BRENDA - Enzyme Database show

Purification and properties of an enantioselective and thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila

Egorova, K.; Trauthwein, H.; Verseck, S.; Antranikian, G.; Appl. Microbiol. Biotechnol. 65, 38-45 (2004)

Data extracted from this reference:

Activating Compound
EC Number
Activating Compound
Commentary
Organism
Structure
3.5.1.4
DTT
20% activation at 1 mM
Pseudonocardia thermophila
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
3.5.1.4
Ba2+
13% inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
Ca2+
15% inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
Co2+
complete inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
Cu2+
74% inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
Fe2+
20% inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
Hg2+
complete inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
iodoacetamide
10% inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
iodoacetate
78% inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
Mg2+
10% inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
additional information
no inhibition by Triton X-100, SDS, EDTA, Mn2+, and Fe3+ at 1 mM
Pseudonocardia thermophila
3.5.1.4
Ni2+
56% inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
Zn2+
7% inhibition at 1 mM
Pseudonocardia thermophila
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
3.5.1.4
0.9
-
2-phenylpropionamide
pH 7.2, 70°C
Pseudonocardia thermophila
3.5.1.4
4.9
-
Benzamide
pH 7.2, 70°C
Pseudonocardia thermophila
3.5.1.4
7.4
-
Propionamide
pH 7.2, 70°C
Pseudonocardia thermophila
3.5.1.4
9.2
-
methacrylamide
pH 7.2, 70°C
Pseudonocardia thermophila
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
3.5.1.4
52000
-
2 * 52000, SDS-PAGE
Pseudonocardia thermophila
3.5.1.4
108000
-
gel filtration
Pseudonocardia thermophila
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
3.5.1.4
Pseudonocardia thermophila
-
thermophilic actinomycete, DSMZ 43832
-
Purification (Commentary)
EC Number
Commentary
Organism
3.5.1.4
native enzyme 48fold to homogeneity by hydrophobic interaction chromatography, ultrafiltration, anion exchange chromatography, and gel filtration
Pseudonocardia thermophila
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
3.5.1.4
19.4
-
purified enzyme, substrate benzamide
Pseudonocardia thermophila
3.5.1.4
23.5
-
purified enzyme, substrate methacrylamide
Pseudonocardia thermophila
3.5.1.4
25.4
-
purified enzyme, substrate propionamide
Pseudonocardia thermophila
3.5.1.4
26.8
-
purified enzyme, substrate acrylamide
Pseudonocardia thermophila
Storage Stability
EC Number
Storage Stability
Organism
3.5.1.4
4°C, purified enzyme, in 50 mM phosphate buffer, more than 6 months without loss of activity
Pseudonocardia thermophila
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3.5.1.4
(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
667278
Pseudonocardia thermophila
(S)-2-phenylpropionic acid + NH3
-
-
-
?
3.5.1.4
2-aminobenzamide + H2O
-
667278
Pseudonocardia thermophila
2-aminobenzoic acid + NH3
-
-
-
?
3.5.1.4
2-hydroxybenzamide + H2O
-
667278
Pseudonocardia thermophila
2-hydroxybenzoic acid + NH3
-
-
-
?
3.5.1.4
2-tolylamide + H2O
-
667278
Pseudonocardia thermophila
? + NH3
-
-
-
?
3.5.1.4
3-indolylacetamide + H2O
-
667278
Pseudonocardia thermophila
3-indolylacetic acid + NH3
-
-
-
?
3.5.1.4
3-tolylamide + H2O
-
667278
Pseudonocardia thermophila
? + NH3
-
-
-
?
3.5.1.4
4-aminobenzamide + H2O
-
667278
Pseudonocardia thermophila
4-aminobenzoic acid + NH3
-
-
-
?
3.5.1.4
4-hydroxybenzamide + H2O
-
667278
Pseudonocardia thermophila
4-hydroxybenzoic acid + NH3
-
-
-
?
3.5.1.4
4-tolylamide + H2O
-
667278
Pseudonocardia thermophila
? + NH3
-
-
-
?
3.5.1.4
acetamide + H2O
-
667278
Pseudonocardia thermophila
acetic acid + NH3
-
-
-
?
