JP2003088384A - New nitrile hydratase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cobalt type nitrile hydratase having cysteinesulfinic acid and cysteinesulfenic acid in the active center. SOLUTION: This cobalt type nitrile hydratase comprises a subunit bound through a region represented by an amino acid sequence Cys-Thr-Leu-Csi-Ser-Cse described in sequence number 1 of the sequence listing (refer to the specification) to a cobalt atom as a constituent element (Csi in the sequence denotes the cysteinesulfinic acid; and Cse denotes the cysteinesulfenic acid in the sequence).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel amino acid sequence of cobalt type nitrile hydratase. Further, a culture solution obtained by culturing a transformant strain transformed with a recombinant plasmid containing the enzyme gene.
The present invention relates to a method for producing a corresponding amide compound from a nitrile compound using a cell / treated cell.

[0002]

2. Description of the Related Art A technique for producing a corresponding amide compound by hydrating a nitrile group of a nitrile compound to convert it to an amide group is known as a chemical treatment in which heat treatment is performed in the presence of an acid or an alkali or in the presence of a copper catalyst. The method was known more than before.

On the other hand, in recent years, a nitrile hydratase, which is an enzyme having a nitrile hydration activity for hydrating a nitrile group and converting it to an amide group, has been discovered, and a nitrile compound is produced using the enzyme or a microbial cell containing the enzyme. Methods for producing the more corresponding amide compounds have already been disclosed. This manufacturing method is known to have advantages such as high conversion and selectivity of a nitrile compound to a corresponding amide compound as compared with conventional chemical methods.

It is already known that nitrile hydratase has a non-heme iron atom or a non-choline nuclear cobalt atom as a prosthetic group in the active center, and iron type nitrile hydratase and cobalt type, respectively. It is distinguished by the name nitrile hydratase.

As the iron-type nitrile hydratase, one derived from Rhodococcus sp. Strain N-771 can be mentioned as a typical example. One of the characteristics of this enzyme is that the enzyme activity is changed by irradiating the bacterium with light. That is, the enzyme activity decreases when the cells are left under dark conditions, but the enzyme activity increases again by irradiating with light. The mechanism is thought to be that nitric oxide (NO) is bound to the non-heme iron center of the inactive enzyme and NO is dissociated from the non-heme iron center by light, and the enzyme is activated ( Endo et al., Bioscience and Industry Vol.56, No.8, pp519
-524, 1998).

Further, the iron type nitrile hydratase has been subjected to X-ray crystal structure analysis, and its three-dimensional structure has been clarified. As a result, the enzyme has a cysteine cluster (Cys-Ser-Leu-Cys) from the 109th position to the 114th position of the α subunit forming the active center.
-Ser-Cys) has been confirmed to be bound to non-heme iron through four amino acid residues. In particular, two cysteine residues in the cysteine cluster (112th and 114th of α subunit) were post-translationally modified into cysteine sulfinic acid and cysteine sulfenic acid, respectively. It is proposed that the photoresponsiveness due to the binding is produced (Japanese Patent Laid-Open No. 11-80194).

On the other hand, as cobalt-type nitrile hydratase, Rhodococcus spp.
(Japanese Patent No. 11379) can be mentioned as a typical example, but X-ray crystal structure analysis has not been reported for cobalt type nitrile hydratase.

[0008]

The present inventors have found Pseudonocardia thermophila as a microorganism having nitrile hydratase activity, and have succeeded in expressing the enzyme in large amounts in Escherichia coli ( JP-A-9
-275978).

Therefore, the present inventors have proposed the MT described in this patent.
The -10822 strain was cultured, nitrile hydratase was purified from the transformed Escherichia coli, and the obtained purified nitrile hydratase was analyzed in detail. As a result, the following findings 1 to 4 have been obtained.

First, using the purified enzyme, ICP
(Inductive Coupled Plasma
The amount of contained metal according to a) was measured. As a result, it was revealed that this enzyme contained a cobalt atom but not an iron atom. That is, this enzyme was confirmed to be a cobalt-type nitrile hydratase.

