EP0444639B1

A gene encoding a polypeptide having nitrile hydratase activity, a transformant containing the gene and a process for the production of amides using the transformant

Abstract

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EP0444639B1, drawing sheet 1 — Sheet 1 of 17

Term

Term ended

Expired 27 February 2011, 15.6 years ago.

8 claims: 1 independent, 7 dependent

1
A gene encoding a polypeptide having nitrile hydratase activity, said polypeptide comprising the α-subunit of the amino acid sequence as defined in the Sequence Listing by SEQ ID:No. 1 and the β-subunit of the amino acid sequence as defined in SEQ ID: No. 2.

100 paragraphs in 8 sections, as filed

0001The present invention relates to a gene which is derived from Pseudomonaschlororaphis B23 and which encodes a polypeptide having nitrile hydratase activity to hydrate nitriles to amides. The invention also relates to a recombinant DNA containing the gene, and a transformant transformed with the recombinant DNA. The present invention further relates to a method of the production of nitrile hydratase using the transformant and of amides using nitrile hydratase.

0002Nitrile hydratase or nitrilase is known as an enzyme that hydrates nitriles to amides. Microorganisms that produce nitrile hydratase include those belonging to the genus Bacillus, the genus Bacteridium, the genus Micrococcus and the genus Brevibacterium (See, JP-B-62-21517/1989, USP No. 4,001,081), the genus Corynebacterium and the genus Nocardia (See, JP-B-56-17918/1989, USP No. 4,248,968), the genus Pseudomonas (See, JP-B-59-37951/1984, USP No. 4,637,982), the genus Rhodococcus, the genus Arthrobacter and the genus Microbacterium (See, JP-A-61-162193/1986, EP-A-0188316), and Rhodococcusrhodochrous (See, JP-A-2-470/1990, EP-A-0307926).

0003Nitrile hydratase has been used to hydrate nitriles to amides. In the invention, microorganisms are engineered to contain multiple copies of a recombinant DNA encoding nitrile hydratase according to a recombinant DNA technology. The recombinant produces a remarkably high level of nitrile hydratase compared with conventional methods.

0004The present inventors previously disclosed a gene derived from Rhodococcus sp. N-774 (FERM BP-1936) which also encodes a polypeptide having nitrile hydratase activity (JP-A-2-119778/1988).

0005In contrast, the present inventors utilized a gene derived from Pseudomonaschlororaphis B23 described in said USP No. 4,637,982 for the production of nitrile hydratase. We isolated the gene encoding nitrile hydratase, inserted the gene into a suitable plasmid vector and transformed an appropriate host with the recombinant plasmid, thus successfully obtained the transformant producing nitrile hydratase.

The present invention relates to

0006<ul id="ul0001" list-style="none"><li>(1) a gene encoding a polypeptide which has nitrile hydratase activity and which comprises the α-subunit of the amino acid sequence as defined in the Sequence Listing by SEQ ID: No. 1, and the β-subunit of the amino acid sequence as defined in the Sequence Listing by SEQ ID: No. 2;</li><li>(2) a gene as described in (1) encoding α- and β-subunits, comprising the α-subunit-encoding sequence as defined in the Sequence Listing by SEQ ID: No. 3, and the β-subunit-encoding sequence as defined in the Sequence Listing by SEQ ID: No. 4;</li><li>(3) a recombinant DNA comprising a vector containing the gene as described in (1) or (2);</li><li>(4) a transformant transformed with the recombinant DNA as described in (3);</li><li>(5) a method for the production of nitrile hydratase which comprises culturing the transformant as described in (4) and recovering nitrile hydratase from the culture;</li><li>(6) a method for the production of amides which comprises hydrating nitriles using the nitrile hydratase as described in (5) to form amides; and</li><li>(7) a method for the production of amides which comprises culturing the transformant as described in (4), and hydrating nitriles using the resultant culture, isolated bacterial cells, treated matter thereof, or a fixed material of them, to form amides.</li></ul>

0007The present invention is described in detail as follows.

(1)

Isolation and Purification of Nitrile Hydratase and

Partial Amino Acid Sequencing of Nitrile Hydratase

0008Nitrile hydratase is isolated and purified from Pseudomonaschlororaphis B23 and separated into α and β subunits using HPLC. N-Terminal amino acid sequence of the subunits is determined and shown in the Sequence Listing by SEQ ID: No. 5 and No. 6, respectively.