3.5.1.4
acrylamide + H2O
-
667278
Pseudonocardia thermophila
acrylic acid + NH3
-
-
-
?
3.5.1.4
adipoamide + H2O
-
667278
Pseudonocardia thermophila
adipic acid + NH3
-
-
-
?
3.5.1.4
benzamide + H2O
-
667278
Pseudonocardia thermophila
benzoic acid + NH3
-
-
-
?
3.5.1.4
cyclohexanoamide + H2O
-
667278
Pseudonocardia thermophila
cyclohexanoic acid + NH3
-
-
-
?
3.5.1.4
DL-lactamide + H2O
-
667278
Pseudonocardia thermophila
DL-lactate + NH3
-
-
-
?
3.5.1.4
DL-methioninamide + H2O
-
667278
Pseudonocardia thermophila
DL-methionine + NH3
-
-
-
?
3.5.1.4
DL-tryptophanamide + H2O
-
667278
Pseudonocardia thermophila
DL-tryptophan + NH3
-
-
-
?
3.5.1.4
formamide + H2O
-
667278
Pseudonocardia thermophila
formate + NH3
-
-
-
?
3.5.1.4
hexanoamide + H2O
-
667278
Pseudonocardia thermophila
hexanoic acid + NH3
-
-
-
?
3.5.1.4
hydroxylamine hydrochloride + H2O
-
667278
Pseudonocardia thermophila
?
-
-
-
?
3.5.1.4
isobutyramide + H2O
-
667278
Pseudonocardia thermophila
isobutyric acid + NH3
-
-
-
?
3.5.1.4
isonicotinamide + H2O
-
667278
Pseudonocardia thermophila
isonicotinic acid + NH3
-
-
-
?
3.5.1.4
L-alanineamide + H2O
-
667278
Pseudonocardia thermophila
L-alanine + NH3
-
-
-
?
3.5.1.4
L-leucineamide + H2O
-
667278
Pseudonocardia thermophila
L-leucine + NH3
-
-
-
?
3.5.1.4
L-prolinamide + H2O
-
667278
Pseudonocardia thermophila
L-proline + NH3
-
-
-
?
3.5.1.4
L-valineamide + H2O
-
667278
Pseudonocardia thermophila
L-valine + NH3
-
-
-
?
3.5.1.4
malonamide + H2O
-
667278
Pseudonocardia thermophila
malonic acid + NH3
-
-
-
?
3.5.1.4
methacrylamide + H2O
-
667278
Pseudonocardia thermophila
methacrylic acid + NH3
-
-
-
?
3.5.1.4
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
667278
Pseudonocardia thermophila
?
-
-
-
-
3.5.1.4
nicotinamide + H2O
-
667278
Pseudonocardia thermophila
nicotinic acid + NH3
-
-
-
?
3.5.1.4
propionamide + H2O
-
667278
Pseudonocardia thermophila
propionic acid + NH3
-
-
-
?
3.5.1.4
pyrazinamide + H2O
-
667278
Pseudonocardia thermophila
? + NH3
-
-
-
?
3.5.1.4
succinamide + H2O
-
667278
Pseudonocardia thermophila
succinic acid + NH3
-
-
-
?
3.5.1.4
trimethylacetamide + H2O
-
667278
Pseudonocardia thermophila
trimethylacetic acid + NH3
-
-
-
?
3.5.1.4
urea + H2O
-
667278
Pseudonocardia thermophila
?
