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Literature summary for 3.4.22.B50 extracted from

  • Dutta, S.; Dattagupta, J.K.; Biswas, S.
    Enhancement of proteolytic activity of a thermostable papain-like protease by structure-based rational design (2013), PLoS ONE, 8, e62619.
    View publication on PubMedView publication on EuropePMC

Activating Compound

Activating Compound Comment Organism Structure
L-cysteine 20 mM used in assay conditions Tabernaemontana divaricata

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli BL21(DE3) cells Tabernaemontana divaricata

Protein Variants

Protein Variants Comment 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 Comment Organism Structure
E-64
-
Tabernaemontana divaricata

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment 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] Comment Organism
40000
-
x * 40000, SDS-PAGE Tabernaemontana divaricata

Organism

Organism UniProt Comment Textmining
Tabernaemontana divaricata P83654
-
-

Purification (Commentary)

Purification (Comment) Organism
Ni-NTA column chromatography Tabernaemontana divaricata

Renatured (Commentary)

Renatured (Comment) Organism
dialysis Tabernaemontana divaricata

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment 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 Comment Substrates Organism Products Comment (Products) Rev. Reac.
azocasein + H2O
-
Tabernaemontana divaricata ?
-
?
Gelatin + H2O
-
Tabernaemontana divaricata ?
-
?
N-benzoyl-Phe-Val-Arg-4-nitroanilide + H2O
-
Tabernaemontana divaricata N-benzoyl-Phe-Val-Arg + 4-nitroaniline
-
?

Subunits

Subunits Comment Organism
? x * 40000, SDS-PAGE Tabernaemontana divaricata

Synonyms

Synonyms Comment Organism
Erv-C
-
Tabernaemontana divaricata
ervatamin-C
-
Tabernaemontana divaricata

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
70
-
-
Tabernaemontana divaricata

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment 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 Comment 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 Comment Organism
6.5 7.5
-
Tabernaemontana divaricata

pH Range

pH Minimum pH Maximum Comment Organism
4.5 8 more than 50% activity between pH 4.5 and 8.0 Tabernaemontana divaricata

IC50 Value

IC50 Value IC50 Value Maximum Comment 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

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment 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