BRENDA - Enzyme Database show

Purification and characterization of a novel thermo-active amidase from Geobacillus subterraneus RL-2a

Mehta, P.; Bhatia, S.; Bhatia, R.; Bhalla, T.; Extremophiles 17, 637-648 (2013)

Data extracted from this reference:

Activating Compound
EC Number
Activating Compound
Commentary
Organism
Structure
3.5.1.4
additional information
presence of mid-chain aliphatic and amino acid amides enhances the enzymatic activity
Geobacillus subterraneus
Cloned(Commentary)
EC Number
Commentary
Organism
3.5.1.4
constitutive enzyme
Geobacillus subterraneus
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
3.5.1.4
Ag+
complete inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Ba2+
11% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Ca2+
10% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Co2+
9% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Cu2+
28.5% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
DTT
21% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
EDTA
21% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Fe3+
87% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Hg2+
complete inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Mg2+
53% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Mn2+
12% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Na+
17% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Pb2+
8% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
PEG
8% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
PMSF
87.5% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Zn2+
11% inhibition at 1 mM
Geobacillus subterraneus
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
3.5.1.4
additional information
-
additional information
Michaelis–Menten kinetics
Geobacillus subterraneus
3.5.1.4
6.02
-
nicotinamide
pH 6.5, 70°C
Geobacillus subterraneus
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
3.5.1.4
37000
-
3 * 37000, SDS-PAGE
Geobacillus subterraneus
3.5.1.4
111000
-
gel filtration
Geobacillus subterraneus
Organic Solvent Stability
EC Number
Organic Solvent
Commentary
Organism
3.5.1.4
Acetone
5-10% activity remaining at 5-10% acetone after 15 min
Geobacillus subterraneus
3.5.1.4
benzene
70-80% activity remaining at 5-10% benzene after 15 min
Geobacillus subterraneus
3.5.1.4
Butanol
15-25% activity remaining at 5-10% butanol after 15 min
Geobacillus subterraneus
3.5.1.4
CCl4
80-90% activity remaining at 5-10% CCl4 after 15 min
Geobacillus subterraneus
3.5.1.4
dimethyl sulfoxide
35-40% activity remaining at 5-10% DMSO after 15 min
Geobacillus subterraneus
3.5.1.4
Ethanol
15% activity remaining at 5-10% ethanol after 15 min
Geobacillus subterraneus
3.5.1.4
Ethylene glycol
60-70% activity remaining at 5-10% ethylene glycol after 15 min
Geobacillus subterraneus
3.5.1.4
propanol
20-25% activity remaining at 5-10% propanol after 15 min
Geobacillus subterraneus
3.5.1.4
toluene
90% activity remaining at 10% toluene after 15 min
Geobacillus subterraneus
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
3.5.1.4
Geobacillus subterraneus
-
isolated from Manikaran thermal spring (Himachal Pradesh, India)
-
3.5.1.4
Geobacillus subterraneus RL-2a
-
isolated from Manikaran thermal spring (Himachal Pradesh, India)
-
Purification (Commentary)
EC Number
Commentary
Organism
3.5.1.4
native enzyme 52.04fold to homogeneity by heat treatment, ammonium sulfate fractionation, anion exchange chromatography, and gel filtration
Geobacillus subterraneus
Source Tissue
EC Number
Source Tissue
Commentary
Organism
Textmining
3.5.1.4
additional information
constitutive enzyme
Geobacillus subterraneus
-
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
48.66
-
purified native enzyme, pH 6.5, 70°C, substrate nicotinamide
Geobacillus subterraneus
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3.5.1.4
benzamide + H2O
-
733808
Geobacillus subterraneus
benzoate + NH3
-
-
-
?
3.5.1.4
butyramide + H2O
high activity
733808
Geobacillus subterraneus
butyrate + NH3
-
-
-
?