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
3.5.1.4
dimer
2 * 52000, SDS-PAGE
Pseudonocardia thermophila
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
3.5.1.4
70
-
assay at, hydrolytic activity
Pseudonocardia thermophila
Temperature Range [°C]
EC Number
Temperature Minimum [°C]
Temperature Maximum [°C]
Commentary
Organism
3.5.1.4
30
80
10% of maximal activity at 30°C and 80°C
Pseudonocardia thermophila
Temperature Stability [°C]
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
3.5.1.4
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
3.5.1.4
70
-
purified enzyme, half-life 1.2 h
Pseudonocardia thermophila
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
3.5.1.4
7.2
-
amidase activity
Pseudonocardia thermophila
pH Range
EC Number
pH Minimum
pH Maximum
Commentary
Organism
3.5.1.4
5
9
high activity within this range
Pseudonocardia thermophila
pI Value
EC Number
Organism
Commentary
pI Value Maximum
pI Value
3.5.1.4
Pseudonocardia thermophila
isoelectric focusing
-
4.2
Activating Compound (protein specific)
EC Number
Activating Compound
Commentary
Organism
Structure
3.5.1.4
DTT
20% activation at 1 mM
Pseudonocardia thermophila
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
3.5.1.4
Ba2+
13% inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
Ca2+
15% inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
Co2+
complete inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
Cu2+
74% inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
Fe2+
20% inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
Hg2+
complete inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
iodoacetamide
10% inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
iodoacetate
78% inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
Mg2+
10% inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
additional information
no inhibition by Triton X-100, SDS, EDTA, Mn2+, and Fe3+ at 1 mM
Pseudonocardia thermophila
3.5.1.4
Ni2+
56% inhibition at 1 mM
Pseudonocardia thermophila
3.5.1.4
Zn2+
7% inhibition at 1 mM
Pseudonocardia thermophila
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
3.5.1.4
0.9
-
2-phenylpropionamide
pH 7.2, 70°C
Pseudonocardia thermophila
3.5.1.4
4.9
-
Benzamide
pH 7.2, 70°C
Pseudonocardia thermophila
3.5.1.4
7.4
-
Propionamide
pH 7.2, 70°C
Pseudonocardia thermophila
3.5.1.4
9.2
-
methacrylamide
pH 7.2, 70°C
Pseudonocardia thermophila
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
3.5.1.4
52000
-
2 * 52000, SDS-PAGE
Pseudonocardia thermophila
3.5.1.4
108000
-
gel filtration
Pseudonocardia thermophila
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
3.5.1.4
native enzyme 48fold to homogeneity by hydrophobic interaction chromatography, ultrafiltration, anion exchange chromatography, and gel filtration
Pseudonocardia thermophila
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
3.5.1.4
19.4
-
purified enzyme, substrate benzamide
Pseudonocardia thermophila
3.5.1.4
23.5
-
purified enzyme, substrate methacrylamide
Pseudonocardia thermophila
3.5.1.4
25.4
-
purified enzyme, substrate propionamide
Pseudonocardia thermophila
3.5.1.4
26.8
-
purified enzyme, substrate acrylamide
Pseudonocardia thermophila
Storage Stability (protein specific)
EC Number
Storage Stability
Organism
3.5.1.4
4°C, purified enzyme, in 50 mM phosphate buffer, more than 6 months without loss of activity
Pseudonocardia thermophila
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3.5.1.4
(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
667278
Pseudonocardia thermophila
(S)-2-phenylpropionic acid + NH3
-
-
-
?
3.5.1.4
2-aminobenzamide + H2O
-
667278
Pseudonocardia thermophila
2-aminobenzoic acid + NH3
-
-
-
?
3.5.1.4
2-hydroxybenzamide + H2O
-
667278
Pseudonocardia thermophila
2-hydroxybenzoic acid + NH3
-
-
-
?
3.5.1.4
2-tolylamide + H2O
-
667278
Pseudonocardia thermophila
? + NH3
-
-
-
?
3.5.1.4
3-indolylacetamide + H2O
-
667278
Pseudonocardia thermophila
3-indolylacetic acid + NH3
-
-
-
?
3.5.1.4
3-tolylamide + H2O
-
667278
Pseudonocardia thermophila
? + NH3
-
-
-
?
3.5.1.4
4-aminobenzamide + H2O
-
667278
Pseudonocardia thermophila
4-aminobenzoic acid + NH3
-
-
-
?
3.5.1.4
4-hydroxybenzamide + H2O
-
667278
Pseudonocardia thermophila
4-hydroxybenzoic acid + NH3
-
-
-
?
3.5.1.4
4-tolylamide + H2O
-
667278
Pseudonocardia thermophila
? + NH3
-
-
-
?
3.5.1.4
acetamide + H2O
-
667278
Pseudonocardia thermophila
acetic acid + NH3
-
-
-
?
3.5.1.4
acrylamide + H2O
-
667278
Pseudonocardia thermophila
acrylic acid + NH3
-
-
-
?
3.5.1.4
adipoamide + H2O
-
667278
Pseudonocardia thermophila
adipic acid + NH3
-
-
-
?
