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Literature summary for 1.10.3.2 extracted from
- Engineering platforms for directed evolution of Laccase from Pycnoporus cinnabarinus (2012), Appl. Environ. Microbiol., 78, 1370-1384.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| functional enzyme expression in Saccharomyces cerevisiae, expression of engineered mutant enzymes in Aspergillus niger, the evolved variants are highly | Trametes cinnabarina |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| A240P | site-directed mutagenesis | Trametes cinnabarina |
| D341N | site-directed mutagenesis the D3 domain coil, surface, the mutant shows H bonding with surrounding residue N340 in contrast to the wild-type enzyme | Trametes cinnabarina |
| additional information | replacement of the native PcL signal peptide by the alpha-factor preproleader, and subjected to six rounds of evolution coupled to a multiscreening assay based on the oxidation of natural and synthetic redox mediators at more neutral pHs. The sequence encoding the evolved alpha-factor preproleader of 7A9 for secretion in Sacchaaromyces cerevisiae is replaced by the 24-amino-acid glucoamylase preprosequence from Aspergillus niger, under the Emericella nidulans gpd promoter and trpC terminator. Total laccase activity is enhanced 8000fold, the evolved alpha-factor preproleader improves secretion levels 40fold, and several mutations in mature laccase provide a 13.7fold increase in kcat. While the pH activity profile is shifted to more neutral values, the thermostability and the broad substrate specificity of PcL are retained. Mutational effect on laccase secretion, overview | Trametes cinnabarina |
| N208S | | site-directed mutagenesis in the D2 domain beta sheet, near D206 (responsible for binding phenolic substrates at the T1 site), the mutant shows increased H bonding with surrounding residues | Trametes cinnabarina |
| N331D | site-directed mutagenesis the D3 domain beta sheet substrate binding loop, contiguous to F332 key residue of the binding pocket, the mutant shows increased H bonding with surrounding residues | Trametes cinnabarina |
| P394H | site-directed mutagenesis | Trametes cinnabarina |
| P394H | site-directed mutagenesis the D3 domain coil substrate binding loop, contiguous to H395, T1 Cu ligand, the mutant shows H bonding with surrounding residue S427 in contrast to the wild-type enzyme | Trametes cinnabarina |
| R280H | | site-directed mutagenesis in the D2 domain end of distal beta sheet, surface, the mutant shows reduced H bonding with surrounding residues | Trametes cinnabarina |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | kinetics of mutant enzymes, overview | Trametes cinnabarina | |
| 0.013 | - |
Sinapic acid | recombinant wild-type enzyme, pH 5.0, temperature not specified in the publication | Trametes cinnabarina |  |
| 0.027 | - |
2,6-dimethoxyphenol | recombinant wild-type enzyme, pH 5.0, temperature not specified in the publication | Trametes cinnabarina | |
| 0.028 | - |
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) | recombinant wild-type enzyme, pH 3.0, temperature not specified in the publication | Trametes cinnabarina | |
| 0.035 | - |
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) | recombinant wild-type enzyme, pH 5.0, temperature not specified in the publication | Trametes cinnabarina | |
| 0.085 | - |
Sinapic acid | recombinant wild-type enzyme, pH 3.0, temperature not specified in the publication | Trametes cinnabarina | |
| 0.324 | - |
2,6-dimethoxyphenol | recombinant wild-type enzyme, pH 3.0, temperature not specified in the publication | Trametes cinnabarina | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Trametes cinnabarina | Q9UVQ2 | - |
- |
| Trametes cinnabarina I-937 | Q9UVQ2 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant wild-type and mutant enzymes from Saccharomyces cerevisiae by tangential ultrafiltration, dialysis, pressure ultrafiltration, anion exchange chromatography, and gel filtration | Trametes cinnabarina |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) + O2 | - |
Trametes cinnabarina | ? | - |
? | |
| 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) + O2 | - |
Trametes cinnabarina I-937 | ? | - |
? | |
| 2,6-dimethoxyphenol + O2 | - |
Trametes cinnabarina | ? | - |
? | |
| 2,6-dimethoxyphenol + O2 | - |
Trametes cinnabarina I-937 | ? | - |
? | |
| additional information | broad substrate specificity | Trametes cinnabarina | ? | - |
? | |
| additional information | broad substrate specificity | Trametes cinnabarina I-937 | ? | - |
? | |
| sinapic acid + O2 | - |
Trametes cinnabarina | ? | - |
? | |
| sinapic acid + O2 | - |
Trametes cinnabarina I-937 | ? | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| PCL | - |
Trametes cinnabarina |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 16.3 | - |
2,6-dimethoxyphenol | recombinant wild-type enzyme, pH 5.0, temperature not specified in the publication | Trametes cinnabarina | |
| 21.6 | - |
Sinapic acid | recombinant wild-type enzyme, pH 5.0, temperature not specified in the publication | Trametes cinnabarina | |
| 26.4 | - |
2,6-dimethoxyphenol | recombinant wild-type enzyme, pH 3.0, temperature not specified in the publication | Trametes cinnabarina | |
| 38 | - |
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) | recombinant wild-type enzyme, pH 5.0, temperature not specified in the publication | Trametes cinnabarina | |
| 52.8 | - |
Sinapic acid | recombinant wild-type enzyme, pH 3.0, temperature not specified in the publication | Trametes cinnabarina | |
| 144.6 | - |
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) | recombinant wild-type enzyme, pH 3.0, temperature not specified in the publication | Trametes cinnabarina | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 2 | - |
wild-type enzyme with substrate 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) | Trametes cinnabarina |
| 3 | - |
wild-type enzyme with substrate 2,6-dimethoxyphenol | Trametes cinnabarina |
| 3 | 5 | mutant optima shift from the wild-type enzyme, overview | Trametes cinnabarina |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 81 | - |
2,6-dimethoxyphenol | recombinant wild-type enzyme, pH 3.0, temperature not specified in the publication | Trametes cinnabarina | |
| 581 | - |
2,6-dimethoxyphenol | recombinant wild-type enzyme, pH 5.0, temperature not specified in the publication | Trametes cinnabarina | |
| 621 | - |
Sinapic acid | recombinant wild-type enzyme, pH 3.0, temperature not specified in the publication | Trametes cinnabarina | |
| 1085 | - |
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) | recombinant wild-type enzyme, pH 5.0, temperature not specified in the publication | Trametes cinnabarina | |
| 1662 | - |
Sinapic acid | recombinant wild-type enzyme, pH 5.0, temperature not specified in the publication | Trametes cinnabarina | |
| 5164 | - |
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) | recombinant wild-type enzyme, pH 3.0, temperature not specified in the publication | Trametes cinnabarina | |
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