JPH0799980A - Gene dna encoding polypeptide having nitrilase activity and production of carboxylic acid from nitriles by transformant containing the same - Google Patents

Abstract

PURPOSE:To obtain a new gene encoding a polypeptide having nitrilase activity derived from Rhodococcus rhodochrous, capable of industrially and advantageously producing a carboxylic acid from a nitrile by a genetics engineering method. CONSTITUTION:Rhodococcus rhodochrous PA-34 (FERM BP-1559) is added to a medium, cultured at 30 deg.C for 24 hours, mixed with glycine and further cultured for 24 hours. The cell is collected, suspended in tris-hydrochloric acid buffer solution (pH 8), treated with lysozyme and peptidase and further ground to collect a gene contained in the cell. Then, the gene is treated with a restriction enzyme and bonded to a vector to prepare a DNA library by a conventional method. The DNA library is screened with a probe containing part of a gene encoding nitrilase to select a clone. A DNA is recovered from the clone and treated with a restriction enzyme to give the objective new gene encoding a polypeptide having nitrilase activity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a gene DNA encoding a polypeptide having nitrilase activity for converting nitriles into corresponding carboxylic acids, which originates from Rhodococcus rhodochrous .
The present invention also relates to a recombinant DNA in which this gene DNA is incorporated into a vector and a transformant transformed with this recombinant DNA. Furthermore, the present invention relates to a method for converting nitriles to the corresponding carboxylic acids by means of this transformant.

[0002]

2. Description of the Related Art An enzyme that hydrates nitriles to convert them into corresponding carboxylic acids is known as nitrilase. Examples of microorganisms producing the enzyme include Fusa.
rium oxysporum f. sp. me lonis ( Biotechnol. Appl.
Biochem., 11 , 581-601 (1989)), Fusarium solani (Bio
chem. J., 167 , 685-692 (1977)), Nocardia sp. (Int.
J. Biochem., 17 , 677-683 (1985)), Arthrobacter sp. (A
ppl. Environ. Microbiol., 51 , 302-306 (1986)), Rhod
ococcus rhodochrous Jl (Eur. J. Biochem., 182, 349-
356 (1989)), Rhodococcus rhodochrous K22 (J. Bacte
riol., 172, 4807-4815 (1990)), Rhodococcus rhodoc
hrous PA-34 (Appl. Microbiol. Biotechnol., 37 , 184-
190 (1992)) and the like. Of these microorganisms, only Rhodococcus rhodochrous PA-34 is known to produce an optically active α-amino acid from α-aminonitrile.

[0003]

The present inventors have cloned a nitrilase gene by a gene recombination operation,
This is incorporated into a vector to form a recombinant DNA, the recombinant DNA is used to transform a microorganism, and a large number of copies of the nitrilase gene are present in the microbial cells to dramatically increase the catalytic activity of the microorganism to thereby improve nitriles. The present inventors have earnestly studied the production of carboxylic acids. As a result, Rhodococcus rhodochr
ous) -derived nitrilase gene, and its base sequence could be determined. And this gene DN
The present inventors have completed the present invention by finding that the corresponding carboxylic acid can be obtained from nitriles in good yield by the above gene recombination operation using A. That is, an object of the present invention is to provide a nitrilase gene.

Another object of the present invention is to provide a method for producing the corresponding carboxylic acids from nitriles by gene recombination using this gene. A further object of the present invention is to provide a recombinant DNA and a transformant used in this gene recombination operation.
According to the method of the present invention, the corresponding optically active α-amino acid can be simply produced particularly from α-aminonitrile.

[0005]

SUMMARY OF THE INVENTION As described above, the present inventors have found that Rhodococcus rhodoc
hrous) PA-34 was isolated from the gene DNA encoding the polypeptide having nitrilase activity, the base sequence was determined, and this was incorporated into a vector to give a recombinant DNA,
Further, the present invention was completed by transforming a microorganism with the recombinant DNA to prepare a transformant, and using this to convert nitriles into carboxylic acids.

The present invention will be described in detail below. Sources of nitrilase gene of the present invention, Rhodococcus having nitrilase activity Rodokurosu (Rhodococcus rh
odochrous) PA-34 is used. The mycological properties of this strain are described in JP-A-4-79894. This strain has been deposited at the Institute for Microbial Technology, Institute of Industrial Science (Biotechnology Institute), as Micro Engineering Research Article No. 1559 (FERM BP-1559). The present invention is carried out by the following steps (1) to (5).