3.5.1.4
cyanoacetamide + H2O
low activity
733808
Geobacillus subterraneus
cyanoacetate + NH3
-
-
-
?
3.5.1.4
formamide + H2O
-
733808
Geobacillus subterraneus
formate + NH3
-
-
-
?
3.5.1.4
formamide + H2O
-
733808
Geobacillus subterraneus RL-2a
formate + NH3
-
-
-
?
3.5.1.4
isobutyramide + H2O
high activity
733808
Geobacillus subterraneus
isobutyrate + NH3
-
-
-
?
3.5.1.4
isobutyramide + H2O
high activity
733808
Geobacillus subterraneus RL-2a
isobutyrate + NH3
-
-
-
?
3.5.1.4
isonicotinamide + H2O
-
733808
Geobacillus subterraneus
isonicotinate + NH3
-
-
-
?
3.5.1.4
L-leucinamide + H2O
-
733808
Geobacillus subterraneus
L-leucine + NH3
-
-
-
?
3.5.1.4
L-leucinamide + H2O
-
733808
Geobacillus subterraneus RL-2a
L-leucine + NH3
-
-
-
?
3.5.1.4
lactamide + H2O
-
733808
Geobacillus subterraneus
lactate + NH3
-
-
-
?
3.5.1.4
methacrylamide + H2O
-
733808
Geobacillus subterraneus
methacrylate + NH3
-
-
-
?
3.5.1.4
additional information
glycinamide, malonamide, acetamide, and acrylamide are poor substrates, while urea, mandelamide, N-methylacetamide, and thioacetamide are no substrates, substrate specificity, overview
733808
Geobacillus subterraneus
?
-
-
-
-
3.5.1.4
additional information
glycinamide, malonamide, acetamide, and acrylamide are poor substrates, while urea, mandelamide, N-methylacetamide, and thioacetamide are no substrates, substrate specificity, overview
733808
Geobacillus subterraneus RL-2a
?
-
-
-
-
3.5.1.4
nicotinamide + H2O
reaction of EC 3.5.1.19, nicotinamidase
733808
Geobacillus subterraneus
nicotinate + NH3
-
-
-
?
3.5.1.4
prolinamide + H2O
-
733808
Geobacillus subterraneus
proline + NH3
-
-
-
?
3.5.1.4
propainamide + H2O
high activity
733808
Geobacillus subterraneus
? + NH3
-
-
-
?
3.5.1.4
propionamide + H2O
best substrate
733808
Geobacillus subterraneus
propionate + NH3
-
-
-
?
3.5.1.4
sulfanilamide + H2O
-
733808
Geobacillus subterraneus
sulfanilate + NH3
-
-
-
?
3.5.1.4
valeramide + H2O
high activity
733808
Geobacillus subterraneus
valerate + NH3
-
-
-
?
