JP2001069978A - Nitrile hydratase gene and amidase gene which are derived from phodococcus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new nitrile hydratase gene which is derived from rhodococcus, contains a DNA sequence coding for a specific amino acid sequence, and is useful for producing the enzyme having position selectivity to an aromatic polynitrile, etc. SOLUTION: This is a new nitrile hydratase gene which is derived from rhodococcus, contains a DNA sequence coding for the amino acid sequence shown by the formula, or a DNA sequence coding for an amino acid sequence modified by deleting, substituting, or adding one amino acid or more from, in, or to the amino acid sequence, and is useful for producing the enzyme which has especially excellent position selectivity to an aromatic polynitrile and allows producing an amide compound and carboxylic acid compound useful as raw materials for medicines and agrochemicals from a nitrile compound, etc. This gene is obtained by preparing a library from chromosomal DNA of Rhodococcus rhodochrous ATCC 39484, followed by carrying out PCR using it as a template and using primers.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a DNA sequence of a nitrile hydratase gene and an amidase gene, which are nitrile-degrading enzyme systems derived from a genus Rhodococcus exhibiting particularly excellent regioselectivity for a nitrile group of an aromatic polynitrile compound. .

[0002]

2. Description of the Related Art Nitrile hydratase and amidase are enzymes that catalyze reactions for converting a nitrile compound into an amide compound and an amide compound into a carboxylic acid compound, respectively. By using nitrile hydratase and amidase, an amide compound or a carboxylic acid compound useful as a raw material for medical and agricultural chemicals can be obtained from the nitrile compound.

A method for converting a nitrile compound into a corresponding amide compound or carboxylic acid compound has been developed by utilizing a biocatalyst, and many microorganisms having such a catalytic ability have been reported (JP-B-56-19918).
JP, JP-B-59-37951, JP-B-61-
No. 162193, Japanese Patent Publication No. 61-21519,
JP-B 64-86889, JP-B 4-19718
No. 9, JP-A-2-470, EP0444464
No. 0).

[0004] Further, nitrile hydratase, amidase or nitrilase is purified from these microorganisms, and their genes have been isolated and their primary structures have been determined in order to utilize these enzymes for genetic engineering.

[0005] Regarding the nitrile hydratase gene,
For example, a gene derived from a bacterium belonging to the genus Rhodococcus is disclosed in U.S. Pat. No. 2,840,253 or EP04445646 (Japanese Unexamined Patent Publication No.
Japanese Patent Application Laid-Open No. 3-251184) discloses a gene derived from Pseudomonas genus bacteria and Japanese Patent Application Laid-Open No. Hei 6-252 discloses a gene derived from Rhizobium bacteria.
No. 96 and JP-A-6-302971,
For the amidase gene, for example, genes derived from Brevibacterium sp. And Rhodococcus sp.
No. 0433117.

A gene derived from Rhodococcus erythropolis is Eur. J. Biochem. 217
(1), 327-336 (1993), a gene derived from a bacterium belonging to the genus Pseudomonas is FEBS Lett. 36
7, 275-279 (1995). Further, an invention relating to a recombinant plasmid containing both nitrile hydratase gene and amidase gene derived from a Rhodococcus bacterium is disclosed in JP-A-5-68566.

In recent years, attempts have been made to apply the ability of such microorganisms to convert nitrile compounds. In particular, an enzyme having excellent stereoselectivity and regioselectivity has been desired for use in producing a compound having high added value. For example, JP-A-2-84198 discloses a microorganism used for producing an optically active α-substituted organic acid.
No. 85 discloses a microorganism used for producing an optically active 2-hydroxycarboxylic acid, and EP 0433117 discloses a microorganism used for producing an optically active ketoprofen.

[0008] Among such microorganisms, Rhodococcus
Rhodocross (Rhodococcus rhodoch)
(Rous) ATCC39484 strain has been reported to have an excellent regioselective hydrolysis ability to an aromatic polynitrile compound having a plurality of nitrile groups (US Pat. No. 5,566,625). A compound having both a nitrile group and an amide functional group or a nitrile group and a carboxyl functional group in a molecule generated by this selective nitrile-decomposing enzyme system is extremely effective as a synthetic block for the production of medical and agricultural chemicals. In order to industrially utilize this property of this bacterium, it is desired to improve the productivity, stability and reaction characteristics of the enzyme that catalyzes the reaction. However, the genes of nitrile hydratase and amidase of the bacterium essential for these modifications have not been clarified.