(1) N-of the amino acid sequence of nitrilase
Determination of terminal partial sequence and preparation of DNA probe Rhodococcus rhodochrous
Nitrilases are extracted and purified from PA-34, and the N-terminal amino acid sequence thereof is determined. A DNA probe having a gene base sequence deduced from the determined amino acid sequence is synthesized.

(2) DNA containing nitrilase gene
Preparation of fragments Rhodococcus rhodochrous
Isolate chromosomal DNA from PA-34, cut with restriction enzyme,
The DNA fragment containing the nitrilase gene is detected by Southern hybridization with the DNA probe prepared in step (1).

(3) Preparation of recombinant DNA library A recombinant DNA library is prepared by incorporating the DNA fragment prepared in step (2) into a vector, and then a transformant is prepared. Transformants containing the nitrilase gene are selected from the prepared transformants by colony hybridization, and confirmed by Southern hybridization.

(4) Determination of nucleotide sequence The nucleotide sequence of the DNA fragment in the recombinant DNA contained in the transformant selected in step (3) is determined.

(5) Expression of nitrilase gene A DNA fragment containing a nitrilase gene in which an unnecessary portion is removed by a restriction enzyme is incorporated into an expression vector, a transformant is prepared and then cultured, and then nitrilase is prepared by nitrilase prepared from cultured cells. To produce carboxylic acids.

The vector used in the above step includes plasmid pUC19, plasmid pMK2, etc., and the host used for transformation is E. coli HB101, E. coli.
Examples thereof include, but are not limited to, E. coli JM109 and E. coli JM109.

Examples of the nitrile compound serving as a substrate for nitrilase include α-amino nitrile compounds and other nitrile compounds described in JP-A-1-317394. For example, α-aminonitrile includes 2-aminopropanenitrile, 2-aminobutanenitrile, 2-amino-3.
-Methylbutanenitrile, 2-amino-4-methylpentanenitrile, 2-amino-3-methylpentanenitrile, 2-amino-3-hydroxypropanenitrile, 2-amino-3-hydroxybutanenitrile, 2-amino-3 -Guanidinopentanenitrile, 2-amino-3-methylcaptopropanenitrile, 2,7-diamino-4,5-dithiaoctanenitrile, 2-
Amino-4-methyloxybutanenitrile, 2-amino-3-
Phenylpropanenitrile, 3- (4-hydroxyphenyl) propanenitrile, 3-amino-3-cyanopropanoic acid, 4-amino-4-cyanobutanoic acid, 3-amino-3-cyanopropanamide, 4-amino-4- Cyanobutanamide, 2,
6-diaminohexanenitrile, 2,6-diamino-5-hydroxyhexanenitrile, 2-amino-3- (3-indolyl) propanenitrile, 2-amino-3- (4-imidazoyl) propanenitrile, 2-cyanopyrrolidine , 2-cyano-4-hydroxypyrrolidine, 2-amino-2-phenylethanenitrile, and the like. Examples of other nitrile compounds include acetonitrile, propionitrile, n-butyronitrile, n-capronitrile, methacrylonitrile, isobutyronitrile, glutaronitrile, triacrylonitrile, crotononitrile, lactonitrile, succinonitrile. , Acrylonitrile, benzonitrile, phenylacetonitrile and the like. However, it is not limited to these nitrile compounds. Further, as the α-nitrile compound, a DL-form, an L-form or a D-form may be used.
When the L-form or D-form is used, an optically active amino acid can be obtained by a simple operation.

For the conversion of nitriles to carboxylic acids, in addition to the above-mentioned enzyme preparation, a transformant culture solution, cells, treated cells, immobilized cells and the like can be used. The conversion reaction of nitriles to carboxylic acid is carried out by bringing the above-mentioned nitrile compound into contact with an enzyme, a bacterial cell or a treated product thereof, and various buffer solutions within a pH range of 6 to 12 and a temperature range of 20 to 50 ° C. You can do it inside. As the buffer solution, a phosphate buffer solution, an ammonia buffer solution, a borate buffer solution, a Tris buffer solution or the like can be used. If necessary, the buffer solution may be a carbon source, a nitrogen source or the like which promotes the growth of the bacterial cells. Can be carried out by adding the components of. An example of these reaction liquid conditions has already been described in JP-A-1-317394 or JP-A-4-79894. It can be performed by adding a carbon source, a nitrogen source and other components. Examples of these reaction conditions have already been described in JP-A-1-317394 and JP-A-4-79894. The obtained carboxylic acid can be separated and collected by applying known means such as phase separation, filtration, extraction and column chromatography.

Hereinafter, the present invention will be described in more detail with reference to examples.

