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Literature summary for 3.5.1.4 extracted from

  • Egorova, K.; Trauthwein, H.; Verseck, S.; Antranikian, G.
    Purification and properties of an enantioselective and thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila (2004), Appl. Microbiol. Biotechnol., 65, 38-45.
    View publication on PubMed

Activating Compound

Activating Compound Comment Organism Structure
DTT 20% activation at 1 mM Pseudonocardia thermophila

Inhibitors

Inhibitors Comment Organism Structure
Ba2+ 13% inhibition at 1 mM Pseudonocardia thermophila
Ca2+ 15% inhibition at 1 mM Pseudonocardia thermophila
Co2+ complete inhibition at 1 mM Pseudonocardia thermophila
Cu2+ 74% inhibition at 1 mM Pseudonocardia thermophila
Fe2+ 20% inhibition at 1 mM Pseudonocardia thermophila
Hg2+ complete inhibition at 1 mM Pseudonocardia thermophila
iodoacetamide 10% inhibition at 1 mM Pseudonocardia thermophila
iodoacetate 78% inhibition at 1 mM Pseudonocardia thermophila
Mg2+ 10% inhibition at 1 mM Pseudonocardia thermophila
additional information no inhibition by Triton X-100, SDS, EDTA, Mn2+, and Fe3+ at 1 mM Pseudonocardia thermophila
Ni2+ 56% inhibition at 1 mM Pseudonocardia thermophila
Zn2+ 7% inhibition at 1 mM Pseudonocardia thermophila

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.9
-
2-phenylpropionamide pH 7.2, 70°C Pseudonocardia thermophila
4.9
-
Benzamide pH 7.2, 70°C Pseudonocardia thermophila
7.4
-
Propionamide pH 7.2, 70°C Pseudonocardia thermophila
9.2
-
methacrylamide pH 7.2, 70°C Pseudonocardia thermophila

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
52000
-
2 * 52000, SDS-PAGE Pseudonocardia thermophila
108000
-
gel filtration Pseudonocardia thermophila

Organism

Organism UniProt Comment Textmining
Pseudonocardia thermophila
-
thermophilic actinomycete, DSMZ 43832
-

Purification (Commentary)

Purification (Comment) Organism
native enzyme 48fold to homogeneity by hydrophobic interaction chromatography, ultrafiltration, anion exchange chromatography, and gel filtration Pseudonocardia thermophila

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
19.4
-
purified enzyme, substrate benzamide Pseudonocardia thermophila
23.5
-
purified enzyme, substrate methacrylamide Pseudonocardia thermophila
25.4
-
purified enzyme, substrate propionamide Pseudonocardia thermophila
26.8
-
purified enzyme, substrate acrylamide Pseudonocardia thermophila

Storage Stability

Storage Stability Organism
4°C, purified enzyme, in 50 mM phosphate buffer, more than 6 months without loss of activity Pseudonocardia thermophila