Secondly, the purified enzyme was crystallized and subjected to X-ray structural analysis. The structural analysis was carried out by a molecular replacement method using the α-chain and β-chain of iron-type nitrile hydratase derived from Rhodococcus sp. Strain N-771 for which X-ray structural analysis has been performed as model molecules. As a result, the cobalt atom was bound to the cysteine cluster site from the 108th cysteine residue to the 113th cysteine residue of the α subunit. Furthermore, 1 of α subunit
An extra electron density was observed around the 11th cysteine residue and the 113th cysteine residue, and both cysteine residues were post-translationally modified to give cysteine sulfinic acid (Cys), similar to iron-type nitrile hydratase. -SO
OH) and cysteine sulfenic acid (Cys-SOH).

Based on the above findings, the present inventors have found that in the cobalt-type nitrile hydratase derived from Pseudonocardia thermophila, the 111th cysteine residue of the α subunit remains in the cysteine sulfinic acid and the 113th cysteine residue. It was concluded that the groups were respectively post-translationally modified by cysteine sulfenic acid, and the polypeptide chain of the α subunit and the cobalt atom were linked via this modified amino acid residue.

That is, the present inventors have found that Pseudonocardia thermophila-derived cobalt-type nitrile hydratase is a non-natural amino acid residue cysteine sulfinic acid (which cannot be deduced from its gene sequence). Hereinafter, when the sequence is described in juxtaposition with other amino acids, it is abbreviated as Csi), and cysteine sulfenic acid (hereinafter, when the sequence is described in juxtaposition with other amino acids, it is abbreviated as Cse). The present invention has been completed based on the finding that it is bonded to a cobalt atom via these residues. That is, the present invention provides cysteine sulfinic acid and a cobalt-type nitrile hydratase having cysteine sulfenic acid as an active center.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The cobalt type nitrile hydratase provided in the present invention means the amino acid sequence (Cys-Thr-Leu-Csi-S) shown in SEQ ID NO: 1 in the sequence listing.
(er-Cse) is not particularly limited as long as it is a protein containing a subunit bound to a cobalt atom through a region containing er-Cse) as a constituent and having the activity of hydrating a nitrile compound.

In particular, in the present invention, the amino acid sequence consisting of the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing and shown in SEQ ID NO: 1 of the sequence listing (Cys-Thr-Leu-Csi-
Ser-Cse) containing a subunit bound to a cobalt atom through a region containing the same as a constituent element, and at the same time containing a subunit represented by the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing as a constituent element. As an example:

Even when the gene having the same nucleotide sequence is transcribed and translated as a template, the type of host into which it is introduced, the components and composition of the nutrient medium used for culture, the temperature and pH during culture, etc. In some cases, one or two or more amino acids near the N-terminal in the sequence listing may be deleted or the N-terminal may be deleted due to modification by the enzyme in the host after gene expression, although the desired enzyme activity is retained. A mutant with one or more amino acids newly added may be produced. Furthermore, due to advances in recombinant DNA technology, it is relatively easy to substitute or delete one or more of the constituent amino acids with other amino acids without substantially changing the action of the enzyme,
You can now delete or insert. Further, by substituting, deleting, deleting or inserting one or more of the constituent amino acids with another amino acid, properties having high industrial utility value such as improvement in organic solvent resistance and change in substrate specificity Attempts have also been made to create a mutant enzyme with added. In view of such a state of the art, the nitrile hydratase referred to in the present invention is not limited to one containing the subunit represented by the amino acid sequences shown in SEQ ID NOs: 3 and 4 as a constituent element as it is, or one or Even if it is a variant containing a subunit represented by an amino acid sequence in which two or more amino acids are substituted, deleted, deleted or inserted with other amino acids as a constituent, it is the amino acid shown in SEQ ID NO: 1 of the sequence listing. Array (C
In the present invention, when a subunit containing a subunit bonded to a cobalt atom through a region containing ys-Thr-Leu-Csi-Ser-Cse) is contained as a component and it has an activity of hydrating a nitrile compound, Shall be included.

More specifically, 6th, 19th, 38th, 77th of the amino acid sequence of SEQ ID NO: 2 in the sequence listing,
90th, 102nd, 106th, 126th, 13
0th, 142nd, 146th, 187th, 194
Amino acid sequence (Cy
Mutant cobalt-type nitrile hydratase containing a mutant subunit bound to a cobalt atom through a region containing s-Thr-Leu-Csi-Ser-Cse) as a constituent element. The other subunit of the mutant cobalt-type nitrile hydratase is the 20th, 21st, 1st amino acid sequence of SEQ ID NO: 3 in the sequence listing.
Mutant cobalt-type nitrile hydratase, which is a mutant subunit obtained by substituting one or more amino acids of the 08th, 200th, and 212th amino acids with another amino acid, is also included.

