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 (Comment) | Organism |
---|---|
recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) | Pseudomonas sp. SE83 |
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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas sp. SE83 | P15557 | - |
- |
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 | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
cephalosporin C + H2O | - |
Pseudomonas sp. SE83 | 7-aminocephalosporanic acid + 2-amino-5-hydroxypentanoate | - |
? |
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 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 | 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 |