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Literature summary for 3.5.1.93 extracted from
- Computational design of thermostable mutants for cephalosporin C acylase from Pseudomonas strain SE83 (2018), Comput. Chem. Eng., 116, 112-121 .
No PubMed abstract available
Application
| Application | Comment | Organism |
|---|---|---|
| synthesis | the enzyme is used for production of the important cephalosporin antibiotic 7-aminocephalosporanic acid (7-ACA). 7-ACA is an important cephalosporin nucleus for the synthesis of many widely used beta-lactam antibiotics | Pseudomonas sp. SE83 |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) | Pseudomonas sp. SE83 |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| C471betaS | site-directed mutagenesis, mutant M3, mutation within the hydrophobic core regions of the alphabetabetaalpha structural motif | Pseudomonas sp. SE83 |
| C471betaS/A114alphaS | site-directed mutagenesis, mutant M10, the mutant shows altered catalytic efficiency compared to wild-type | Pseudomonas sp. SE83 |
| C471betaS/A114alphaS/A38betaS | site-directed mutagenesis, mutant M12 | Pseudomonas sp. SE83 |
| C471betaS/A114alphaS/A38betaS/L180betaF | site-directed mutagenesis, mutant M13, the mutant shows altered catalytic efficiency compared to wild-type | Pseudomonas sp. SE83 |
| C471betaS/A38betaS/L154betaF | site-directed mutagenesis, mutant M14, the mutant shows altered catalytic efficiency compared to wild-type | Pseudomonas sp. SE83 |
| C471betaS/A38betaS/L154betaF/L180betaF | site-directed mutagenesis, mutant M15, the mutant shows altered catalytic efficiency compared to wild-type | Pseudomonas sp. SE83 |
| C47C471betaS/A38betaS | site-directed mutagenesis, mutant M11, the mutant shows altered catalytic efficiency compared to wild-type | Pseudomonas sp. SE83 |
| L154betaF | site-directed mutagenesis, mutant M4, mutation within the hydrophobic core regions of the alphabetabetaalpha structural motif | Pseudomonas sp. SE83 |
| L180betaF | site-directed mutagenesis, mutant M5, mutation within the hydrophobic core regions of the alphabetabetaalpha structural motif | Pseudomonas sp. SE83 |
| additional information | computational design of thermostable mutants for cephalosporin C acylase from Pseudomonas strain SE83, overview. Redesign to enhance its stability by repacking the hydrophobic core regions and reconstructing the protein-protein interactions in the segment interface regions. Single point mutations Asn2betaThr, Asn2betaVal, Cys470betaSer, Leu154betaPhe, and Leu180betaPhe in hydrophobic core regions, and Ala100alphaSer and Ala37betaSer in segment-segment interface regions, increase the Tm by 4.7-19.7°C, while combining these confirmed single mutations increases the Tm by up to 20.5°C. Construction of six multiple-point variants with negative calculated folding free energy changes. At a reaction temperature of 37°C, the catalytic efficiencies of the design template, and mutants M1, M11, and M13 are 2.72, 2.48, 0.42, and 0.64 U/mg/ mM, respectively. At 50°C, the catalytic efficiencies are 0.475, 0.70, 0.80, and 0.92 U/mg/mM, respectively | Pseudomonas sp. SE83 |
| N3betaT | site-directed mutagenesis, mutant M1, mutation within the hydrophobic core regions of the alphabetabetaalpha structural motif | Pseudomonas sp. SE83 |
| N3betaV | site-directed mutagenesis, mutant M2, mutation within the hydrophobic core regions of the alphabetabetaalpha structural motif | Pseudomonas sp. SE83 |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| cephalosporin C + H2O | Pseudomonas sp. SE83 | - |
7-aminocephalosporanic acid + 2-amino-5-hydroxypentanoate | - |
? |  |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Pseudomonas sp. SE83 | P15557 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and desalting gel filtration | Pseudomonas sp. SE83 |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| cephalosporin C + H2O | - |
Pseudomonas sp. SE83 | 7-aminocephalosporanic acid + 2-amino-5-hydroxypentanoate | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| AcyI | - |
Pseudomonas sp. SE83 |
| acylase ACY 1 proenzyme | UniProt | Pseudomonas sp. SE83 |
| CCA | - |
Pseudomonas sp. SE83 |
| cephalosporin C acylase | - |
Pseudomonas sp. SE83 |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| additional information | - |
single point mutations Asn2betaThr, Asn2betaVal, Cys470betaSer, Leu154betaPhe, and Leu180betaPhe in hydrophobic core regions, and Ala100alphaSer and Ala37betaSer in segment-segment interface regions, increase the Tm by 4.7-19.7°C, while combining these confirmed single mutations increases the Tm by up to 20.5°C. Construction of six multiple-point variants with negative calculated folding free energy changes | Pseudomonas sp. SE83 |
General Information
| General Information | Comment | Organism |
|---|---|---|
| physiological function | cephalosporin C acylase is the key enzyme catalyst for the hydrolysis of cephalosporin C (CPC), which directly produces the important cephalosporin antibiotic 7-aminocephalosporanic acid (7-ACA). 7-ACA is an important cephalosporin nucleus for the synthesis of many widely used beta-lactam antibiotics | Pseudomonas sp. SE83 |
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