3.5.1.4
valeramide + H2O
high activity
733808
Geobacillus subterraneus RL-2a
valerate + NH3
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
3.5.1.4
trimer
3 * 37000, SDS-PAGE
Geobacillus subterraneus
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
3.5.1.4
70
-
-
Geobacillus subterraneus
Temperature Range [°C]
EC Number
Temperature Minimum [°C]
Temperature Maximum [°C]
Commentary
Organism
3.5.1.4
40
90
activity range
Geobacillus subterraneus
Temperature Stability [°C]
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
3.5.1.4
70
-
purified native enzyme, half-life is 354 min
Geobacillus subterraneus
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
3.5.1.4
6.5
-
-
Geobacillus subterraneus
pH Range
EC Number
pH Minimum
pH Maximum
Commentary
Organism
3.5.1.4
4.5
11.5
activity range
Geobacillus subterraneus
Activating Compound (protein specific)
EC Number
Activating Compound
Commentary
Organism
Structure
3.5.1.4
additional information
presence of mid-chain aliphatic and amino acid amides enhances the enzymatic activity
Geobacillus subterraneus
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
3.5.1.4
constitutive enzyme
Geobacillus subterraneus
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
3.5.1.4
Ag+
complete inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Ba2+
11% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Ca2+
10% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Co2+
9% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Cu2+
28.5% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
DTT
21% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
EDTA
21% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Fe3+
87% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Hg2+
complete inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Mg2+
53% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Mn2+
12% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Na+
17% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Pb2+
8% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
PEG
8% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
PMSF
87.5% inhibition at 1 mM
Geobacillus subterraneus
3.5.1.4
Zn2+
11% inhibition at 1 mM
Geobacillus subterraneus
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
3.5.1.4
additional information
-
additional information
Michaelis–Menten kinetics
Geobacillus subterraneus
3.5.1.4
6.02
-
nicotinamide
pH 6.5, 70°C
Geobacillus subterraneus
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
3.5.1.4
37000
-
3 * 37000, SDS-PAGE
Geobacillus subterraneus
3.5.1.4
111000
-
gel filtration
Geobacillus subterraneus
Organic Solvent Stability (protein specific)
EC Number
Organic Solvent
Commentary
Organism
3.5.1.4
Acetone
5-10% activity remaining at 5-10% acetone after 15 min
Geobacillus subterraneus
3.5.1.4
benzene
70-80% activity remaining at 5-10% benzene after 15 min
Geobacillus subterraneus
3.5.1.4
Butanol
15-25% activity remaining at 5-10% butanol after 15 min
Geobacillus subterraneus
3.5.1.4
CCl4
80-90% activity remaining at 5-10% CCl4 after 15 min
Geobacillus subterraneus
3.5.1.4
dimethyl sulfoxide
35-40% activity remaining at 5-10% DMSO after 15 min
Geobacillus subterraneus
3.5.1.4
Ethanol
15% activity remaining at 5-10% ethanol after 15 min
Geobacillus subterraneus
3.5.1.4
Ethylene glycol
60-70% activity remaining at 5-10% ethylene glycol after 15 min
Geobacillus subterraneus
3.5.1.4
propanol
20-25% activity remaining at 5-10% propanol after 15 min
Geobacillus subterraneus
3.5.1.4
toluene
90% activity remaining at 10% toluene after 15 min
Geobacillus subterraneus
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
3.5.1.4
native enzyme 52.04fold to homogeneity by heat treatment, ammonium sulfate fractionation, anion exchange chromatography, and gel filtration
Geobacillus subterraneus
Source Tissue (protein specific)
EC Number
Source Tissue
Commentary
Organism
Textmining
3.5.1.4
additional information
constitutive enzyme
Geobacillus subterraneus
-
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
48.66
-
purified native enzyme, pH 6.5, 70°C, substrate nicotinamide
Geobacillus subterraneus
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
benzamide + H2O
-
733808
Geobacillus subterraneus
benzoate + NH3
-
-
-
?
3.5.1.4
butyramide + H2O
high activity
733808
Geobacillus subterraneus
butyrate + NH3
-
-
-
?
3.5.1.4
cyanoacetamide + H2O
low activity
733808
Geobacillus subterraneus
cyanoacetate + NH3
-
-
-
?
3.5.1.4
formamide + H2O
-
733808
Geobacillus subterraneus
formate + NH3
-
-
-
?
3.5.1.4
formamide + H2O
-
733808
Geobacillus subterraneus RL-2a
formate + NH3
-
-
-
?
3.5.1.4
isobutyramide + H2O
high activity
733808
Geobacillus subterraneus
isobutyrate + NH3
-
-
-
?
3.5.1.4
isobutyramide + H2O
high activity
733808
Geobacillus subterraneus RL-2a
isobutyrate + NH3
-
-
-
?
3.5.1.4
isonicotinamide + H2O
-
733808
Geobacillus subterraneus
isonicotinate + NH3
-
-
-
?
3.5.1.4
L-leucinamide + H2O
-
733808
Geobacillus subterraneus
L-leucine + NH3
-
-
-
?
