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Literature summary for 3.5.1.4 extracted from

  • Makhongela, H.S.; Glowacka, A.E.; Agarkar, V.B.; Sewell, B.T.; Weber, B.; Cameron, R.A.; Cowan, D.A.; Burton, S.G.
    A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity (2007), Appl. Microbiol. Biotechnol., 75, 801-811.
    View publication on PubMed
Search for more literature for 3.5.1.4

Activating Compound

Activating Compound Comment Organism Structure
dithiothreitol activation to 150% original activity results from addition of dithiothreitol at millimolar concentration Aeribacillus pallidus

Application

Application Comment Organism
synthesis the amidase has potential for application under high temperature conditions as a biocatalyst for D-selective amide hydrolysis producing enantiomerically pure carboxylic acids and for production of novel amides by acyl transfer Aeribacillus pallidus

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli Aeribacillus pallidus
expression in Escherichia coli Aeribacillus pallidus

General Stability

General Stability Organism
binding on Eupergit C beads at 4°C and on Amberlite-XAD57 results in low protein binding and low activity, but immobilization on Eupergit C beads at 25°C with cross-linking results in high protein binding yield and high immobilized specific activity (80% of non-immobilized activity) Aeribacillus pallidus

Inhibitors

Inhibitors Comment Organism Structure
2-mercaptoethanol complete inhibition at 5% (v/v) 2-mercaptoethanol Aeribacillus pallidus
acetamide substrate inhibition Aeribacillus pallidus
acetone 5% v/v, 86% inhibition; 86% decreased activity at 5% (v/v) Aeribacillus pallidus
acetonitrile 5% v/v, 93% inhibition; 93% decreased activity at 5% (v/v) Aeribacillus pallidus
Acrylamide substrate inhibition Aeribacillus pallidus
Co2+ 89-48% residual activity at 1 mM Aeribacillus pallidus
Cu2+ 89-48% residual activity at 1 mM Aeribacillus pallidus
Dimethyl sulfide 5% v/v, complete inhibition; complete inhibition at 5% (v/v) Aeribacillus pallidus
dimethyl sulfoxide 5% v/v, complete inhibition; complete inhibition at 5% (v/v) Aeribacillus pallidus
dithiothreitol 80% residual activity at 1 mM Aeribacillus pallidus
ethanol 58% decreased activity at 5% (v/v); 5% v/v, 58% inhibition Aeribacillus pallidus
Fe3+ 89-48% residual activity at 1 mM Aeribacillus pallidus
H2O2 5% v/v, complete inhibition; complete inhibition at 5% (v/v) H2O2 Aeribacillus pallidus
Isopropanol 5% v/v, 60% inhibition; 60% decreased activity at 5% (v/v) Aeribacillus pallidus
mercaptoethanol 5% v/v, complete inhibition Aeribacillus pallidus
methanol 50% decreased activity at 5% (v/v); 5% v/v, 50% inhibition Aeribacillus pallidus
n-butanol 48% decreased activity at 5% (v/v); 5% v/v, 48% inhibition Aeribacillus pallidus
Zn2+ 89-48% residual activity at 1 mM Aeribacillus pallidus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
22.7
-
 acetamide pH 7.2 Aeribacillus pallidus
43.8
-
 Acrylamide pH 7.2 Aeribacillus pallidus

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
38000
-
 6 * 38000, SDS-PAGE Aeribacillus pallidus
218000
-
 gel filtration Aeribacillus pallidus
228000
-
 SDS-PAGE Aeribacillus pallidus

Organism

Organism UniProt Comment Textmining
Aeribacillus pallidus
-
-
-
Aeribacillus pallidus RAPc8
-
-
-

Oxidation Stability

Oxidation Stability Organism
after incubation for 24 days at 20°C, buffered samples of amidase without reducing agents are completely inactivated. Samples incubated in the presence of 1 mM DTT show activities of approximately 80% of initial levels. At 20 mM DTT under otherwise identical conditions, acyl transfer activity is 200% higher than activities measured at time zero Aeribacillus pallidus

Purification (Commentary)

Purification (Comment) Organism
-
 Aeribacillus pallidus
heat precipitation and S-300 gel filtration Aeribacillus pallidus

