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Information on EC 4.2.1.84 - nitrile hydratase and Organism(s) Pseudomonas putida and UniProt Accession P97052
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4.2.1.84 nitrile hydrataseIUBMB Comments
Acts on short-chain aliphatic nitriles, converting them into the corresponding amides. Does not act on these amides or on aromatic nitriles. cf. EC 3.5.5.1 nitrilase.
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Word Map
- 4.2.1.84
- rhodococcus
- amidase
- acrylamide
- rhodochrous
- erythropolis
- synthesis
-
feiii
-
low-spin
-
fe-type
-
non-heme
-
sulfenic
- benzonitrile
- pseudonocardia
-
sulfinate
- propionamide
-
cysteine-sulfinic
-
neonicotinoid
- ruber
- propionitrile
-
cgmcc
- thiacloprid
-
carboxamido
- aldoxime
- indole-3-acetonitrile
- chlororaphis
-
metallochaperone
- industry
- pharmacology
- degradation
- environmental protection
- analysis
The taxonomic range for the selected organisms is: Pseudomonas putida
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
nhase, nitrile hydratase, nilco, l-nhase, h-nhase, co-type nhase, cobalt-containing nitrile hydratase, fe-nhase, ctnhase, anhase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
3-cyanopyridine hydratase
-
-
-
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acrylonitrile hydratase
-
-
-
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aliphatic nitrile hydratase
-
-
-
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H-NHase
-
-
-
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H-nitrilase
-
-
-
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hydratase, nitrile
-
-
-
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L-Nhase
-
-
-
-
L-nitrilase
-
-
-
-
NHase
NI1 NHase
-
-
-
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nitrilase
-
-
-
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NHase
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-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C-O bond cleavage by elimination of water
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-
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PATHWAY SOURCE
PATHWAYS
KEGG
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-, -, -, -
SYSTEMATIC NAME
IUBMB Comments
aliphatic-amide hydro-lyase (nitrile-forming)
Acts on short-chain aliphatic nitriles, converting them into the corresponding amides. Does not act on these amides or on aromatic nitriles. cf. EC 3.5.5.1 nitrilase.
CAS REGISTRY NUMBER
COMMENTARY
82391-37-5
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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
2(R)-(4-chlorophenyl)-3-methylbutyronitrile + H2O
2(R)-(4-chloro-phenyl)-3-methyl-butyramide
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enantioselective hydration
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?
2(S)-(4-chlorophenyl)-3-methylbutyronitrile + H2O
2(S)-(4-chloro-phenyl)-3-methyl-butyramide
2(S)-(4-chlorophenyl)-3-methylbutyronitrile + H2O
2(S)-(4-chloro-phenyl)-3-methyl-butyramide
-
-
-
?
2(S)-(4-chlorophenyl)-3-methylbutyronitrile + H2O
2(S)-(4-chloro-phenyl)-3-methyl-butyramide
-
-
-
?
2(S)-(4-chlorophenyl)-3-methylbutyronitrile + H2O
2(S)-(4-chloro-phenyl)-3-methyl-butyramide
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enantioselective hydration
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?
additional information
?
-
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S enantiomer conversion is 14 times greater than the rate of R enantiomer conversion
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-
?
additional information
?
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roles of second- and third-shell residues of the active site structure in catalysis, overview. Three of the predicted second-shell residues, alpha-Asp164, beta-Glu56, and beta-His147, and one predicted third-shell residue, beta-His71, have significant effects on the catalytic efficiency of the enzyme, while one of the predicted residues, alpha-Glu168, and the three residues not predicted, alpha-Arg170, alpha-Tyr171, and beta-Tyr215, do not have any significant effects on the catalytic efficiency of the enzyme
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-
?
additional information
?
