Cloned (Comment) | Organism |
---|---|
gene azoR, DNA and amino acid sequence determination and analysis, phylogenetic analysis, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) from three different vectors pET32a, pET28a and pET20bI. pET32a contains an N-terminal Trx-tag encoding for a 109-amino-acid highly soluble polypeptide thioredoxin, TrxA, the pET28a is an original vector without special fusion protein, while pET20bI (similar to pET22b) contains an N-terminal pelB signal sequence for potential periplasmic localization resulting in recombinant enzymes of molecular weight of 40.4 kDa, 26.2 kDa and 25.8 kDa, respectively. Recombinant AzrS_32 is soluble by 98%, AzrS_28 by 90%, while AzrS_20 is mostly accumulated as inclusion bodies, that have no activity, although pelB leads the target protein to the periplasmic space, where there is a more favorable environment for folding and disulfide bond formation. By contrast, the protein amount of AzrS_28 (i.e. 278 mg/l) is slightly higher than that of AzrS_32 (i.e. 253 mg/l). Therefore, the plasmid pET28a is considered as the ideal vector for production of AzrS | Shewanella xiamenensis |
General Stability | Organism |
---|---|
AzrS is an oxygen-insensitive, FMN-dependent, robust against to organic solvent and has extremely high and stable activity at room temperature | Shewanella xiamenensis |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
iodoacetamide | loss of 30.6% activity at 10% v/v | Shewanella xiamenensis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten steady-state kinetics | Shewanella xiamenensis | |
0.1056 | - |
Methyl orange | recombinant enzyme, pH 6.5, 22°C | Shewanella xiamenensis | |
0.1593 | - |
Methyl red | recombinant enzyme, pH 6.5, 22°C | Shewanella xiamenensis | |
0.2792 | - |
Congo red | recombinant enzyme, pH 6.5, 22°C | Shewanella xiamenensis |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Ca2+ | activates at 5 mM | Shewanella xiamenensis | |
Co2+ | activates at 5 mM | Shewanella xiamenensis | |
Cr3+ | activates at 5 mM | Shewanella xiamenensis | |
Cu2+ | activates at 5 mM | Shewanella xiamenensis | |
Fe3+ | activates at 5 mM | Shewanella xiamenensis | |
K+ | activates at 5 mM | Shewanella xiamenensis | |
Mg2+ | 41% activation at 5 mM, best activating cation | Shewanella xiamenensis | |
Mn2+ | activates at 5 mM | Shewanella xiamenensis | |
Na+ | activates at 5 mM | Shewanella xiamenensis | |
Zn2+ | activates at 5 mM | Shewanella xiamenensis |
Organic Solvent | Comment | Organism |
---|---|---|
1-butanol | loss of 85.4% activity at 10% v/v | Shewanella xiamenensis |
1-propanol | loss of 40.9% activity at 10% v/v | Shewanella xiamenensis |
Acetone | at 10% v/v, the enzyme is completely stable | Shewanella xiamenensis |
chloroform | loss of 55.6% activity at 10% v/v | Shewanella xiamenensis |
DMSO | loss of 12.2% activity at 10% v/v | Shewanella xiamenensis |
Ethanol | loss of 7.6% activity at 10% v/v | Shewanella xiamenensis |
isobutanol | loss of 73.8% activity at 10% v/v | Shewanella xiamenensis |
isopropanol | gain of 5.8% activity at 10% v/v | Shewanella xiamenensis |
Methanol | at 10% v/v, the enzyme is completely stable | Shewanella xiamenensis |
additional information | AzrS is an oxygen-insensitive, FMN-dependent, robust against to organic solvent and has extremely high and stable activity at room temperature | Shewanella xiamenensis |
N,N-dimethylformamide | loss of 3.0% activity at 10% v/v | Shewanella xiamenensis |
tert-Butanol | loss of 19.0% activity at 10% v/v | Shewanella xiamenensis |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Shewanella xiamenensis | A0A073KHN9 | - |
- |
Shewanella xiamenensis BC01 | A0A073KHN9 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography | Shewanella xiamenensis |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
254 | - |
purified recombinant enzyme, pH 6.5, 22°C, substrate congo red | Shewanella xiamenensis |
