JPH08266277A - Production of amide compound using transformant - Google Patents

Abstract

PURPOSE: To obtain a nitrile hydratase useful for the production of an amide compound from a corresponding nitrile compound. CONSTITUTION: This nitrile hydratase is produced by the culture of Achromobacter xerosis IFO 12668, etc. The enzyme is constructed of an α- subunit having the amino acid sequence of formula I and a β-subunit having the amino acid sequence of formula II. It has high activity and enables the use of α-hydroxyisobutyronitrile as a substrate. It can be mass-produced by a transformant.

Description

Detailed Description of the Invention

[0001]

The present invention relates to nitrile hydratase,
A gene encoding the α and β subunits of the enzyme, a recombinant plasmid containing the gene, and a transformant transformed by the recombinant plasmid are provided. From another aspect, the present invention uses the above transformant. The present invention relates to a method for producing a nitrile hydratase and a method for producing a corresponding amide compound from a nitrile compound using a culture solution, a separated bacterial cell, or a treated product of the bacterial cell obtained by culturing the above transformant.

[0002]

2. Description of the Related Art In the technology for producing a corresponding amide compound by hydrating a nitrile group of a nitrile compound to convert it to an amide group, a method of heat treatment in the presence of an acid or an alkali as a catalyst for the hydration reaction or copper is used. A method of heat treatment using a catalyst has been known.

On the other hand, in recent years, a method for producing an amide by using a nitrile hydratase, which is an enzyme having a function of catalyzing the hydration reaction, by using a microorganism has been known (Japanese Patent Laid-Open No. Sho 5).
9-2693, 61-162193, 62-91189, 64-86889
Nos. 56-17918, 59-37951, 62-21519, etc.). Since the production method using this microorganism can perform the hydration reaction at room temperature and has a high conversion rate from nitrile to amide, it is superior to other chemical methods. However, when a microorganism is used as a catalyst for a hydration reaction, the nitrile hydration activity inherent in the microorganism is not sufficient, so the cost of the microbial catalyst in the amide production cost cannot be ignored, and it is at an industrially applicable level. It is important to reduce the cost to Therefore, in order to improve the nitrile hydration activity per microbial cell weight, the expression amount of nitrile hydratase per microbial cell weight may be improved. In recent years, in order to improve the expression level of nitrile hydratase, a method of expressing a large amount of nitrile hydratase in cells using genetic engineering and converting the nitrile compound to a corresponding amide compound by utilizing the cells was examined. Has been done. For example, in Japanese Patent Publication No. 3-54558, 2 derived from a microorganism belonging to the genus Rhodococcus
Nitrile hydratase consisting of two hetero subunits is described at the enzyme level, and JP-A-2-119778 describes a nitrile hydratase gene consisting of two hetero subunits derived from a microorganism belonging to the genus Rhodococcus. In Kaihei 3-251184, 2 derived from a microorganism belonging to the genus Pseudomonas
One of the heterologous been nitrile hydratase gene composed of subunits disclosure, also European Patent Unexamined Publication 455,646 Rhodococcus rhodochrous (Rhodococcus rh
A nitrile hydratase gene consisting of two hetero subunits derived from odochrous ) is disclosed. However, the activity expression level of nitrile hydratase in Escherichia coli transformed with an expression plasmid having the nitrile hydratase gene described in these publications as an insert fragment is low in any case, and the amount of nitrile hydratase per weight of transformant is low. Nitrile hydration activity is lower than that of the original microorganism from which the gene was derived (Ikehata, O., Nishiyama, M., Horinouch
i, S. and Beppu, T. `` Primary structure of nitrile
hydratase deduced from the nucleotide sequence of
a Rhodococcus speices N-774 and its expression in
Escherichia coli '' Eur. J. Biochem. 181 (1989): 563
-570, Nishiyama, M., Horinouchi, S., Kobayashi, M., N
agasawa, T., Yamada, H. and Beppu, T. `` Cloning and
Characterization of Genes Responsible for Metaboli
sm of Nitrile Compounds from Pseudomonas chlororap
his B23 '' Journal of bacteriology 173 (1991): 2465-
2472, Kobayashi, M., Nishiyama, M., Nagasawa, T., Hor
inouchi, S., Beppu, T. and Yamada, H. `` Cloning nucl
eotide sequence and expression in Escherichia coli
of two cobalt-containing nitrile hydratase genes f
rom Rhodococcus rhodochrous J1 '' Biochimica et Bi
Ophysica Acta. 1129 (1991): 23-33). Thus, expression of nitrile hydratase activity in Escherichia coli is possible, but it is never easy to obtain a transformant having a high nitrile hydration activity that can be utilized for industrial amide production. .