【Example】

(1) Determination of N-terminal partial sequence of nitrilase amino acid sequence and preparation of DNA probe Rhodococcus rhodochrous PA-34 was added to a medium (glucose, 10 g; Na ₂ HPO ₄ · 12H ₂ O, 2.5 g; KH ₂ PO ₄ , 2.0g; MgSO
_{4 · 7H 2 O, 0.5g;} FeSO 4 · 7H 2 O, 0.03g; CaCl 2 · 2H 2 O,
0.06g; yeast extract, 0.1g; isobutyl nitrile, 5ml;
In addition to water (1 l; pH 7.2), the cells were cultured at 30 ° C for 28 hours. Then, the bacteria are collected from the culture solution by centrifugation, and the bacterial cells are washed with 0.1 M phosphate buffer containing 5 mM dithiothreitol (hereinafter, the phosphate buffer has the above composition) to obtain the phosphate buffer. It was suspended in the liquid. The microbial cells in the obtained suspension were crushed with a French press and centrifuged for 20 minutes at an additional load of 13000 g, and the resulting supernatant was used as an enzyme solution. Literature (App
l. Microbiol. Biotechnol., 37 , 184-190 (1992)), after fractionating the elementary enzyme solution with sulfuric acid, Rhodoc
Sephacr nitrilase from occusrhodochrous PA-34
yl S-300 HR, DEAE Toyopearl 650S column chromatography for separation and purification. Using the isolated nitrilase, its N-terminal amino acid sequence was analyzed by an amino acid sequencer (manufactured by Applied Biosystems). The determined 11 amino acid sequence from the N-terminus is shown in SEQ ID NO: 3 in the Sequence Listing. Then, a synthetic DNA having a nucleotide sequence predicted from this amino acid sequence was prepared with an automatic DNA synthesizer (manufactured by Beckman), and further, one of the 5'-terminal acids of the synthetic DNA was designated as T4 polynucleotide kinase [ A DNA probe labeled with the radioisotope ³² P was prepared using γ- ³² P] ATP. The nucleotide sequence of the DNA probe is shown in SEQ ID NO: 4 of the Sequence Listing, except for 32 nucleotide sequences except for one nucleotide at the 5'end. Two types of codons are expected
For Glu, Tyr, Asn, Phe, and Lys, two types of bases are expected, and four types of codons are expected, Thr and Val.
In regard to 1), a plurality of predicted synthetic DNAs were prepared by using 1 type of base containing inosinic acid (I). In the following steps, a mixture composed of the synthetic DNA having each of the above base sequences was used as a DNA probe.

(2) Preparation of DNA fragment containing nitrilase gene Rhodococcus rhodochrous
PA-34 (Microtechnical Laboratory Article No. 1559) was added to the medium (glucose, 10
g; Na ₂ HPO ₄・ 12H ₂ O, 2.5g; KH ₂ PO ₄ , 2g; MgSO ₄・ 7H ₂ O
, 0.5g; FeSO ₄・ 7H ₂ O, 0.03g; CaCl ₂・ 2H ₂ O, 0.06g;
Yeast extract, 0.1 g; isobutyronitrile, 5 ml; water, 1 l;
After culturing in pH 7.2) at 30 ° C. for 24 hours, glycine was added to 0.2 M to culture for 24 hours. Contains 10 mM EDTA after harvest
Wash with 10 mM Tris-HCl buffer (PH8) and absorb at 50
10 mM Tris-HCl buffer containing 10 mM EDTA (PH
8). Lysozyme and achromobacter endopeptidase were added to the prepared bacterial suspension at 10 mg / m 2 each.
l, 5mg / l, and leave at 58 ℃ for 2 hours.
Add DS (Sodium dodecyl sulfate) to 2% and add 5
It was left at 8 ° C for 2 hours. After the above treatment, the cells were
(Biochim. Biophys. Acta, 72, 619-629 (1963)), crushing to collect the gene contained in the bacterium, and total DNA containing the nitrilase gene derived from the bacterium from the gene. Was prepared. After digesting the prepared total DNA with various restriction enzymes, the obtained DNA fragment and the DNA probe prepared in the above step (1) having the nucleic acid sequence shown in SEQ ID NO: 4 in the Sequence Listing were used to prepare a literature ((B
asic methods in molecular biology, Elsevier, New Yo
Southern hybridization was performed according to the method described in rk (1986)). The temperature of prehybridization and hybridization in 2X SSC buffer is 56
The washing temperature was 50 ° C. As a result of the autoradiogram, a fragment of about 0.9 kbp of the EcoR I degradation product, a fragment of about 2.6 kbp of the BamHI degradation product, and a fragment of about 9.5 kbp of the Pst I degradation product,
It was confirmed that these three DNA fragments hybridized with the above DNA probe. The three DNA fragments are
It was found to have a nucleic acid base sequence encoding an 11 amino acid sequence from the N-terminal of the bacterium-derived nitrilase shown in SEQ ID NO: 3 in the Sequence Listing, and each was isolated.