(2)

Preparation of a DNA Probe for a Nitrile Hydratase Gene

0009A DNA probe is prepared from JM105/pYUK121 (FERM BP-1937) as described in JP-A-2-119778/1990 due to the high degree of homology in the amino acid sequence between the nitrile hydratase β subunit of Rhodococcus sp. N-774 described in said Japanese Patent official gazette and that of Pseudomonaschlororaphis B23. Plasmid pYUK121 containing nitrile hydratase gene derived from Rhodococcus sp. N-774 is prepared from a JM105/pYUK121 culture. pYUK121 DNA is digested with SphI and SalI. The SphI-SalI fragment contains the nitrile hydratase gene (shown in the Sequence Listing as defined by SEQ ID: No. 7) of Rhodococcus sp. N-774. The DNA fragment is radiolabeled to give a probe.

(3)

Detection of a DNA Segment Containing a Nitrile

Hydratase Gene from the Chromosome of

Pseudomonas chlororaphis B 23

0010Chromosomal DNA is prepared from a Pseudomonaschlororaphis B23 culture. Chromosomal DNA is digested with restriction enzymes and hybridized to the probe described in (2), according to the Southern hybridization method [Southern, E.M., J. Mol. Biol. 98, 503 (1975)]. Two DNA fragments of different length are screened.

(4)

Construction of a Recombinant Plasmid

0011A recombinant plasmid is constructed by inserting the chromosomal DNA fragment as prepared in (3) into a plasmid vector.

(5)

Transformation and Screening for a Transformant Containing the Recombinant Plasmid

0012Transformants are prepared using the recombinant plasmid as described in (4). The transformant containing the recombinant plasmid is selected using the probe as described in (2) according to the colony hybridization method [R. Bruce Wallace et al., Nuc. Aci. Res. 9, 879 (1981)]. Additionally, the presence of the nitrile hydratase gene in the recombinant plasmid is confirmed using the Southern hybridization method. The plasmids thus selected are designated as pPCN1 and pPCN3.

(6)

Isolation and Purification of Plasmid DNA and Construction of the Restriction Map

0013Plasmid DNAs of pPCN1 and pPCN3 as described in (5) are isolated and purified. The restriction maps of the DNAs are constructed (Fig. 1) to determine the region containing nitrile hydratase gene.

(7)

DNA Sequencing

0014The extra segment of the inserted DNA fragment in pPCN1 and pPCN3 is excised using an appropriate restriction enzyme. The inserted DNA fragment is then used for sequencing. The nucleotide sequence of the DNA fragment (see the Sequence Listing as defined by SEQ ID: No. 8) reveals that it contains the sequence deduced from the amino acid sequence as described in (1).

(8)

Insertion of the DNA Fragment into an Expression Vector and Transformation

0015The DNA fragment is cleaved from pPCN1 and pPCN3 using appropriate restriction enzymes. These two fragments are ligated and inserted into the expression vector pUC19. The construct is used for transformation of E. coli JM105 (Amersham), and the transformant is designated as JM105/pPCN4.

(9)

Production of Nitrile Hydratase Using the Transformant and Conversion of Nitriles to Amides

0016The transformant as described in (8) is cultured. The bacterial cells are mixed with nitriles, a substrate of nitrile hydratase, and amides are produced.

0017Pseudomonaschlororaphis B23 was deposited with Fermentation Research Institute, Agency of Industrial Science and Technology and was assigned the accession number FERM BP-187. A transformant JM105/pPCN4 was deposited with the same and assigned the accession number FERM BP-2779.

0018Any vector including a plasmid vector (e.g., pAT153, pMP9, pHC624, pKC7, etc.), a phage vector (e.g., λgt11 (Toyobo), Charon 4A (Amersham), etc.) may be used for the present invention. Enzymes which may be used include SphI, SalI, SacI, BamHI, EcoRI, PstI and the like, which are commercially available (Takara Shuzo). Various hosts may be used for transformation including but not limited to E.coli JM105 and TGl. Culture media for the transformant are those ordinarily used in the art.

0019Conversion of nitriles to amides is carried out using nitrile hydratase, crude nitrile hydratase, the culture of the transformant, the isolated bacterial cells or treated matter thereof, and the like, prepared from the culture of the transformant.

0020Suitable nitriles include those having 2-4 carbon atoms such as acetonitrile, propionitrile, acrylonitrile, methacrylonitrile, n-butyronitrile and isobutyronitrile, and acrylonitrile is preferred.

0021Fig. 1 shows restriction maps of recombinant plasmids, pPCN1, pPCN3 and pPCN4.

0022The present invention discloses the amino acid sequence and nucleotide sequence of the α- and β-subunits of nitrile hydratase derived from Pseudomonaschlororaphis B23. The gene encoding nitrile hydratase is inserted into an expression vector and the recombinant vector is used for transformation. The transformant contains multiple copies of the gene and produces a higher level of nitrile hydratase than conventionally used microorganisms.

