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Information on EC 3.5.5.1 - nitrilase and Organism(s) Aspergillus niger and UniProt Accession A9QXE0

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EC Tree
     3 Hydrolases
         3.5 Acting on carbon-nitrogen bonds, other than peptide bonds
             3.5.5 In nitriles
                3.5.5.1 nitrilase
IUBMB Comments
Acts on a wide range of aromatic nitriles including (indol-3-yl)acetonitrile, and also on some aliphatic nitriles, and on the corresponding acid amides. cf. EC 4.2.1.84 nitrile hydratase.
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This record set is specific for:
Aspergillus niger
UNIPROT: A9QXE0
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The taxonomic range for the selected organisms is: Aspergillus niger
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Reaction Schemes
hide
a nitrile
+
2
H2O
=
a carboxylate
+
NH3
+
2
=
+
Synonyms
nitrilase, nitrilase 1, nitrilase bll6402, cyc705, benzonitrilase a, nlase, nitrile aminohydrolase, nitras-atii, nitmc-fb, nitrilase i, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
acetonitrilase
-
-
-
-
Arylacetonitrilase
-
-
-
-
benzonitrilase
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
nitrile aminohydrolase
Acts on a wide range of aromatic nitriles including (indol-3-yl)acetonitrile, and also on some aliphatic nitriles, and on the corresponding acid amides. cf. EC 4.2.1.84 nitrile hydratase.
CAS REGISTRY NUMBER
COMMENTARY hide
157297-79-5
Arabidopsis thaliana columbiana and Lansberg gene NIT2
157575-01-4
Arabidopsis thaliana columbiana gene NIT3
157575-02-5
Arabidopsis thaliana columbiana gene NIT4
205331-43-7
Arabidopsis thaliana nit2 isoenzyme2
205394-78-1
Arabidopsis thaliana nit3 isoenzyme3
205394-80-5
Arabidopsis thaliana nit1 isoenzyme1
9024-90-2
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-cyanopyridine + 2 H2O
pyridine 2-carboxylic acid + NH3
show the reaction diagram
-
-
-
?
3-chlorobenzonitrile + 2 H2O
3-chlorobenzoic acid + NH3
show the reaction diagram
-
-
-
?
3-cyanopyridine + 2 H2O
nicotinic acid + NH3
show the reaction diagram
-
-
-
?
4-chlorobenzonitrile + 2 H2O
4-chlorobenzoic acid + NH3
show the reaction diagram
-
-
-
?
4-cyanopyridine + 2 H2O
pyridine 4-carboxylic acid + NH3
show the reaction diagram
-
-
-
?
4-cyanopyridine + H2O
pyridine 4-carboxylic acid + NH3
show the reaction diagram
410.7% activity compared to benzonitrile
-
-
?
benzonitrile + 2 H2O
benzoic acid + NH3
show the reaction diagram
100% activity
-
-
?
phenylacetonitrile + 2 H2O
phenylacetic acid + NH3
show the reaction diagram
low activity with the native enzyme, poor activity with the recombinant enzyme
-
-
?
(2S)-1-[(4-methylphenyl)sulfonyl]piperidine-2-carbonitrile + H2O
?
show the reaction diagram
-
poor substrate
-
-
?
(3R)-1-[(4-methylphenyl)sulfonyl]piperidine-3-carbonitrile + H2O
?
show the reaction diagram
-
poor substrate
-
-
?
(3R)-3-methyl-1-[(4-methylphenyl)sulfonyl]pyrrolidine + H2O
?
show the reaction diagram
-
preferred substrate, amides are by-products of the nitrilase-catalyzed reaction
-
-
?
(R,S)-2-phenylpropionitrile + H2O
?
show the reaction diagram
-
1% activity compared to benzonitrile
-
-
?
1,3-dicyanobenzene + H2O
?
show the reaction diagram
-
8.4% activity compared to benzonitrile
-
-
?
1,4-dicyanobenzene + H2O
?
show the reaction diagram
-
79.5% activity compared to benzonitrile
-
-
?