Among nitrile hydratase, α-
There is little known that hydroxyisobutyronitrile can be used as a substrate, and α-hydroxyisobutyronitrile can be converted into α-hydroxyisobutyl using Escherichia coli transformed with an expression plasmid having a nitrile hydratase gene as an insert fragment. No method for producing amides has been known so far.

[0005]

As described above, when the corresponding amide is industrially produced from nitrile, a microorganism having a high nitrile hydratase activity per cell weight to the extent that it can be used in the production is obtained. Was needed, but no such thing was known.

An object of the present invention is to provide a nitrile hydratase having a high nitrile hydration activity, particularly a nitrile hydratase capable of using α-hydroxyisobutyronitrile as a substrate, and another object of the present invention. Is to provide a gene encoding nitrile hydratase having high nitrile hydration activity, a recombinant plasmid containing the gene, and a transformant transformed with the recombinant plasmid. And, another object of the present invention is to produce a corresponding amide from nitrile by using a transformant expressing high nitrile hydration activity.

[0007]

[Means for Solving the Problems] As a result of intensive studies under such circumstances, the present inventor has shown that nitrile hydratase having extremely high nitrile hydration activity in Achromobacter xerosis IFO 12668. (Sometimes referred to as AxNH hereinafter), the gene encoding the enzyme was cloned by DNA recombination technology, and the resulting gene was incorporated into an expression vector to prepare an E. coli transformant using the resulting expression plasmid. Then
The obtained transformant succeeded in producing a large amount of AxNH protein, and completed the present invention.

That is, the present invention is the α subunit obtained by deleting, substituting, modifying or inserting a part of the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing, and SEQ ID NO: of the sequence listing: 2. A nitrile hydratase consisting of a β subunit obtained by deleting, substituting, modifying or inserting a part of the amino acid sequence of 2 or the amino acid sequence, a gene encoding the α and β subunits of the enzyme, and the gene A recombinant plasmid containing the same, a transformant transformed with the recombinant plasmid, a method for producing nitrile hydratase using the transformant, and a method for producing amide using the transformant. .

The nitrile hydratase of the present invention comprises α, β
It is a protein consisting of two subunits of Achromobacter xerosis IFO 12
First, the nitrile hydratase derived from 668) is exemplified. The above strains are stored in the Fermentation Research Institute (2-17-85, 13 Sanhonmachi, Osaka City, Osaka Prefecture) under the number IFO 12668 and freely distributed to anyone by request. The enzyme consists of 193 alpha subunits and 220 beta subunits of 220 amino acid residues. And in the present invention, a part of the amino acid sequence of the subunit is deleted, replaced,
Those obtained by modification or insertion are also included in the nitrile hydratase of the present invention as long as they have nitrile hydration activity. The nitrile hydratase of the present invention is considered to have one α subunit and one β subunit bonded. In order to obtain the nitrile hydratase of the present invention, Achromobacter xylosis is cultured and may be separated from the bacterial cells by a usual method, but it is preferably separated from the transformant obtained by the method described below. can do.