0023The present invention is illustrated by the following Example.

0024The following abbreviations are used in the Example. <dl id="dl0001"><dt>TE:</dt><dd>Tris-HCl (10 mM; pH 7.8), EDTA (1 mM, pH 8.0)</dd><dt>TNE:</dt><dd>Tris-HCl (50 mM; pH 8.0), EDTA (1 mM, pH 8.0), NaCl (50 mM)</dd><dt>STE:</dt><dd>Tris-HCl (50 mM; pH 8.0), EDTA (5 mM, pH 8.0), Sucrose (35 mM)</dd><dt>2xYT medium:</dt><dd>1.6% Trypton; 1.0% Yeast extract, 0.5% NaCl</dd></dl>

Example

(1)

Isolation and Purification of Nitrile Hydratase and the Amino Acid Sequencing of a Portion of Nitrile Hydratase

0025Pseudomonaschlororaphis B23 was cultured in a medium (10 g/ℓ of sucrose, 4 g/ℓ of methacrylamide, 0.5 g/ℓ of KH₂PO₄, 0.5 g/ℓ of K₂HPO₄, 0.5 g/ℓ of MgSO₄·7H₂O, 0.01 g/ℓ of FeSO₄·7H₂O, pH 7.0) at 25°C for 28 hours. The bacterial cells were harvested. 100 g of the bacterial cells was disrupted and fractionated with ammonium sulfate. The sample was dialyzed and the dialysate was centrifuged. The supernatant was removed and loaded on DEAE-Sephacel chromatography, Octyl-Sepharose CL-4B chromatography, Phenyl-Sepharose CL-4B chromatography and Sephadex G-150 chromatography. Active fractions were collected and dialyzed. The dialysate containing the enzyme was loaded on a high performance liquid chromatography using a reversed phase column (Senshu Pak VP-304-1251, Senshu Kagaku), and two subunits (α and β) were obtained. The N-terminal amino acid sequence of α- and β-subunits was determined using an amino acid sequencer (Applied Biosystems model 470A). N-terminal amino acid sequences of α- and β-subunits are shown in the Sequence Listing as defined by SEQ ID: No. 5 and SEQ ID: No. 6 respectively.

(2)

Preparation of a DNA Probe for Nitrile Hydratase Gene

0026E.coli JM105 containing pYUK121 (FERM BP-1937) was cultured in 100 ml of 2×YT medium containing 50 µg/ml of ampicillin at 30°C overnight (12 hours). The bacterial cells were harvested and TNE was added to the cells. The cell suspension was then centrifuged. 8 ml of STE and 10 mg of lysozyme were added to the pellet. The mixture was incubated at 0°C for five minutes followed by the addition of 4 ml of 0.25M EDTA. 2 ml of 10% SDS and 5 ml of 5M NaCl were then added to the mixture at room temperature. The resultant mixture was left standing at 0-4°C for three hours and then ultracentrifuged. 1/2 volume of 30% PEG 6000 was added to the supernatant. The mixture was allowed to stand at 0-4°C overnight (12 hours) and centrifuged. TNE was added to the pellet to bring the volume to 7.5 ml and CsCl was then added to the suspension. The mixture was centrifuged to remove proteins. Then, 300-500 mg/ml of ethidium bromide was added to the supernatant. The mixture was transferred to a centrifuge tube. The tube was heat-sealed and then ultracentrifuged. cccDNA was extracted using a peristaltic pump. A bit more than equal volume of isopropyl alcohol saturated with water was added to the extract to remove ethidium bromide. The sample was dialyzed against TE. About 3 ml of purified pYUK121 was obtained.

0027pYUK121 DNA was digested with SphI and SalI, resulting in a 2.07 kb DNA fragment containing a nitrile hydratase gene derived from Rhodococcus sp. N-774. The fragment was radiolabeled with ³P to produce a probe. The nucleotide sequence of the probe is shown in the Sequence Listing as defined by SEQ ID: No. 7.