(3) Preparation of recombinant DNA library About 0.9 kbp fragment of the EcoR I degradation product, about 2.6 kbp fragment of BamH I degradation product, and about 9.5 kbp fragment of Pst I degradation product,
That is, the three DNA fragments collected in the above step (2) were treated with restriction enzymes EcoR I, BamH I and Pst I, respectively, and then ligated to a plasmid vector pUC19 treated with Escherichia coli alkaline phosphatase (Takara Shuzo). Built in. As a result of this operation, a recombinant plasmid vector into which the approximately 0.9 kbp fragment of the EcoR I degradation product, the approximately 2.6 kbp fragment of the BamHI degradation product, and the approximately 9.5 kbp fragment of the Pst I degradation product were respectively incorporated is shown in FIG. The three recombinants of pNLE, pNLB and pNLP shown were prepared respectively. These operations and the operation for preparing a DNA library using the recombinant plasmid vector described below are described in the literature (Molecular cloning:
a laboratory manual, 2nd edition. Cold Spring Harb
or, New York (1989)). Escherichia coli E. coli HB101 was transformed with the prepared recombinant plasmid vector, and from the ampicillin-resistant colonies obtained by growing the transformed strain on a plate, only colonies having the recombinant plasmid vector were obtained by colony hybridization. Selected. Next, the DNA contained in the recombinant plasmid vector is extracted from the selected colonies, and Southern hybridization is performed according to the method described in the above step (2) to obtain the target recombinant DNA,
That is, the fungus Rhodococcus rhod described in SEQ ID NO: 3 in the Sequence Listing
ochrous PA-34-derived nitrilase having the acid-base sequence encoding 11 amino acid sequences from the N-terminus, and a fragment of EcoR I degradation product of about 0.9 kbp and a BamH I degradation product of about 2.6 kbp, respectively. , And a Pst I degradation product was confirmed to be a fragment of about 9.5 kbp. Using the recombinant DNA prepared by this operation, the restriction enzyme map of each recombinant plasmid vector was examined. It was confirmed that the three recombinants of pNLE, pNLB and pNLP each had the restriction enzyme map of FIG. Do you get it. Plasmid pNLE is a plasmid containing a fragment of about 0.9 kbp of EcoR I degradation product, and plasmid pNLB is about 2.
The plasmid containing the 6 kbp fragment, plasmid pNLP is Pst I
It is a plasmid containing a fragment of about 9.5 kbp of the degradation product, and further, according to the restriction enzyme map shown in FIG. 1, the fragment of about 9.5 kbp of the Pst I degradation product contained in the plasmid pNLP was digested with the restriction enzyme Ecor I.
The two types of fragments obtained by digestion with BamH I and BamH I are the same as the approximately 0.9 kbp fragment of the Ecor I degradation product contained in the plasmid pNLE and the approximately 2.6 kbp fragment of the BamH I degradation product contained in pNLB, respectively. It was found by hybridization. By the above operation, three recombinant plasmids pNLE,
A recombinant DNA library obtained from pNLB and pNLP was prepared.

(4) Determination of nucleotide sequence From the DNA fragment in the recombinant plasmid obtained in step (3), a part of the nucleic acid nucleotide sequence was analyzed by the dideoxy method (Prode).
c. Natl. Acad. Sci., 74, 5463-5467 (1977)). Pst obtained from the recombinant plasmid pNLP
The DNA fragment obtained by cleaving an about 9.5 kbp fragment of the I degradation product with the restriction enzyme Hind III has the nucleotide sequence shown in SEQ ID NO: 5 in the Sequence Listing, which is predicted from the amino acid sequence determined in step (I). The nucleic acid base sequence was confirmed. That is, the sequence listing SEQ ID NO: 2 contained in the base sequence shown in the sequence listing SEQ ID NO: 5
It was confirmed that the nucleobase sequence portion shown in SEQ ID NO: 1 encodes the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing, and Rhodococcus rh
It was found to be a gene for nitrilase derived from odochrous PA-34.