The cobalt-type nitrile hydratase in the present invention can be exemplified by those derived from Pseudonocardia thermophila, but it is a cobalt-type nitrile hydratase derived from a microorganism strain other than the strain. Also, the protein has an amino acid sequence (Cys-Thr-Leu-Csi-Se) shown in SEQ ID NO: 1 in the sequence listing.
The present invention includes any protein which has a subunit bound to a cobalt atom through a region containing r-Cse) as a constituent and which has an activity of hydrating a nitrile compound. . Such microbial strains include Nocardia , Corynebacterium
(Corynebacterium) genus Bacillus (Bacillus) genus, thermophilic Bacillus, Pseudomonas (Pseudomonas) genus Micrococcus (Micrococcus) genus rhodochrous (Rhodochrou
s) Rodokokkkasu typified by the species (Rhodococcus) genus Acinetobacter (Acinetobacter) genus Xanthobacter Enterobacter
(Xanthobacter) genus Streptomyces (Streptomyces)
Genus Rhizobium (Rhizobium) genus, Klebsiella (Klebsiel
la) genus, Enterobacter (Enterobacter) genus, Erwinia (Erwinia) genus, Earomonasu (Aeromonas) genus, Citrobacter (Citrobacter) sp., Achromobacter (Achromob
Preferable examples include strains belonging to the genus Pseudonocardia other than the genus acter, the genus Agrobacterium , the genus Amycolatopsis, or the thermophila sp. JCM3095 strain. it can.

The cobalt-type nitrile hydratase in the present invention can be exemplified by those derived from Pseudonocardia thermophila, but due to recent advances in molecular biology and genetic engineering, Pseudonocardia By analyzing the molecular biological properties and amino acid sequence of cobalt-type nitrile hydratase of a microbial strain represented by Thermophila, the gene of the protein is obtained from the microbial strain, and necessary for the gene and expression. It became possible, and relatively easy, to construct a recombinant plasmid having a control region inserted therein and introduce this into an arbitrary host to produce a recombinant bacterium expressing the protein. In view of the state of the art, even in the case of a cobalt type nitrile hydratase produced by a gene recombinant bacterium in which such a gene of cobalt type nitrile hydratase is introduced into an arbitrary host, the protein is SEQ ID NO: 1 in the sequence listing. The amino acid sequence shown in (Cys-Thr-Leu-
The present invention includes a protein containing a subunit bonded to a cobalt atom through a region containing Csi-Ser-Cse) as a constituent and having a nitrile compound hydrating activity. And

The control region required for expression herein means a promoter sequence (including an operator sequence that controls transcription), a ribosome binding sequence (SD sequence), a transcription termination sequence and the like. Regarding the sequence order of these control regions on the recombinant plasmid, it is desirable that the promoter sequence, the ribosome binding sequence, the gene encoding the cobalt-type nitrile hydratase, and the transcription termination sequence are arranged in this order from the 5 ′ end side upstream. In addition, as a specific example of the plasmid,
PBR3 having a region capable of autonomous replication in Escherichia coli
22, pUC18, Bluescript II SK
(+), PKK223-3, pSC101, and pUB110, p having a region capable of autonomous replication in Bacillus subtilis
TZ4, pC194, ρ11, φ1, φ105, etc. can be used as a vector. In addition, as an example of a plasmid capable of autonomous replication in two or more types of hosts, p
HV14, TRp7, YEp7 and pBS7 can be used as vectors.

Escherichia coli is a representative example of the arbitrary host as described later, but it is not particularly limited to Escherichia coli and Bacillus subtilis or the like. Other microbial strains such as Bacillus, yeast and actinomycetes are also included.

A method for constructing the recombinant plasmid of the present invention by inserting the gene encoding the nitrile hydratase of the present invention into the plasmid vector into a plasmid vector having a region required for expression of the gene, The method for transforming into a desired host and the method for producing nitrile hydratase in the transformed cells include:
"Molecular Cloning 2nd Ed
edition ”(T. Maniatis et al .; Cold S
pring Harbor Laboratory P
molecular biology as described in Res., 1989).
General methods and hosts known in the field of biotechnology and genetic engineering may be used. Although LB medium, M9 medium and the like are generally used as a medium for culturing the transformed strain, it is more preferable that 0.1 μg / ml or more of Co ion is present in such a medium component.