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the Fe-type NHase exhibits broad substrate specificity
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-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Co3+
Co3+
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CD spectrum suggests low-spin Co3+ in tetragonally-distorted octahedral ligand field
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
NHase activator protein
the activator gene encding activator protein might be involved in protein folding of the alpha- and beta-subunits of NHase
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P14 protein
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protein encoded in an open frame of the structural frames that is essential for optimal activity of NHase over-produced in Escherichia coli
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
recombinant wild-type and mutant enzymes, kinetics analysis, overview
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36.3
3-Cyanopyridine
25°C, pH not specified in the publication, beta-subunit mutant enzyme S189E
62.9
3-Cyanopyridine
25°C, pH not specified in the publication, subunit-fused wild-type nitrile hydratase
64.9
3-Cyanopyridine
25°C, pH not specified in the publication, beta-subunit mutant enzyme T173Y
72.8
3-Cyanopyridine
25°C, pH not specified in the publication, beta-subunit mutant enzyme M150C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
720.6
3-Cyanopyridine
25°C, pH not specified in the publication, subunit-fused wild-type nitrile hydratase
922.3
3-Cyanopyridine
25°C, pH not specified in the publication, beta-subunit mutant enzyme M150C
1112
3-Cyanopyridine
25°C, pH not specified in the publication, beta-subunit mutant enzyme T173Y
8935.6
3-Cyanopyridine
25°C, pH not specified in the publication, beta-subunit mutant enzyme S189E
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
11.5
3-Cyanopyridine
25°C, pH not specified in the publication, subunit-fused wild-type nitrile hydratase
12.7
3-Cyanopyridine
25°C, pH not specified in the publication, beta-subunit mutant enzyme M150C
17.1
3-Cyanopyridine
25°C, pH not specified in the publication, beta-subunit mutant enzyme T173Y
25.8
3-Cyanopyridine
25°C, pH not specified in the publication, beta-subunit mutant enzyme S189E
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
109
-
cell extract supernatant containing recombinant His-tagged EAK-16 N-terminal fusion enzyme variant, pH 7.5, 20°C
110
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cell extract supernatant containing recombinant His-tagged wild-type enzyme, pH 7.5, 20°C
13
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cell extract supernatant containing recombinant His-tagged ELK-16 N-terminal fusion enzyme variant, pH 7.5, 20°C
26.61
purified recombinant enzyme, pH 8.0, 25°C, substrate 3-cyanopyridine
372
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purified recombinant His-tagged ELK-16 N-terminal fusion enzyme variant, pH 7.5, 20°C
426
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purified recombinant His-tagged EAK-16 N-terminal fusion enzyme variant, pH 7.5, 20°C
429
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purified recombinant His-tagged EAK-16 C-terminal fusion enzyme variant, pH 7.5, 20°C
439
535
purified recombinant enzyme, pH 8.0, 25°C, substrate valeronitrile
553
purified recombinant enzyme, pH 8.0, 25°C, substrate 4-chlorobutyronitrile
775
purified recombinant enzyme, pH 8.0, 25°C, substrate isobutyronitrile
941
purified recombinant enzyme, pH 8.0, 25°C, substrate acrylonitrile
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
because of the absence of sequence similarity between the Co- and Fe-type NHase activators, the two types of NHases might be assembled and maturated by different molecular mechanisms
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
22982
23000
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1 * 23000, about, alpha-subunit + 1 x 23000, about, beta-subunit, Co-type NHases are bacterial heterodimers, consisting of nonhomologous alpha- and beta-subunits
24108
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2 * 22982, alpha + 2 * 24108, beta, amino acid sequence, gel filtration
54000
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chromatography on Sephacryl S-400 and Sepharose CL-6B, alpha-beta form
22982
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2 * 22982, alpha + 2 * 24108, beta, amino acid sequence, gel filtration
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
heterodimer
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1 * 23000, about, alpha-subunit + 1 x 23000, about, beta-subunit, Co-type NHases are bacterial heterodimers, consisting of nonhomologous alpha- and beta-subunits
tetramer
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2 * 22982, alpha + 2 * 24108, beta, amino acid sequence, gel filtration
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified recombinant wild-type and mutant enzymes, hanging drop vapour diffusion method, 25°C, from 20 mg/mL ppNHase and a reservoir containing 22% polyacrylic acid sodium salt 5100, 100 mM HEPES, pH 7.5, 20 mM magnesium chloride, and 4% acetone, single crystals are transferred to 17.6% polyacrylic acid sodium salt 5100 and 20% glycerol in 100 mM HEPES, pH 7.5, as cryoprotectant, X-ray diffraction structure determination and analysis
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
M150C
beta-subunit mutant enzyme, 32% increase in half-life at 50°C, the kcat/Km value is 1.1fold higher than the kcat/Km value of the wild-type enzyme
S189E
beta-subunit mutant enzyme, 107% increase in half-life at 50°C, the kcat/Km value is 2.2fold higher than the kcat/Km value of the wild-type enzyme
T173Y
beta-subunit mutant enzyme, 7% increase in half-life at 50°C, the kcat/Km value is 1.5fold higher than the kcat/Km value of the wild-type enzyme
additional information
