Cloned (Comment) | Organism |
---|---|
gene pac, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Thermus thermophilus |
Protein Variants | Comment | Organism |
---|---|---|
I57F | site-directed mutagenesis, the substrate specificity of the mutant shifts from penicillin K to penicillin G, Km for penicillin G is 4.5fold, while the catalytic efficiency remains at wild-type value | Thermus thermophilus |
L188F | site-directed mutagenesis, the substrate specificity of the mutant shifts from penicillin K to penicillin G, penicillin G binding is 10fold improved compared to the wild-type enzyme | Thermus thermophilus |
L188R | site-directed mutagenesis, the substrate specificity of the mutant shifts from penicillin K to penicillin G | Thermus thermophilus |
L188R/S189F | site-directed mutagenesis, the substrate specificity of the mutant shifts from penicillin K to penicillin G | Thermus thermophilus |
L24F | site-directed mutagenesis, the substrate specificity of the mutant shifts from penicillin K to penicillin G, penicillin G binding is 12fold improved compared to the wild-type enzyme | Thermus thermophilus |
L24F/I57F | site-directed mutagenesis, the substrate specificity of the mutant shifts from penicillin K to penicillin G | Thermus thermophilus |
L24F/I57F/L188F | site-directed mutagenesis, the substrate specificity of the mutant shifts from penicillin K to penicillin G | Thermus thermophilus |
L24F/I57F/L188R/S189F | site-directed mutagenesis, inactive mutant | Thermus thermophilus |
additional information | substrate specificity change through engineering: generation of a set of enzyme variants containing between one and four amino acid replacements, with altered catalytic properties in the hydrolyses of penicillins K and G. The introduction of a single phenylalanine residue in position alpha188, alpha189, or beta24 improves the Km for penicillin G between 9 and 12fold, and the catalytic efficiency of these variants for penicillin G was improved up to 6.6fold | Thermus thermophilus |
S189F | site-directed mutagenesis, the substrate specificity of the mutant shifts from penicillin K to penicillin G, penicillin G binding is 9fold improved compared to the wild-type enzyme | Thermus thermophilus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetics, overview | Thermus thermophilus | |
0.05 | - |
penicillin K | pH 5.5, 65°C, recombinant mutant L24F/I57F | Thermus thermophilus | |
0.1 | - |
penicillin K | pH 5.5, 65°C, recombinant mutants L188F and S189F | Thermus thermophilus | |
0.12 | - |
penicillin K | pH 5.5, 65°C, recombinant mutant L24F | Thermus thermophilus | |
0.15 | - |
penicillin K | pH 5.5, 65°C, recombinant mutant L188F/L24F/I57F | Thermus thermophilus | |
0.27 | - |
penicillin K | pH 5.5, 65°C, recombinant mutant I57F | Thermus thermophilus | |
0.32 | - |
penicillin K | pH 5.5, 65°C, recombinant wild-type enzyme | Thermus thermophilus | |
13.4 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant L24F | Thermus thermophilus | |
15.5 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant L188F | Thermus thermophilus | |
17.8 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant S189F | Thermus thermophilus | |
36.5 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant I57F | Thermus thermophilus | |
37.6 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant L24F/I57F | Thermus thermophilus | |
43.3 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant L188F/S189F | Thermus thermophilus | |
44.4 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant L188F/L24F/I57F | Thermus thermophilus | |
164 | - |
penicillin G | pH 5.5, 65°C, recombinant wild-type enzyme | Thermus thermophilus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
penicillin G + H2O | Thermus thermophilus | - |
phenylacetate + 6-aminopenicillanate | - |
? | |
penicillin K + H2O | Thermus thermophilus | - |
octanoic acid + 6-aminopenicillanate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Thermus thermophilus | Q72G76 | gene pac | - |
Purification (Comment) | Organism |
---|---|