Further, in order to produce the corresponding amide compound from a nitrile compound using the cobalt type nitrile hydratase or transformant strain of the present invention, the desired nitrile compound is added to a culture solution of the transformant strain, or the transformant. It may be brought into contact with a transformed bacterium obtained by culturing the strain, a treated product of the transformed bacterium, or a nitrile hydratase isolated and purified from the transformed bacterium in an aqueous medium. The contact temperature is not particularly limited, but is preferably within a temperature range in which the nitrile hydratase is not inactivated, and more preferably 0 ° C to 50 ° C. The nitrile compound is not particularly limited as long as it is a compound to which the nitrile hydratase of the present invention can act as a substrate, but preferably acetonitrile, propionitrile, acrylonitrile, methacrylonitrile, n-butyronitrile, isobutyronitrile, crotono. Typical examples thereof include nitrile compounds having 2 to 4 carbon atoms such as nitrile and α-hydroxyisobutyronitrile. The concentration of the nitrile compound in the aqueous medium is not particularly limited as long as it does not exceed the maximum solubility of the nitrile compound in the aqueous medium, but preferably the loss of the enzyme by the nitrile compound. Considering the activity, it is 5% by weight or less, more preferably 2% by weight or less.

[0024]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In the following examples, the nitrile hydratase activity was measured as follows. The sample solution was added to a 20 mM Tris.Cl buffer (pH 7.5:
Hereinafter, it is appropriately diluted with buffer A), 1% by weight of acrylonitrile is added thereto, and the mixture is mixed at 10 ° C. for 10 minutes.
Let react for minutes. The same amount of 1 M phosphoric acid aqueous solution as this was added to the reaction solution to stop the reaction, and the concentration of acrylamide produced was measured by HPLC analysis. The HPLC column is ULTRON 80HG (length 50 mm x diameter 8 mm)
Was used as a developing solution. Acrylamide was detected by the absorbance at 220 nm.

[Example 1] Purification of nitrile hydratase derived from Pseudonocardia thermophila JCM3095 strain and measurement of contained metal atoms 100 ml of a medium having the following composition was prepared in a 500 ml Erlenmeyer flask with a baffle, and 121 ° C at 20 ° C. After sterilizing by autoclaving for 30 minutes, 30 ampicillin-added substances were prepared so that the final concentration was 50 μg / ml. Add MT-10822 strain (F
ERM BP-5785) was inoculated with one white bacterium ear and 37
The culture was performed at 130 ° C. for 20 hours. After collecting the culture solution in each baffled Erlenmeyer flask together, centrifuge (15000 G × 15 minutes) to separate only the bacterial cells from the culture solution, and then resuspend the bacterial cells in 50 ml of physiological saline. After turbidity, centrifuge again to obtain 120g of wet cells.
Got

[0026]   Medium composition Yeast extract 5.0 g / L             Polypeptone 10.0 g / L             NaCl 5.0 g / L             Cobalt chloride hexahydrate 10.0 mg / L             Ferric sulfate heptahydrate 40.0 mg / L               pH 7.5