additional information
-
homologous protein fragment swapping method is used for the improvement of the stability of NHase from Pseudomonas putida NRRL-18668, site targeted amino recombination software and molecular dynamics are used for determination of the crossover sites for fragment recombination, overview. One thermophilic NHase fragment M1 to G98 from Comamonas testosteroni 5-MGAM-4D and two fragments K165-V196 and K165-D209 from Pseudonocardia thermophila JCM3095 are selected to swap the corresponding fragments of Pseudomonas putida NHase. The chimeric NHases show 1.4 to 3.5fold enhancement in thermostability, some show reduced activity and product inhibition compared to wild-type Pseudomonas putida NHase. But mutants 3AB and 3ABC show increased activity due to altered secondary structure compared to the wild-type
additional information
-
the thermostability and product tolerance of nitrile hydratase are enhanced by fusing with two of the self-assembling amphipathic peptides, EAK16 (AEAEAKAKAEAEAKAK) at the N- and C-terminus and ELK16 (LELELKLKLELELKLK) at the N-terminus. When self-assembling amphipathic peptide ELK16 is fused to the N-terminus of the enzymes beta-subunit, the resultant enzyme (SAP-NHase-2) becomes an active inclusion body, while EAK16 does not affect the enzyme solubility when fused to the enzyme's C-terminus (SAP-NHase-10) or N-termninus (AP-NHase-1) of the beta-subunit
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
20
purified recombinant enzyme, over 60% activity remaining after 3 h
20 - 45
purified recombinant enzyme, 50% of maximal activity within this range. Half-lives of NHase F1 at 20°C, 25°C, 30°C, and 35°C are calculated as 5.1 h, 3.8 h, 2.2 h, and 1.2 h, respectively
50
55
Tm value of subunit-fused wild-type nitrile hydratase is 54.5°C, Tm value of beta-subunit mutant enzyme T173Y is 55°C
50
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wild-type enzyme, 30 min, deactivation, enzyme fused to self-assembling amphipathic peptide ELK16 (LELELKLKLELELKLK) retains 30% activity, and SAP-NHase-1 N-terminally fused to self-assembling amphipathic peptide EAK16 (AEAEAKAKAEAEAKAK) retains 45% activity, C-terminally fused enzyme SAP-NHase-10 50% activity. Treatment with buffer containing 10% acrylamide increases the thermostability so that the wild-type retains 30% activity, and the fused enzymes SAP-NHase-1, SAP-NHase-2, and SAP-NHase-10 retain 52%, 42%, and 55% activity, respectively
50
t1/2: 26 min (subunit-fused wild-type nitrile hydratase), 36 min (beta-subunit mutant enzyme A119D), 32 min (beta-subunit mutant enzyme V139A), 34 min (beta-subunit mutant enzyme N141G), 13 min (beta-subunit mutant enzyme T148G), 32 min (beta-subunit mutant enzyme R149G), 40 min (beta-subunit mutant enzyme M150C), 29 min (beta-subunit mutant enzyme G156M), 30 min (beta-subunit mutant enzyme G156S), 25 min (beta-subunit mutant enzyme G156Q), 24 min (beta-subunit mutant enzyme G156N), 32 min (beta-subunit mutant enzyme G156E), 23 min (beta-subunit mutant enzyme H165G), 32 min (beta-subunit mutant enzyme T173Y), 38 min (beta-subunit mutant enzyme T173F), 24 min (beta-subunit mutant enzyme V188G), above 60 min (beta-subunit mutant enzyme S189E), 28 min (beta-subunit mutant enzyme Q198G)
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
chromatography on DEAE-Cellulose, hydroxyapatite, phenyl-Sepharose and Sephacryl S-400
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recombinant His-tagged wild-type enzyme and peptide fused enzyme variants from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration
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recombinant His6-tagged subunits 2.6fold from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and desalting gel filtration
recombinant soluble wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by anion exchange chromatography, ammonium sulfate fractionation, and hydrophobic interaction chromatography, followed by ultrafiltration and another step of anion exchange chromatography
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of His-tagged wild-type enzyme and peptide fused enzyme variants in Escherichia coli strain BL21(DE3)
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expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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gene Pput_2728, gene cluster and phylogenetic analysis, functional expression of His6-tagged subunits in Escherichia coli strain BL21(DE3) by assisting of a putative activator gene adjacent to beta-subunit region. Maximal enzyme activity is obtained when the structural and activator genes are transcribed as one unit in plasmid pCDFDuet-1 at 18°C. The expressed product does not show any NHase activity when the downstream activator gene is ignored, and the product completely exists in insoluble inclusion body
gene Pput_2729, gene cluster and phylogenetic analysis, functional expression of His6-tagged subunits in Escherichia coli strain BL21(DE3) by assisting of a putative activator gene adjacent to beta-subunit region. Maximal enzyme activity is obtained when the structural and activator genes are transcribed as one unit in plasmid pCDFDuet-1 at 18°C. The expressed product does not show any NHase activity when the downstream activator gene is ignored, and the product completely exists in insoluble inclusion body
genetic organization of wild-type and chimeric mutant enzymes, expression of wild-type and recombinant chimeric mutants in Escherichia coli strain JM109
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
industrial production of highly purified acrylamide and nicotinamide. The thermostability and catalytic efficiency of the subunit-fused nitrile hydratase is improved by semi-rational engineering
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Payne, M.S.; Wu, S.; Fallon, R.D.; Tudor, G.; Stieglitz, B.; Turner, I.M., Jr.; Nelson, M.J.