(5) Expression of nitrilase gene A Hind III-Pst I fragment (about 4.7 kbp) containing the entire nitrilase gene was subcloned from the fragment in the plasmid pNLP, and the obtained fragment was treated with restriction enzyme Bal 31 and then 2.2k
Expression vector pMK2 in which the bp fragment was treated with the restriction enzyme Sma I
(JP-A 64-51088), the plasmid pNLK
Named 2. FIG. 6 shows the restriction map of the plasmid pNLK2 and the location of the nitrilase gene. Then, Escherichia coli E. coli HB 101 and E. coli JM 109 were transformed, and colonies showing ampicillin resistance were obtained from each of them. The obtained colonies were cultured in 2xYT medium at 30 ° C overnight, and then the cells were collected, washed twice with 0.1 M phosphate buffer containing 5 mM dithiothreitol, and then suspended in the same buffer. Reference (Appl. M
icrobiol. Biotechnol., 37, 184-190 (1992)), 75 mM α-aminoisocapronitrile was added to 1 ml of the obtained bacterial suspension, and the mixture was reacted at 30 ° C. for 1 hour to give a substrate. By measuring the amount of L-leucine produced from α-aminoisocapronitrile, the nitrilase activity of resting cells obtained from each colony was measured, and E. coli HB 101 and E. coli HB 101 and E.
From the transformed strains of E. coli JM 109, one strain was selected and one colony showing the highest nitrilase activity was selected. Based on the Budapest Treaty, E.coli HB 101
Transformant E. coli HB 101 (pNLK2) is FERM BP-4404
E. Ccoli, a transformant of E. coli JM 109
JM 109 (pNLK2) has been deposited as FERM BP-4405.

Next, colonies of each of these two strains were added to 100 ml of 2xYT containing isopropyl-β-D-thiogalactopyranoside (ITPG) as a tac inducer.
After culturing in the medium at 30 ° C. overnight, the cells were collected, washed twice with 0.1 M phosphate buffer containing 5 mM dithiothreitol, and then suspended in the same buffer. The bacterial suspension was treated with a French press to obtain a crude enzyme solution, which was then prepared in the literature (Appl. Microbiol. Biotechnol., 37, 1
84-190 (1992)), the nitrilase activity of resting cells was measured using α-aminoisocapronitrile as a substrate. The results are shown in Table 1. Table 1 also shows the results using a crude enzyme extract of Rhodococcus rhodochrous PA-34 as a reference example. From Table 1, it can be seen that the two transformants are Rhodococcus rhodochrous.
It is found that PA-34-derived nitrilase is produced in resting cells and exhibits nitrilase activity that the host strain originally does not have. Furthermore, from the recombinant DNAs incorporated in the two transformants, the target Rhodococcus
It can be seen that a nitrilase polypeptide derived from rhodochrous PA-34 is produced. The α-amino acid corresponding to α-aminonitrile produced by the enzymatic activity of this polypeptide is described in the literature (Appl. Microbi
ol. Biotechnol., 37, described 184-190 (1992)) Rho
Like the nitrilase derived from dococcus rhodochrous PA-34, only one of the optically active substances is produced.
That is, an α-amino acid with high optical purity is produced.

[0021]

[Table 1] Nitrilase production by E. coli HB 101 recombinant and E. coli JM 109 recombinant Strain Protein Units Specific activity (mg / l) HB 101 (pNLK2) 3266 19.6 0.006 JM 109 (pNLK2) 949 7.6 0.008 PA-34 994 242.5 68.9 Reference example 1 unit is 1 minute from α-aminoisocapronitrile
It shows that μmol of leucine is produced.

[0022]

INDUSTRIAL APPLICABILITY According to the present invention, a DNA sequence encoding a polypeptide having a nitrilase activity for producing an amino acid from α-aminonitrile, and a microorganism transformed with an expression vector having the DNA sequence produce the polypeptide. And the produced polypeptide is α-
It has been shown to produce amino acids from amino nitriles.

[0023]

[Sequence list]