110 g of the cells were added to 50 mM Tris.C.
1 It was suspended in 540 mL of buffer solution (pH 7.5) and crushed with an ultrasonic crusher for 10 minutes while cooling with ice. After confirming that the cells were completely crushed by a microscope, the crude enzyme extract was obtained by centrifugation (25,000 G × 20 minutes). The crude enzyme extract was heat-treated by bathing it in a hot water bath at 50 ° C for 30 minutes, and then centrifuged (2500
(0G × 20 minutes) to obtain a supernatant. Ammonium sulfate was added to the supernatant so that the saturation concentration was 40%.
Stir for 0 minutes at low temperature. Centrifuge (25000G x 10
For 10 minutes, ammonium sulfate was added again to the supernatant so that the saturated concentration became 70%, and the mixture was stirred at low temperature for 30 minutes. Then, a precipitate was obtained by centrifugation (25000 G × 10 minutes). The precipitate was added to a 50 mM Tris.Cl buffer (p
H7.5) After suspending in 20 mL, dialysis treatment was performed against buffer A for desalting. The precipitate was dissolved in 1 ml of KPB and dialyzed against 2 L of the same solution at 4 ° C. for 48 hours. The dialyzed solution was subjected to anion exchange chromatography using DEAE-Sepharose manufactured by Amersham Pharmacia. Buffer A was used as a developing solution, and the fraction containing nitrile hydratase activity was obtained by increasing with potassium chloride at a constant gradient from 0 M to 1.0 M. Next, the active fraction solution was dialyzed against buffer A, and then Phenyl-Toyo manufactured by Tosoh Corporation.
It was subjected to hydrophobic chromatography using a pearl.
Buffer A was used as the developing solution, and the ammonium sulfate concentration was adjusted to 2
The active fraction was obtained by a constant gradient reduction from 0% saturation to 0% saturation. Further, the active fraction solution was dialyzed against buffer A and then subjected to anion exchange chromatography using a MonoQ column (HR5 / 5) manufactured by Amersham Pharmacia. Buffer A was used as a developing solution, and the fraction containing nitrile hydratase activity was obtained by increasing with potassium chloride at a constant gradient from 0 M to 1.0 M. At the last stage, the active fraction solution was dialyzed against buffer A and then concentrated with Centricon 30 manufactured by Amicon to obtain a purified enzyme solution having a protein concentration of 8.0 mg / mL. Next, 50 μL of the purified enzyme solution and 50 μL of pure water were dispensed into a microtube, the lid of the microtube was opened, and the aluminum foil was covered at 80 ° C.
Dry Thermo Unit DTU-28 (manufactured by TAITEC Co., Ltd.) was allowed to stand for 3 hours to dry. Then, add 30% each of 61% nitric acid, and use Dry Thermo Unit DTU-28 (manufactured by TAITEC) at 80 ° C.
After leaving it to stand for 18 hours to completely dry it, add 1N of 0.1N nitric acid.
After adding mL, the amount of contained metal was measured using an SPS 1200VR plasma spectrometer (manufactured by Seiko). as a result,
It was found that about 1.62 Co atoms were included in one molecule (α ₂ β ₂ ) of this enzyme. Moreover, the iron atom was not included. In this measurement, buffer A was used as a control instead of the purified enzyme solution, and
As a standard, a 1 ppm Fe solution (ferric chloride hexahydrate) and a Co solution (cobalt chloride hexahydrate solution) were prepared. The two types of metals were detected from the measurement sample.

[Example 2] Crystallization and X-ray structure analysis A purified enzyme solution of Pseudonocardia thermophila JCM3095 strain-derived nitrile hydratase obtained in Example 1, 1.4 M sodium citrate and 0.1 M Reservoir solution consisting of sodium HEPES (pH
7.5) was mixed in a ratio of 1: 1 and crystals of the enzyme were produced at 5 ° C by a vapor equilibrium diffusion method. The obtained crystal was diffracted by X-ray up to a high resolution of 1.8Å and the cell size a
= B = 65.501 nanometers, c = 184.614
It belonged to the P3 ₂ 21 space group with nanometers.
One αβ heterodimer was present in the asymmetric unit of the crystal. The crystal structure of the enzyme is Rhodococcus sp. N-771.
Based on the crystal structure (PDB file code: 2AHJ) of the nitrile hydratase derived from, it was analyzed by the molecular replacement (MR) method. Cobalt atom is described in JP-A-9-27597.
No. 8, cysteine clusters (Cys-Thr-Leu-Cys-) located at positions 108 to 113 of the amino acid sequence of the α subunit of the enzyme.
Ser-Cys) site. More specifically, three Cs of 108th, 111th and 113th
It was bonded to the sulfur atom of the ys residue and the nitrogen atom of the main chain of the 112th Ser residue and the 113th Cys residue. In addition, extra electron density was observed around the 111th and 113th Cys residues. In the Fo-Fc map, two electron densities were observed around the 111th Cys residue and one electron density was observed around the 113th Cys residue. That is, like the iron-type nitrile hydratase, the 111th Cys residue was post-translationally modified with sulfinic acid (Cys-SO2H) and the 113th Cys residue was modified with sulfenic acid (Cys-SOH). . That is, the amino acid sequence of the cysteine cluster site located at positions 108 to 113 of the α subunit of the present enzyme is Cys-Thr-Leu-Csi-Se.
r-Cse (Csi in the sequence represents cysteine sulfinic acid, and Cse represents cysteine sulfenic acid).