[0009]

SUMMARY OF THE INVENTION An object of the present invention is to efficiently produce nitrile hydratase and amidase from a Rhodococcus bacterium using genetic engineering techniques or to improve them using protein engineering techniques. It is an object of the present invention to provide a necessary DNA sequence of a nitrile hydratase gene and an amidase gene derived from Rhodococcus bacteria.

[0010]

Means for Solving the Problems The present invention provides the following (1) to
It has the configuration of (6). (1) A nitrile hydratase gene derived from a bacterium belonging to the genus Rhodococcus, comprising a DNA sequence encoding the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. (2) A nitrile hydratase gene derived from a bacterium belonging to the genus Rhodococcus, comprising a DNA sequence encoding an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. (3) An amidase gene derived from a bacterium belonging to the genus Rhodococcus, comprising a DNA sequence encoding the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing. (4) An amidase gene derived from a bacterium belonging to the genus Rhodococcus, comprising a DNA sequence encoding an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing.

(5) An amidase gene derived from a bacterium belonging to the genus Rhodococcus, comprising a DNA sequence encoding the amino acid sequence represented by SEQ ID NO: 6 in the sequence listing. (6) An amidase gene derived from a bacterium belonging to the genus Rhodococcus, comprising a DNA sequence encoding an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 6 in the sequence listing. (7) The bacterium belonging to the genus Rhodococcus is Rhodococcus rhodochrous.
The nitrile hydratase gene according to (1) or (2), which is ATCC 39484 strain. (8) The genus Rhodococcus bacterium is Rhodococcus rhodochrous.
s) The amidase gene according to (3) or (4), which is the ATCC 39484 strain. (9) Rhodococcus bacterium belonging to the genus Rhodococcus rhodochrous
s) The amidase gene according to (5) or (6), which is strain ATCC39484.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
Rhodococcus Rhodococcus
rhodochrous) ATCC39484 strain chromosomal DNA can be obtained, for example, by the method of Saito et al. (Bioche).
m. Biophys. Acta. 72, 619
(1963)).

Chromosome DN used for gene cloning
The A library can be prepared using a plasmid vector such as pUC18. Cloning of nitrile hydratase gene and amidase gene
For example, Saiki et al., Polymerase Chai
n Reaction (PCR) method (Science23)
0, 1350 (1985)). At this time, one of the PCR primers to be used is a universal primer (forward or reverse), and the other is to analyze the N-terminal sequence of the target enzyme protein or the peptide terminal sequence obtained by hydrolyzing the target enzyme protein, and analyze it. An appropriate sequence is selected from the sequence to be encoded.

By combining these primers and performing PCR using a chromosomal DNA library as a template, a coding sequence fragment of the target enzyme can be obtained. By using the DNA fragment of the nitrile hydratase coding sequence or the amidase coding sequence obtained by the anchor PCR method as a probe for screening the entire gene region, Rhodococcus rhodoch can be obtained.
rous (R. rhodochrous) ATCC39
Recombinant DN containing nitrile hydratase gene and amidase gene from chromosome DNA library of strain 484
A can be obtained.

The DNA sequences of the nitrile hydratase coding sequence fragment and the amidase coding sequence fragment are Sang.
er et al. (Proc. Nat).
l. Acad. Sci. U. S. A. 74, 5
463 (1997)).

[0016]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

(Example 1) Preparation of chromosomal DNA R. cultivated on nutrient (L broth) agar plate medium for 24 hours. rho
dochrous ATCC39484 strain loopful basal medium _{(KH 2 PO 4 1.5g / l} , Na 2 HPO 4 · 2H
_{2 O 0.75g / l, MgSO 4} · 7H 2 O 0.2g
_{/ L, CaSO 4 · 2H 2} O 10mg / l, FeSO 4
・ A medium 300 containing 5 mg / l of 7H ₂ O, 20 mg / l of yeast extract and 1 g / l of glucose and 1 g / l of urea.
The cells were cultured at 30 ° C. for 1 day.