The genes encoding the α and β subunits of the nitrile hydratase of the present invention include Achromobacter xerosis IFO 1266
The nitrile hydratase gene derived from 8) is first exemplified. Examples of such a gene include those having the nucleotide sequences shown in SEQ ID NO: 3 or: 4 in the sequence listing, which are 582 bp and 663 bp in total length, respectively. In addition, a part of the nucleotide sequence of the gene is deleted or replaced,
The protein obtained by modification or insertion is also included in the gene of the present invention as long as the encoded protein has nitrile hydration activity. In order to obtain a gene encoding the α and β subunits of nitrile hydratase, the following method can be used, for example. That is, chromosomal DNA obtained by preparing chromosomal DNA from a microorganism having nitrile hydratase activity
The method of T. Maniatis et al. (Molecular Cloning 2nd Editio
n; Cold Spring Harbor Laboratory Press; 1989) and the like, and introduced into E. coli to prepare a plasmid library. By culturing the obtained Escherichia coli, selecting a positive clone of Escherichia coli by measuring the nitrile hydration activity of the cultured bacterial cell (or culture solution), and extracting DNA from the selected positive clone by a conventional method. A DNA encoding the gene of interest is obtained. The origin of the chromosomal DNA in preparing the plasmid library may be any one that encodes the gene of the present invention, and as such, Achromobacter xerosis ( Ac
hromobacter xerosis IFO 12668) can be exemplified. In order to determine the nucleotide sequences of the genes encoding the α and β subunits of nitrile hydratase obtained as described above, the following method may be used. That is, from the positive colonies containing the gene of interest, the method of T. Maniatis et al. (Molecular Cloning 2nd Edition; Cold Spring Harbor
DNA was prepared by Laboratory Press; 1989), cleaved with an appropriate restriction enzyme, and then cloned into a vector plasmid such as pUC18 (Pharmacia).
y method (Proceedings of National Academy Science USA; 7
4,5463, 1977) to determine the nucleotide sequence.

The recombinant plasmid of the present invention contains a gene encoding the α and β subunits of the above-mentioned nitrile hydratase.
A region that regulates the expression of the gene and / or a selectable marker is included. A plasmid having an ori region derived from ColE1 is preferably used as an expression vector. Ampicillin, tet as selection markers
It is easy to handle if it encodes a drug resistance gene against antibiotics such as racycline and kanamycin. The region that regulates the expression of the gene encoding the α and β subunits of nitrile hydratase includes a promoter and a ribosome binding site (SD sequence). When E. coli is used as the host for the recombinant plasmid, the expression plasmid is The promoter preferably has any of lac, trp, tac, and trc, and the ribosome binding site is also preferably of E. coli type. The promoter should be ligated to a position where the gene of the present invention can be transcribed, and the order of ligation is from the 5 ′ side to the 3 ′ side of DNA in the order of the promoter, the ribosome binding site, and the gene of the present invention.

The recombinant expression plasmid constructed as described above is introduced into host cells such as Escherichia coli, yeast, other microorganisms and animal cells by a known transformation method. The obtained transformant was cultured in Molecular Cloning 2nd Edition.
(Cold Spring Harbor Laboratory Press; 1989). Culture temperature is 28-42
℃ is suitable. The nitrile hydratase of the present invention can be separated and purified from the cells thus cultured.

In order to produce a corresponding amide from a nitrile compound using the transformant of the present invention or the above-mentioned nitrile hydratase, a nitrite hydratase, a transformant culture solution, or an isolated cell obtained from the culture solution is used. The nitrile may be reacted in an aqueous medium in the presence of a treated product of the cells. Although the reaction temperature is not particularly limited, it is a temperature within which nitrile hydratase is not inactivated and nitrile hydratase is sufficiently exhibited.

The nitrile compound used in the production method of the present invention has 2 carbon atoms such as acetonitrile, propionitrile, acrylonitrile, methacrylonitrile, n-butyronitrile, isobutyronitrile, α-hydroxyisobutyronitrile and the like. ~ 4 nitrile compounds. The amount of these raw materials added to the reaction solution is determined in consideration of the inactivation of the enzyme by the raw material substrate.