 SEQ ID NO: 1 Sequence Length: 380 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence 1 5 10 15 Met Val Glu Tyr Thr Asn Thr Phe Lys Val Ala Ala Val Gln Ala 20 25 30 Gln Pro Val Trp Phe Asp Ala Ala Lys Thr Val Asp Lys Thr Val 35 40 45 Ser Ile Ile Ala Glu Ala Ala Arg Asn Gly Cys Glu Leu Val Ala 50 55 60 Phe Pro Glu Val Phe Ile Pro Gly Tyr Pro Tyr His Ile Trp Val 65 70 75 Asp Ser Pro Leu Ala Gly Met Ala Lys Phe Ala Val Arg Tyr His 80 85 90 Glu Asn Ser Leu Thr Met Asp Ser Pro His Val Gln Arg Leu Leu 95 100 105 Asp Ala Ala Arg Asp His Asn Ile Ala Val Val Val Gly Ile Ser 110 115 120 Glu Arg Asp Gly Gly Ser Leu Tyr Met Thr Gln Leu Ile Ile Asp 125 130 135 Ala Asp Gly Gln Leu Val Ala Arg Arg Arg Lys Leu Lys Pro Thr 140 145 150 His Val Glu Arg Ser Val Tyr Gly Glu Gly Asn Gly Ser Asp Ile 155 160 165 Ser Val Tyr Asp Met Pro Phe Ala Arg Leu Gly Ala Leu Asn Cys 170 175 180 Trp Glu His Phe Gln Thr Leu Thr Lys Tyr Ala Met Tyr Ser Met 185 190 195 His Glu Gln Val His Val Ala Ser Trp Pro Gly Met Ser Leu Tyr 200 205 210 Gln Pro Glu Val Pro Ala Phe Gly Val Asp Ala Gln Leu Thr Ala 215 220 225 Thr Arg Met Tyr Ala Leu Glu Gly Gln Thr Phe Val Val Cys Thr 230 235 240 Thr Gln Val Val Thr Pro Glu Ala His Glu Phe Phe Cys Glu Asn 245 250 255 Glu Glu Gln Arg Lys Leu Ile Gly Arg Gly Gly Gly Phe Ala Arg 260 265 270 Ile Ile Gly Pro Asp Gly Arg Asp Leu Ala Thr Pro Leu Ala Glu 275 280 285 Asp Glu Glu Gly Ile Leu Tyr Ala Asp Ile Asp Leu Ser Ala Ile 290 295 300 Thr Leu Ala Lys Gln Ala Ala Asp Pro Val Gly His Tyr Ser Arg 305 310 315 Pro Asp Val Leu Ser Leu Asn Phe Asn Gln Arg Arg Thr Thr Pro 320 325 330 Val Asn Thr Pro Leu Ser Thr Ile His Ala Thr His Thr Phe Val 335 340 345 Pro Gln Phe Gly Ala Leu Asp Gly Val Arg Glu Leu Asn Gly Ala 350 355 360 Asp Glu Gln Arg Ala Leu Pro Ser Thr His Ser Asp Glu Thr Asp 365 370 375 Arg Ala Thr Ala Pro Ser Asp Ser Gly Ala Pro Val Ala Pro Pro 380 Lys Arg His Gly Val

SEQ ID NO: 2 Sequence length: 1130 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Other nucleic acid Sequence ATG GTC GAA TAC ACA AAC ACA TTC AAA GTT GCT GCG GTG CAG GCA 45 CAG CCT GTG TGG TTC GAC GCG GCC AAA ACG GTC GAC AAG ACC GTG 90 TCC ATC ATC GCG GAA GCA GCC CGG AAC GGG TGC GAG CTC GTT GCG 135 TTT CCC GAG GTA TTC ATC CCG GGG TAC CCG TAC CAC ATC TGG GTC 180 GAC AGC CCG CTC GCC GGA ATG GCG AAG TTC GCC GTG CGC TAC CAC 225 GAG AAT TCC CTG ACG ATG GAC AGC CCG CAC GTA CAG CGG TTG CTC 270 GAT GCC GCC CGC GAC CAC AAC ATC GCC GTA GTG GTG GGA ATC AGC GAG CGG GAT GGC GGC AGC TTG TAC ATG ACC CAG CTC ATC ATC GAC 360 GCC GAT GGG CAA CTG GTC GCC CGA CGC CGC AAG CTC AAG CCC ACC 405 CAC GTC GAG CGT TCG GTA TAC GGA GAA GGA AAC GGC TCG GAT ATC 450 TCC GTC TAC GAC ATG CCT TTC GCA CGG CTT GGC GCG CTC AAC TGC 495 TGG GAG CAT TTC CAG ACG CTC ACC AAG TAC GCA ATG TAC TCG ATG 540 CAC GAG CAG GTG CAC GTC GCG AGC TGG CCT GGC ATG TCG CTG TAC 585 CAG CCG GAG GTC CCC GCA TTC GGT GTC GAT GCC CAG CTC ACG GCC 630 ACG CGT ATG TAC GCA CTC GAG GGA CAA ACC TTC GTG GTC TGC ACC 675 ACC CAG GTG GTC ACA CCG GAG GCC CAC GAG TTC TTC TGC GAG AAC 720 GAG CAG CGA AAG TTG ATC GGC CGA GGC GGA GGT TTC GCG CGC 765 ATC ATC GGG CCC GAC GGC CGC GAT CTC GCA ACT CCT CTC GCC GAA 810 GAT GAG GAG GGG ATC CTC TAC GCC GAC ATC GAT CTG TCT GCG ATC 855 AACC GTG CAG GCC GCT GAC CCC GTG GGC CAC TAC TCA CGG 900 CCG GAT GTG CTG TCG CTG AAC TTC AAC CAG CGC CGC ACC ACG CCC 945 GTC AAC ACC CCA CTT TCC ACC ATC CAT GCC ACG CAC ACG TTC GTG 990 CCG CAG TTC GGG GCA GAC GGC GTC CGT GAG CTC AAC GGA GCG 1035 GAC GAA CAG CGC GCA TTG CCC TCC ACA CAT TCC GAC GAG ACG GAC 1080 CGG GCG ACA GCA CCC TCT GAC TCG GGC GCA CCC GTG GCG CCT CCG 1125 AAG CGC CAC GGT GTG