1-[(4-methylphenyl)sulfonyl]piperidine-4-carbonitrile + H2O
?
show the reaction diagram
-
preferred substrate
-
-
?
2 benzonitrile + 3 H2O
benzoic acid + benzamide + NH3
show the reaction diagram
-
100% activity
-
-
?
2-cyanopyridine + 2 H2O
pyridine-2-carboxylic acid + NH3
show the reaction diagram
-
14.2% activity compared to benzonitrile
-
-
?
2-phenylacetonitrile + 2 H2O
2-phenylacetic acid + NH3
show the reaction diagram
-
10.8% activity compared to benzonitrile
-
-
?
3-chlorobenzonitrile + H2O
3-chlorobenzoic acid + NH3
show the reaction diagram
-
41% activity compared to benzonitrile
-
-
?
3-cyanopyridine + 2 H2O
pyridine-3-carboxylic acid + NH3
show the reaction diagram
-
32.4% activity compared to benzonitrile
-
-
?
3-hydroxybenzonitrile + H2O
3-hydroxybenzoate + NH3
show the reaction diagram
-
8.4% activity compared to benzonitrile
-
-
?
3-tolunitrile + H2O
3-methylbenzoic acid + NH3
show the reaction diagram
-
5.5% activity compared to benzonitrile
-
-
?
4-chlorobenzonitrile + H2O
4-chlorobenzoate + NH3
show the reaction diagram
-
29.8% activity compared to benzonitrile
-
-
?
4-cyanopyridine + 2 H2O
4-pyridinecarboxylic acid + NH3
show the reaction diagram
-
-
-
-
?
4-cyanopyridine + 2 H2O
pyridine-4-carboxylic acid + NH3
show the reaction diagram
-
410.7% activity compared to benzonitrile
-
-
?
4-tolunitrile + H2O
4-methylbenzoic acid + NH3
show the reaction diagram
-
3.4% activity compared to benzonitrile
-
-
?
benzyl (2S)-2-cyanopiperidine-1-carboxylate + H2O
?
show the reaction diagram
-
poor substrate
-
-
?
benzyl (3R)-3-methylpyrrolidine-1-carboxylate + H2O
?
show the reaction diagram
-
preferred substrate
-
-
?
benzyl 4-cyanopiperidine-1-carboxylate + H2O
?
show the reaction diagram
-
preferred substrate
-
-
?
benzyl [(1R,3R)-3-cyanocyclohexyl]carbamate + H2O
?
show the reaction diagram
-
preferred substrate
-
-
?
benzyl [(1S,3R)-3-cyanocyclohexyl]carbamate + H2O
?
show the reaction diagram
-
preferred substrate
-
-
?
butyronitrile + H2O
butyric acid + NH3
show the reaction diagram
-
17.6% activity compared to benzonitrile
-
-
?
N-[(1R,3R)-3-cyanocyclopentyl]-4-methylbenzenesulfonamide + H2O
?
show the reaction diagram
-
poor substrate
-
-
?
N-[(1S,3R)-3-cyanocyclohexyl]-4-methylbenzenesulfonamide + H2O
?
show the reaction diagram
-
preferred substrate
-
-
?
N-[(1S,3R)-3-cyanocyclohexyl]benzamide + H2O
?
show the reaction diagram
-
poor substrate
-
-
?
N-[(1S,3R)-3-cyanocyclopentyl]-4-methylbenzenesulfonamide + H2O
?
show the reaction diagram
-
preferred substrate
-
-
?
propionitrile + H2O
propionic acid + NH3
show the reaction diagram
-
6.9% activity compared to benzonitrile
-
-
?
thiophen-2-acetonitrile + H2O
thiophen-2-ylacetic acid + NH3
show the reaction diagram
-
56.1% activity compared to benzonitrile
-
-
?
valeronitrile + H2O
valeric acid + NH3
show the reaction diagram
-
19.6% activity compared to benzonitrile
-
-
?
additional information
?