The cultured cells are collected, and 5 mM EDTA is added.
The cells were washed with 100 ml of the solution. 30 ml of this cell
Buffer (20 mM Tris-HCl buffer (pH 7.
1)), 60 mg of lysozyme was added, and the mixture was incubated at 37 ° C. for 2 hours. The suspension is centrifuged (5,0
(00 rpm, 7 minutes) to collect the cells.
l of TE Buffer and resuspended in 10% SDS0.
6 ml, and proteinase K (manufactured by Merck) to a concentration of 100 μg / ml.
Shake gently at 5 ° C. This solution was extracted with phenol and precipitated with ethanol to prepare chromosomal DNA.

Example 2 Preparation of Chromosome Library 20 μg of the obtained chromosomal DNA was subjected to restriction enzyme Sau3.
Partial digestion was performed using AI. That is, 4 μg of chromosomal DNA is placed in each of five tubes, and a restriction enzyme Sau3AI (Takara Shuzo Co., Ltd., 4 to 12) is placed in a reaction volume of 100 μl.
(U / μl) was added and the reaction was carried out at 37 ° C., one tube was taken every 10 seconds, and EDTA was added to a final concentration of 20 mM to stop the reaction.

The partially digested fragment solution of the chromosomal DNA thus prepared was subjected to agarose gel electrophoresis,
A 10 kb DNA fragment was recovered by precipitation extraction and ethanol precipitation, and dissolved in 30 μl of a TE solution. After ligation of 9 μl of this sample and 1 μg of BUCHI-digested BAP-treated pUC18 (pUC18 / BamHI manufactured by Takara Shuzo Co., Ltd.) using T4 DNA ligase (Takara Shuzo Ligation Kit ver. 2) in a total volume of 20 μl, E. coli The JM109 strain was transformed. In order to prepare an amplification library of this library, E. coli transformants were inoculated into L broth (pH 7.0) containing 50 ppm of ampicillin, and cultured overnight. A plasmid used as a template was extracted from the cells by the alkali-SDS method.

(Example 3) Purification of nitrile hydratase and amidase One primer derived from an enzyme sequence necessary for anchor PCR has an appropriate Tm from the terminal sequence of the enzyme peptide prepared as follows. The sequence was selected and created.

The nitrile hydratase activity or the amidase activity was measured using benzonitrile 10 mM or benzamide 10 mM, potassium phosphate buffer (pH
7.0) 1 ml of the reaction mixture containing 30 mM and a predetermined amount of the bacterial cell extract was reacted at 25 ° C. for 30 minutes, and the produced benzamide or benzoic acid was converted to H.
Performed qualitatively by detection by PLC.

R. rhodochrous ATCC3
9484 strain was added to the basal medium of Example 1 as an induction substrate at 1 g.
The cells were inoculated into 6 l of a nitrile-degrading enzyme group induction medium supplemented with 1 / l of benzonitrile and cultured with shaking at 30 ° C.

The culture solution cultured overnight is centrifuged (8000 rpm).
pm, 15 minutes) to collect the cells, and wet 32 g of the obtained cells with a 100 mM potassium phosphate buffer (pH 7.0, 1 mM EDTA and 2 mM DDT).
After washing with 50 ml, the same buffer 200 m
l. This was subjected to a wet mill to disrupt the cells, and then centrifuged (12,000 rpm, 20 minutes) to obtain 180 ml of a supernatant (crude enzyme extract).

Ammonium sulfate was added to the cell-free extract to a 45% saturation concentration, and the mixture was stirred at 4 ° C. for 1 hour, and the formed precipitate was removed by centrifugation. Ammonium sulfate was further added to the separated supernatant to a concentration of 60% saturation, and the mixture was stirred at 4 ° C. for 1 hour, and the precipitate was recovered by centrifugation. The resulting precipitate was confirmed to have nitrile hydratase activity and amidase activity. The obtained precipitate is washed with a 100 mM potassium phosphate buffer (pH 7.0, 1
10 ml containing mM EDTA and 2 mM DDT)
And dialyzed against the same buffer.

A 100 mM potassium phosphate buffer (p
The dialyzed crude enzyme solution was applied to a DEAE-Sephacel column (2 cm × 20 cm) equilibrated with H7.0, 1 mM EDTA and 2 mM DDT, and washed with the equilibration buffer until the UV 280 nm of the eluate was reduced. Subsequently, the eluate was washed with the same buffer to which 0.1 M KCl was added until the UV280 nm of the eluate was lowered, and further washed with a buffer having a KCl concentration increased to 0.2 M until the UV280 nm of the eluate was lowered.