5152 | - |
purified recombinant enzyme, pH 6.5, 22°C, substrate methl orange | Shewanella xiamenensis |
97153 | - |
purified recombinant enzyme, pH 6.5, 22°C, substrate methl red | Shewanella xiamenensis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
Congo red + NADH + H+ | - |
Shewanella xiamenensis | ? + NAD+ | - |
? | |
Congo red + NADH + H+ | - |
Shewanella xiamenensis BC01 | ? + NAD+ | - |
? | |
methyl orange + NADH + H+ | - |
Shewanella xiamenensis | ? + NAD+ | - |
? | |
methyl orange + NADH + H+ | - |
Shewanella xiamenensis BC01 | ? + NAD+ | - |
? | |
methyl red + 2 NADH + 2 H+ | i.e. 4-dimethylaminoazobenzene-2'-carboxylic acid | Shewanella xiamenensis | anthranilate + 4-amino-N,N-dimethylaniline + 2 NAD+ | - |
? | |
methyl red + 2 NADH + 2 H+ | i.e. 4-dimethylaminoazobenzene-2'-carboxylic acid | Shewanella xiamenensis BC01 | anthranilate + 4-amino-N,N-dimethylaniline + 2 NAD+ | - |
? | |
additional information | substrate specificity: the catalytic efficiency of AzrS follows the descending order of methyl red, methyl orange, and congo red. The enzyme has a wide substrate range of azo dyes | Shewanella xiamenensis | ? | - |
? | |
additional information | substrate specificity: the catalytic efficiency of AzrS follows the descending order of methyl red, methyl orange, and congo red. The enzyme has a wide substrate range of azo dyes | Shewanella xiamenensis BC01 | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
? | x * 21300, about, sequence calculation, x * 40400, recombinant AzrS_32 with TrxA, DS-PAGE, x * 26200, recombinant untagged AzrS_28, SDS-PAGE, x * 25800, recombinant AzrS_20 with pelB signal peptide | Shewanella xiamenensis |
Synonyms | Comment | Organism |
---|---|---|
aerobic azoreductase | - |
Shewanella xiamenensis |
AzoR | - |
Shewanella xiamenensis |
AzrS | - |
Shewanella xiamenensis |
FMN-dependent NADH-azo compound oxidoreductase | UniProt | Shewanella xiamenensis |
FMN-dependent NADH-azoreductase | UniProt | Shewanella xiamenensis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
20 | 30 | - |
Shewanella xiamenensis |
Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
---|---|---|---|
10 | 50 | activity range, profile overview | Shewanella xiamenensis |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
10 | 50 | most stable at 20°C, loss of activity at 60°C | Shewanella xiamenensis |
22 | - |
AzrS is an oxygen-insensitive, FMN-dependent, robust against to organic solvent and has extremely high and stable activity at room temperature | Shewanella xiamenensis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6.5 | - |
- |
Shewanella xiamenensis |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
5 | 9 | activity range, profile overview | Shewanella xiamenensis |
pH Stability | pH Stability Maximum | Comment | Organism |
---|---|---|---|
6 | 10 | most stable at pH 7.0-8.0, loss of activity at pH 5.0 and below | Shewanella xiamenensis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FMN | dependent on, best at 0.125 mM | Shewanella xiamenensis | |
additional information | AzrS shows activity with both NADH and NADPH. The maximum relative activity of AzrS with 0.875 mM NADPH or NADH is 28.1% and 100%, respectively, indicating that AzrS shows a preference for NADH rather than NADPH | Shewanella xiamenensis | |
NADH | preferred cofactor | Shewanella xiamenensis | |
NADPH | low activity | Shewanella xiamenensis |
Organism | Comment | pI Value Maximum | pI Value |
---|---|---|---|
Shewanella xiamenensis | sequence calculation | - |
5.23 |
General Information | Comment | Organism |
---|---|---|
additional information | homologous modeling and molecular docking of AzrS for protein-ligand interactions analysis, using the the azoreductase structure from Escherichia coli strain JM109, PDB ID 1v4b. Methyl red and methyl orange formed the hydrogen bonds with His143, Gly140, Gly141 and Asn176 | Shewanella xiamenensis |