(3)

Preparation of a DNA Fragment Containing a Nitrile Hydratase Gene of Chromosome

0028Pseudomonaschlororaphis B23 was cultured in 100 ml of the medium as described in (1). The bacterial cells were harvested and washed with TNE. The pellet was then suspended in 10 ml of TE. 4 ml of 0.25M EDTA, 10-20 mg of lysozyme, 10-20 mg of achromoprotease and 10 ml of 10% SDS were added to the suspension. The mixture was incubated at 37°C for three hours. 15 ml of phenol was added to the mixture. The mixture was incubated at room temperature for 15 minutes and then centrifuged. To 15 ml of the upper layer, 0.7 ml of 2.5M sodium acetate and diethyl ether were added and the mixture was centrifuged. The upper layer was discarded. Two volumes of ethanol were added to the bottom layer and DNA was removed with a glass rod. DNA was rinsed with TE:ethanol 2:8, 1:9, and 0:10 (v/v) for five minutes each. DNA was then resuspended in 2-4 ml of TE (37°C). 10 µℓ of a mixture of RNase A and T1 was added to the suspension and the mixture was incubated at 30°C . An equal volume of phenol was added to the mixture. The mixture was then centrifuged. After centrifugation 2-4 ml of the upper layer was removed. More than equal amount of ether was added to the removed portion. The mixture was centrifuged. After centrifugation, the upper layer was discarded. The bottom layer was dialyzed overnight against 2 ℓ of TE containing a small amount of chloroform and further dialyzed against fresh TE for 3-4 hours. 4ml of crude chromosomal DNA was obtained. Enzymatic digestion of chromosomal DNA was carried out as follows: <ul id="ul0002" list-style="none"><li>(a) 2 µℓ of SacI + 3 µℓ of reaction buffer (10×) + 15 µℓ of chromosomal DNA + 10 µℓ of TE</li><li>(b) 2 µℓ of BamHI + 2 µℓ of SalI + 3 µℓ of reaction buffer (10×) + 15 µℓ of chromosomal DNA + 8 µℓ of TE</li></ul>

0029The mixtures were incubated at 37°C for an hour and electrophoresed on an agarose gel at 60 V for three hours. The Southern hybridization of chromosomal DNA was carried out using the probe as described in (2). About 4.6 kb and 4.7 kb fragments were found to show a strong hybridization.

003015 µℓ of chromosomal DNA was digested with SacI, and BamHI and SalI and electrophoresed on an agarose gel as described above. 4.6 kb and 4.7 kb DNA fragments were cut out from the gel and placed in three volumes of 8M NaClO₄. The solution was dotted on a GF/C (Whatman) filter paper (6 mm in diameter). Ten drops (≃ 100 µℓ) of TE containing 6M NaClO₄ and then ten drops (≃ 100 µℓ) of 95% ethanol were added to the filter paper. The paper was air-dried and placed in 0.5 ml Eppendorf tube. 40 µℓ of TE was added to the tube and the tube was incubated at 37°C for 30 minutes. The tube was then centrifuged. About 40 µℓ of the supernatant was obtained which contained 4.6 kb and 4.7 kb DNA fragments containing a nitrile hydratase gene of chromosomal DNA.

(4)

Insertion of the Chromosomal DNA Fragment into a Vector

(a) 4.6 kb DNA Fragment

00312 µℓ of SacI, 3 µℓ of reaction buffer (10×) and 10 µℓ of TE were added to 10 µℓ of pUC18 DNA. The mixture was incubated at 37°C for an hour. 2 µℓ of 0.25M EDTA was added to the mixture to stop the reaction. Then 7 µℓ of 1M Tris-HCl (pH 9) and 3 µℓ of BAP (bacterial alkaline phosphatase) were added to the mixture. The mixture was incubated at 65°C for an hour. TE was then added to the mixture to bring the volume to 100 µℓ. The mixture was extracted 3× with an equal volume of phenol. An equal volume of ether was added to the extract. The bottom layer was removed and 10 µℓ of 3M sodium acetate and 250 µℓ of ethanol were added to the bottom layer. The mixture was incubated at -80°C for 30 minutes, centrifuged, dried, and resuspended in TE.

00325 µℓ of pUC18 DNA thus obtained and 40 µℓ of the 4.6 kb DNA fragment as described in (3) were mixed. 6 µℓ of ligation buffer, 6 µℓ of ATP (6 mg/ml) and 3 µℓ of T4 DNA ligase were added to the mixture. The mixture was incubated at 4°C overnight (12 hours) to produce the recombinant plasmid containing the 4.6 kb DNA fragment in the SacI site of pUC18.

(b) 4.7 kb DNA Fragment

0033pUC18 DNA was digested with BamHI and SalI. The 4.7 kb DNA fragment was inserted into the BamHI-SalI site of pUC18 in a similar manner as described in (4)-(a). The recombinant plasmid containing the 4.7 kb DNA fragment in the BamHI-SalI site of pUC18 was obtained.

