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Literature summary for 3.4.24.26 extracted from
- The role of N-glycosylation sites in the activity, stability, and expression of the recombinant elastase expressed by Pichia pastoris (2014), Enzyme Microb. Technol., 54, 32-37.
Application
| Application | Comment | Organism |
|---|---|---|
| synthesis | the Pseudomonas aeruginosa elastase, produced by Pichia pastoris, is a promising biocatalyst for peptide synthesis in organic solvents | Pseudomonas aeruginosa |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| recombinant enzyme expression in Pichia pastoris with heterogeneous N-glycosylation, expression of enzyme glycosylation site mutants | Pseudomonas aeruginosa |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| additional information | mutation of any potential N-glycosylation site was detrimental to its expression in Pichia pastoris with 23.9% decrease in expression of the N43Q mutant, 63.6% of the N212Q mutant, and 63.7% of the N280Q mutant compared with the wild type | Pseudomonas aeruginosa |
| N212Q | site-directed mutagenesis, the mutant enzyme shows similar activity and slightly decreased thermostability compared to the wild-type enzyme | Pseudomonas aeruginosa |
| N212Q/N280Q | site-directed mutagenesis, 90.6% decreased activity compared to the wild-type enzyme | Pseudomonas aeruginosa |
| N280Q | site-directed mutagenesis, the mutant enzyme shows similar activity and slightly decreased thermostability compared to the wild-type enzyme | Pseudomonas aeruginosa |
| N43Q | site-directed mutagenesis, the mutant enzyme shows similar activity and slightly decreased thermostability compared to the wild-type enzyme | Pseudomonas aeruginosa |
| N43Q/N212Q | site-directed mutagenesis, 68.7% decreased activity compared to the wild-type enzyme | Pseudomonas aeruginosa |
| N43Q/N212Q/N280Q | site-directed mutagenesis, 90.6% decreased activity compared to the wild-type enzyme | Pseudomonas aeruginosa |
| N43Q/N280Q | site-directed mutagenesis, 73.6% decreased activity compared to the wild-type enzyme | Pseudomonas aeruginosa |
General Stability
| General Stability | Organism |
|---|---|
| the first and second disulfide bonds are essential for the stability and activity of the enzyme, respectively | Pseudomonas aeruginosa |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Ca2+ | a calcium ion is also required for enzyme activity, and stabilizes its tertiary structure. Contact with the calcium ion is made by the carboxyl groups of Asp136, Glu172, Glu175, and Asp183, the carbonyl group of Leu185, and one water molecule | Pseudomonas aeruginosa |  |
| Zn2+ | the enzyme is a metalloprotease that requires a zinc atom, bound to His140, His144, and Glu164, for its activity | Pseudomonas aeruginosa | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| Elastin + H2O | Pseudomonas aeruginosa | - |
? | - |
? | |
Organic Solvent Stability
| Organic Solvent | Comment | Organism |
|---|---|---|
| chloroform | pH 7.0, 30°C, 4 days, 95.6% activity remaining for the nonglycosylated recombinant enzyme, 91.5% for the glycosylated recombinant enzyme | Pseudomonas aeruginosa |
| DMSO | pH 7.0, 30°C, 4 days, 95.1% activity remaining for the nonglycosylated recombinant enzyme, 83.2% for the glycosylated recombinant enzyme | Pseudomonas aeruginosa |
| Ethanol | pH 7.0, 30°C, 4 days, 71.5% activity remaining for the nonglycosylated recombinant enzyme, 62.1% for the glycosylated recombinant enzyme | Pseudomonas aeruginosa |
| isopropanol | pH 7.0, 30°C, 4 days, 39.2% activity remaining for the nonglycosylated recombinant enzyme, 17.5% for the glycosylated recombinant enzyme | Pseudomonas aeruginosa |
| Methanol | pH 7.0, 30°C, 4 days, 102% activity remaining for the nonglycosylated recombinant enzyme, 96.5% for the glycosylated recombinant enzyme | Pseudomonas aeruginosa |
| n-Butanol | pH 7.0, 30°C, 4 days, 89.5% activity remaining for the nonglycosylated recombinant enzyme, 78.5% for the glycosylated recombinant enzyme | Pseudomonas aeruginosa |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Pseudomonas aeruginosa | - |
- |
- |
Posttranslational Modification