Sequence Listing Free Text Sequence No. 1: Cobalt Atom Bonding Region in Cobalt-Type Nitrile Hydratase Sequence No. 2: Pseudonocardia thermophila JC
Amino acid sequence of α subunit of nitrile hydratase derived from M3095 strain SEQ ID NO: 3: Pseudonocardia thermophila JC
Amino acid sequence of β subunit of nitrile hydratase derived from M3095 strain

[0030]

INDUSTRIAL APPLICABILITY According to the present invention, the amino acid sequence of the cobalt atom binding region in the cobalt-type nitrile hydratase (C
ys-Thr-Leu-Csi-Ser-Cse or Cys-Ser-Leu-Csi-Ser-Cs
e) is provided. In the sequence, Csi represents cysteine sulfinic acid and Cse represents cysteine sulfenic acid.

[0031]

[Sequence Listing] SEQUENCE LISTING <110> MITSUI CHEMICALS, INC. <120><160> 9 <210> 1 <211> 6 <212> PRT <213> Artificial Sequece <220><223> Cobalt atom binding domain in cobalt -type nitrile hydratase <220><221> OTHER <222> 4 <223> Cystein-sulfinic acid <220><221> OTHER <222> 6 <223> Cystein-sulfenic acid <400> 1 Cys Thr Leu Xaa Ser Xaa 1 5 <210> 2 <211> 205 <212> PRT <213> Pseudonocardia thermophila JCM3095 <220><223> Arufa-subunit amino acid sequence of cobalt-type nitrile hydratase <220><221> OTHER <222> 111 <223> Cystein-sulfinic acid <220><221> OTHER <222> 113 <223> Cystein-sulfenic acid <400> 2 Met Thr Glu Asn Ile Leu Arg Lys Ser Asp Glu Glu Ile Gln Lys Glu 5 10 15 Ile Thr Ala Arg Val Lys Ala Leu Glu Ser Met Leu Ile Glu Gln Gly 20 25 30 Ile Leu Thr Thr Ser Met Ile Asp Arg Met Ala Glu Ile Tyr Glu Asn 35 40 45 Glu Val Gly Pro His Leu Gly Ala Lys Val Val Val Lys Ala Trp Thr 50 55 60 Asp Pro Glu Phe Lys Lys Arg Leu Leu Ala Asp Gly Thr Glu Ala Cys 65 70 75 80 Lys Glu Leu Gly Ile Gly Gly Leu Gln Gly Glu As p Met Met Trp Val 85 90 95 Glu Asn Thr Asp Glu Val His His Val Val Val Cys Thr Leu Xaa Ser 100 105 110 Xaa Tyr Pro Trp Pro Val Leu Gly Leu Pro Pro Asn Trp Phe Lys Glu 115 120 125 Pro Gln Tyr Arg Ser Arg Val Val Arg Glu Pro Arg Gln Leu Leu Lys 130 135 140 Glu Glu Phe Gly Phe Glu Val Pro Pro Ser Lys Glu Ile Lys Val Trp 145 150 155 160 Asp Ser Ser Ser Glu Met Arg Phe Val Val Leu Pro Gln Arg Pro Ala 165 170 175 Gly Thr Asp Gly Trp Ser Glu Glu Glu Leu Ala Thr Leu Val Thr Arg 180 185 190 Glu Ser Met Ile Gly Val Glu Pro Ala Lys Ala Val Ala 195 200 205 <210> 3 <211> 233 <212> PRT <213> Pseudonocardia thermophila JCM3095 <220><223> Beta-subunit amino acid sequence of cobalt-type nitrile hydratase <400> 3 Met Asn Gly Val Tyr Asp Val Gly Gly Thr Asp Gly Leu Gly Pro Ile 5 10 15 Asn Arg Pro Ala Asp Glu Pro Val Phe Arg Ala Glu Trp Glu Lys Val 20 25 30 Ala Phe Ala Met Phe Pro Ala Thr Phe Arg Ala Gly Phe Met Gly Leu 35 40 45 Asp Glu Phe Arg Phe Gly Ile Glu Gln Met Asn Pro Ala Glu Tyr Leu 50 55 60 Glu Ser Pro Tyr Tyr Trp His Trp Ile Arg Thr Tyr Ile His His Gly 65 70 75 80 Val Arg Thr Gly Lys Ile Asp Leu Glu Glu Leu Glu Arg Arg Thr Gln 85 90 95 Tyr Tyr Arg Glu Asn Pro Asp Ala Pro Leu Pro Glu His Glu Gln Lys 100 105 110 Pro Glu Leu Ile Glu Phe Val Asn Gln Ala Val Tyr Gly Gly Leu Pro 115 120 125 Ala Ser Arg Glu Val Asp Arg Pro Pro Lys Phe Lys Glu Gly Asp Val 130 135 140 Val Arg Phe Ser Thr Ala Ser Pro Lys Gly His Ala Arg Arg Ala Arg 145 150 155 160 Tyr Val Arg Gly Lys Thr Gly Thr Val Val Lys His His Gly Ala Tyr 165 170 175 Ile Tyr Pro Asp Thr Ala Gly Asn Gly Leu Gly Glu Cys Pro Glu His 180 185 190 Leu Tyr Thr Val Arg Phe Thr Ala Gln Glu Leu Trp Gly Pro Glu Gly 195 200 205 Asp Pro Asn Ser Ser Val Tyr Tyr Asp Cys Trp Glu Pro Tyr Ile Glu 210 215 220 Leu Val Asp Thr Lys Ala Ala Ala Ala 225 230 233