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(R,S)-2-phenylpropionamide + H2O the enzyme is highly S-stereoselective for 2-phenylpropionamide, and the racemic amide is converted to the corresponding S-acid with an enantiomeric excess of over 95% at 50% conversion of the substrate Pseudonocardia thermophila (S)-2-phenylpropionic acid + NH3
-
?
2-aminobenzamide + H2O
-
Pseudonocardia thermophila 2-aminobenzoic acid + NH3
-
?
2-hydroxybenzamide + H2O
-
Pseudonocardia thermophila 2-hydroxybenzoic acid + NH3
-
?
2-tolylamide + H2O
-
Pseudonocardia thermophila ? + NH3
-
?
3-indolylacetamide + H2O
-
Pseudonocardia thermophila 3-indolylacetic acid + NH3
-
?
3-tolylamide + H2O
-
Pseudonocardia thermophila ? + NH3
-
?
4-aminobenzamide + H2O
-
Pseudonocardia thermophila 4-aminobenzoic acid + NH3
-
?
4-hydroxybenzamide + H2O
-
Pseudonocardia thermophila 4-hydroxybenzoic acid + NH3
-
?
4-tolylamide + H2O
-
Pseudonocardia thermophila ? + NH3
-
?
acetamide + H2O
-
Pseudonocardia thermophila acetic acid + NH3
-
?
acrylamide + H2O
-
Pseudonocardia thermophila acrylic acid + NH3
-
?
adipoamide + H2O
-
Pseudonocardia thermophila adipic acid + NH3
-
?
benzamide + H2O
-
Pseudonocardia thermophila benzoic acid + NH3
-
?
cyclohexanoamide + H2O
-
Pseudonocardia thermophila cyclohexanoic acid + NH3
-
?
DL-lactamide + H2O
-
Pseudonocardia thermophila DL-lactate + NH3
-
?
DL-methioninamide + H2O
-
Pseudonocardia thermophila DL-methionine + NH3
-
?
DL-tryptophanamide + H2O
-
Pseudonocardia thermophila DL-tryptophan + NH3
-
?
formamide + H2O
-
Pseudonocardia thermophila formate + NH3
-
?
hexanoamide + H2O
-
Pseudonocardia thermophila hexanoic acid + NH3
-
?
hydroxylamine hydrochloride + H2O
-
Pseudonocardia thermophila ?
-
?
isobutyramide + H2O
-
Pseudonocardia thermophila isobutyric acid + NH3
-
?
isonicotinamide + H2O
-
Pseudonocardia thermophila isonicotinic acid + NH3
-
?
L-alanineamide + H2O
-
Pseudonocardia thermophila L-alanine + NH3
-
?
L-leucineamide + H2O
-
Pseudonocardia thermophila L-leucine + NH3
-
?
L-prolinamide + H2O
-
Pseudonocardia thermophila L-proline + NH3
-
?
L-valineamide + H2O
-
Pseudonocardia thermophila L-valine + NH3
-
?
malonamide + H2O
-
Pseudonocardia thermophila malonic acid + NH3
-
?
methacrylamide + H2O
-
Pseudonocardia thermophila methacrylic acid + NH3
-
?
additional information the enzyme is enantioselective, the enzyme also shows acyl transferase and hydrolase activities, no activity with N-methylurea, fumaramide, 4-aminobenzosulfonamide, N-phenylurea, acetanilide, benzylcarbamate, and o-tolylamide, broad substrate spectrum, including aliphatic, aromatic and amino acid amides, the presence of a double bond or a methyl group near the carboxamide group of aliphatic and amino acid amides enhances the enzymatic activity, among aromatic amides with substitutions at the 2-, 3-, or 4-position, the 4-substituted amides are the preferred substrates, detailed overview Pseudonocardia thermophila ?
-
?
nicotinamide + H2O
-
Pseudonocardia thermophila nicotinic acid + NH3
-
?
propionamide + H2O
-
Pseudonocardia thermophila propionic acid + NH3
-
?
pyrazinamide + H2O
-
Pseudonocardia thermophila ? + NH3
-
?
succinamide + H2O
-
Pseudonocardia thermophila succinic acid + NH3
-
?
trimethylacetamide + H2O
-
Pseudonocardia thermophila trimethylacetic acid + NH3
-
?
urea + H2O
-
Pseudonocardia thermophila ?
-
?

Subunits

Subunits Comment Organism
dimer 2 * 52000, SDS-PAGE Pseudonocardia thermophila

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
70
-
assay at, hydrolytic activity Pseudonocardia thermophila

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
30 80 10% of maximal activity at 30°C and 80°C Pseudonocardia thermophila

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
60
-
purified enzyme, pH 7.2, the enzyme is heat stable, no loss of activity within the first 9 h, 20% loss of activity after 25 h Pseudonocardia thermophila
70
-
purified enzyme, half-life 1.2 h Pseudonocardia thermophila

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.2
-
amidase activity Pseudonocardia thermophila

pH Range

pH Minimum pH Maximum Comment Organism
5 9 high activity within this range Pseudonocardia thermophila

pI Value

Organism Comment pI Value Maximum pI Value
Pseudonocardia thermophila isoelectric focusing
-
4.2