[0015]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

[Example 1] (1) Preparation of chromosomal DNA of Achromobacter xerosis Achromobacter xerosis
Chromosomal DNA of IFO 12668) was prepared according to a conventional method. That is, Achromobacter xerosis
sis: IFO12668) was added to 1 liter of LB medium (Bactotryptone 1
0 g / l, yeast extract 5 g / l, NaCl 10 g /
(1), pH 7.2), the cells were cultured and then collected by centrifugation. 160 ml of 0.15 M NaCl aqueous solution (pH 8.0) containing 50 mM EDTA
Suspended in water, added 160 mg of lysozyme and added at 37 ° C for 2
After gently stirring for 0 minutes, 4 ml of 20% SDS was added, and the mixture was kept at 65 ° C. for 20 minutes. Another 8 mg of Proteina
se K was added and the mixture was kept at 37 ° C. for 1 hour. Then TE (1
0 mM Tris-HCl, 1 mM EDTA, pH
Add 160 ml of phenol saturated with 8.0),
Stir gently. Chloroform 1 in the upper layer after centrifugation
It re-extracted by adding 60 ml. Furthermore, this is centrifuged to collect the upper layer, 320 ml of ethanol is added, and the DNA is wound with a glass rod, and the recovered DNA is 70%, 80%, 90%.
Was sequentially dehydrated with ethanol. Next, 40 ml of DNA
And 10 μg / ml RNase A aqueous solution to 200 μl.
1 and the mixture was kept at 37 ° C. for 1.5 hours. Then, phenol extraction and chloroform extraction were performed, and DNA was collected by ethanol precipitation. As a result, 2 mg of Achromobacter xerosis chromosomal DNA was finally obtained.

(2) Include the AxNH protein gene in its insert.
Screening of clones 100 μg of the above-mentioned chromosomal DNA preparation was digested with restriction enzyme XhoI (manufactured by Takara Shuzo Co., Ltd.) and subjected to sucrose density gradient centrifugation to separate and collect DNA fragments of 5 kb or more. About 100 ng of recovered DNA fragment and plasmid vector pBluescri
pt SK + (Stratagene) DNA digested with the restriction enzyme XhoI was further dephosphorylated at the 5'end with bacterial alkaline phosphatase (Takara Shuzo) (BAP), and then phenol / chloroform. PBluescript SK + / XhoI obtained by extraction and ethanol precipitation
About 30 ng of BAP was subjected to a ligation reaction using a DNA ligation kit (Takara Shuzo). Escherichia coli DH5 competent cells (Competent High DH5: manufactured by Toyobo Co., Ltd.) were transformed with the ligation product obtained as described above, and ampicillin containing 14 cm in diameter (100
The bacterial solution was applied to a petri dish containing μg / ml) LB agar medium. As a result of keeping the dish at 28 ° C. overnight, 2000 to 3000 colonies appeared per sheet. The colonies thus generated were used as a screening library. A part of the colony of the library was scraped, suspended in 100 μl of 50 mM potassium phosphate buffer (pH 7.0) containing 2.5% acrylonitrile, and incubated at 10 ° C. overnight. The supernatant obtained by centrifugation is diluted 10-fold with distilled water, and subjected to HPLC (High Perforation).
Acrylamide was detected by mance Liquid Chromatography). A positive colony in which acrylamide was detected was suspended in LB medium and applied to a petri dish containing ampicillin (100 μg / ml) LB agar medium having a diameter of 8 cm so that about 100 colonies would appear per plate,
Positive clones were purified by performing similar screening again.

[Example 2] Subclonin of the inserted DNA fragment of the obtained positive clone
Plasmid DNA pBAN-7 from grayed and sequencing purified by positive clones (Fig. 1) were prepared using the alkali method, after a small fragmented appropriate restriction enzyme the necessary DNA region, a plasmid vector pUC1
Subcloning into 8 and 19 (Pharmacia) was performed. The plasmid DNA from the obtained subclone is QIApr
It was prepared by ep-spin (manufactured by DIAGEN), and the nucleotide sequence was determined using a sequencing kit manufactured by abi and auto sequencer 373A. As a result, the nucleotide sequences shown in SEQ ID NOs: 3 and 4: in the sequence listing were obtained.

