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Literature summary for 3.4.21.62 extracted from

  • Oskarsson, K.R.; Kristjansson, M.M.
    Improved expression, purification and characterization of VPR, a cold active subtilisin-like serine proteinase and the effects of calcium on expression and stability (2019), Biochim. Biophys. Acta, 1867, 152-162 .
    View publication on PubMed
Search for more literature for 3.4.21.62

Cloned(Commentary)

Cloned (Comment) Organism
gene vpr, recombinant expression of extracellular enzyme in Escherichia coli strain XL10-Gold from pET 11a vector, high-level expression of the cold adapted subtilase, VPR, utilizing the rhamnose titratable T7 system of Lemo21 in presence of Ca2+ ions (best at about 100 mM) and rhamnose (best at about 0.076 mM) resulting in a dramatic increase of soluble protein compared to the other systems. Method evaluation, overview. In the absence of rhamnose, toxic effects of the proteinase are clearly observed, as cultures grow slower and end up at lower cell density. Addition of Ca2+ increases the yield of soluble protein. The calcium present in the growth medium is not only aiding in correct folding and stabilization of VPR but is also increasing the resistance of Lemo21 against the toxic effects caused by enzyme VPR Bacillus subtilis

Inhibitors

Inhibitors Comment Organism Structure
additional information enzyme destabilization in the presence of EDTA is due to chelation of a tightly bound calcium ion by EDTA, rather than destabilizing effects caused by EDTA on the structure Bacillus subtilis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information Michaelis-Menten kinetics and thermodynamics Bacillus subtilis
0.115
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide recombinant enzyme, 5°C, pH 8.6 Bacillus subtilis
0.133
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide recombinant enzyme, 25°C, pH 9.4 Bacillus subtilis
0.178
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide recombinant enzyme, 25°C, pH 8.6 Bacillus subtilis
0.198
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide recombinant enzyme, 25°C, pH 7.4 Bacillus subtilis
0.227
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide recombinant enzyme, 45°C, pH 8.6 Bacillus subtilis

Localization

Localization Comment Organism GeneOntology No. Textmining
extracellular
-
 Bacillus subtilis
-
-

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ importance of calcium in the medium after enzyme induction, both for stability of the proteinase and cell health. The two calcium binding sites have apparent binding constants in the mM range. Binding of calcium to the weaker of those two sites only affects resistance of the enzyme against irreversible thermal inactivation. Kinetics Bacillus subtilis

Organism

Organism UniProt Comment Textmining
Bacillus subtilis P29141
-
-
Bacillus subtilis 168 P29141
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant extracellular enzyme 7.8fold by ammonium sulfate fractionation, N-carbobenzoxy-D-phenylalanyl-triethylenetetramine-Sepharose (z-D-Phe-TETA) affinity chromatography, hydrophobic interaction chromatography, and anion exchange chrmatography, all in presence of 10 mM Ca2+ Bacillus subtilis

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
876
-
 purified recombinant enzyme, pH 8.6, 25°C Bacillus subtilis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide + H2O
-
 Bacillus subtilis N-succinyl-Ala-Ala-Pro-Phe + 4-nitroaniline
-
 ?
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide + H2O
-
 Bacillus subtilis 168 N-succinyl-Ala-Ala-Pro-Phe + 4-nitroaniline
-
 ?

Synonyms

Synonyms Comment Organism
cold active subtilisin-like serine proteinase
-
 Bacillus subtilis
vPR
-
 Bacillus subtilis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
65
-
 the activity steadily increases up to 65°C Bacillus subtilis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
40.8
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide recombinant enzyme, 5°C, pH 8.6 Bacillus subtilis
193.1
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide recombinant enzyme, 25°C, pH 7.4 Bacillus subtilis
225.7
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide recombinant enzyme, 25°C, pH 8.6 Bacillus subtilis
248.7
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide recombinant enzyme, 25°C, pH 9.4 Bacillus subtilis
552.3
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide recombinant enzyme, 45°C, pH 8.6 Bacillus subtilis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.4 8.6 assay at Bacillus subtilis

pH Range

pH Minimum pH Maximum Comment Organism
7.4 9.4 the catalytic efficiency (kcat/Km) increases with pH from 7.4 to 9.4 when measured at 25°C, both as a result of increased kcat and lowered Km Bacillus subtilis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
354.8
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide recombinant enzyme, 5°C, pH 8.6 Bacillus subtilis
975.25
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide recombinant enzyme, 25°C, pH 7.4 Bacillus subtilis
1268
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide recombinant enzyme, 25°C, pH 8.6 Bacillus subtilis
1869.9
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide recombinant enzyme, 25°C, pH 9.4 Bacillus subtilis
2433
-
 N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide recombinant enzyme, 45°C, pH 8.6 Bacillus subtilis