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Information on EC 3.5.5.1 - nitrilase and Organism(s) Alcaligenes faecalis and UniProt Accession P20960
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3.5.5.1 nitrilaseIUBMB 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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Word Map
- 3.5.5.1
-
enantioselectivity
- amidase
-
hydratase
- rhodococcus
-
biocatalyst
- synthesis
- rhodochrous
- benzonitrile
- mandelonitrile
-
indole-3-acetic
- alcaligenes
- indole-3-acetonitrile
- 3-cyanopyridine
-
dinitriles
- phenylacetonitrile
- acrylonitrile
- bromoxynil
-
r-mandelic
- nhase
-
acidovorax
- ozaenae
- facilis
- industry
-
iminodiacetic
- gibberella
- analysis
The taxonomic range for the selected organisms is: Alcaligenes faecalis
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
nitrilase, nitrilase 1, cyc705, nitrilase bll6402, nitrile aminohydrolase, benzonitrilase a, nlase, nitras-atii, nitmc-fb, nitrilase i, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
acetonitrilase
-
-
-
-
Arylacetonitrilase
-
-
-
-
benzonitrilase
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
PATHWAY SOURCE
PATHWAYS
KEGG
-
-, -, -
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
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)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(R)-2-chloromandelonitrile + H2O
(R)-2-chloromandelic acid + (S)-2-chloromandelic acid + NH3
-
the wild type enzyme yields 76% (R)-2-chloromandelic acid and 9% (S)-2-chloromandelic acid at pH 7.5, as well as 12% (R)-2-chloromandelic acid and no (S)-2-chloromandelic acid at pH 4.5
-
?
2-phenylpropionitrile + 2 H2O
2-phenylpropionic acid + NH3
-
-
-
?
2 (R,S)-mandelonitrile + 4 H2O
(R)-mandelic acid + NH3 + (S)-mandelonitrile
-
-
-
-
?
2,4-dichlorophenyl acetonitrile + 2 H2O
2,4-dichlorophenyl acetic acid + NH3
-
-
-
-
?
2-amino-4-methylbenzonitrile + 2 H2O
2-amino-4-methylbenzoic acid + NH3
-
-
-
-
?
2-amino-5-chloro-benzonitrile + 2 H2O
2-amino-5-chloro-benzoic acid + NH3
-
-
-
-
?
2-amino-6-chloro-benzonitrile + 2 H2O
2-amino-6-chloro-benzoic acid + NH3
-
good substrate, when the cells are induced by benzonitrile
-
-
?
2-hydroxy-2-phenylpropionitrile + 2 H2O
2-hydroxy-2-phenylpropionic acid + NH3
-
acetophenone cyanohydrin, the substrate is transformed in cells to the corresponding acid (atrolactate) and amide (atrolactamide) at a ratio of about 3.4:1. The (R)-acid and the (S)-amide are formed preferentially from acetophenone cyanohydrin
i.e. atrolactic acid
-
?
2-thiophenacetonitrile + H2O
2-thiophenacetic acid + NH3
-
strain JM3
-
-
?
3,4,5-trimethoxybenzonitrile + 2 H2O
3,4,5-trimethoxybenzoic acid + NH3
-
-
-
-
?
4,5-dimethoxybenzonitrile + 2 H2O
4,5-dimethoxybenzoic acid + NH3
-
-
-
-
?
4-methoxy-2-nitrobenzonitrile + 2 H2O
4-methoxy-2-nitrobenzoic acid + NH3
-
-
-
-
?
acrylonitrile + 2 H2O
acrylic acid + NH3
-
preferred substrate, acrylonitrile is also a good inducer of enzyme production
-
-
?
mandelonitrile + 2 H2O
mandelic acid + NH3
-
best substrate of strain ATCC 8750, low activity in strain JM3
-
-
?
2-methyl-2-phenylpropionitrile + 2 H2O
2-methyl-2-phenylpropionic acid + NH3
-
-
-
-
?
2-methyl-2-phenylpropionitrile + 2 H2O
2-methyl-2-phenylpropionic acid + NH3
-
substrate contains a quaternary carbon atom in the alpha-position toward the nitrile group
-
-
?
benzonitrile + 2 H2O
benzoic acid + NH3
-
preferred substrate, when the cells are induced by benzonitrile
-
-
?
additional information
?