Thereafter, the KCl concentration was increased to 0.3M.
00 mM potassium phosphate buffer (pH 7.0, 1 m
(Including M EDTA and 2 mM DDT) to elute nitrile hydratase and amidase. The fractions showing the activity were collected and subjected to ultrafiltration (excluding the molecular weight limit of 30,0).
00) was used to concentrate the enzyme protein.

A 100 mM potassium phosphate buffer (p
H7.0, containing 10% saturated ammonium sulfate)
Phenyl-Sepharose CL equilibrated with
The concentrated active fraction to which a 10% saturated concentration of ammonium sulfate was added was supplied to a -4B column (2 cm × 40 cm) to adsorb the enzyme. The eluate was washed with the equilibration buffer until the UV 280 nm of the eluate was reduced. Thereafter, nitrile hydratase and amidase were eluted with an elution buffer (100 mM potassium phosphate buffer (pH 7.0)). The active fraction was collected and the enzyme protein was concentrated using an ultrafiltration membrane (exclusion molecular weight limit: 30,000).

The concentrated nitrile hydratase active fraction was subjected to a 100 mM potassium phosphate buffer (pH 7.0).
Seph equilibrated with 0, 0.5 M NaCl)
acryl S-300 super fine column (2
cm × 60 cm), separation was performed using the same buffer, and the eluate was fractionated by about 0.5 ml each. The fraction having the highest nitrile hydratase and amidase activities and the fractions before and after the fraction were collected. About 1.5 ml of each fraction was concentrated using an ultrafiltration membrane (exclusion molecular weight limit: 30,000).

A part of each concentrated solution was subjected to SDS-PAG
When analyzed by E (12.5% polyacrylamide gel), one main band and a plurality of minor bands were confirmed. Therefore, the protein was blotted on a PVDF membrane, and the N-terminal sequence of the main band was analyzed by Edman degradation, but a plurality of amino acids were detected and the sequence could not be determined. Therefore, hydrolysis of the enzyme protein was performed as follows, and analysis was performed after the peptide was formed.

Example 4 Determination of Peptide Terminal Sequence The obtained nitrile hydratase and amidase enzyme proteins were hydrolyzed by the cyanogen bromide (BrCN) method. Subsequently, the generated peptide was separated under the following conditions.

Main unit; LC-9A (Shimadzu Corporation) Column; Asahipak ODP-50 6D (Shodex) Eluent: Acetonitrile 0 to 80% (linear concentration gradient, 60 minutes) 0.1% trifluoroacetic acid, flow rate 0.5 ml / min SPD-6AV UV-VIS Spectro-photometer (Shimadzu Corp.), 215 nm Column temperature; 25 ° C. Among a plurality of peptides obtained from the nitrile hydratase active fraction, a relatively well-separated sample was selected, and N was determined by Edman degradation. When the terminal sequence was analyzed, the following sequences having high homology to the sequence of the existing nitrile hydratase were confirmed. Glu (E) ・ Tyr (Y) ・ Arg (R) ・ Ser (S) ・ Arg (R) ・ Val (V) ・
Val (V) This sequence and the Rhodococcus genus Codon
In consideration of Usage, a primer for nitrile hydratase was prepared. 5′-GAG TAC CGG TCC CGA-3 ′ (and its complementary chain) Similarly, among a plurality of peptides obtained from the amidase active fraction, a sample with relatively good separation was selected, and N-terminal sequence analysis by Edman degradation was performed. As a result, the following sequences having high homology to the existing amidase sequence were confirmed. Ala (A) ・ Val (V) ・ Gly (G) ・ Gly (G) ・ Asp (D) ・ Gln (Q) ・ Gl
y (G) This sequence and Rhodococcus spp.
In consideration of Usage, primers for amidase were prepared. 5'-GCA GTC GGC GGC GAC-3 '(and its complementary strand)

Example 5 Anchor PCR The PCR was performed under the following reaction conditions. Reaction solution composition: rhodochrous ATCC39484 chromosomal DNA library 1 μg Universal primer 100 pmol Enzyme peptide terminal primer 100 pmol dNTP solution 1 mM each 10 × reaction buffer 10 μl ExTaq DNA polymerase (Takara Shuzo) 2.5 Unit Total 50 μl Reaction conditions: heat denaturation 94 ° C., 45 seconds annealing 42-57 ° C. , 60 seconds extension 72 ° C, 60-90 seconds Number of cycles 24

Of the reactions under various conditions performed in this manner, the reaction solution in which a fragment to be specifically amplified was
% Agarose gel electrophoresis, cut out the gel containing the fragment, and used ESAYTRAP ver. 2 (Takara Shuzo).