-
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ag+
-
complete inhibition at 0.1 mM
Al2(SO4)3
-
complete inhibition at 5 mM
ammonium sulfate
-
75% residual activity at 800 mM
Benzamide
-
66% residual activity at 25 mM
Ca2+
-
96% residual activity at 5 mM
Co2+
-
62% residual activity at 5 mM
Cr3+
-
7% residual activity at 5 mM
Cu2+
-
1% residual activity at 0.1 mM
dithiothreitol
-
58% residual activity at 1 mM
DL-cysteine
-
97% residual activity at 1% (w/v)
EDTA
-
93% residual activity at 15 mM
Fe2+
-
60% residual activity at 5 mM
Fe3+
-
71% residual activity at 5 mM
H2O2
-
4% residual activity at 5 mM
Hg2+
-
complete inhibition at 0.1 mM
iodoacetamide
-
67% residual activity at 1 mM
Mg2+
-
91% residual activity at 5 mM
NaN3
-
88% residual activity at 5 mM
Ni2+
-
44% residual activity at 5 mM
p-hydroxymercuribenzoate
-
complete inhibition at 1 mM
Pb2+
-
52% residual activity at 0.1 mM
phenylhydrazine
-
43% residual activity at 1 mM
Zn2+
-
complete inhibition at 5 mM
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Urea
-
19% increase of activity at 1% (w/v)
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.9
-
crude extract, at pH 8.0 and 45°C
91.6
-
after 18.7fold purification, at pH 8.0 and 45°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2 - 8.5
-
-
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
native and recombinant Escherichia coli-expressed enzymes differ in substrate specificity, acid/amide ratio, reaction optima and stability
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
CHT_ASPNG
356
0
40022
Swiss-Prot
-
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38500
42700
x * 42700, recombinant enzyme, SDS-PAGE, x * 38500, native enzyme, SDS-PAGE, the multimeric nitrilase is composed of 12-16 subunits, mass spectrometry, analytical centrifugation, and dynamic light scattering, modelling
650000
above 650000 Da
38500
-
SDS-PAGE, stained with Coomassie Brilliant Blue R-250
38560
-
predicted protein size
60000
-
SDS-PAGE, stained with silver nitrate
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodecamer or homododecamer
10 * 38500 or 12 * 38500
multimer
x * 42700, recombinant enzyme, SDS-PAGE, x * 38500, native enzyme, SDS-PAGE, the multimeric nitrilase is composed of 12-16 subunits, mass spectrometry, analytical centrifugation, and dynamic light scattering, modelling
additional information
homology modelling and molecular dynamics, overview
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
the mature enzyme is reduced in size by about 4 kDa compared to the unprocessed enzyme, cleavage of the C-terminal peptide
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 10
-
a sharp activity decrease is observed at slightly acidic pH values, only a trace activity being observed at pH 5.5, 70% and 17% of the activity being retained at pH 9 and 10, respectively
684594
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
-70
-
after five freeze-thaw cycles and subsequent storage for 5 days at -70°C, the activity of the enzyme decreases to about one-fifth of the original activity
30 - 55
-
stable at 30°C, highly active at 48°C (with 92% of maximum activity), but its activity declines sharply at higher temperatures (to 53% and 19% at 50°C and 55°C, respectively), at 30°C and pH 7.2-9.0 the enzyme half-life is about 11 h, at 35°C and 40°C, the half-life decreases to 6.2 h and 2.8 h, respectively
40
-
the enzyme exhibits a half-life of 87 h at 40°C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the purified native and recombinant enzyme are stabilized by glycine at 1% w/v, sucrose at 10% w/v, D-glucose at 10% w/v, trehalose at 10% w/v, D-sorbitol at 10% w/v, xylitol at 10% w/v, D-myo-inositol at 10%, D-glycerol at 10% w/v, and bovine serum albumin at 0.1-1.0% w/v, in different extents, overview
the enzyme stability is markedly improved in the presence of D-sorbitol and xylitol (20% w/v), or myo-inositol (10% w/v)
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme 2fold from Escherichia coli strain BL21-Gold(DE3)/pOK101/pTf16, further purification of the refolded recombinant enzyme after 1 month storage by gel filtration
ammonium sulfate precipitation, Hi-Prep Sephacryl S-200 gel filtration, and Hi-Prep 16/10 Q-Sepharose column chromatography
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, sequence comparison, recombinant expression in Escherichia coli strain BL21-Gold(DE3)/pOK101/pTf16, no cleavage of the C-terminal peptide of the enzyme in the recombinant bacteria
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme is fully denatured in 6 M guanidine-HCl and 2 M Tris-carboxyethylphosphine and refolded in vitro
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
industry
the high chemical specificity and frequent enantioselectivity of nitrilases makes them attractive biocatalysts for the production of fine chemicals and pharmaceutical intermediates. Nitrilases are also used in the treatment of toxic industrial effluent and cyanide remediation
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Kaplan, O.; Vejvoda, V.; Plihal, O.; Pompach, P.; Kavan, D.; Bojarova, P.; Bezouska, K.; Mackova, M.; Cantarella, M.; Jirku, V.; Kren, V.; Martinkova, L.
