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Literature summary for 3.4.21.66 extracted from

  • Toplak, A.; Wu, B.; Fusetti, F.; Quaedflieg, P.J.; Janssen, D.B.
    Proteolysin, a novel highly thermostable and cosolvent-compatible protease from the thermophilic bacterium Coprothermobacter proteolyticus (2013), Appl. Environ. Microbiol., 79, 5625-5632.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.4.21.66

Activating Compound

Activating Compound Comment Organism Structure
cetyltrimethylammonium bromide CTAB, 5% v/v, activates to 178% activity Coprothermobacter proteolyticus
DTT
-
 Coprothermobacter proteolyticus

Cloned(Commentary)

Cloned (Comment) Organism
gene aprE, DNA and amino acid sequence determination and analysis, phylogenetic tree, recombinant expression of wild-type and mutant N-terminally maltose-binding protein-tagged and C-terminally His6-tagged enzymes in Escherichia coli strain C43(DE3) Coprothermobacter proteolyticus

Protein Variants

Protein Variants Comment Organism
C182A/C201A site-directed mutagenesis, elimination of the disulfide bond, the mutant shows increased activity but reduced thermostability compared to the wild-type enzyme Coprothermobacter proteolyticus

Inhibitors

Inhibitors Comment Organism Structure
EDTA 87% inhibition at 10 mM Coprothermobacter proteolyticus
guanidinium hydrochloride 6 M guanidinium hydrochloride gives a 40% reduction of activity after 1 h of incubation at 40°C Coprothermobacter proteolyticus
H2O2 15% v/v, 90% inhibition Coprothermobacter proteolyticus
additional information the enzyme shows good activity and stability in the presence of organic solvents, detergents, and dithiothreitol, and it remains active in 6 M guanidinium hydrochloride Coprothermobacter proteolyticus
PMSF completely abolishes the activity of proteolysin Coprothermobacter proteolyticus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.33
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, recombinant mutant C182A/C201A Coprothermobacter proteolyticus
0.41
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, recombinant wild-type enzyme Coprothermobacter proteolyticus
0.57
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 70°C, recombinant wild-type enzyme Coprothermobacter proteolyticus
0.66
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 70°C, recombinant mutant C182A/C201A Coprothermobacter proteolyticus
1.58
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, recombinant wild-type enzyme, in presence of DTT Coprothermobacter proteolyticus
1.96
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, recombinant mutant C182A/C201A, in presence of DTT Coprothermobacter proteolyticus

Localization

Localization Comment Organism GeneOntology No. Textmining
extracellular
-
 Coprothermobacter proteolyticus
-
-

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ required, two calcium sites (Ca1 and Ca2) are present in thermitase are likely to be present in proteolysin as well Coprothermobacter proteolyticus
additional information bivalent metal cations increase the activity of proteolysin Coprothermobacter proteolyticus

Organism

Organism UniProt Comment Textmining
Coprothermobacter proteolyticus B5Y6G0 gene aprE
-

Posttranslational Modification

Posttranslational Modification Comment Organism
additional information recombinant proteolysin processing and maturation requies a heat treatment step. The processing can be performed at 70°C for 12 h or at 80°C for 3 h Coprothermobacter proteolyticus

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant N-terminally maltose-binding protein-tagged and C-terminally His6-tagged enzymes from Escherichia coli strain C43(DE3) to near homogeneity from crude cell lysate by heat treatment at 80°C for 3 h, and ultrafiltration Coprothermobacter proteolyticus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
azocasein + H2O proteinolytic activity Coprothermobacter proteolyticus ?
-
 ?
additional information proteolytic activities of cell extracts and purified enzyme samples are monitored on 5% skim milk-LB agar plates or by azocasein assey Coprothermobacter proteolyticus ?
-
 ?
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide + H2O amidolytic activity Coprothermobacter proteolyticus N-succinyl-Ala-Ala-Pro-Phe + 4-nitroaniline
-
 ?
oxidized insulin B chain + H2O peptidolytic activity, primary cleavage site of proteolysin is located between Ala14 and Leu15. Prolonged incubation of 24 h reveals additional cleavage sites after Phe1, Asn3, Gln4, Leu17, Cys19, Phe24, Phe25, Tyr25, and Lys29, which shows that proteolysin has a relaxed cleavage site specificity Coprothermobacter proteolyticus ?
-
 ?

Synonyms

Synonyms Comment Organism
PrlA
-
 Coprothermobacter proteolyticus
proteolysin
-
 Coprothermobacter proteolyticus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
40 70 substrate N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide Coprothermobacter proteolyticus
85
-
 substrate azocasein Coprothermobacter proteolyticus

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
20 80 activity range, recombinant enzyme Coprothermobacter proteolyticus

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
70
-
 purified recombinant enzyme, substrate N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide, 20 h, 35% activity remaining Coprothermobacter proteolyticus
80 100 thermostability profiles of the purified recombinant enzyme with different substrates, overview Coprothermobacter proteolyticus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
25
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, recombinant wild-type enzyme Coprothermobacter proteolyticus
37
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, recombinant wild-type enzyme, in presence of DTT Coprothermobacter proteolyticus
110
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 70°C, recombinant wild-type enzyme Coprothermobacter proteolyticus
111
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, recombinant mutant C182A/C201A Coprothermobacter proteolyticus
124
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, recombinant mutant C182A/C201A, in presence of DTT Coprothermobacter proteolyticus
248
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 70°C, recombinant mutant C182A/C201A Coprothermobacter proteolyticus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
-
 Coprothermobacter proteolyticus

pH Range

pH Minimum pH Maximum Comment Organism
7 9 activity range Coprothermobacter proteolyticus

General Information

General Information Comment Organism
evolution proteolysin is a serine protease of the thermitase subgroup of subtilases, phylogenetic tree Coprothermobacter proteolyticus
additional information structure homology modelling, overview Coprothermobacter proteolyticus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
62
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, recombinant wild-type enzyme Coprothermobacter proteolyticus
65
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 70°C, recombinant wild-type enzyme Coprothermobacter proteolyticus
69
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, recombinant wild-type enzyme, in presence of DTT Coprothermobacter proteolyticus
126
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, recombinant mutant C182A/C201A, in presence of DTT Coprothermobacter proteolyticus
189
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 70°C, recombinant mutant C182A/C201A Coprothermobacter proteolyticus
335
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide pH 7.5, 40°C, recombinant mutant C182A/C201A Coprothermobacter proteolyticus