With respect to this DNA fragment,
The DNA sequence was determined by the method. As a result of confirming that the translated amino acid sequence has homology to the terminal sequence of the enzyme protein and the known nitrile hydratase and amidase, the resulting fragments 4, 14, and 56 contained, The presence of the open reading frames as shown in 3, 5 was confirmed.

The open reading frame encoding 80 amino acids of SEQ ID NO: 1 in the sequence listing is a DNA sequence encoding the α subunit of nitrile hydratase, and the open reading frame encoding 287 amino acids of SEQ ID NO: 3 contains amidase. D to code
It was found to be an NA sequence, and that the open reading frame encoding 157 amino acids of SEQ ID NO: 5 was a DNA sequence encoding an amidase different from SEQ ID NO: 3.

[0037]

Industrial Applicability The present invention provides a DNA partial sequence of nitrile hydratase and amidase genes of Rhodococcus bacteria having excellent regioselective hydrolytic ability for an aromatic polynitrile compound having a plurality of nitrile groups. . These DNA sequences are indispensable for efficient production of nitrile hydratase and amidase using genetic engineering techniques and improvement of enzymes using protein engineering techniques, and the enzymes thus obtained are useful. The compound can be expected to be applied to industrial production.

[0038]

[Sequence List] SEQUENCE LISTING <110> SHOWA DENKO K.K. <120> Nucleic acid encoding nitrile hydratase and amidase from Rhodococcus sp. <130> 11H110213 <160> 6 <170> PatentIn Ver. 2.0

<210> 1 <211> 578 <212> DNA <213> Rhodococcus sp. <220> <221> CDS <222> (1) .. (243) <400> 1 gag tac cgg tcc cga gtg gta gca gac cct cgt gga gta ctc aag cgc 48 Glu Tyr Arg Ser Arg Val Val Ala Asp Pro Arg Gly Val Leu Lys Arg 1 5 10 15 gat ttc ggg ttc gac atc ccc gat gag gtg gag gtc agg gtt tgg gac 96 Asp Phe Gly Phe Asp Ile Pro Asp Glu Val Glu Val Arg Val Trp Asp 20 25 30 agc agc tcc gaa atc cgc tac atc gtc atc ccg gaa cgg ccg gcc ggc 144 Ser Ser Ser Glu Ile Arg Tyr Ile Val Ile Pro Glu Arg Pro Ala Gly 35 40 45 acc gac ggt tgg tcc gag gac gag ctg gcg aag ctg gtg agt cgg gac 192 Thr Asp Gly Trp Ser Glu Asp Glu Leu Ala Lys Leu Val Ser Arg Asp 50 55 60 tcg atg atc ggt gtc agt aat gcg ctc acacc ca gaa gtg atc gta 240 Ser Met Ile Gly Val Ser Asn Ala Leu Thr Pro Gln Glu Val Ile Val 65 70 75 80 tga gtgaagacac actcactgat cggctcccgg cgactgggac cgccgcgccaccg 293 ccccgcgaca atggcgagct tgtattc gagcgcgcgc gcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgccggccgggcgcgcgccggcgcgccggccggcgccggcggcggcgccggccggcgccggcgggcggcggcgggcggcggcgg ggca gcgtctcatt 413 cactcgatcg ctgaggccaa cggttgcgag gcatactacg agagctggac aaaggcgctc 473 gaggccagcg tggtcgattc ggggctgatc accgaagatg agatccgcga gcgcat cgg tcgccatgc gcgca tgc gcg cg tg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg tg cg cg cg tg

<210> 2 <211> 80 <212> PRT <213> Rhodococcus sp. <400> 2 Glu Tyr Arg Ser Arg Val Val Ala Asp Pro Arg Gly Val Leu Lys Arg 1 5 10 15 Asp Phe Gly Phe Asp Ile Pro Asp Glu Val Glu Val Arg Val Trp Asp 20 25 30 Ser Ser Ser Glu Ile Arg Tyr Ile Val Ile Pro Glu Arg Pro Ala Gly 35 40 45 Thr Asp Gly Trp Ser Glu Asp Glu Leu Ala Lys Leu Val Ser Arg Asp 50 55 60 Ser Met Ile Gly Val Ser Asn Ala Leu Thr Pro Gln Glu Val Ile Val 65 70 75 80