(5)

Transformation and Screening of Transformants

0034E.coli JM105 (Amersham) was inoculated into 10 ml of 2xYT medium and incubated at 37°C for 12 hours. After incubation, the culture was added to a fresh 2×YT medium to a final concentration of 1%, and the mixture was incubated at 37°C for two hours. The culture was centrifuged and the pellet was suspended in 5 ml of cold 50 mM CaCl₂. The suspension was placed on 0°C for 40 minutes and then centrifuged. 0.25 ml of cold 50 mM CaCl₂ and 60 µℓ each of the recombinant plasmids as prepared in (4)(a) and (b) were added to the pellet in a separate tube. The mixture was incubated on ice for 40 minutes, heat-shocked at 42°C for two minutes, placed on ice for five minutes, and added to 10 ml of 2×YT medium. The mixture was incubated at 37°C for 90 minutes with shaking, then centrifuged. The pellet was suspended in 1 ml of 2×YT medium, and 10 µℓ of the suspension was plated on a 2×YT agar plate containing 50 µg/ml of ampicillin. The plate was incubated at 37°C. The colony grown on the plate was selected by the colony hybridization method: The colony was transferred to a nitrocellulose filter and lysed. The DNA fixed on the filter was hybridized to the probe as described in (2). The filter was autoradiographed and a recombinant colony was selected. Additionally, the presence of a nitrile hydratase gene in the transformant was confirmed according to the Southern hybridization method.

(6)

Isolation and Purification of Recombinant Plasmid and Construction of the Restriction Map of the Inserted DNA Fragments

0035The transformant selected as described in (5) was grown in 100 ml of a 2xYT medium containing 50 µg/ml of ampicillin at 37°C overnight (12 hours). The bacterial cells were harvested and TNE was added to the cells. The cells were collected again by centrifugation, and 8 ml of STE and 10 mg of lysozyme were added to the cells. The mixture was incubated at 0°C for five minutes. 4 ml of 0.25M EDTA, 2 ml of 10% SDS (at room temperature) and 5 ml of 5M NaCl were added to the mixture. The mixture was incubated at 0-4°C for three hours, and ultracentrifuged. 1/2 volume of 30% PEG 6000 was added to the supernatant. The mixture was incubated at 0-4°C overnight (12 hours) and centrifuged again. TNE was added to the pellet to bring the volume up to 7.5 ml. CsCl was added to the mixture. The mixture was centrifuged to rid of proteins. Then, 300-500 mg/ml of ethidium bromide was added to the supernatant and the mixture was transferred to a centrifuge tube. The tube was heat-sealed and ultracentrifuged. cccDNA was removed using a peristaltic pump. A bit more than equal volume of isopropyl alcohol saturated with water was added to cccDNA to remove ethidium bromide. The DNA sample was dialyzed against TE, resulting in about 3 ml of recombinant DNA. The recombinant plasmid containing a 4.6 kb DNA fragment was designated as pPCN1 (The recombinant plasmid containing a 4.7 kb DNA fragment was designated as pPCN3).

0036These plasmid DNAs were digested with EcoRI, BamHI, PstI, SacI and SalI. The restriction maps were constructed and are shown in Fig. 1.

(7)

DNA Sequencing

0037The location of a nitrile hydratase gene in the DNA fragments of pPCN1 and pPCN3 was determined according to the restriction map constructed and to the Southern hybridization method. Based on the results, the 2.456 kb BamHI-HincII DNA fragment was sequenced by the Sanger method [Sanger, F., Science 214: 1205-1210 (1981)] using M13 phage vector. The 2.456 kb DNA fragment from Pseudomonaschlororaphis B23 is shown in the Sequence Listing as defined by SEQ ID: No. 8.

0038All the nucleotide sequence deduced from the amino acid sequence as determined in (1) was found in the sequence as determined above. The sequence analysis also revealed that the DNA fragment contained the sequence coding for the α- and β-subunits.

(8)

Insertion of the BamHI-HincII DNA Fragment into an Expression Vector and Transformation

00392.2 kb SphI-BamHI fragment of pPCN1 and 4.7 kb BamHI-SalI fragment of pPCN3 were cleaved and both fragments were ligated (Fig. 1). The ligated fragment was inserted into the SphI-SalI site of an expression vector pUC19 and the construct was designated as pPCN4.

0040E.coli JM105 was transformed with pPCN4 in a similar manner as in (5) and the transformant was designated as JM105/pPCN4 (FERM BP-2779).