-
-
the enzyme hydrolyzes aliphatic, heterocyclic and aromatic nitriles with different substitutions, and is efficient in conversion of both aliphatic and aromatic nitriles. Substrate specificity in vivo is dependent on the inducer component, overview
-
-
?
additional information
?
-
-
the enzyme catalyzes the reactions of nitrilase, EC 3.5.5.1, and arylacetonitrilase, EC 3.5.5.5, substrate specificity and enantioselectivity, overview. Steric hindrance with amino acid residue Tyr54 impairs the binding or conversion of sterically demanding substrates, homology modelling
-
-
?
additional information
?
-
-
the enzyme hydrolyzes aliphatic, heterocyclic and aromatic nitriles with different substitutions, and is efficient in conversion of both aliphatic and aromatic nitriles. No activity with any inducer on malononitrile
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2,4-dichlorophenyl acetonitrile + 2 H2O
2,4-dichlorophenyl acetic acid + NH3
-
-
-
-
?
2-amino-4-methylbenzonitrile + 2 H2O
2-amino-4-methylbenzoic acid + NH3
-
-
-
-
?
2-amino-5-chloro-benzonitrile + 2 H2O
2-amino-5-chloro-benzoic acid + NH3
-
-
-
-
?
2-amino-6-chloro-benzonitrile + 2 H2O
2-amino-6-chloro-benzoic acid + NH3
-
good substrate, when the cells are induced by benzonitrile
-
-
?
2-methyl-2-phenylpropionitrile + 2 H2O
2-methyl-2-phenylpropionic acid + NH3
-
-
-
-
?
3,4,5-trimethoxybenzonitrile + 2 H2O
3,4,5-trimethoxybenzoic acid + NH3
-
-
-
-
?
4,5-dimethoxybenzonitrile + 2 H2O
4,5-dimethoxybenzoic acid + NH3
-
-
-
-
?
4-methoxy-2-nitrobenzonitrile + 2 H2O
4-methoxy-2-nitrobenzoic acid + NH3
-
-
-
-
?
acrylonitrile + 2 H2O
acrylic acid + NH3
-
preferred substrate, acrylonitrile is also a good inducer of enzyme production
-
-
?
benzonitrile + 2 H2O
benzoic acid + NH3
-
preferred substrate, when the cells are induced by benzonitrile
-
-
?
additional information
?
-
-
the enzyme hydrolyzes aliphatic, heterocyclic and aromatic nitriles with different substitutions, and is efficient in conversion of both aliphatic and aromatic nitriles. Substrate specificity in vivo is dependent on the inducer component, overview
-
-
?
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
kinetic and chiral analysis, overview
-
8.34
mandelonitrile
-
recombinant enzyme in cross-linked enzyme aggregates
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.083
mandelonitrile
-
recombinant enzyme in cross-linked enzyme aggregates
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.1
the specific activity is less than 0.1 micromol/min/mg, wild type enzyme, using (R)-2-chloromandelonitrile as substrate, in 50 mM potassium phosphate buffer, at pH 4.5 and 30°C
0.14
mutant enzyme A198V/I290F/H135Y/Y213H/T350S/Y177C/A285T, using (R)-2-chloromandelonitrile as substrate, in 50 mM potassium phosphate buffer, at pH 7.5 and 30°C
0.52
mutant enzyme A198V/I290F/H135Y/Y213H/T350S/Y177C/A285T, using (R)-2-chloromandelonitrile as substrate, in 50 mM potassium phosphate buffer, at pH 4.5 and 30°C
0.62
wild type enzyme, using (R)-2-chloromandelonitrile as substrate, in 50 mM potassium phosphate buffer, at pH 7.5 and 30°C
0.4
-
enzyme in cells, substrate 2-methyl-2-phenylpropionitrile in Na/K-phosphate buffer, pH 7.0, 30°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7
-
with substrate 2-methyl-2-phenylpropionitrile in Na/K-phosphate buffer
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
-
optimization of enzyme production, best conditions are glucose 1.0%, acrylonitrile 0.1%, pH 7.0
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
generation of a model of the nitrilase by homology modeling, overview
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
32000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A198V
the mutant enzyme shows 2.4fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 compared to the wild type enzyme
A198V/I290F
the mutant enzyme shows 1.2fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 and 140% activity at pH 4.5 compared to the wild type enzyme
A198V/I290F/H135Y
the mutant enzyme shows 1.7fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 and 150% activity at pH 4.5 compared to the wild type enzyme
A198V/I290F/H135Y/Y213H/T350S/Y177C/A285T
the mutant enzyme shows 2.8fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 and 740% activity at pH 4.5 compared to the wild type enzyme
