, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
73.63
-
mutant enzyme S32T, with azocasein as substrate, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
86.95
-
mutant enzyme A67Y, with azocasein as substrate, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ID
azocasein + H2O
-
732725
Tabernaemontana divaricata
?
-
-
-
?
Gelatin + H2O
-
732725
Tabernaemontana divaricata
?
-
-
-
?
N-benzoyl-Phe-Val-Arg-4-nitroanilide + H2O
-
732725
Tabernaemontana divaricata
N-benzoyl-Phe-Val-Arg + 4-nitroaniline
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
?
x * 40000, SDS-PAGE
Tabernaemontana divaricata
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
70
-
-
Tabernaemontana divaricata
Temperature Stability [°C] (protein specific)
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
40
80
after 10 incubation, the enzyme shows 100% activity between 40 and 55°C, about 97% activity at 60°C, about 65% activity at 70°C, 50% activity at 72°C, about 25% activity at 75°C, and no activity at 80°C
Tabernaemontana divaricata
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.23
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
wild type enzyme, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
1.43
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
mutant enzyme S32T/A67Y, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
2.23
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
mutant enzyme S32T, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
2.65
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
mutant enzyme A67Y, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
6.5
7.5
-
Tabernaemontana divaricata
pH Range (protein specific)
pH Minimum
pH Maximum
Commentary
Organism
4.5
8
more than 50% activity between pH 4.5 and 8.0
Tabernaemontana divaricata
KCat/KM [mM/s]
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.8
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
wild type enzyme, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
14.75
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
mutant enzyme A67Y, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
14.76
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
mutant enzyme S32T/A67Y, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
14.87
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
mutant enzyme S32T, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.8
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
wild type enzyme, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
14.75
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
mutant enzyme A67Y, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
14.76
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
mutant enzyme S32T/A67Y, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
14.87
-
N-benzoyl-Phe-Val-Arg-4-nitroanilide
mutant enzyme S32T, in 50 mM Na-acetate buffer pH 5.0, at 60°C
Tabernaemontana divaricata
Other publictions for EC 3.4.22.B50
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
Synonyms
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)
752517
Chu
Porcine epidemic diarrhea vir ...
porcine epidemic diarrhea virus
Antiviral Res.
158
199-205
2018
-
-
1
1
1
-
2
4
-
-
1
-
-
1
-
-
-
-
-
-
-
-
3
1
-
-
-
-
4
-
-
-
-
1
-
2
-
-
1
-
1
1
-
2
2
1
4
-
-
1
-
-
-
-
-
-
-
-
-
3
1
-
-
-
4
-
-
-
-
-
-
-
-
4
4
731149
Baez-Santos
The SARS-coronavirus papain-li ...
Severe acute respiratory syndrome-related coronavirus
Antiviral Res.
115
21-38
2015
-
1
-
1
1
-
20
-
-
2
-
-
-
5
-
-
-
1
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
1
-
16
-
1
-
-
1
1
-
16
20
1
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
2
-
-
-
732186
Kong
Structural view and substrate ...
infectious bronchitis virus
J. Biol. Chem.
290
7160-7168
2015
-
-
1
1
-
-
-
2
-
-
-
1
-
1
-
-
1
-
-
-
-
-
4
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
1
-
-
-
-
-
2
-
-
-
1
-
-
-
1
-
-
-
-
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
754545
Mielech
Murine coronavirus ubiquitin- ...
Murine coronavirus
J. Virol.
89
4907-4917
2015
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
755638
van Kasteren
In vivo assessment of equine ...
equine arteritis virus, equine arteritis virus Bucyrus
Vet. Microbiol.
178
132-137
2015
-
1
-
-
1
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
731069
Chou
Structural basis for catalysis ...
Severe acute respiratory syndrome-related coronavirus
Acta Crystallogr. Sect. D
70
572-581
2014
-
-
1
1
9
-
3
9
-
-
-
-
-
3
-
-
1
-
-
-
-
-
2
-
3
1
-
-
8
1
-
-
-
2
-
-
-
-
1
-
1
9
-
-
3
2
9
-
-
-
-
-
-
-
1
-
-
-
-
2
-
1
-
-
8
1
-
-
-
-
3
3
-
8
8
732257
Xing
The papain-like protease of po ...
porcine epidemic diarrhea virus
J. Gen. Virol.
94
1554-1567
2013
-
-
1
-
3
-
-
-
-
-
-
3
-
1
-
-
-
-
-
-
-
-
3
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
3
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
732725
Dutta
Enhancement of proteolytic act ...
Tabernaemontana divaricata
PLoS ONE
8
e62619
2013
1
-
1
-
3
-
1
4
-
-
1
-
-
3
-
-
1
-
1
-
4
-
3
1
2
1
-
1
4
1
1
-
-
-
-
4
1
-
1
-
-
3
-
4
1
-
4
-
-
1
-
-
-
-
1
1
-
4
-
3
1
1
-
1
4
1
1
-
-
-
-
-
-
4
4
732827
van Kasteren
Deubiquitinase function of art ...
equine arteritis virus
Proc. Natl. Acad. Sci. USA
110
E838-E847
2013
-
-
1
1
4
-
1
-
-
-
-
1
-
1
-
-
1
-
-
-
-
-
3
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
1
4
-
-
1
-
-
-
-
-
1
-
-
-
1
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
2
2
-
6
6
718000
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