| Posttranslational Modification | Comment | Organism |
|---|---|---|
| glycoprotein | the recombinant elastase contains three potential N-glycosylation sites N43, N212, and N280 (Asn-Xaa-Ser/Thr consensus sequences), potential role of N-glycosylation in the activity and stability. Non- and glycosylated isoforms of rPAE display similar kinetic parameters for hydrolyzing casein in aqueous medium, and when catalyzing bipeptide synthesis in 50% v/v DMSO, they exhibit identical substrate specificity and activity, and produce similar yields. The N-linked oligosaccharides of Pichia pastoris-secreted glycoproteins are a high-mannose type (Man8GlcNAc2 or Man9GlcNAc2) with molecular weights close to 2 kDa | Pseudomonas aeruginosa |
| proteolytic modification | the enzyme is synthesized in the cytoplasm as a pre-proenzyme consisting of a 2.4-kDa signal peptid, an 18.1-kDa pro-peptide and the 33.1-kDa mature protein. The signal peptide is cleaved from the pre-proenzyme during translocation across the inner membrane, leaving a 51.2-kDa proenzyme (consisting of the pro-peptide and the mature protein). In the periplasm, the proenzymeis folded, guided by the pro-peptide, and a disulfide bond between Cys270 and Cys297 is formed. The pro-peptide is then removed by autoproteolysis, but remains non-covalently attached to mature pseudolysin. A second disulfide bond between Cys30 and Cys58 of the enzyme is then formed. The pro-peptide and mature enzyme are secreted from the cell together, where they dissociate, and the liberated pro-peptide is degraded by the active enzyme | Pseudomonas aeruginosa |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 2389 | - |
recombinant glycosylated enzyme, pH 7.5, 60°C, substrate casein | Pseudomonas aeruginosa |
| 2401 | - |
recombinant nonglycosylated enzyme, pH 7.5, 60°C, substrate casein | Pseudomonas aeruginosa |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| casein + H2O | - |
Pseudomonas aeruginosa | ? | - |
? | |
| Elastin + H2O | - |
Pseudomonas aeruginosa | ? | - |
? | |
| additional information | non- and glycosylated isoforms of rPAE display similar kinetic parameters for hydrolyzing casein in aqueous medium, and when catalyzing bipeptide synthesis in 50% v/v DMSO, they exhibit identical substrate specificity and activity, and produce similar yields | Pseudomonas aeruginosa | ? | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| elastase | - |
Pseudomonas aeruginosa |
| PAE | - |
Pseudomonas aeruginosa |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 60 | - |
- |
Pseudomonas aeruginosa |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 60 | - |
stable up to, recombinant enzyme | Pseudomonas aeruginosa |
| 70 | - |
half-lives of the glycosylated and non-glycosylated forms of recombinant enzyme are 32.2 and 23.1 min, respectively | Pseudomonas aeruginosa |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8 | - |
- |
Pseudomonas aeruginosa |
pH Stability
| pH Stability | pH Stability Maximum | Comment | Organism |
|---|---|---|---|
| 6 | 9.5 | stable at, recombinant enzyme | Pseudomonas aeruginosa |
pI Value
| Organism | Comment | pI Value Maximum | pI Value |
|---|---|---|---|
| Pseudomonas aeruginosa | - |
- |
5.9 |
General Information
| General Information | Comment | Organism |
|---|---|---|
| additional information | the enzyme is synthesized in the cytoplasm as a pre-proenzyme consisting of a 2.4-kDa signal peptid, an 18.1-kDa pro-peptide and the 33.1-kDa mature protein. The signal peptide is cleaved from the pre-proenzyme during translocation across the inner membrane, leaving a 51.2-kDa proenzyme (consisting of the pro-peptide and the mature protein). In the periplasm, the proenzymeis folded, guided by the pro-peptide, and a disulfide bond between Cys270 and Cys297 is formed. The pro-peptide is then removed by autoproteolysis, but remains non-covalently attached to mature pseudolysin. A second disulfide bond between Cys30 and Cys58 of the enzyme is then formed. The pro-peptide and mature enzyme are secreted from the cell together, where they dissociate, and the liberated pro-peptide is degraded by the active enzyme | Pseudomonas aeruginosa |
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