Cloned (Comment) | Organism |
---|---|
recombinant overexpression of wild-type and mutant enzymes in Escherichia coli | Bacillus thermoproteolyticus |
Protein Variants | Comment | Organism |
---|---|---|
N116A | site-directed mutagenesis, the mutant shows slightly decreased activity compared to the wild-type enzyme | Bacillus thermoproteolyticus |
N116D | site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme | Bacillus thermoproteolyticus |
N116Q | site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme | Bacillus thermoproteolyticus |
N116T | site-directed mutagenesis, the mutant shows slightly decreased activity compared to the wild-type enzyme | Bacillus thermoproteolyticus |
General Stability | Organism |
---|---|
Ca2+ stablizes the enzyme | Bacillus thermoproteolyticus |
important role of Asn116 in the activity and stability of thermolysin presumably by stabilizing the beta-hairpin structure. In the N-terminal domain of thermolysin, two antiparallel beta-strands, Asn112-Ala113-Phe114-Trp115 and Ser118-Gln119-Met120-Val121-Tyr122 are connected by an Asn116-Gly117 turn to form a beta-hairpin structure | Bacillus thermoproteolyticus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten kinetics | Bacillus thermoproteolyticus | |
0.38 | - |
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester | purified recombinant mutant N116Q, pH 7.5, 25°C | Bacillus thermoproteolyticus | |
0.39 | - |
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester | purified recombinant wild-type enzyme, pH 7.5, 25°C | Bacillus thermoproteolyticus | |
0.41 | - |
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester | purified recombinant mutant N116A, pH 7.5, 25°C | Bacillus thermoproteolyticus | |
0.48 | - |
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester | purified recombinant mutant N116D, pH 7.5, 25°C | Bacillus thermoproteolyticus | |
0.49 | - |
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester | purified recombinant mutant N116T, pH 7.5, 25°C | Bacillus thermoproteolyticus |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Ca2+ | stablizes the enzyme | Bacillus thermoproteolyticus |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
34600 | - |
x * 34600, recombinant mature enzyme, SDS-PAGE | Bacillus thermoproteolyticus |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Bacillus thermoproteolyticus | P00800 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant mature wild-type and mutant enzymes 7.2-11fold from Escherichia coli by hydrophobic interaction and affinity chromatography | Bacillus thermoproteolyticus |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
one proteolytic unit is defined as the amount which liberates a quantity of acid-soluble peptides that corresponds to an increase in A275 of 0.0074 (A275 of 1 mg of tyrosine)/min | Bacillus thermoproteolyticus |
5300 | - |
purified recombinant mutant N116A, pH 7.5, 25°C, substrate N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide | Bacillus thermoproteolyticus |
5400 | - |
purified recombinant mutant N116T, pH 7.5, 25°C, substrate N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide | Bacillus thermoproteolyticus |
6800 | - |
purified recombinant mutant N116Q, pH 7.5, 25°C, substrate N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide | Bacillus thermoproteolyticus |
7900 | - |
purified recombinant mutant N116D, pH 7.5, 25°C, substrate N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide | Bacillus thermoproteolyticus |
12000 | - |
purified recombinant wild-type enzyme, pH 7.5, 25°C, substrate N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide | Bacillus thermoproteolyticus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
casein + H2O | from bovine milk | Bacillus thermoproteolyticus | L-tyrosine + ? | - |
? | |
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester + H2O | - |
Bacillus thermoproteolyticus | ? | - |
? | |
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide + H2O | - |
Bacillus thermoproteolyticus | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
? | x * 34600, recombinant mature enzyme, SDS-PAGE | Bacillus thermoproteolyticus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Bacillus thermoproteolyticus |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
80 | - |
first-order rate constant of the thermal inactivation at 80°C in the presence of 1-100mM CaCl2 | Bacillus thermoproteolyticus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
5.4 | - |
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester | purified recombinant mutant N116T, pH 7.5, 25°C | Bacillus thermoproteolyticus | |
6.7 | - |
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester | purified recombinant mutant N116A, pH 7.5, 25°C | Bacillus thermoproteolyticus | |
7.1 | - |
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester | purified recombinant mutant N116Q, pH 7.5, 25°C | Bacillus thermoproteolyticus | |
7.7 | - |
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester | purified recombinant wild-type enzyme, pH 7.5, 25°C | Bacillus thermoproteolyticus | |
12 | - |
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester | purified recombinant mutant N116D, pH 7.5, 25°C | Bacillus thermoproteolyticus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Bacillus thermoproteolyticus |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
5 | 8.5 | activity range, profile overview | Bacillus thermoproteolyticus |
General Information | Comment | Organism |
---|---|---|
additional information | structure analsis using enzyme structure PDB ID 8TLN. Catalytically important residues Glu143 and His231. Important role of Asn116 in the activity and stability of thermolysin presumably by stabilizing the beta-hairpin structure. In the N-terminal domain of thermolysin, two antiparallel beta-strands, Asn112-Ala113-Phe114-Trp115 and Ser118-Gln119-Met120-Val121-Tyr122 are connected by an Asn116-Gly117 turn to form a beta-hairpin structure | Bacillus thermoproteolyticus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
11 | - |
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester | purified recombinant mutant N116T, pH 7.5, 25°C | Bacillus thermoproteolyticus | |
16 | - |
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester | purified recombinant mutant N116A, pH 7.5, 25°C | Bacillus thermoproteolyticus | |
19 | - |
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester | purified recombinant mutant N116Q, pH 7.5, 25°C | Bacillus thermoproteolyticus | |
20 | - |
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester | purified recombinant wild-type enzyme, pH 7.5, 25°C | Bacillus thermoproteolyticus | |
26 | - |
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide | purified recombinant mutant N116T, pH 7.5, 25°C | Bacillus thermoproteolyticus | |
27 | - |
N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester | purified recombinant mutant N116D, pH 7.5, 25°C | Bacillus thermoproteolyticus | |
31 | - |
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide | purified recombinant mutant N116A, pH 7.5, 25°C | Bacillus thermoproteolyticus | |
37 | - |
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide | purified recombinant mutant N116Q, pH 7.5, 25°C | Bacillus thermoproteolyticus | |
37 | - |
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide | purified recombinant wild-type enzyme, pH 7.5, 25°C | Bacillus thermoproteolyticus | |
117 | - |
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide | purified recombinant mutant N116D, pH 7.5, 25°C | Bacillus thermoproteolyticus |