(9)

Production of Nitrile Hydratase Using the Transformant and Conversion of Nitriles to Amides Using Nitrile Hydratase

0041JM105/pPCN4 was inoculated into 10 ml of 2×YT medium containing 50 µg/ml of ampicillin and incubated at 26.5°C overnight (12 hours). 100 µℓ of the resultant culture was added to 10 ml of 2×YT medium (50 µg/ml of ampicillin, 50 mg/ℓ of FeSO₄·7H₂O, 10 mg/ℓ of MgSO₄·7H₂O, 1 mg/ℓ of pyrroloquinolinequinone). The mixture was incubated at 26.5°C for five hours. IPTG was added to the mixture to a final concentration of 1 mM. The mixture was incubated at 26.5°C for 10 hours. After harvesting the cells, the cells were suspended in 5 ml of 1/20 M phosphate buffer (pH 7.7). 10 µℓ of the substrate solution (1M acrylonitrile) was added to 0.1 ml of the suspension. The mixture was incubated at 20°C for 20 minutes. As a control, the mixture obtained by the same procedure as described above but from E. coli JM105 was used. The reaction mixture was tested for the presences of acrylamide (the product of the enzymatic reaction) and acrylonitrile using HPLC. Acrylamide but no acrylonitrile was found in the reaction mixture of JM105/pPCN4, whereas acrylonitrile but no acrylamide was found in the reaction mixture of JM105.

(1)

INFORMATION FOR SEQ ID NO: 1

0042<ul id="ul0003" list-style="none"><li>(i) SEQUENCE CHARACTERISTICS: <ul id="ul0004" list-style="none"><li>(A) LENGTH: 200 amino acids</li><li>(B) TYPE: Amino acid</li><li>(C) STRANDEDNESS: -</li><li>(D) TOPOLOGY: Linear</li></ul></li><li>(ii) MOLECULE TYPE: Peptide</li><li>(vi) ORIGINAL SOURCE <ul id="ul0005" list-style="none"><li>(A) ORGANISM: Pseudomonas chlororaphis</li><li>(B) STRAIN: B23 (FERM BP-187)</li></ul></li><li>(ix) FEATURES <ul id="ul0006" list-style="none"><li>(A) OTHER INFORMATION α-subunit</li></ul></li><li>(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1<img file="EP0444639B1_D0001.tif" /><img file="EP0444639B1_D0002.tif" /></li></ul>

(2)

INFORMATION FOR SEQ ID NO: 2

0043<ul id="ul0007" list-style="none"><li>(i) SEQUENCE CHARACTERISTICS: <ul id="ul0008" list-style="none"><li>(A) LENGTH: 220 amino acids</li><li>(B) TYPE: Amino acid</li><li>(C) STRANDEDNESS: -</li><li>(D) TOPOLOGY: Linear</li></ul></li><li>(ii) MOLECULE TYPE: Peptide</li><li>(vi) ORIGINAL SOURCE <ul id="ul0009" list-style="none"><li>(A) ORGANISM: Pseudomonas chlororaphis</li><li>(B) STRAIN: B23 (FERM BP-187)</li></ul></li><li>(ix) FEATURES <ul id="ul0010" list-style="none"><li>(A) OTHER INFORMATION β-subunit</li></ul></li><li>(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2<img file="EP0444639B1_D0003.tif" /><img file="EP0444639B1_D0004.tif" /></li></ul>

(3)

INFORMATION FOR SEQ ID NO: 3

0044<ul id="ul0011" list-style="none"><li>(i) SEQUENCE CHARACTERISTICS: <ul id="ul0012" list-style="none"><li>(A) LENGTH: 600 base pairs</li><li>(B) TYPE: Nucleic acid</li><li>(C) STRANDEDNESS: Single</li><li>(D) TOPOLOGY: Linear</li></ul></li><li>(ii) MOLECULE TYPE: Genomic DNA</li><li>(vi) ORIGINAL SOURCE <ul id="ul0013" list-style="none"><li>(A) ORGANISM: Pseudomonas chlororaphis</li><li>(B) STRAIN: B23 (FERM BP-187)</li></ul></li><li>(ix) FEATURES <ul id="ul0014" list-style="none"><li>(A) OTHER INFORMATION α-subunit</li></ul></li><li>(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3<img file="EP0444639B1_D0005.tif" /></li></ul>

(4)

INFORMATION FOR SEQ ID NO: 4

0045<ul id="ul0015" list-style="none"><li>(i) SEQUENCE CHARACTERISTICS: <ul id="ul0016" list-style="none"><li>(A) LENGTH: 660 base pairs</li><li>(B) TYPE: Nucleic acid</li><li>(C) STRANDEDNESS: Single</li><li>(D) TOPOLOGY: Linear</li></ul></li><li>(ii) MOLECULE TYPE: Genomic DNA</li><li>(vi) ORIGINAL SOURCE <ul id="ul0017" list-style="none"><li>(A) ORGANISM: Pseudomonas chlororaphis</li><li>(B) STRAIN: B23 (FERM BP-187)</li></ul></li><li>(ix) FEATURES <ul id="ul0018" list-style="none"><li>(A) OTHER INFORMATION β-subunit</li></ul></li><li>(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4<img file="EP0444639B1_D0006.tif" /></li></ul>