recombinant enzyme from Escherichia coli strain BL21(DE3) by heat shock treatment of 20 min at 65°C, and immobilized metal ion affinity chromatography | Thermus thermophilus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | the acylase shows a marked preference for penicillin K over penicillin G, substrate specificity, overview | Thermus thermophilus | ? | - |
? | |
penicillin G + H2O | - |
Thermus thermophilus | phenylacetate + 6-aminopenicillanate | - |
? | |
penicillin K + H2O | - |
Thermus thermophilus | octanoic acid + 6-aminopenicillanate | - |
? | |
penicillin K + H2O | preferred substrate for the wild-type enzyme | Thermus thermophilus | octanoic acid + 6-aminopenicillanate | - |
? |
Synonyms | Comment | Organism |
---|---|---|
penicillin acylase | - |
Thermus thermophilus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
65 | - |
assay at | Thermus thermophilus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.02 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant L188F/S189F | Thermus thermophilus | |
0.13 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant L188F/L24F/I57F | Thermus thermophilus | |
0.3 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant L24F/I57F | Thermus thermophilus | |
0.32 | - |
penicillin K | pH 5.5, 65°C, recombinant mutant L24F | Thermus thermophilus | |
0.36 | - |
penicillin K | pH 5.5, 65°C, recombinant mutant S189F | Thermus thermophilus | |
0.43 | - |
penicillin K | pH 5.5, 65°C, recombinant mutant L188F | Thermus thermophilus | |
0.44 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant I57F | Thermus thermophilus | |
0.54 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant L188F | Thermus thermophilus | |
0.65 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant S189F | Thermus thermophilus | |
0.75 | - |
penicillin K | pH 5.5, 65°C, recombinant mutant L24F/I57F | Thermus thermophilus | |
0.85 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant L24F | Thermus thermophilus | |
1.43 | - |
penicillin K | pH 5.5, 65°C, recombinant mutant L188F/L24F/I57F | Thermus thermophilus | |
1.56 | - |
penicillin G | pH 5.5, 65°C, recombinant wild-type enzyme | Thermus thermophilus | |
4.91 | - |
penicillin K | pH 5.5, 65°C, recombinant mutant I57F | Thermus thermophilus | |
5.6 | - |
penicillin K | pH 5.5, 65°C, recombinant wild-type enzyme | Thermus thermophilus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
5.5 | - |
assay at | Thermus thermophilus |
General Information | Comment | Organism |
---|---|---|
additional information | three-dimensional modeling with identification of the catalytic residues and the substrate binding pocket, overview. The substrate binding and active site orientation is predited by docking and molecular homology structure modeling | Thermus thermophilus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0005 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant L188F/S189F | Thermus thermophilus | |
0.003 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant L188F/L24F/I57F | Thermus thermophilus | |
0.008 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant L24F/I57F | Thermus thermophilus | |
0.009 | - |
penicillin G | pH 5.5, 65°C, recombinant wild-type enzyme | Thermus thermophilus | |
0.012 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant I57F | Thermus thermophilus | |
0.034 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant L188F | Thermus thermophilus | |
0.036 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant S189F | Thermus thermophilus | |
0.063 | - |
penicillin G | pH 5.5, 65°C, recombinant mutant L24F | Thermus thermophilus | |
3.6 | - |
penicillin K | pH 5.5, 65°C, recombinant mutant S189F | Thermus thermophilus | |
4.2 | - |
penicillin K | pH 5.5, 65°C, recombinant mutant L24F | Thermus thermophilus | |
4.3 | - |
penicillin K | pH 5.5, 65°C, recombinant mutant L188F | Thermus thermophilus | |
9.5 | - |
penicillin K | pH 5.5, 65°C, recombinant mutant L188F/L24F/I57F | Thermus thermophilus | |
15 | - |
penicillin K | pH 5.5, 65°C, recombinant mutant L24F/I57F | Thermus thermophilus | |
16.1 | - |
penicillin K | pH 5.5, 65°C, recombinant wild-type enzyme | Thermus thermophilus | |
18.2 | - |
penicillin K | pH 5.5, 65°C, recombinant mutant I57F | Thermus thermophilus |