Example 3 Construction of AxNH Protein Expression Plasmid and Transformant Plasmid p containing the AxNH protein gene DNA obtained in Example 2
BAN-7 DNA 100ng with restriction enzymes KpnI (Takara Shuzo) and SacII
After digestion with (Takara Shuzo), by agarose gel electrophoresis, a DNA fragment of about 3 kbp containing the AxNH protein gene and a DN of about 2.9 kbp containing the vector (pBluescript SK +).
After separating the A fragment, the DNA fragment mixture was concentrated by ethanol precipitation and 5 μl of TE (10 mM Tris-HC
1, 1 mM EDTA, pH 8.0). The fragments were subjected to a ligation reaction using a DNA ligation kit (Takara Shuzo). By transforming Escherichia coli DH5 competent cells using this reaction solution, a transformant MT10770 containing pBAN-2 (Fig. 2), which is an expression plasmid into which a DNA fragment containing the AxNH protein gene was inserted, was obtained. Obtained. MT10770 has been deposited with the deposit number FERM P-14756 at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Technology, 1-3-1 Higashi, Tsukuba, Ibaraki.

[Example 4] Production of AxNH protein using MT10770 and nitrile compound
From the amide compound to 100 μg of the transformant MT10770 obtained in Example 3
ml ampicillin and 40μg / ml FeSO ₄ · 7
H ₂ O, containing 10 μg / ml CoCl ₂ 2H ₂ O 10
It was inoculated into ml of LB medium and cultured at 28 ° C. overnight (about 15 hours). After completion of the culture, the collected cells obtained by centrifugation were washed with physiological saline, and then 2 ml of 50 mM was added.
It was suspended in a potassium phosphate buffer (pH 7.0). 200 μl of this suspension was added to 50 mM potassium phosphate buffer (pH 7.0) containing 3% (w / v) acrylonitrile.
It was suspended in 0 μl and reacted at 10 ° C. for 1 hour. After the reaction was completed, the reaction solution was analyzed using HPLC. As a result, only acrylamide was detected in the reaction solution, and the presence of acrylonitrile and acrylic acid was not observed. The conversion efficiency was 100%. MT10 obtained above in the same manner
200 μl of a suspension of 770 was added to a 50 mM potassium phosphate buffer (pH 7.0) containing 4% (v / v) α-hydroxyisobutyronitrile and 25% (v / v) acetone.
It was suspended in 00 μl and reacted at 10 ° C. for 1 hour. After the reaction was completed, the reaction solution was analyzed by HPLC. As a result, only α-hydroxyisobutyramide was detected in the reaction solution, and the presence of α-hydroxyisobutyronitrile was not observed. The conversion efficiency was 100%. On the other hand, instead of the above expression plasmid pBAN-2, a transformant containing only the vector portion pBluescript SK + (DH5 (pB
When I performed a similar experiment using luescript SK +)),
Acrylamide and α-hydroxyisobutyramide could not be detected in the reaction solution after the reaction was completed.
The addition of metal components to the medium is important, and MT10770 was cultured in a medium without addition, and a similar experiment was conducted.As a result, acrylamide and α-hydroxy in the reaction solution after the reaction were completed. The detected amount of isobutyramide was extremely small.

Comparative Example 1 Nitrile of MT10770 and Achromobacter xylosis
Comparison of Hydration Activity Using the MT10770 harvested cells obtained in Example 4 and the Achromobacter xerosis harvested cells obtained in Example 1, experiments were conducted to compare the hydration activity of acrylonitrile. 50 including 2.7% (w / v) acrylonitrile
2 mg of bacterial cells were suspended in 10 ml of mM potassium phosphate buffer (pH 7.0) and reacted at 10 ° C for 1 hour. After completion of the reaction, the amount of acrylamide contained in the reaction solution was quantified using HPLC. The results are shown in Fig. 3. From this result, MT10770 is the original stock of Achromobacter.
Approximately 20 per cell weight compared to Xerosis IFO12668
It was shown to have double the acrylonitrile hydration activity.

[0022]

According to the present invention, a nitrile hydratase having a high nitrile hydratase activity can be obtained. Further, according to the present invention, the corresponding amide is industrially produced from nitrile,
In particular, a method of converting isobutyronitrile to an amide using a transformant is provided. Achromobacter xerosis IFO126 consisting of two polypeptides, α subunit and β subunit, which have nitrile hydratase activity
A new DNA fragment encoding the AxNH protein derived from 68 was obtained. Furthermore, by using Escherichia coli transformed with the expression plasmid constructed in the present invention, it became possible to convert a nitrile compound into its corresponding amide compound. Moreover, by using the method of the present invention, it becomes possible to efficiently produce an amide compound from a nitrile compound.