(5)

INFORMATION FOR SEQ ID NO: 5

0046<ul id="ul0019" list-style="none"><li>(i) SEQUENCE CHARACTERISTICS: <ul id="ul0020" list-style="none"><li>(A) LENGTH: 25 amino acids</li><li>(B) TYPE: Amino acid</li><li>(C) STRANDEDNESS: -</li><li>(D) TOPOLOGY: Linear</li></ul></li><li>(ii) MOLECULE TYPE: Peptide</li><li>(vi) ORIGINAL SOURCE <ul id="ul0021" list-style="none"><li>(A) ORGANISM: Pseudomonas chlororaphis</li><li>(B) STRAIN: B23 (FERM BP-187)</li></ul></li><li>(ix) FEATURES <ul id="ul0022" list-style="none"><li>(A) OTHER INFORMATION α-subunit: α₁</li></ul></li><li>(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5<img file="EP0444639B1_D0007.tif" /></li></ul>

(6)

INFORMATION FOR SEQ ID NO: 6

0047<ul id="ul0023" list-style="none"><li>(i) SEQUENCE CHARACTERISTICS: <ul id="ul0024" list-style="none"><li>(A) LENGTH: 23 amino acids</li><li>(B) TYPE: Amino acid</li><li>(C) STRANDEDNESS: -</li><li>(D) TOPOLOGY: Linear</li></ul></li><li>(ii) MOLECULE TYPE: Peptide</li><li>(vi) ORIGINAL SOURCE <ul id="ul0025" list-style="none"><li>(A) ORGANISM: Pseudomonas chlororaphis</li><li>(B) STRAIN: B23 (FERM BP-187)</li></ul></li><li>(ix) FEATURES <ul id="ul0026" list-style="none"><li>(A) OTHER INFORMATION β-subunit: β₁</li></ul></li><li>(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6<img file="EP0444639B1_D0008.tif" /></li></ul>

(7)

INFORMATION FOR SEQ ID NO: 7

0048<ul id="ul0027" list-style="none"><li>(i) SEQUENCE CHARACTERISTICS: <ul id="ul0028" list-style="none"><li>(A) LENGTH: 2070 base pairs</li><li>(B) TYPE: Nucleic acid</li><li>(C) STRANDEDNESS: Single</li><li>(D) TOPOLOGY: Linear</li></ul></li><li>(ii) MOLECULE TYPE: Genomic DNA</li><li>(vi) ORIGINAL SOURCE <ul id="ul0029" list-style="none"><li>(A) ORGANISM: Rhodococcus</li><li>(B) STRAIN: sp. N-774 (FERM BP-1936)</li></ul></li><li>(ix) FEATURES from nucleotide No. 675 to 1295: subunit α from nucleotide No. 1325 to 1960: subunit β</li><li>(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7<img file="EP0444639B1_D0009.tif" /><img file="EP0444639B1_D0010.tif" /><img file="EP0444639B1_D0011.tif" /><img file="EP0444639B1_D0012.tif" /></li></ul>

(8)

INFORMATION FOR SEQ ID NO: 8

0049<ul id="ul0030" list-style="none"><li>(i) SEQUENCE CHARACTERISTICS: <ul id="ul0031" list-style="none"><li>(A) LENGTH: 2456 base pairs</li><li>(B) TYPE: Nucleic acid</li><li>(C) STRANDEDNESS: Single</li><li>(D) TOPOLOGY: Linear</li></ul></li><li>(ii) MOLECULE TYPE: Genomic DNA</li><li>(vi) ORIGINAL SOURCE <ul id="ul0032" list-style="none"><li>(A) ORGANISM: Pseudomonas chlororaphis</li><li>(B) STRAIN: B23 (FERM BP-187)</li></ul></li><li>(ix) FEATURES from nucleotide No. 1021 to 1620: subunit a from nucleotide No. 1666 to 2325: subunit β</li><li>(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8<img file="EP0444639B1_D0013.tif" /><img file="EP0444639B1_D0014.tif" /><img file="EP0444639B1_D0015.tif" /><img file="EP0444639B1_D0016.tif" /></li></ul>

