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Literature summary extracted from
- Overexpression of a nitrile hydratase from Klebsiella oxytoca KCTC 1686 in Escherichia coli and its biochemical characterization (2015), Biotechnol. Bioprocess Eng., 20, 995-1004 .
No PubMed abstract available
Activating Compound
| EC Number | Activating Compound | Comment | Organism | Structure |
|---|---|---|---|---|
| 4.2.1.84 | activator 17K | the putative activator (17K, MW 17 kDa) gene next to the beta-subunit gene is proven to be important for the functional expression of recombinant NHaseK in Escherichia coli | Klebsiella oxytoca |
Cloned(Commentary)
| EC Number | Cloned (Comment) | Organism |
|---|---|---|
| 4.2.1.84 | the nitrile hydratase (NHaseK, consisting of alpha- and beta-subunits) genes and the putative activator (17K) gene adjacent to the beta subunit region are cloned from Klebsiella oxytoca KCTC 1686. 17K is essential for the functional expression of recombinant NHaseK in Escherichia coli. The expression level of 17K is very low when the 17K gene and NHaseK structural genes are expressed as a gene cluster in Escherichia coli BL21(DE3). To improve the 17K expression level and NHaseK activity, the expression cassette is redesigned by placing the 17K gene and NHaseK structural genes under the control of different promoters in the pETDuet-1 expression vector, co-expressing the 17K gene with the gene cluster in a double plasmid or a single plasmid with a double promoter, and introducing an efficient Shine-Dalgarno sequence 5' to the 17K gene. The specific activity of NHaseK is improved when 17K is co-expressed with the gene cluster, whereas the production of NHaseK protein decreases. The maximum activity is achieved when an efficient Shine-Dalgarno sequence is introduced 5' to the 17K gene. The expression level of 17K is significantly improved and the expression level of NHaseK does not decrease significantly | Klebsiella oxytoca |
Protein Variants
| EC Number | Protein Variants | Comment | Organism |
|---|---|---|---|
| 4.2.1.84 | up | the putative activator (17K) gene next to the beta-subunit gene is proven to be important for the functional expression of recombinant NHaseK in Escherichia coli | Klebsiella oxytoca |
KM Value [mM]
| EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 4.2.1.84 | 48 | - |
3-Cyanopyridine | 35°C, pH 6.5 | Klebsiella oxytoca |  |
Metals/Ions
| EC Number | Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|---|
| 4.2.1.84 | Co2+ | the enzyme requires non-corrin cobalt for its catalytic activity. Cobalt ions are necessary for the expression and activity of NHaseK in Escherichia coli | Klebsiella oxytoca | |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 4.2.1.84 | Klebsiella oxytoca | A0A0H3HE78 AND A0A0H3H964 | A0A0H3HE78: alpha-subunit, A0A0H3H964: beta-subunit | - |
| 4.2.1.84 | Klebsiella oxytoca DSM 4798 | A0A0H3HE78 AND A0A0H3H964 | A0A0H3HE78: alpha-subunit, A0A0H3H964: beta-subunit | - |
Purification (Commentary)
| EC Number | Purification (Comment) | Organism |
|---|---|---|
| 4.2.1.84 | - |
Klebsiella oxytoca |
Specific Activity [micromol/min/mg]
| EC Number | Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|---|
| 4.2.1.84 | 45.2 | - |
substrate: 3-cyanopyridine, 35°C, pH 6.5 | Klebsiella oxytoca |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 4.2.1.84 | 2,2-dimethylcyclopropanecarbonitrile + H2O | 29% of the activity compared to 3-cyanopyridine | Klebsiella oxytoca | ? | - |
? | |
| 4.2.1.84 | 2-cyanopyridine + H2O | 46% of the activity compared to 3-cyanopyridine | Klebsiella oxytoca | pyridine-2-carbamide | - |
? | |
| 4.2.1.84 | 2-cyanopyridine + H2O | 46% of the activity compared to 3-cyanopyridine | Klebsiella oxytoca DSM 4798 | pyridine-2-carbamide | - |
? | |
| 4.2.1.84 | 3-cyanopyridine + H2O | - |
Klebsiella oxytoca | pyridine-3-carbamide | - |