SEQ ID NO: 3 Sequence length: 11 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Val Glu Tyr Thr Asn Thr Phe Lys Val Ala 1 5 10

SEQ ID NO: 4 Sequence length: 32 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA

SEQ ID NO: 5 Sequence length: 1341 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Senomic DNA Origin organism name: Rhodococcus rhod
ochrous) strain name: PA-34 sequence CAC GGG TGG CGC AGA ATG CCA GGA CCC GTG TCA TTC CAC GTC AAT 45 TCA CGC GCC TTT TCA CCT CGT ACT GTC CTG CCA AAC ACA AGC AAC 90 GGA GGT ACG GAC ATG GTC GAA TAC ACA AAC ACA TTC AAA GTT GCT 135 Met Val Glu Tyr Thr Asn Thr Phe Lys Val Ala GCG GTG CAG GCA CAG CCT GTG TGG TTC GAC GCG GCC AAA ACG GTC 180 Ala Val Gln Ala Gln Pro Val Trp Phe Asp Ala Ala Lys Thr Val GAC AAG ACC GTG TCC ATC ATC GCG GAA GCA GCC CGG AAC GGG TGC 225 Asp Lys Thr Val Ser Ile Ile Ala Glu Ala Ala Arg Asn Gly Cys GAG CTC GTT GCG TTT CCC GAG GTA TTC ATC CCG GGG TAC CCG TAC 270 Glu Leu Val Ala Phe Pro Glu Val Phe Ile Pro Gly Tyr Pro Tyr CAC ATC TGG GTC GAC AGC CCG CTC GCC GGA ATG GCG AAG TTC GCC 315 His Ile Trp Val Asp Ser Pro Leu Ala Gly Met Ala Lys Phe Ala GTG CGC TAC CAC GAG AAT TCC CTG ACG ATG GAC AGC CCG CAC GTA 360 Val Arg Tyr His Glu Asn Ser Leu Thr Met Asp Ser Pro His Val CAG CGG TTG CTC GAT GCC GCC CGC GAC CAC AAC ATC GCC GTA GTG 405 Gln Arg Leu Leu Asp Ala Ala Arg Asp His Asn Ile Ala Val Val GTG GGA ATC AGC GAG CGG GAT GGC GGC AGC TTG TAC ATG ACC CAG 450 Val Gly Ile Ser Glu Arg Asp Gly Gly Ser Leu Tyr Met Thr Gln CTC ATC ATC GAC GCC GAT GGG CAA CTG GTC GCC CGA CGC CGC AAG 495 Leu Ile Ile Asp Ala Asp Gly Gln Leu Val Ala Arg Arg Arg Lys CTC AAG CCC ACC CAC GTC GAG CGT TCG GTA TAC GGA GAA GGA AAC 540 Leu Lys Pro Thr His Val Glu Arg Ser Val Tyr Gly Glu Gly Asn GGC TCG GAT ATC TCC GTG TAC GAC ATG CCT TTC GCA CGG CTT GGC 585 Gly Ser Asp Ile Ser Val Tyr Asp Met Pro Phe Ala Arg Leu Gly GCG CTC AAC TGC TGG GAG CAT TTC CAG ACG CTC ACC AAG TAC GCA 630 Ala Leu Asn Cys Trp Glu His Phe Gln Thr Leu Thr Lys Tyr Ala ATG TAC TCG ATG CAC GAG CAG GTG CAC GTC GCG AGC TGG CCT GGC 675 Met Tyr Ser Met His Glu Gln Val His Val Ala Ser Trp Pro Gly ATG TCG CTG TAC CAG CCG GAG GTC CCC GCA TTC GGT GTC GAT GCC 720 Met Ser Leu Tyr Gln Pro Glu Val Pro Ala Phe Gly Val Asp Ala CAG CTC ACG GCC ACG CGT ATG TAC GCA CTC GAG GGA CAA ACC TTC 765 Gln Leu Thr Ala Thr Arg Met Tyr Ala Leu Glu Gly Gln Thr Phe GTG GTC TGC ACC ACC CAG GTG GTC ACA CCG GAG GCC CAC GAG TTC 810 Val Val Cys Thr Thr Gln Val Val Thr Pro Glu Ala His Glu Phe TTC TGC GAG AAC GAG GAA CAG CGA AAG TTG ATC GGC CGA GGC GGA 855 Phe Cys Glu Asn Glu Glu Gln Arg Lys Leu Ile Gly Arg Gly Gly GGT TTC GCG CGC ATC ATC GGG CCC GAC GGC CGC GAT CTC GCA ACT 900 Gly Phe Ala Arg Ile Ile Gly Pro Asp Gly Arg Asp Leu Ala Thr CCT CTC GCC GAA GAT GAG GAG GGG ATC CTC TAC GCC GAC ATC GAT 945 Pro Leu Ala Glu Asp Glu Glu Gly Ile Leu Tyr Ala Asp Ile Asp CTG TCT GCG ATC ACC TTG GCG AAG CAG GCC GCT GAC CCC GTG GGC 990 Leu Ser Ala Ile Thr Leu Ala Lys Gln Ala Ala Asp Pro Val Gly CAC TAC TCA CGG CCG GAT GTG CTG TCG CTG AAC TTC AAC CAG CGC 1035 His Tyr Ser Arg Pro Asp Val Leu Ser Leu Asn Phe Asn Gln Arg CGC ACC ACG CCC GTC AAC ACC CCA CTT TCC ACC ATC CAT GCC ACG 1080 Arg Thr Thr Pro Val Asn Thr Pro Leu Ser Thr Ile His Ala Thr CAC ACG TTC GTG CCG CAG TTC GGG GCA CTC GAC GGC GTC CGT GAG 1125 His Thr Phe Val Pro Gln Phe Gly Ala Leu Asp Gly Val Arg Glu CTC AAC GGA GCG GAC GAA CAG CGC GCA TTG CCC TCC ACA CAT TCC 1170 Leu Asn Gly Ala Asp Glu Gln Arg Ala Leu Pro Ser Thr His Ser GAC GAG ACG GAC CGG GCG ACA GCA CCC TCT GAC TCG GGC GCA CCC 1215 Asp Glu Thr Asp Arg Ala Thr Ala Pro Ser Asp Ser Gly Ala Pro GTG GCG CCT CCG AAG CGC CAC GGT GTG TGA AGG GGC GAG ACA GGG 1260 Val Ala Pro Pro Lys Arg His Gly Val * GAA TCG GAG GAT CAC CGA GTA CAC CAT CGT CGA TCG CAG CGA GTA 1305 GCC CGC CCG TAC CCC GAT AGG TCC ACC CCA CGT ATC