Contents8

Every citation, both ways

Citations
Document	Relation	Office	Cited during
US4637982A	Cites	United States of America	Examiner
EP0093782A	Cites	European Patent Office (EPO)	–
FR2633938A	Cites	France	–
US4637982A	Cites	United States of America	–
M. Kobayashi et al., TIBTECH NOVEMBER 1992, vol. 10, pp. 402-408	Non-patent	–	Examiner
EUROPEAN JOURNAL OF BIOCHEMISTRY vol. 162, no. 3, February 1987, BERLIN, DE pages 691 - 698; NAGASAWA, T. ET AL.: 'Nitrile hydratase of Pseudomonas chlororaphis B23; purification and characterization'	Non-patent	–	–
AGRICULTURAL AND BIOLOGICAL CHEMISTRY. vol. 46, no. 5, May 1982, TOKYO JP pages 1183 - 1189; ASANO, Y. ET AL.: 'A new enzymatic method of acrylamide production'	Non-patent	–	–
AGRICULTURAL AND BIOLOGICAL CHEMISTRY. vol. 50, no. 11, November 1986, TOKYO JP pages 2859 - 2865; YAMADA, H. ET AL.: 'Optimum culture conditions for production by Pseudomonas chlororaphis B23 of nitrile hydratase'	Non-patent	–	–
M. Kobayashi et al., TIBTECH NOVEMBER 1992, vol. 10, pp. 402-408	Non-patent	–	–

17 members in 8 offices; this record represents the family

This recordMember, by publication datePriority claim

Priority claims4

Priority claims
Document	Office	Kind	Date
4807990	Japan	A	1990-02-28
4807990	Japan	–	1990-02-28
JP19900048079	–	–	–
4807990	–	–	–

Members17

Family members, oldest first
Document	Office	Kind
EP0444639A2	European Patent Office (EPO)	A2	1991-09-04
CN1054614A	China	A	1991-09-18
AU7129591A	Australia	A	1991-09-26
JPH03251184A	Japan	A	1991-11-08
KR910021479A	Republic of Korea	A	1991-12-20
EP0444639A3	European Patent Office (EPO)	A3	1992-07-08
AU636008B2	Australia	B2	1993-04-08
KR950000888B1	Republic of Korea	B1	1995-02-03
RU2028380C1	Russian Federation	C1	1995-02-09
EP0444639B1This record	European Patent Office (EPO)	B1	1996-01-31
DE69116704D1	Germany	D1	1996-03-14
DE69116704T2	Germany	T2	1996-08-08
RU2082761C1	Russian Federation	C1	1997-06-27
US5648256A	United States of America	A	1997-07-15
US5789211A	United States of America	A	1998-08-04
JP2907479B2	Japan	B2	1999-06-21
CN1052509C	China	C	2000-05-17

24 legal events, as 4 offices reported them to INPADOC

Over the term

Point at a mark for the event

Events

Legal events, newest first
	Event	Code	Office
2013-06-28	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2011-07-29	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2011-03-23	Patent expired after termination of 20 yearsExpiredPE20	PE20	GB
2011-02-27	Expiry of rightR071	R071	DE
2010-06-30	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2010-06-30	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2010-05-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2002-01-01	European patent in force as of 2002-01-01IF02	IF02	GB
1999-06-04	Transmission of propertyTP	TP	FR
1998-12-30	Amendments to the register in respect of changes of name or changes affecting rights (sect. 32/1977)732E	732E	GB
1997-01-22	No opposition filedOpposition26N	26N	EP
1996-12-06	No opposition filed within time limitOppositionORIGINAL CODE: 0009261PLBE	PLBE	EP
1996-12-06	Information on the status of an ep patent application or granted ep patentGrantedSTATUS: NO OPPOSITION FILED WITHIN TIME LIMITSTAA	STAA	EP
1996-03-15	Fr: translation filedET	ET	EP
1996-03-14	Corresponds to:REF	REF	EP
1996-01-31	Designated contracting statesAK	AK	EP
1996-01-04	(expected) grantORIGINAL CODE: 0009210GRAA	GRAA	EP
1994-06-29	First examination report despatched17Q	17Q	EP
1992-11-19	Request for examination filed17P	17P	EP
1992-07-08	Designated contracting statesAK	AK	EP
1992-05-16	Search report despatchedORIGINAL CODE: 0009013PUAL	PUAL	EP
1992-03-04	Designated contracting states (corrected)RBV	RBV	EP
1991-09-04	Designated contracting statesAK	AK	EP
1991-07-18	Public reference made under article 153(3) epc to a published international application that has entered the european phaseORIGINAL CODE: 0009012PUAI	PUAI	EP