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Literature summary 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

Application

EC Number Application Comment Organism
3.4.24.27 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
3.4.24.27 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
3.4.24.27 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
3.4.24.27 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)

EC Number Cloned (Comment) Organism
3.4.24.27 genes npr and nprT, DNA and amino acid sequence determination and analysis, expression in Escherichia coli and Bacillus subtilis Bacillus thermoproteolyticus
3.4.24.27 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)

EC Number Crystallization (Comment) Organism
3.4.24.27 crystal structure determination Bacillus thermoproteolyticus

Protein Variants

EC Number Protein Variants Comment Organism
3.4.24.27 A4T/G8C/T56A/G58A/N60C/T63F/S65P/A69P the mutant shows altered thermodynamics Bacillus thermoproteolyticus
3.4.24.27 A4T/T56A/G58A/T63F/S65P/A69P the mutant shows altered thermodynamics Bacillus thermoproteolyticus
3.4.24.27 E143R inactive mutant Bacillus thermoproteolyticus
3.4.24.27 E143S inactive mutant Bacillus thermoproteolyticus
3.4.24.27 E143W inactive mutant Bacillus thermoproteolyticus
3.4.24.27 G8C/N60C the mutant shows altered thermodynamics Bacillus thermoproteolyticus
3.4.24.27 H231A the mutant shows 500fold decreased catalytic efficiency compared to the wild-type enzyme Bacillus thermoproteolyticus
3.4.24.27 L144S/D150W/N227H the mutant shows 10fold decreased catalytic efficiency compared to the wild-type enzyme Bacillus thermoproteolyticus
3.4.24.27 L155S the mutant shows increased stability at 80°C compared to the wild-type enzyme Bacillus thermoproteolyticus
3.4.24.27 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
3.4.24.27 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
3.4.24.27 N112D the mutant shows an altered pKa value Bacillus thermoproteolyticus
3.4.24.27 N116D/Q119R/D150Q/Q225R the mutant shows 4fold decreased catalytic efficiency compared to the wild-type enzyme Bacillus thermoproteolyticus
3.4.24.27 R203A the mutant shows 5fold decreased catalytic efficiency compared to the wild-type enzyme Bacillus thermoproteolyticus
3.4.24.27 R203M the mutant shows 2300fold decreased catalytic efficiency compared to the wild-type enzyme Bacillus thermoproteolyticus
3.4.24.27 S103A the mutant shows 3fold decreased catalytic efficiency compared to the wild-type enzyme Bacillus thermoproteolyticus
3.4.24.27 W115L inactive mutant Bacillus thermoproteolyticus
3.4.24.27 W115V inactive mutant Bacillus thermoproteolyticus

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.4.24.27 bacitracin
-
 Geobacillus stearothermophilus
3.4.24.27 Gly-D-Phe
-
 Geobacillus stearothermophilus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.4.24.27 0.4
-
 N-carbobenzoxy-L-Asp-L-Phe methyl ester pH 7.5, 25°C, recombinant wild-type enzyme Bacillus thermoproteolyticus
3.4.24.27 0.52
-
 N-carbobenzoxy-L-Asp-L-Phe methyl ester pH 7.5, 25°C, native wild-type enzyme Bacillus thermoproteolyticus

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
3.4.24.27 cytosol secretion from cytosol via periplasmic space Geobacillus stearothermophilus 5829
-
3.4.24.27 extracellular secretion from cytosol via periplasmic space Geobacillus stearothermophilus
-
-
3.4.24.27 extracellular secretion mechanism, overview Bacillus thermoproteolyticus
-
-
3.4.24.27 periplasm pro-enzyme form, folding and autocleavage takes place in the periplasmic space, overview Bacillus thermoproteolyticus
-
-
3.4.24.27 periplasm secretion from cytosol via periplasmic space Geobacillus stearothermophilus
-
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.4.24.27 Ca2+
-
 Geobacillus stearothermophilus
3.4.24.27 Ca2+ four Ca2+ per enzyme molecule are required for enzyme stability Bacillus thermoproteolyticus
3.4.24.27 Zn2+ a zinc metalloproteinase Geobacillus stearothermophilus
3.4.24.27 Zn2+ a zinc metalloproteinase that contains a HEXXH motif, one Zn2+ per enzyme molecule is required for activity Bacillus thermoproteolyticus

Organism

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

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
3.4.24.27 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
3.4.24.27 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)

EC Number Purification (Comment) Organism
3.4.24.27 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)

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

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.4.24.27 N-(4-methoxyphenylazoformyl)-Leu-Leu-OH + H2O a synthetic substrate Geobacillus stearothermophilus ?
-
 ?
3.4.24.27 N-(4-methoxyphenylazoformyl)-Leu-Leu-OH + H2O a synthetic substrate Geobacillus stearothermophilus MK232 ?
-
 ?
3.4.24.27 N-carbobenzoxy-L-Asp-L-Phe methyl ester + H2O
-
 Geobacillus stearothermophilus ?
-
 ?
3.4.24.27 N-carbobenzoxy-L-Asp-L-Phe methyl ester + H2O
-
 Bacillus thermoproteolyticus ?
-
 ?
3.4.24.27 N-carbobenzoxy-L-Asp-L-Phe methyl ester + H2O
-
 Geobacillus stearothermophilus MK232 ?
-
 ?

Subunits

EC Number Subunits Comment Organism
3.4.24.27 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

EC Number Synonyms Comment Organism
3.4.24.27 NprM
-
 Geobacillus stearothermophilus
3.4.24.27 NprM
-
 Bacillus thermoproteolyticus
3.4.24.27 thermolysin-like protease
-
 Geobacillus stearothermophilus
3.4.24.27 thermolysin-like protease
-
 Bacillus thermoproteolyticus
3.4.24.27 TLP-ste
-
 Geobacillus stearothermophilus
3.4.24.27 TLP-ste
-
 Bacillus thermoproteolyticus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.4.24.27 25
-
 assay at Geobacillus stearothermophilus
3.4.24.27 25
-
 assay at Bacillus thermoproteolyticus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.4.24.27 3.8
-
 N-carbobenzoxy-L-Asp-L-Phe methyl ester pH 7.5, 25°C, recombinant wild-type enzyme Bacillus thermoproteolyticus
3.4.24.27 4.3
-
 N-carbobenzoxy-L-Asp-L-Phe methyl ester pH 7.5, 25°C, native wild-type enzyme Bacillus thermoproteolyticus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.4.24.27 7.5
-
 assay at Geobacillus stearothermophilus
3.4.24.27 7.5
-
 assay at Bacillus thermoproteolyticus