[Brief description of drawings]

[Figure 1] Pseudonocardia thermophila JCM
The Fo-Fc map of the cobalt atom binding region of the nitrile hydratase derived from strain 3095 is shown. In the figure, in the skeleton of the amino acid, open carbon atoms, filled carbon atoms, fringes oxygen atoms, and lattice fringes sulfur atoms, respectively.
The dotted line shows the coordination state of cobalt, and the shaded area is Fo.
-Shows an Fc map. + Indicates a water molecule.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 9/88 C12N 15/00 ZNAA C12P 13/02 5/00 AF term (reference) 4B024 AA03 BA07 CA04 DA05 EA04 GA11 4B050 CC01 CC03 DD02 LL05 4B064 AE02 CA21 4B065 AA01Y AA26X AB01 AC14 BA02 CA27

Claims (8)

[Claims] 1. An amino acid sequence (Cys-Thr-Leu-Csi-Ser-Cs) described in SEQ ID NO: 1 in the sequence listing.
A cobalt-type nitrile hydratase containing, as a constituent, a subunit bonded to a cobalt atom through a region represented by e). (Csi in the sequence represents cysteine sulfinic acid, and Cse represents cysteine sulfenic acid.) 2. An amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, which is represented by SEQ ID NO: 1 in the sequence listing (Cys-Thr-Leu-Csi-Ser-Cse).
The cobalt-type nitrile hydratase according to claim 1, which contains a subunit bonded to a cobalt atom through a region represented by the formula (1) as a constituent. (Csi in the sequence represents cysteine sulfinic acid, and Cse represents cysteine sulfenic acid.) 3. A part of the amino acid sequence excluding the 108th to 113th positions in SEQ ID NO: 2 in the sequence listing is substituted or deleted,
It consists of the amino acid sequence obtained by deletion or insertion, and has the amino acid sequence (Cys-Th
The cobalt-type nitrile hydratase according to claim 1, which contains a subunit bound to a cobalt atom through a region represented by r-Leu-Csi-Ser-Cse) as a constituent element. (Csi in the sequence represents cysteine sulfinic acid, and Cse represents cysteine sulfenic acid.) 4. The cobalt type according to claim 1, which contains the subunit according to claim 2 or claim 3 and the subunit consisting of the amino acid sequence of SEQ ID NO: 3 in the sequence listing as constituent elements. Nitrile hydratase. 5. From the amino acid sequence obtained by substituting, deleting, deleting or inserting a part of the subunit according to claim 2 or 3 and the amino acid sequence according to SEQ ID NO: 3 in the sequence listing. The cobalt-type nitrile hydratase according to claim 1, which comprises the following subunit as a constituent. 6. The cobalt-type nitrile hydratase according to claim 1, which is derived from Pseudonocardia the rmophila JCM3095. 7. A strain (including a transformant) containing the cobalt-type nitrile hydratase according to any one of claims 1 to 6, a culture solution of the strain, and a treated product thereof. 8. The strain according to claim 7 (including a transformant), a culture solution of the strain, a treated product thereof, or a cobalt-type nitrile hydratase and a nitrile compound obtained from them, in an aqueous medium. And a method for producing an amide compound corresponding to the nitrile compound.