[0023]

[Sequence list]

SEQ ID NO: 1 Sequence length: 193 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin organism name: Achromobacter xerosis Strain name: IFO12668 Direct origin Clone name: pBAN-2 Sequence characteristics Method of characterizing: E Other information: AxNH protein α subunit

SEQ ID NO: 2 Sequence length: 220 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin organism name: Achromobacter xerosis Strain name: IFO12668 Direct origin Clone name: pBAN-2 Sequence features Method of characterizing: E Other information: AxNH protein β subunit

SEQ ID NO: 3 Sequence length: 582 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Achromobacter xerosis IFO12668 Direct origin Clone name: pBAN -2 methods to determine the characteristics features of the sequence: E other information: AxNH protein α subunit sequence ATGACGGCAA CTTCAACCCC TGGTGAGCGC GCACGCGCAC TGTTTGCAGT GCTCAAGCGC 60 AAAGACCTCA TCCCCGAGGG CTACATCGAA CAGCTCACCC AGCTGATGGA ACACGGCTGG 120 AGCCCGGAAA ACGGCGCGCG CATCGTCGCC AAGGCCTGGG TCGATCCGCA GTTTCGCGAG 180 CTGCTGCTCA AGGACGGTAC GGCCGCCTGC GCCCAGTTCG GCTTCACCGG CCCACAGGGT 240 GAATACATCG TCGCCCTGAA AAACACCCCG CAGTTGAAAA ACGTGATCGT CTGCAGCCTG 300 TGCTCCTGCA CGAACTGGAC GGTGATGGGA CTGCCGCCTG AGTGGTACAA GGGCTTCGAG 360 TTCCGTGAGC GGTTGGTCCG GAAGGGCCGC ACGGTATTGC GCGAGCTGGG CACCGAGTTG 420 CCCGGTGACA TGGTGGTCAA GGTCTGGGAT ACCAGCGCTG AAAGCCGCTA CCTGGTGCTG 480 CCGCAACGGC CAGCGGGATC AGAGCATATG AACGAAGAGC AGTTGCGGCA ACTGGTCACC 540 AAGGATGTGC TGATCG GCGT CGCCCTGCCC CGCGTTGGCT GA 582

SEQ ID NO: 4 Sequence length: 663 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Achromobacter xerosis Strain name: IFO12668 Direct origin clone name: pBAN-2 sequence method to determine the characteristics features: E other information: AxNH protein β subunit sequence ATGGATGGCT TTCACGATCT CGGCGGTTTC CAGGGCTTTG GCAAAGTGTC CCACCGCATC 60 AACAGCCTGA GCTACAAGCA GGTGTTCAAG CAGGACTGGG AACACCTGGC CTACAGCCTG 120 ATGTTCATCG GCGTCGACCA CCTGAACAAG TTCAGCGTCG ATGAAATACG TCATGCCGTC 180 GAACGCATTG ACGTGCGCCA GCACGTCGGC ACCGAATACT ACGAACGTTA TGTGATCGCC 240 ACTGCCACGC TGCTGGTCGA AACAGGCGTC ATCACCCAGG CCGAACTGGA TGAAGCACTC 300 GGCTCGCACT TCAAGCTGGC CAACCCCGCC CATGCGCAAG GGCGTGCTGC AATTACCGGG 360 CGAGCGCCTT TTGAAGTGGG CGACCGGGTC ATCGTACGCG ATGAATACGT GGCCGGGCAT 420 GTGCGCATGC CTGCATATGT GCGCGGCAAG CAAGGCGTAG TGCTGCACCG GACCACTGAA 480 CAGTGGCCGT TTCCGGACGC GATTGGCCAT GGCGACCAGA ACGCTGCGCA TCAACCGACC 540 TACCATGT CG AGTTCCGCGT GCGGGACCTG TGGGGCGATG CCGCAGACGA CGGCCTGGTG 600 GTGGTAGACC TGTTTGAAAG CTATCTGGAC AGGGTCGAAA GCCCGCGAGT GGTGCGCGCA 660 TGA 663

[Brief description of drawings]

FIG. 1 shows a restriction enzyme cleavage map of plasmid pBAN-7.

