Application | Comment | Organism |
---|---|---|
industry | the enzyme is used for synthesis of N-carbobenzyloxy L-Asp-L-Phe methyl ester, a precursor of the artificial sweetener aspartam | Geobacillus stearothermophilus |
nutrition | the enzyme is used for synthesis of N-carbobenzyloxy L-Asp-L-Phe methyl ester, a precursor of the artificial sweetener aspartam | Bacillus thermoproteolyticus |
synthesis | the enzyme is used for synthesis of N-carbobenzyloxy L-Asp-L-Phe methyl ester, a precursor of the artificial sweetener aspartam | Geobacillus stearothermophilus |
synthesis | the enzyme is used for synthesis of N-carbobenzyloxy L-Asp-L-Phe methyl ester, a precursor of the artificial sweetener aspartam | Bacillus thermoproteolyticus |
Cloned (Comment) | Organism |
---|---|
genes npr and nprT, DNA and amino acid sequence determination and analysis, expression in Escherichia coli and Bacillus subtilis | Bacillus thermoproteolyticus |
genes npr and nprT, DNA and amino acid sequence determination and analysis, expression in Escherichia coli and Bacillus subtilis, different methods, expression of tagged mature enzyme, proenzyme, or mutant enzyme in inclusion bodies or in the cytosol or medium as soluble protein, overview | Geobacillus stearothermophilus |
Crystallization (Comment) | Organism |
---|---|
crystal structure determination | Bacillus thermoproteolyticus |
Protein Variants | Comment | Organism |
---|---|---|
A4T/G8C/T56A/G58A/N60C/T63F/S65P/A69P | the mutant shows altered thermodynamics | Bacillus thermoproteolyticus |
A4T/T56A/G58A/T63F/S65P/A69P | the mutant shows altered thermodynamics | Bacillus thermoproteolyticus |
E143R | inactive mutant | Bacillus thermoproteolyticus |
E143S | inactive mutant | Bacillus thermoproteolyticus |
E143W | inactive mutant | Bacillus thermoproteolyticus |
G8C/N60C | the mutant shows altered thermodynamics | Bacillus thermoproteolyticus |
H231A | the mutant shows 500fold decreased catalytic efficiency compared to the wild-type enzyme | Bacillus thermoproteolyticus |
L144S/D150W/N227H | the mutant shows 10fold decreased catalytic efficiency compared to the wild-type enzyme | Bacillus thermoproteolyticus |
L155S | the mutant shows increased stability at 80°C compared to the wild-type enzyme | Bacillus thermoproteolyticus |
additional information | generation of an engineered enzyme with a higher activity in the synthesis of N-carbobenzyloxy L-Asp-L-Phe methyl ester | Geobacillus stearothermophilus |
additional information | generation of an engineered enzyme with a higher activity in the synthesis of N-carbobenzyloxy L-Asp-L-Phe methyl ester | Bacillus thermoproteolyticus |
N112D | the mutant shows an altered pKa value | Bacillus thermoproteolyticus |
N116D/Q119R/D150Q/Q225R | the mutant shows 4fold decreased catalytic efficiency compared to the wild-type enzyme | Bacillus thermoproteolyticus |
R203A | the mutant shows 5fold decreased catalytic efficiency compared to the wild-type enzyme | Bacillus thermoproteolyticus |
R203M | the mutant shows 2300fold decreased catalytic efficiency compared to the wild-type enzyme | Bacillus thermoproteolyticus |
S103A | the mutant shows 3fold decreased catalytic efficiency compared to the wild-type enzyme | Bacillus thermoproteolyticus |
W115L | inactive mutant | Bacillus thermoproteolyticus |
W115V | inactive mutant | Bacillus thermoproteolyticus |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
bacitracin | - |
Geobacillus stearothermophilus | |
Gly-D-Phe | - |
Geobacillus stearothermophilus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.4 | - |
N-carbobenzoxy-L-Asp-L-Phe methyl ester | pH 7.5, 25°C, recombinant wild-type enzyme | Bacillus thermoproteolyticus | |
0.52 | - |
N-carbobenzoxy-L-Asp-L-Phe methyl ester | pH 7.5, 25°C, native wild-type enzyme | Bacillus thermoproteolyticus |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