A198V/I290F/Y177C/A285T
the mutant enzyme shows 0.9fold conversion rate of 2-phenylpropionitrile at pH 7.5 and 240% activity at pH 4.5 compared to the wild type enzyme
A198V/I290F/Y213H/T350S
the mutant enzyme shows 2fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 and 200% activity at pH 4.5 compared to the wild type enzyme
A198V/Q197H
the mutant enzyme shows 2.9fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 compared to the wild type enzyme
A198V/Q197H/L176M
the mutant enzyme shows 6.5fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 compared to the wild type enzyme
A198V/Q197H/L176M/V306I
the mutant enzyme shows 8fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 compared to the wild type enzyme
I290F/Q3L
the mutant enzyme shows 2.3fold increased conversion rate of 2-phenylpropionitrile at pH 7.5 and 100% activity at pH 4.5 compared to the wild type enzyme
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4 - 7.5
the wild type enzyme shows no activity after 30 min incubation at pH 4.5, about 10% activity after 30 min incubation at pH 5.0, about 20% activity after 30 min incubation at pH 5.5, about 60% activity after 30 min incubation at pH 6.0, and 100% activity after 30 min incubation at pH 6.5-7.5
711870
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30 - 50
-
soluble recombinant nitrilase has a half-life of 1732.5 min at 30°C and 128.3 min at 50°C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the nitrilase activity in cross-linked enzyme aggregates is at 30°C and 60°C about 5times more stable than in soluble preparations
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Acetone
-
extent of nitrile hydrolysis and enantiomeric purity of product are significantly enhanced in the range of 5-15%
dimethyl acetamide
-
extent of nitrile hydrolysis and enantiomeric purity of product are significantly enhanced at 5%
dimethyl formamide
-
extent of nitrile hydrolysis and enantiomeric purity of product are significantly enhanced in the range of 5-25%
dimethyl sulfoxide
-
signifantly enhances the synthetic potential of the enzyme as well as its enantioselectivity. Extent of nitrile hydrolysis and enantiomeric purity of product are significantly enhanced in the range of 525%
dioxane
-
extent of nitrile hydrolysis and enantiomeric purity of product are significantly enhanced in the range of 5-10%
Ethanol
-
extent of nitrile hydrolysis and enantiomeric purity of product are significantly enhanced in the range of 5-20%
isopropanol
-
extent of nitrile hydrolysis and enantiomeric purity of product are significantly enhanced in the range of 5-15%
Methanol
-
extent of nitrile hydrolysis and enantiomeric purity of product are significantly enhanced in the range of 5-20%
n-propanol
-
extent of nitrile hydrolysis and enantiomeric purity of product are significantly enhanced in the range of 5-15%
additional information
-
addition of organic solvent up to 10%-20% leads to an enhancement in reaction rate, any further increase beyond the critical concentration in the latter leads to the decrease in catalytic efficiency of the enzyme. The solvent dielectric constant shows a linear correlation with the critical concentration of the solvent used and the extent of nitrile hydrolysis. Unlike alcohols, the reaction rate in case of aprotic solvents can be linearly correlated to solvent log P. The affinity of the enzyme for its substrate is highly dependent upon the aprotic solvent used
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression under the control of a rhamnose-inducible promoter in Escherichia coli strain JM109
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
induction by epsilon-caprolactam, 3-cyanopyridine, acrylonitrile, and benzonitrile
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
synthesis
-
attractive as green, mild, and selective catalysts for setting stereogenic centers in fine-chemical synthesis and enantiospecific synthesis of a variety of carboxylic acid derivatives
synthesis
-
recombinant cells expressing the enzyme are promising catalysts for the synthesis of stable chiral quaternary carbon centers from ketones
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
O'Reilly, C.; Turner, P.D.