FIG. 2 shows a restriction enzyme digestion map of plasmid pBAN-2.

FIG. 3 is a graph showing the experimental results comparing the hydration activities of MT10770 and Achromobacter xerosis IFO 12668 for acrylonitrile.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location // (C12N 9/88 C12R 1: 025) (C12N 9/88 C12R 1:19) (C12N 1 / 21 C12R 1:19) (C12P 13/02 C12R 1:19)

Claims (8)

[Claims] 1. An α subunit obtained by deleting, substituting, modifying or inserting a part of the amino acid sequence of SEQ ID NO: 1 in the sequence listing, or the SEQ ID NO: 2 of the sequence listing. A nitrile hydratase comprising a β subunit obtained by deleting, substituting, modifying or inserting an amino acid sequence or a part of the amino acid sequence. 2. Achromobacter Xerosis
The nitrile hydratase according to claim 1, which is derived from bacterium xerosis IFO 12668). 3. A nitrile hydratase α subunit represented by the nucleotide sequence of SEQ ID NO: 3 in the sequence listing or a nucleotide sequence obtained by deleting, substituting, modifying or inserting a part of the nucleotide sequence. Gene to do. 4. A nitrile hydratase β subunit represented by a nucleotide sequence represented by SEQ ID NO: 4 in the sequence listing or a nucleotide sequence obtained by deleting, substituting, modifying or inserting a part of the nucleotide sequence is encoded. Gene to do. 5. A nitrile hydratase α subunit represented by a nucleotide sequence represented by SEQ ID NO: 3 in the sequence listing or a nucleotide sequence obtained by deleting, substituting, modifying or inserting a part of the nucleotide sequence is encoded. Gene and a gene encoding the nitrile hydratase β subunit represented by the nucleotide sequence of SEQ ID NO: 4 in the sequence listing or the nucleotide sequence obtained by deleting, substituting, modifying or inserting a part of the nucleotide sequence A recombinant plasmid containing and. 6. A nitrile hydratase α subunit represented by a nucleotide sequence represented by SEQ ID NO: 3 in the sequence listing or a nucleotide sequence obtained by deleting, substituting, modifying or inserting a part of the nucleotide sequence is encoded. Gene and a gene encoding the nitrile hydratase β subunit represented by the nucleotide sequence of SEQ ID NO: 4 in the sequence listing or the nucleotide sequence obtained by deleting, substituting, modifying or inserting a part of the nucleotide sequence A transformant transformed with a recombinant plasmid containing 7. A nitrile hydratase α subunit represented by the nucleotide sequence of SEQ ID NO: 3 in the sequence listing or a nucleotide sequence obtained by deleting, substituting, modifying or inserting a part of the nucleotide sequence is encoded. Gene and a gene encoding the nitrile hydratase β subunit represented by the nucleotide sequence of SEQ ID NO: 4 in the sequence listing or the nucleotide sequence obtained by deleting, substituting, modifying or inserting a part of the nucleotide sequence A method for producing a nitrile hydratase by culturing a transformant transformed with a recombinant expression plasmid containing and. 8. A nitrile hydratase α subunit represented by a nucleotide sequence represented by SEQ ID NO: 3 in the sequence listing or a nucleotide sequence obtained by deleting, substituting, modifying or inserting a part of the nucleotide sequence is encoded. Gene and a gene encoding the nitrile hydratase β subunit represented by the nucleotide sequence of SEQ ID NO: 4 in the sequence listing or the nucleotide sequence obtained by deleting, substituting, modifying or inserting a part of the nucleotide sequence A culture solution obtained by culturing a transformant transformed with a recombinant expression plasmid containing and a amide compound corresponding to a nitrile compound in the presence of a bacterium isolated from the culture and a treated product of the bacterium. Method of manufacturing.