A stereoselective cobalt-containing nitrile hydratase
Biochemistry
36
5447-5454
1997
Pseudomonas putida, Pseudomonas putida NRRL-18668
Wu, S.; Fallon, R.D.; Payne, M.S.
Over-production of stereoselective nitrile hydratase from Pseudomonas putida 5B in Escherichia coli: activity requires a novel downstream protein
Appl. Microbiol. Biotechnol.
48
704-708
1997
Pseudomonas putida, Pseudomonas putida 5B
Kobayashi, M.; Shimizu, S.
Metalloenzyme nitrile hydratase: structure, regulation, and application to biotechnology
Nat. Biotechnol.
16
733-736
1998
Pseudomonas chlororaphis, Pseudomonas chlororaphis B23, Pseudomonas putida, Rhodococcus rhodochrous, Rhodococcus rhodochrous J1, Rhodococcus sp., Rhodococcus sp. N-774, Rhodococcus sp. R312
Brodkin, H.R.; Novak, W.R.; Milne, A.C.; DAquino, J.A.; Karabacak, N.M.; Goldberg, I.G.; Agar, J.N.; Payne, M.S.; Petsko, G.A.; Ondrechen, M.J.; Ringe, D.
Evidence of the participation of remote residues in the catalytic activity of Co-type nitrile hydratase from Pseudomonas putida
Biochemistry
50
4923-4935
2011
Pseudomonas putida, Pseudomonas putida NRRL-18668
Cui, Y.; Cui, W.; Liu, Z.; Zhou, L.; Kobayashi, M.; Zhou, Z.
Improvement of stability of nitrile hydratase via protein fragment swapping
Biochem. Biophys. Res. Commun.
450
401-408
2014
Comamonas testosteroni (Q5XPL4), Comamonas testosteroni (Q5XPL5), Comamonas testosteroni 5-MGAM-4D (Q5XPL4), Comamonas testosteroni 5-MGAM-4D (Q5XPL5), Comamonas testosteroni 5-MGAM-4D, Pseudomonas putida, Pseudomonas putida NRRL-18668, Pseudonocardia thermophila (Q7SID2), Pseudonocardia thermophila (Q7SID3), Pseudonocardia thermophila JCM 3095 (Q7SID2), Pseudonocardia thermophila JCM 3095 (Q7SID3)
Liu, Y.; Cui, W.; Liu, Z.; Cui, Y.; Xia, Y.; Kobayashi, M.; Zhou, Z.
Enhancement of thermo-stability and product tolerance of Pseudomonas putida nitrile hydratase by fusing with self-assembling peptide
J. Biosci. Bioeng.
118
249-252
2014
Pseudomonas putida, Pseudomonas putida NRRL-18668
Pei, X.; Yang, L.; Xu, G.; Wang, Q.; Wu, J.
Discovery of a new Fe-type nitrile hydratase efficiently hydrating aliphatic and aromatic nitriles by genome mining
J. Mol. Catal. B
99
26-33
2014
Pseudomonas putida (A5W402)
-
Xia, Y.; Cui, W.; Cheng, Z.; Peplowski, L.; Liu, Z.; Kobayashi, M.; Zhou, Z.
Improving the thermostability and catalytic efficiency of the subunit-fused nitrile hydratase by semi-rational engineering
ChemCatChem
10
1370-1375
2018
Pseudomonas putida (P97051 AND P97052), Pseudomonas putida NRRL-18668 (P97051 AND P97052)
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