Literature summary for 4.2.1.84 extracted from

Guo, F.; Wu, J.; Yang, L.; Xu, G.
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

Activating Compound	Comment	Organism	Structure
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)

Cloned (Comment)	Organism
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

Cloned (Comment)

Organism

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

Protein Variants	Comment	Organism
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]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
48	-	3-Cyanopyridine	35°C, pH 6.5	Klebsiella oxytoca

Metals/Ions

Metals/Ions	Comment	Organism	Structure
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

Organism	UniProt	Comment	Textmining
Klebsiella oxytoca	A0A0H3HE78 AND A0A0H3H964	A0A0H3HE78: alpha-subunit, A0A0H3H964: beta-subunit	-
Klebsiella oxytoca DSM 4798	A0A0H3HE78 AND A0A0H3H964	A0A0H3HE78: alpha-subunit, A0A0H3H964: beta-subunit	-

Purification (Commentary)

Purification (Comment)	Organism
-	Klebsiella oxytoca

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
45.2	-	substrate: 3-cyanopyridine, 35°C, pH 6.5	Klebsiella oxytoca

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
2,2-dimethylcyclopropanecarbonitrile + H2O	29% of the activity compared to 3-cyanopyridine	Klebsiella oxytoca	?	-	?
2-cyanopyridine + H2O	46% of the activity compared to 3-cyanopyridine	Klebsiella oxytoca	pyridine-2-carbamide	-	?
2-cyanopyridine + H2O	46% of the activity compared to 3-cyanopyridine	Klebsiella oxytoca DSM 4798	pyridine-2-carbamide	-	?
3-cyanopyridine + H2O	-	Klebsiella oxytoca	pyridine-3-carbamide	-	?
3-cyanopyridine + H2O	-	Klebsiella oxytoca DSM 4798	pyridine-3-carbamide	-	?
4-cyanopyridine + H2O	74% of the activity compared to 3-cyanopyridine	Klebsiella oxytoca	isonicotinamide	-	?
4-cyanopyridine + H2O	74% of the activity compared to 3-cyanopyridine	Klebsiella oxytoca DSM 4798	isonicotinamide	-	?
acrylonitrile + H2O	79% of the activity compared to 3-cyanopyridine	Klebsiella oxytoca	acrylamide	-	?
acrylonitrile + H2O	79% of the activity compared to 3-cyanopyridine	Klebsiella oxytoca DSM 4798	acrylamide	-	?
alpha-methylbenzyl cyanide + H2O	65% of the activity compared to 3-cyanopyridine	Klebsiella oxytoca	2-phenylpropanamide	-	?
benzonitrile + H2O	82% of the activity compared to 3-cyanopyridine	Klebsiella oxytoca	benzamide	-	?
benzyl cyanide + H2O	85% of the activity compared to 3-cyanopyridine	Klebsiella oxytoca	2-phenylacetamide	-	?
butyronitrile + H2O	248% of the activity compared to 3-cyanopyridine	Klebsiella oxytoca	butyramide	-	?
isobutyronitrile + H2O	215% of the activity compared to 3-cyanopyridine	Klebsiella oxytoca	isobutyramide	-	?
mandelonitrile + H2O	31% of the activity compared to 3-cyanopyridine	Klebsiella oxytoca	?	-	?
methacrylonitrile + H2O	96% of the activity compared to 3-cyanopyridine	Klebsiella oxytoca	methylacrylamide	-	?
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	?	-	?
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	?	-	?
propionitrile + H2O	113% of the activity compared to 3-cyanopyridine	Klebsiella oxytoca	propionamide	-	?

Subunits

Subunits	Comment	Organism
dimer	1 * 22000 (alpha-subunit) + 1 * 24000 (beta-subunit), SDS-PAGE	Klebsiella oxytoca

Synonyms

Synonyms	Comment	Organism
NHaseK	-	Klebsiella oxytoca

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
35	-	-	Klebsiella oxytoca

Temperature Range [°C]

Temperature Minimum [°C]	Temperature Maximum [°C]	Comment	Organism
15	50	15°C: about 50% of maximal activity, 50°C: about 60% of maximal activity	Klebsiella oxytoca

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
30	-	6 h, about 10% loss of activity. 14 h, about 20% loss of activity	Klebsiella oxytoca
35	-	6 h, about 30% loss of activity. 14 h, about 45% loss of activity	Klebsiella oxytoca
40	-	2 h, about 50% loss of activity. 8 h, about 80% loss of activity	Klebsiella oxytoca
45	-	30 min, about 55% loss of activity. 5 h, about 85% loss of activity	Klebsiella oxytoca

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6.5	-	-	Klebsiella oxytoca

pH Range

pH Minimum	pH Maximum	Comment	Organism
5.5	9	pH 5.5: about 60% of maximal activity, pH 9.0: about 60% of maximal activity	Klebsiella oxytoca