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Literature summary for 3.4.24.27 extracted from

  • Inouye, K.; Kusano, M.; Hashida, Y.; Minoda, M.; Yasukawa, K.
    Engineering, expression, purification, and production of recombinant thermolysin (2007), Biotechnol. Annu. Rev., 13, 43-64.
    View publication on PubMed
Search for more literature for 3.4.24.27

Application

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(Commentary)

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 (Commentary)

Crystallization (Comment) Organism
crystal structure determination Bacillus thermoproteolyticus

Protein Variants

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

Inhibitors Comment Organism Structure
bacitracin
-
 Geobacillus stearothermophilus
Gly-D-Phe
-
 Geobacillus stearothermophilus

KM Value [mM]

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

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

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

Organism UniProt Comment Textmining
Bacillus thermoproteolyticus
-
-
-
Geobacillus stearothermophilus
-
 genes npr and nprT
-
Geobacillus stearothermophilus MK232
-
 genes npr and nprT
-

Posttranslational Modification

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 (Commentary)

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 (Commentary)

Renatured (Comment) Organism
solubilization and refolding of recombinant enzyme from inclusion bodies after expression in Escherichia coli Geobacillus stearothermophilus

Substrates and Products (Substrate)

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

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

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 [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Geobacillus stearothermophilus
25
-
 assay at Bacillus thermoproteolyticus

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Geobacillus stearothermophilus
7.5
-
 assay at Bacillus thermoproteolyticus