3.5.1.4
benzamide + H2O
-
667278
Pseudonocardia thermophila
benzoic acid + NH3
-
-
-
?
3.5.1.4
cyclohexanoamide + H2O
-
667278
Pseudonocardia thermophila
cyclohexanoic acid + NH3
-
-
-
?
3.5.1.4
DL-lactamide + H2O
-
667278
Pseudonocardia thermophila
DL-lactate + NH3
-
-
-
?
3.5.1.4
DL-methioninamide + H2O
-
667278
Pseudonocardia thermophila
DL-methionine + NH3
-
-
-
?
3.5.1.4
DL-tryptophanamide + H2O
-
667278
Pseudonocardia thermophila
DL-tryptophan + NH3
-
-
-
?
3.5.1.4
formamide + H2O
-
667278
Pseudonocardia thermophila
formate + NH3
-
-
-
?
3.5.1.4
hexanoamide + H2O
-
667278
Pseudonocardia thermophila
hexanoic acid + NH3
-
-
-
?
3.5.1.4
hydroxylamine hydrochloride + H2O
-
667278
Pseudonocardia thermophila
?
-
-
-
?
3.5.1.4
isobutyramide + H2O
-
667278
Pseudonocardia thermophila
isobutyric acid + NH3
-
-
-
?
3.5.1.4
isonicotinamide + H2O
-
667278
Pseudonocardia thermophila
isonicotinic acid + NH3
-
-
-
?
3.5.1.4
L-alanineamide + H2O
-
667278
Pseudonocardia thermophila
L-alanine + NH3
-
-
-
?
3.5.1.4
L-leucineamide + H2O
-
667278
Pseudonocardia thermophila
L-leucine + NH3
-
-
-
?
3.5.1.4
L-prolinamide + H2O
-
667278
Pseudonocardia thermophila
L-proline + NH3
-
-
-
?
3.5.1.4
L-valineamide + H2O
-
667278
Pseudonocardia thermophila
L-valine + NH3
-
-
-
?
3.5.1.4
malonamide + H2O
-
667278
Pseudonocardia thermophila
malonic acid + NH3
-
-
-
?
3.5.1.4
methacrylamide + H2O
-
667278
Pseudonocardia thermophila
methacrylic acid + NH3
-
-
-
?
3.5.1.4
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
667278
Pseudonocardia thermophila
?
-
-
-
-
3.5.1.4
nicotinamide + H2O
-
667278
Pseudonocardia thermophila
nicotinic acid + NH3
-
-
-
?
3.5.1.4
propionamide + H2O
-
667278
Pseudonocardia thermophila
propionic acid + NH3
-
-
-
?
3.5.1.4
pyrazinamide + H2O
-
667278
Pseudonocardia thermophila
? + NH3
-
-
-
?
3.5.1.4
succinamide + H2O
-
667278
Pseudonocardia thermophila
succinic acid + NH3
-
-
-
?
3.5.1.4
trimethylacetamide + H2O
-
667278
Pseudonocardia thermophila
trimethylacetic acid + NH3
-
-
-
?
3.5.1.4
urea + H2O
-
667278
Pseudonocardia thermophila
?
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
3.5.1.4
dimer
2 * 52000, SDS-PAGE
Pseudonocardia thermophila
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
3.5.1.4
70
-
assay at, hydrolytic activity
Pseudonocardia thermophila
Temperature Range [°C] (protein specific)
EC Number
Temperature Minimum [°C]
Temperature Maximum [°C]
Commentary
Organism
3.5.1.4
30
80
10% of maximal activity at 30°C and 80°C
Pseudonocardia thermophila
Temperature Stability [°C] (protein specific)
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
3.5.1.4
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
3.5.1.4
70
-
purified enzyme, half-life 1.2 h
Pseudonocardia thermophila
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
3.5.1.4
7.2
-
amidase activity
Pseudonocardia thermophila
pH Range (protein specific)
EC Number
pH Minimum
pH Maximum
Commentary
Organism
3.5.1.4
5
9
high activity within this range
Pseudonocardia thermophila
pI Value (protein specific)
EC Number
Organism
Commentary
pI Value Maximum
pI Value
3.5.1.4
Pseudonocardia thermophila
isoelectric focusing
-
4.2