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
37.5
-
 reombinant enzyme in crude extract, at pH 7 and 50°C Aeribacillus pallidus
221
-
-
 Aeribacillus pallidus
221
-
 recombinant enzyme after 6fold purification, at pH 7 and 50°C Aeribacillus pallidus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
acetamide + H2O
-
 Aeribacillus pallidus acetate + NH3
-
 ?
acetamide + H2O
-
 Aeribacillus pallidus RAPc8 acetate + NH3
-
 ?
acetamide + hydroxylamine
-
 Aeribacillus pallidus acetylhydroxamic acid + NH3
-
 ?
acrylamide + H2O 102% of the activity with acetamide Aeribacillus pallidus acrylate + NH3
-
 ?
acrylamide + hydroxylamine
-
 Aeribacillus pallidus acryloylhydroxamic acid + NH3
-
 ?
D-lactamide + H2O 17% of the activity with acetamide, D-selectivity towards lactamide Aeribacillus pallidus D-lactate + NH3
-
 ?
D-lactamide + hydroxylamine
-
 Aeribacillus pallidus ?
-
 ?
diacetamide + H2O 14% of the activity with acetamide Aeribacillus pallidus ?
-
 ?
fluoroacetamide + H2O 165% of the activity with acetamide Aeribacillus pallidus fluoroacetate + NH3
-
 ?
formamide + H2O 30% of the activity with acetamide Aeribacillus pallidus formate + NH3
-
 ?
formamide + H2O 30% of the activity with acetamide Aeribacillus pallidus RAPc8 formate + NH3
-
 ?
isobutyramide + H2O 16% of the activity with acetamide Aeribacillus pallidus isobutanoate + NH3
-
 ?
isobutyramide + hydroxylamine
-
 Aeribacillus pallidus isobutyrylhydroxamic acid + NH3
-
 ?
L-alaninamide + H2O 3% of the activity with acetamide Aeribacillus pallidus L-Ala + NH3
-
 ?
additional information acyl transfer reactions are demonstrated with acetamide, propionamide, isobutyramide, and acrylamide as substrates and hydroxylamine as the acyl acceptor. The highest reaction rate being with isobutyramide. Immobilization by entrapment in polyacrylamide gels, covalent binding on Eupergit C beads at 4°C and on Amberlite-XAD57 results in low protein binding and low activity, but immobilization on Eupergit C beads at 25°C with cross-linking resulted in high protein binding yield and high immobilized specific activity. No activity with: nicotinamide, isonicotinamide, L-asparagine, D-asparagine, DL-phenylalanine, L-prolinamide, hexanamide Aeribacillus pallidus ?
-
 ?
additional information not active on L-lactamide Aeribacillus pallidus ?
-
 ?
additional information acyl transfer reactions are demonstrated with acetamide, propionamide, isobutyramide, and acrylamide as substrates and hydroxylamine as the acyl acceptor. The highest reaction rate being with isobutyramide. Immobilization by entrapment in polyacrylamide gels, covalent binding on Eupergit C beads at 4°C and on Amberlite-XAD57 results in low protein binding and low activity, but immobilization on Eupergit C beads at 25°C with cross-linking resulted in high protein binding yield and high immobilized specific activity. No activity with: nicotinamide, isonicotinamide, L-asparagine, D-asparagine, DL-phenylalanine, L-prolinamide, hexanamide Aeribacillus pallidus RAPc8 ?
-
 ?
additional information not active on L-lactamide Aeribacillus pallidus RAPc8 ?
-
 ?
propionamide + H2O 67% of the activity with acetamide Aeribacillus pallidus propionate + NH3
-
 ?
propionamide + hydroxylamine
-
 Aeribacillus pallidus propionylhydroxamic acid + NH3
-
 ?
urea + H2O 1.5% of the activity with acetamide Aeribacillus pallidus ?
-
 ?
urea + H2O 1.5% of the activity with acetamide Aeribacillus pallidus RAPc8 ?
-
 ?

Subunits

Subunits Comment Organism
hexamer 6 * 38000, SDS-PAGE Aeribacillus pallidus
homohexamer 6 * 38000, SDS-PAGE Aeribacillus pallidus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
50
-
-
 Aeribacillus pallidus
50
-
 soluble enzyme and enzyme immobilized on Eupergit C beads at 25°C with cross-linking Aeribacillus pallidus

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
40 80 40°C: about 70% of maximal activity, soluble enzyme. 40°C: about 30% of maximal activity, enzyme immobilized on Eupergit C beads. 80°C: about 50% of maximal activity, soluble enzyme. 80°C: about 55% of maximal activity, enzyme immobilized on Eupergit C beads Aeribacillus pallidus

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
50
-
 half-life: more than 5 h for soluble enzyme Aeribacillus pallidus
50 80 high thermal stability at 50°C and 60°C with half-lives greater than 5 h at both temperatures, the half-life values are 43 min and 10 min at 70°C and 80°C Aeribacillus pallidus
60
-
 half-life: more than 5 h for soluble enzyme Aeribacillus pallidus
70
-
 half-life: 43 min for soluble enzyme, 52 min for enzyme immobilized on Eupergit C beads at 25°C with cross-linking Aeribacillus pallidus
80
-
 half-life: 10 min for soluble enzyme, 30 min for enzyme immobilized on Eupergit C beads at 25°C with cross-linking Aeribacillus pallidus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
38.7
-
 Acrylamide pH 7.2 Aeribacillus pallidus
2668
-
 acetamide pH 7.2 Aeribacillus pallidus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6 8 enzyme immobilized on Eupergit C beads Aeribacillus pallidus
7
-
-
 Aeribacillus pallidus
7
-
 soluble enzyme Aeribacillus pallidus

pH Range

pH Minimum pH Maximum Comment Organism
4 9 pH 4.0: about 60% of maximal activity, soluble enzyme. pH 4.0: about 75% of maximal activity, enzyme immobilized on Eupergit C beads. and enzyme immobilized on Eupergit C beads. pH 9.0: about 40% of maximal activity, soluble enzyme. pH 9.0: about 90% of maximal activity, enzyme immobilized on Eupergit C beads Aeribacillus pallidus

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
2.3
-
 Acrylamide pH 7.2 Aeribacillus pallidus
2.3
-
 Acrylamide at pH 7 and 50°C Aeribacillus pallidus
12.1
-
 acetamide pH 7.2 Aeribacillus pallidus
12.1
-
 acetamide at pH 7 and 50°C Aeribacillus pallidus