? | |
| 4.2.1.84 | 3-cyanopyridine + H2O | - |
Klebsiella oxytoca DSM 4798 | pyridine-3-carbamide | - |
? | |
| 4.2.1.84 | 4-cyanopyridine + H2O | 74% of the activity compared to 3-cyanopyridine | Klebsiella oxytoca | isonicotinamide | - |
? | |
| 4.2.1.84 | 4-cyanopyridine + H2O | 74% of the activity compared to 3-cyanopyridine | Klebsiella oxytoca DSM 4798 | isonicotinamide | - |
? | |
| 4.2.1.84 | acrylonitrile + H2O | 79% of the activity compared to 3-cyanopyridine | Klebsiella oxytoca | acrylamide | - |
? | |
| 4.2.1.84 | acrylonitrile + H2O | 79% of the activity compared to 3-cyanopyridine | Klebsiella oxytoca DSM 4798 | acrylamide | - |
? | |
| 4.2.1.84 | alpha-methylbenzyl cyanide + H2O | 65% of the activity compared to 3-cyanopyridine | Klebsiella oxytoca | 2-phenylpropanamide | - |
? | |
| 4.2.1.84 | benzonitrile + H2O | 82% of the activity compared to 3-cyanopyridine | Klebsiella oxytoca | benzamide | - |
? | |
| 4.2.1.84 | benzyl cyanide + H2O | 85% of the activity compared to 3-cyanopyridine | Klebsiella oxytoca | 2-phenylacetamide | - |
? | |
| 4.2.1.84 | butyronitrile + H2O | 248% of the activity compared to 3-cyanopyridine | Klebsiella oxytoca | butyramide | - |
? | |
| 4.2.1.84 | isobutyronitrile + H2O | 215% of the activity compared to 3-cyanopyridine | Klebsiella oxytoca | isobutyramide | - |
? | |
| 4.2.1.84 | mandelonitrile + H2O | 31% of the activity compared to 3-cyanopyridine | Klebsiella oxytoca | ? | - |
? | |
| 4.2.1.84 | methacrylonitrile + H2O | 96% of the activity compared to 3-cyanopyridine | Klebsiella oxytoca | methylacrylamide | - |
? | |
| 4.2.1.84 | additional information | recombinant NHaseK can hydrolyze a wide range of aliphatic, aromatic, and heterocyclic nitriles, and converts racemic nitriles to the corresponding S-amides, with E values ranging from 9 to 17 | Klebsiella oxytoca | ? | - |
? | |
| 4.2.1.84 | additional information | recombinant NHaseK can hydrolyze a wide range of aliphatic, aromatic, and heterocyclic nitriles, and converts racemic nitriles to the corresponding S-amides, with E values ranging from 9 to 17 | Klebsiella oxytoca DSM 4798 | ? | - |
? | |
| 4.2.1.84 | propionitrile + H2O | 113% of the activity compared to 3-cyanopyridine | Klebsiella oxytoca | propionamide | - |
? | |
Subunits
| EC Number | Subunits | Comment | Organism |
|---|---|---|---|
| 4.2.1.84 | dimer | 1 * 22000 (alpha-subunit) + 1 * 24000 (beta-subunit), SDS-PAGE | Klebsiella oxytoca |
Synonyms
| EC Number | Synonyms | Comment | Organism |
|---|---|---|---|
| 4.2.1.84 | NHaseK | - |
Klebsiella oxytoca |
Temperature Optimum [°C]
| EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|---|
| 4.2.1.84 | 35 | - |
- |
Klebsiella oxytoca |
Temperature Range [°C]
| EC Number | Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
|---|---|---|---|---|
| 4.2.1.84 | 15 | 50 | 15°C: about 50% of maximal activity, 50°C: about 60% of maximal activity | Klebsiella oxytoca |
Temperature Stability [°C]
| EC Number | Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|---|
| 4.2.1.84 | 30 | - |
6 h, about 10% loss of activity. 14 h, about 20% loss of activity | Klebsiella oxytoca |
| 4.2.1.84 | 35 | - |
6 h, about 30% loss of activity. 14 h, about 45% loss of activity | Klebsiella oxytoca |
| 4.2.1.84 | 40 | - |
2 h, about 50% loss of activity. 8 h, about 80% loss of activity | Klebsiella oxytoca |
| 4.2.1.84 | 45 | - |
30 min, about 55% loss of activity. 5 h, about 85% loss of activity | Klebsiella oxytoca |
pH Optimum
| EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|---|
| 4.2.1.84 | 6.5 | - |
- |
Klebsiella oxytoca |
pH Range
| EC Number | pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|---|
| 4.2.1.84 | 5.5 | 9 | pH 5.5: about 60% of maximal activity, pH 9.0: about 60% of maximal activity | Klebsiella oxytoca |
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