3.5.1.4
L-leucinamide + H2O
-
733808
Geobacillus subterraneus RL-2a
L-leucine + NH3
-
-
-
?
3.5.1.4
lactamide + H2O
-
733808
Geobacillus subterraneus
lactate + NH3
-
-
-
?
3.5.1.4
methacrylamide + H2O
-
733808
Geobacillus subterraneus
methacrylate + NH3
-
-
-
?
3.5.1.4
additional information
glycinamide, malonamide, acetamide, and acrylamide are poor substrates, while urea, mandelamide, N-methylacetamide, and thioacetamide are no substrates, substrate specificity, overview
733808
Geobacillus subterraneus
?
-
-
-
-
3.5.1.4
additional information
glycinamide, malonamide, acetamide, and acrylamide are poor substrates, while urea, mandelamide, N-methylacetamide, and thioacetamide are no substrates, substrate specificity, overview
733808
Geobacillus subterraneus RL-2a
?
-
-
-
-
3.5.1.4
nicotinamide + H2O
reaction of EC 3.5.1.19, nicotinamidase
733808
Geobacillus subterraneus
nicotinate + NH3
-
-
-
?
3.5.1.4
prolinamide + H2O
-
733808
Geobacillus subterraneus
proline + NH3
-
-
-
?
3.5.1.4
propainamide + H2O
high activity
733808
Geobacillus subterraneus
? + NH3
-
-
-
?
3.5.1.4
propionamide + H2O
best substrate
733808
Geobacillus subterraneus
propionate + NH3
-
-
-
?
3.5.1.4
sulfanilamide + H2O
-
733808
Geobacillus subterraneus
sulfanilate + NH3
-
-
-
?
3.5.1.4
valeramide + H2O
high activity
733808
Geobacillus subterraneus
valerate + NH3
-
-
-
?
3.5.1.4
valeramide + H2O
high activity
733808
Geobacillus subterraneus RL-2a
valerate + NH3
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
3.5.1.4
trimer
3 * 37000, SDS-PAGE
Geobacillus subterraneus
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
3.5.1.4
70
-
-
Geobacillus subterraneus
Temperature Range [°C] (protein specific)
EC Number
Temperature Minimum [°C]
Temperature Maximum [°C]
Commentary
Organism
3.5.1.4
40
90
activity range
Geobacillus subterraneus
Temperature Stability [°C] (protein specific)
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
3.5.1.4
70
-
purified native enzyme, half-life is 354 min
Geobacillus subterraneus
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
3.5.1.4
6.5
-
-
Geobacillus subterraneus
pH Range (protein specific)
EC Number
pH Minimum
pH Maximum
Commentary
Organism
3.5.1.4
4.5
11.5
activity range
Geobacillus subterraneus
General Information
EC Number
General Information
Commentary
Organism
3.5.1.4
evolution
peptide sequences determination by MALDI-TOF analysis reveals 97% similarity with that of glutamyl-tRNA(Gln) amidotransferase subunit A (Geobacillus kaustophilus HTA426) and amidase of Geobacillus sp. C56-T3, also 68% similarity to amidase of Geobacillus sp. Y412MC61
Geobacillus subterraneus
3.5.1.4
additional information
the enzyme is a wide-spectrum thermostable amidase
Geobacillus subterraneus
General Information (protein specific)
EC Number
General Information
Commentary
Organism
3.5.1.4
evolution
peptide sequences determination by MALDI-TOF analysis reveals 97% similarity with that of glutamyl-tRNA(Gln) amidotransferase subunit A (Geobacillus kaustophilus HTA426) and amidase of Geobacillus sp. C56-T3, also 68% similarity to amidase of Geobacillus sp. Y412MC61
Geobacillus subterraneus
3.5.1.4
additional information
the enzyme is a wide-spectrum thermostable amidase
Geobacillus subterraneus