<210> 3 <211> 861 <212> DNA <213> Rhodococcus sp. <220> <221> CDS <222> (1) .. (861) <400> 3 gca gtc ggc ggc gac cag ggc ggt tcg atc cgc atc ccc gcc gcg ttc 48 Ala Val Gly Gly Asp Gln Gly Gly Ser Ile Arg Ile Pro Ala Ala Phe 1 5 10 15 tgc ggc atc gtc gga cac aaa ccc acc cac gga ctg gtc ccc tat acg Gly I Val Gly His Lys Pro Thr His Gly Leu Val Pro Tyr Thr 20 25 30 gga gca ttt ccc atc gaa cga acc atc gac cac ctc ggt ccg atg acg 144 Gly Ala Phe Pro Ile Glu Arg Thr Ile Asp His Leu Gly Pro Met Thr 35 40 45 cgc acg gtc agc gac gcc gcc gca atg ctc acc gtc ctc gcc ggc acc 192 Arg Thr Val Ser Asp Ala Ala Ala Met Leu Thr Val Leu Ala Gly Thr 50 55 60 gac ggc ctc gat ccc cga cag acc cac cgg atc gaa ccg gtg gac tac 240 Asp Gly Leu Asp Pro Arg Gln Thr His Arg Ile Glu Pro Val Asp Tyr 65 70 75 80 ctc gcg gcg ctg gcc gaa ccc gca tcg ggt ctg cgc gtg ggt gtg gtc 288 Leu Ala Ala Lela Ala Ser Gly Leu Arg Val Gly Val Val 85 90 95 acc gaa ggc ttc gac acc cct gtc tcc gac gc t gcc gtc gac aat gcc 336 Thr Glu Gly Phe Asp Thr Pro Val Ser Asp Ala Ala Val Asp Asn Ala 100 105 110 gtg cgc acc gcc atc ggc gta ctg cgc tcg gcc gga ctt acc gtc gaa 384 Val Arg Thr Ala Ile Gly Val Leu Arg Ser Ala Gly Leu Thr Val Glu 115 120 125 gag gtc tcg atc ccc tgg cac ctc gat gcg atg gcc gtc tgg aac gtg 432 Glu Val Ser Ile Pro Trp His Leu Asp Ala Met Ala Val Trp Asn Val 130 135 140 atc gcc acc gag gga gcg gcc tac cag atg ctc gac ggc aat gcc tac 480 Ile Ala Thr Glu Gly Ala Ala Tyr Gln Met Leu Asp Gly Asn Ala Tyr 145 150 155 160 ggc atg aac act gat ggc ttc tac gat ccc gaa ctg ttc 528 Gly Met Asn Thr Asp Gly Phe Tyr Asp Pro Glu Leu Ile Ala His Phe 165 170 175 tcc cgt caa cga ctc gag cac ggt cac caa ctg tcg aag acg gtc aaa 576 Ser Arg Gln Arg Leu Glu His Gly His Gln Leu Ser Lys Thr Val Lys 180 185 190 ctc gtc ggg atg tcc ggg cgc tac aca ttg gag gta ggc ggc ggc aag 624 Leu Val Gly Met Ser Gly Arg Tyr Thr Leu Glu Val Gly Gly Gly Lys 195 200 205 tac tac gcc atg gcc cgca c tc gtc ccc gaa gtc cgc gcc gcc tac 672 Tyr Tyr Ala Met Ala Arg Gln Leu Val Pro Glu Val Arg Ala Ala Tyr 210 215 220 gac gcc gcc ttg gct cgg tac gac gtg ctg gtg atg ccc acc ctc ccc Leap Ala Alapla Ala Arg Tyr Asp Val Leu Val Met Pro Thr Leu Pro 225 230 235 240 tac acc gcc acc aag atc ccg acc acg gac att ccg ttg gcc gac tat 768 Tyr Thr Ala Thr Lys Ile Pro Thr Thr Asp Ile Pro Leu Ala Asp Tyr 245 250 255 ctg gac acc gca ctg tcg atg atc atc aac acc gca cca ttc gac gtc 816 Leu Asp Thr Ala Leu Ser Met Ile Ile Asn Thr Ala Pro Phe Asp Val 260 265 270 acc ggt cac ccc gcc tgc agt gtc ccc gct gac ctg gtc cac ggg 861 Thr Gly His Pro Ala Cys Ser Val Pro Ala Asp Leu Val His Gly 275 280 285