[Brief description of drawings]

FIG. 1 shows restriction enzyme maps of three recombinant plasmids pNLE, pNLB, and pNLP obtained in the examples.

FIG. 2 shows a restriction enzyme map of the expression plasmid pNLK2 obtained in the example.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12P 13/04 2121-4B // (C12N 15/09 ZNA C12R 1:01) (C12N 1/21 C12R 1:19) (C12P 13/04 C12R 1:19) C12R 1:01)

Claims (7)

[Claims] 1. A gene DNA encoding a polypeptide represented by the amino acid sequence of SEQ ID NO: 1 in the sequence listing having nitrilase activity. 2. The gene DNA according to claim 1, wherein the DNA sequence encoding nitrilase is the DNA sequence of SEQ ID NO: 2 in the Sequence Listing. 3. A recombinant DNA in which the gene DNA according to claim 1 or 2 is incorporated into a vector. 4. A transformant transformed with the recombinant DNA according to claim 3. 5. The transformant is FERM BP-4404 or FERM B.
The transformant according to claim 4, which is P-4405. 6. A method for producing a carboxylic acid, which comprises culturing the transformant according to claim 4 or 5 and allowing the resulting bacterial cell or a treated product thereof to act on nitriles to produce a corresponding carboxylic acid. . 7. The nitrile is α-aminonitrile,
The method for producing a carboxylic acid according to claim 6, wherein the carboxylic acid is an α-amino acid.