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 | 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 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 | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
extracellular | - |
Bacillus subtilis | - |
- |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Bacillus subtilis | P29141 | - |
- |
Bacillus subtilis 168 | P29141 | - |
- |
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 Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
876 | - |
purified recombinant enzyme, pH 8.6, 25°C | Bacillus subtilis |
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 | Comment | Organism |
---|---|---|
cold active subtilisin-like serine proteinase | - |
Bacillus subtilis |
vPR | - |
Bacillus subtilis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
65 | - |
the activity steadily increases up to 65°C | Bacillus subtilis |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.4 | 8.6 | assay at | Bacillus subtilis |
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 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 |