cytosol | secretion from cytosol via periplasmic space | Geobacillus stearothermophilus | 5829 | - |
extracellular | secretion from cytosol via periplasmic space | Geobacillus stearothermophilus | - |
- |
extracellular | secretion mechanism, overview | Bacillus thermoproteolyticus | - |
- |
periplasm | pro-enzyme form, folding and autocleavage takes place in the periplasmic space, overview | Bacillus thermoproteolyticus | - |
- |
periplasm | secretion from cytosol via periplasmic space | Geobacillus stearothermophilus | - |
- |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Ca2+ | - |
Geobacillus stearothermophilus | |
Ca2+ | four Ca2+ per enzyme molecule are required for enzyme stability | Bacillus thermoproteolyticus | |
Zn2+ | a zinc metalloproteinase | Geobacillus stearothermophilus | |
Zn2+ | a zinc metalloproteinase that contains a HEXXH motif, one Zn2+ per enzyme molecule is required for activity | Bacillus thermoproteolyticus |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Bacillus thermoproteolyticus | - |
- |
- |
Geobacillus stearothermophilus | - |
genes npr and nprT | - |
Geobacillus stearothermophilus MK232 | - |
genes npr and nprT | - |
Posttranslational Modification | Comment | Organism |
---|---|---|
proteolytic modification | the pre-pro-enzyme contains a signal peptide and a prosequence, the prosequence acts as an intramolecular chaperone for autocatalytic cleavage of the linking peptide bond | Geobacillus stearothermophilus |
proteolytic modification | the pre-pro-enzyme contains a signal peptide and a prosequence, the prosequence acts as an intramolecular chaperone for autocatalytic cleavage of the linking peptide bond | Bacillus thermoproteolyticus |
Purification (Comment) | Organism |
---|---|
recombinant enzyme, different methods, e.g. by Gly-D-Phe or bacitracin affinity, ion exchange, and hydrophobic interaction chromatography, gel filtration and ammonium sulfate fractionation, detailed overview | Geobacillus stearothermophilus |
Renatured (Comment) | Organism |
---|---|
solubilization and refolding of recombinant enzyme from inclusion bodies after expression in Escherichia coli | Geobacillus stearothermophilus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
N-(4-methoxyphenylazoformyl)-Leu-Leu-OH + H2O | a synthetic substrate | Geobacillus stearothermophilus | ? | - |
? | |
N-(4-methoxyphenylazoformyl)-Leu-Leu-OH + H2O | a synthetic substrate | Geobacillus stearothermophilus MK232 | ? | - |
? | |
N-carbobenzoxy-L-Asp-L-Phe methyl ester + H2O | - |
Geobacillus stearothermophilus | ? | - |
? | |
N-carbobenzoxy-L-Asp-L-Phe methyl ester + H2O | - |
Bacillus thermoproteolyticus | ? | - |
? | |
N-carbobenzoxy-L-Asp-L-Phe methyl ester + H2O | - |
Geobacillus stearothermophilus MK232 | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | the enzyme consists of a beta-rich N-terminal domain and an alpha-helix C-terminal domain connected by a central alpha-helix, which is located at the bottom of the active site cleft | Bacillus thermoproteolyticus |
Synonyms | Comment | Organism |
---|---|---|
NprM | - |
Geobacillus stearothermophilus |
NprM | - |
Bacillus thermoproteolyticus |
thermolysin-like protease | - |
Geobacillus stearothermophilus |
thermolysin-like protease | - |
Bacillus thermoproteolyticus |
TLP-ste | - |
Geobacillus stearothermophilus |
TLP-ste | - |
Bacillus thermoproteolyticus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Geobacillus stearothermophilus |
25 | - |
assay at | Bacillus thermoproteolyticus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
3.8 | - |
N-carbobenzoxy-L-Asp-L-Phe methyl ester | pH 7.5, 25°C, recombinant wild-type enzyme | Bacillus thermoproteolyticus | |
4.3 | - |
N-carbobenzoxy-L-Asp-L-Phe methyl ester | pH 7.5, 25°C, native wild-type enzyme | Bacillus thermoproteolyticus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Geobacillus stearothermophilus |
7.5 | - |
assay at | Bacillus thermoproteolyticus |