<210> 4 <211> 287 <212> PRT <213> Rhodococcus sp. <400> 4 Ala Val Gly Gly Asp Gln Gly Gly Ser Ile Arg Ile Pro Ala Ala Phe 1 5 10 15 Cys Gly Ile Val Gly His Lys Pro Thr His Gly Leu Val Pro Tyr Thr 20 25 30 Gly Ala Phe Pro Ile Glu Arg Thr Ile Asp His Leu Gly Pro Met Thr 35 40 45 Arg Thr Val Ser Asp Ala Ala Ala Met Leu Thr Val Leu Ala Gly Thr 50 55 60 Asp Gly Leu Asp Pro Arg Gln Thr His Arg Ile Glu Pro Val Asp Tyr 65 70 75 80 Leu Ala Ala Leu Ala Glu Pro Ala Ser Gly Leu Arg Val Gly Val Val 85 90 95 Thr Glu Gly Phe Asp Thr Pro Val Ser Asp Ala Ala Val Asp Asn Ala 100 105 110 Val Arg Thr Ala Ile Gly Val Leu Arg Ser Ala Gly Leu Thr Val Glu 115 120 125 Glu Val Ser Ile Pro Trp His Leu Asp Ala Met Ala Val Trp Asn Val 130 135 140 Ile Ala Thr Glu Gly Ala Ala Tyr Gln Met Leu Asp Gly Asn Ala Tyr 145 150 155 160 Gly Met Asn Thr Asp Gly Phe Tyr Asp Pro Glu Leu Ile Ala His Phe 165 170 175 Ser Arg Gln Arg Leu Glu His Gly His Gln Leu Ser Lys Thr Val Lys 180 185 190 Leu Val Gly Met Ser Gly Arg Tyr Thr Leu Glu Val Gly Gly Gly Lys 195 200 205 Tyr Tyr Ala Met Ala Arg Gln Leu Val Pro Glu Val Arg Ala Ala Tyr 210 215 220 Asp Ala Ala Ala Leu Ala Arg Tyr Asp Val Leu Val Met Pro Thr Leu Pro 225 230 235 240 Tyr Thr Ala Thr Lys Ile Pro Thr Thr Asp Ile Pro Leu Ala Asp Tyr 245 250 255 Leu Asp Thr Ala Leu Ser Met Ile Ile Asn Thr Ala Pro Phe Asp Val 260 265 270 Thr Gly His Pro Ala Cys Ser Val Pro Ala Asp Leu Val His Gly 275 280 285

<210> 5 <211> 471 <212> DNA <213> Rhodococcus sp. <220> <221> CDS <222> (1) .. (471) <400> 5 aag gcc gnc gtg ctc gac aac gca cga gcn atn gcc aac atg ntg ntc 48 Lys Ala Xaa Val Leu Asp Asn Ala Arg Xaa Xaa Ala Asn Met Xaa Xaa 1 5 10 15 ggn atg aag gcg ggn ctg ccn ggn ctg gan ctg gtg gtg ttc ccn gag Xaa 96a Ala Xaa Leu Xaa Xaa Leu Xaa Leu Val Val Phe Xaa Glu 20 25 30 tan tcg act ctg gga atc atg tan gac aan cac gag atg tan gcc acn 144 Xaa Ser Thr Leu Gly Ile Met Xaa Asp Xaa His Glu Met Xaa Ala Xaa 35 40 45 gcn gcn acc atc ccc ggn gac gaa acn gac atc ttt tcg nag gcn tgc 192 Xaa Xaa Thr Ile Pro Xaa Asp Glu Xaa Asp Ile Phe Ser Xaa Xaa Cys 50 55 60 cgg gan gcc aac acg tgg ggc gtc ttc tcg atc acc ggn gag cgc cat 240 Arg Xaa Ala Asn Thr Trp Gly Val Phe Ser Ile Thr Xaa Glu Arg His 65 70 75 80 gan gan cac ccg nac aag ccn ccn tac aac acc ctg ntn ctg ntn aac 288 Xaa Xaa His Pro Xaa Lys Xaa Xaa Tyr Asn Thr Leu Xaa Leu Xaa Asn 85 90 95 aat cag ggn gan atc gtg cag aag tac cgn aa g att ctg ccc tgg anc 336 Asn Gln Xaa Xaa Ile Val Gln Lys Tyr Xaa Lys Ile Leu Pro Trp Xaa 100 105 110 ccg atc gan ggc tgg tan ccn ggg gnc nnc acc nan gtg acc gac ggt 384 Pro Ile Xaa Gly Trp Xaa Xaa Gly Xaa Xaa Thr Xaa Val Thr Asp Gly 115 120 125 ccc aag gga ctg aag atc tcc ctg atc atc tgc gac gac ggc aac tac 432 Pro Lys Gly Leu Lys Ile Ser Leu Ile Ile Cys Asp Asp Gly Asn Tyr 130 135 140 ccg gan atc tgg cgn gan tgc gcg atg aag ggc gcc gac 471 Pro Xaa Ile Trp Xaa Xaa Cys Ala Met Lys Gly Ala Asp 145 150 155

