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Literature summary for 1.7.1.6 extracted from

  • Zhang, X.; Ng, I.; Chang, J.
    Cloning and characterization of a robust recombinant azoreductase from Shewanella xiamenensis BC01 (2015), J. Taiwan Inst. Chem. Eng., 61, 97-105 .
No PubMed abstract available

Cloned(Commentary)

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

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

Inhibitors Comment Organism Structure
iodoacetamide loss of 30.6% activity at 10% v/v Shewanella xiamenensis

KM Value [mM]

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

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 Stability

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

Organism UniProt Comment Textmining
Shewanella xiamenensis A0A073KHN9
-
-
Shewanella xiamenensis BC01 A0A073KHN9
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Shewanella xiamenensis

Specific Activity [micromol/min/mg]

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 and Products (Substrate)

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

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

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 [°C] Temperature Optimum Maximum [°C] Comment Organism
20 30
-
Shewanella xiamenensis

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
10 50 activity range, profile overview Shewanella xiamenensis

Temperature Stability [°C]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
6.5
-
-
Shewanella xiamenensis

pH Range

pH Minimum pH Maximum Comment Organism
5 9 activity range, profile overview Shewanella xiamenensis

pH Stability

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

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

pI Value

Organism Comment pI Value Maximum pI Value
Shewanella xiamenensis sequence calculation
-
5.23

General Information

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