Purification and characterization of a nitrilase from Aspergillus niger K10
Appl. Microbiol. Biotechnol.
73
567-575
2006
Aspergillus niger, Aspergillus niger K10
Manually annotated by BRENDA team
Malandra, A.; Cantarella, M.; Kaplan, O.; Vejvoda, V.; Uhnakova, B.; Stepankova, B.; Kubac, D.; Martinkova, L.
Continuous hydrolysis of 4-cyanopyridine by nitrilases from Fusarium solani O1 and Aspergillus niger K10
Appl. Microbiol. Biotechnol.
85
277-284
2009
Aspergillus niger, Aspergillus niger K10, Fusarium solani, Fusarium solani O1
Manually annotated by BRENDA team
Thuku, R.; Brady, D.; Benedik, M.; Sewell, B.
Microbial nitrilases: Versatile, spiral forming, industrial enzymes
J. Appl. Microbiol.
106
703-727
2009
Aeribacillus pallidus (Q0PIV8), Aeribacillus pallidus Dac521 (Q0PIV8), Arabidopsis thaliana (P32961), Arthrobacter sp., Aspergillus niger (A9QXE0), Aspergillus niger K10 (A9QXE0), Bradyrhizobium japonicum, Bradyrhizobium japonicum USDA 110, Brassica napus, Fusarium oxysporum f. sp. melonis, Fusarium solani, Fusarium solani IMI 196840, Fusarium solani O1, Rhodococcus rhodochrous, Rhodococcus rhodochrous ATCC 39484, Rhodococcus rhodochrous PA-34, Rhodococcus sp., Rhodococcus sp. NCIMB 11215, Rhodococcus sp. NCIMB 11216
Manually annotated by BRENDA team
Winkler, M.; Kaplan, O.; Vejvoda, V.; Klempier, N.; Martinkova, L.
Biocatalytic application of nitrilases from Fusarium solani O1 and Aspergillus niger K10
J. Mol. Catal. B
59
243-247
2009
Aspergillus niger, Fusarium solani, Fusarium solani O1, Aspergillus niger K10
-
Manually annotated by BRENDA team
Kaplan, O.; Bezouska, K.; Plihal, O.; Ettrich, R.; Kulik, N.; Vanek, O.; Kavan, D.; Benada, O.; Malandra, A.; Sveda, O.; Vesela, A.B.; Rinagelova, A.; Slamova, K.; Cantarella, M.; Felsberg, J.; Duskova, J.; Dohnalek, J.; Kotik, M.; Kren, V.; Martinkova, L.
Heterologous expression, purification and characterization of nitrilase from Aspergillus niger K10
BMC Biotechnol.
11
2
2011
Aspergillus niger (A9QXE0), Aspergillus niger K10 (A9QXE0), Aspergillus niger K10
Manually annotated by BRENDA team