<210> 6 <211> 157 <212> PRT <213> Rhodococcus sp. <400> 6 Lys Ala Xaa Val Leu Asp Asn Ala Arg Xaa Xaa Ala Asn Met Xaa Xaa 1 5 10 15 Xaa Met Lys Ala Xaa Leu Xaa Xaa Leu Xaa Leu Val Val Phe Xaa Glu 20 25 30 Xaa Ser Thr Leu Gly Ile Met Xaa Asp Xaa His Glu Met Xaa Ala Xaa 35 40 45 Xaa Xaa Thr Ile Pro Xaa Asp Glu Xaa Asp Ile Phe Ser Xaa Xaa Cys 50 55 60 Arg Xaa Ala Asn Thr Trp Gly Val Phe Ser Ile Thr Xaa Glu Arg His 65 70 75 80 Xaa Xaa His Pro Xaa Lys Xaa Xaa Tyr Asn Thr Leu Xaa Leu Xaa Asn 85 90 95 Asn Gln Xaa Xaa Ile Val Gln Lys Tyr Xaa Lys Ile Leu Pro Trp Xaa 100 105 110 Pro Ile Xaa Gly Trp Xaa Xaa Gly Xaa Xaa Thr Xaa Val Thr Asp Gly 115 120 125 Pro Lys Gly Leu Lys Ile Ser Leu Ile Ile Cys Asp Asp Gly Asn Tyr 130 135 140 Pro Xaa Ile Trp Xaa Xaa Cys Ala Met Lys Gly Ala Asp 145 150 155

Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court II (Reference) (C12N 9/80 C12R 1:19) (C12N 9/88 C12R 1:19)

Claims (9)

[Claims] 1. A nitrile hydratase gene derived from a bacterium belonging to the genus Rhodococcus, comprising a DNA sequence encoding an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. 2. A nitrile hydratase gene derived from a bacterium belonging to the genus Rhodococcus, comprising a DNA sequence encoding an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. . 3. An amidase gene derived from a bacterium belonging to the genus Rhodococcus, comprising a DNA sequence encoding an amino acid sequence represented by SEQ ID NO: 4 in the sequence listing. 4. An amidase gene derived from a bacterium belonging to the genus Rhodococcus, comprising a DNA sequence encoding an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing. 5. An amidase gene derived from a bacterium belonging to the genus Rhodococcus, comprising a DNA sequence encoding an amino acid sequence represented by SEQ ID NO: 6 in the sequence listing. 6. An amidase gene derived from a bacterium of the genus Rhodococcus, comprising a DNA sequence encoding an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 6 in the sequence listing. 7. The microorganism of the genus Rhodococcus rhodococcus rhodochr.
ou) ATCC39484 strain.
The nitrile hydratase gene described. 8. The method according to claim 8, wherein the bacterium belonging to the genus Rhodococcus is Rhodococcus rhodochr.
ow) The strain is ATCC39484 strain.
The described amidase gene. 9. The bacterium belonging to the genus Rhodococcus rhodococcus rhodochr.
ous) ATCC39484 strain.
The described amidase gene.