The nitrilase family of CN hydrolyzing enzymes - a comparative study
J. Appl. Microbiol.
95
1161-1174
2003
Acidovorax facilis, Acidovorax facilis 72W, Acinetobacter sp., Acinetobacter sp. AK226, Aeribacillus pallidus, Aeribacillus pallidus Dac521, Alcaligenes faecalis, Alcaligenes faecalis JM3, Arabidopsis thaliana, Bacillus sp. (in: Bacteria), Bacillus sp. (in: Bacteria) OxB-1, Brassica rapa, Comamonas testosteroni, Fusarium oxysporum, Fusarium solani, Fusarium solani IMI196840, Hordeum vulgare, Klebsiella pneumoniae, Nocardia sp., Pseudomonas sp., Rhodococcus rhodochrous, Rhodococcus rhodochrous K22, Rhodococcus sp., Rhodococcus sp. ATCC 39484
Petersen, M.; Kiener, A.
Biocatalysis - preparation and functionalization of N-heterocycles
Green Chem.
2
99-106
1999
Alcaligenes faecalis
-
Kaul, P.; Stolz, A.; Banerjee, U.C.
Cross-linked amorphous nitrilase aggregates for enantioselective nitrile hydrolysis
Adv. Synth. Catal.
349
2167-2176
2007
Alcaligenes faecalis
-
Singh, R.; Sharma, R.; Tewari, N.; Tewari, N.; Rawat, D.S.
Nitrilase and its application as a green catalyst
Chem. Biodivers.
3
1279-1287
2006
Acidovorax facilis, Acidovorax facilis 72W, Acinetobacter sp., Acinetobacter sp. AK226, Aeribacillus pallidus, Aeribacillus pallidus Dac521, Alcaligenes faecalis, Arabidopsis thaliana, Bacillus sp. (in: Bacteria), Bacillus sp. (in: Bacteria) OxB-1, Comamonas testosteroni, Fusarium oxysporum, Fusarium solani, Fusarium solani IMI196840, Hordeum vulgare, Klebsiella pneumoniae, Penicillium multicolor, Pseudomonas sp., Rhodococcus rhodochrous, Rhodococcus rhodochrous K22, Rhodococcus sp.
Kaul, P.; Banerjee, U.C.
Predicting enzyme behavior in nonconventional media: correlating nitrilase function with solvent properties
J. Ind. Microbiol. Biotechnol.
35
713-720
2008
Alcaligenes faecalis, Alcaligenes faecalis MTCC 126
Schreiner, U.; Hecher, B.; Obrowsky, S.; Waich, K.; Klempier, N.; Steinkellner, G.; Gruber, K.; Rozzell, J.; Glieder, A.; Winkler, M.
Directed evolution of Alcaligenes faecalis nitrilase
Enzyme Microb. Technol.
47
140-146
2010
Alcaligenes faecalis (P20960), Alcaligenes faecalis JM3 (P20960)
-
Baum, S.; Williamson, D.S.; Sewell, T.; Stolz, A.
Conversion of sterically demanding alpha,alpha-disubstituted phenylacetonitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191
Appl. Environ. Microbiol.
78
48-57
2012
Alcaligenes faecalis, Alcaligenes faecalis ATCC 8750, Pseudomonas fluorescens (Q5EG61), Pseudomonas fluorescens, Pseudomonas fluorescens EBC191 (Q5EG61), Pseudomonas fluorescens EBC191, Rhodococcus sp., Synechocystis sp.
Nageshwar, Y.V.; Sheelu, G.; Shambhu, R.R.; Muluka, H.; Mehdi, N.; Malik, M.S.; Kamal, A.
Optimization of nitrilase production from Alcaligenes faecalis MTCC 10757 (IICT-A3): effect of inducers on substrate specificity
Bioprocess Biosyst. Eng.
34
515-523
2011
Alcaligenes faecalis, Alcaligenes faecalis MTCC 10757
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