EP0719862A2

A regulatory factor for expression of nitrilase gene and a gene thereof

Abstract

The invention relates to a two component regulatory factor which activates a nitrilase gene promoter, comprising a polypeptide having the amino acid sequence of SEQ ID No: 1 and a polypeptide having the amino acid sequence of SEQ ID No: 2. Nitrilase can be produced by introducing the DNA coding for the regulatory factor together with a nitrilase gene containing a promoter region into a microorganism of the genus Rhodococcus.

EP0719862A2, drawing sheet 1 — Sheet 1 of 20

Term

Term ended

Projected expiry passed 27 December 2015, 10.7 years ago.

8 claims: 1 independent, 7 dependent

1
A two component regulatory factor which activates a nitrilase gene promoter, comprising a polypeptide having the amino acid sequence of SEQ ID No:1 and a polypeptide having the amino acid sequence of SEQ ID No: 2.

17 paragraphs in 7 sections, as filed

FIELD OF THE INVENTION

The present invention relates to a regulatory factor involved in expression of a nitrilase gene and a DNA coding for the same and particularly to a regulatory factor derived from the strain Rhodococcus erythropolis SK92 and activating a nitrilase gene promoter, as well as to DNAs coding for the same, a recombinant plasmid containing the DNAs and a transformant transformed with said recombinant plasmid.

BACKGROUND OF THE INVENTION

As known processes of producing organic acids by conversion from their corresponding nitriles, mention may be made of chemical synthetic means and biological means. The latter involves the use of a microorganism or a microorganism-derived enzyme as a catalyst to hydrolyze nitriles, so this means is advantageous in that organic acids can be produced under mild conditions. Microorganisms belonging to the genus Rhodococcus are known as such catalysts for use in production of amides or organic acids by hydration or hydrolysis of their corresponding nitriles (see Japanese Laid-Open Patent Publication Nos. 251,192/1991, 91,189/1987, 470/1990, and 84,198/1990).

As compared with the above-mentioned conventional processes, the use of a nitrilase gene cloned for hydrolysis of nitriles by genetic recombination is expected to drastically improve the catalytic ability of the microorganism to hydrate nitriles because the microorganism can be engineered to contain multiple copies of the same gene. To obtain such a catalyst organism with higher catalytic activity, the present inventors successfully cloned a nitrilase gene from the strain Rhodococcuserythropolis SK92 and constructed a plasmid by inserting said gene into a region downstream of an E. coli lactose promoter. By introducing this plasmid into E. coli, the organism came to exhibit higher nitrilase activity during incubation in the presence of IPTG (isopropyl-β-D-thiogalactoside). The present inventors further attempted to obtain a transformant of the genus Rhodococcus to attain higher performance as a catalyst organism. In this attempt, the nitrilase gene was inserted into a Rhodococcus-E. coli hybrid plasmid vector (see Japanese Laid-Open Patent Publication Nos. 64,589/1993 and 68,566/1993), and the vector thus constructed was introduced into a microorganism of the genus Rhodococcus. However, no nitrilase activity was expressed, and there is demand for a method of permitting the expression of nitrilase activity in a transformant of the genus Rhodococcus.

SUMMARY OF THE INVENTION

The present inventors speculated that the gene derived from the genus Rhodococcus is not expressed because the promoter for the nitrilase gene fails to function, and that a gene coding for a regulatory factor that allows the promoter to function might be present somewhere on the chromosomal DNA derived from SK92. Through screening, the present inventors found it in a region upstream of the nitrilase structural gene and succeeded thereby in expression of nitrilase activity in a transformant of the genus Rhodococcus.

That is, the present invention relates to a regulatory factor consisting of 2 components i.e. a polypeptide having the amino acid sequence of SEQ ID No: 1 and a polypeptide having the amino acid sequence of SEQ ID No: 2 to activate the nitrilase gene promoter, as well as to DNAs coding for them.

Introduction of the gene coding for the regulatory factor of the invention along with the nitrilase gene containing its promoter permits a microorganism of the genus Rhodococcus to produce nitrilase.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows a schematic drawing of deletion plasmids, where the arrows on the DNA fragment from SK92 indicate the location and direction of the gene coding for the regulatory factor of the invention and the gene coding for nitrilase, respectively.

Fig. 2 shows a restriction enzyme map of recombinant plasmid pSK108.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention is described in detail. The present invention is practiced in the following steps. <ul id="ul0001" list-style="none" compact="compact"><li>(1) Preparation of chromosomal DNA from the strain SK92: Chromosomal DNA is isolated from Rhodococcuserythropolis SK92. </li><li>(2) Construction of a DNA Library: The chromosomal DNA is cleaved with restriction enzymes, and a DNA fragment containing the target gene is detected by Southern hybridization using the nitrilase gene of SK92 as probe. This fragment is inserted into a hybrid plasmid vector capable of replicating in cells of E.coli and the genus Rhodococcus to prepare a library. </li><li>(3) Transformation of E. coli and selection of recombinant DNA: The recombinant library constructed in step (2) is used to prepare transformants. They are subjected to colony hybridization using the probe obtained in step (2) to select a colony carrying the target recombinant DNA. </li><li>(4) Preparation of recombinant plasmid: A plasmid is prepared from the recombinant obtained in step (3). </li><li>(5) Transformation of a microorganism of the genus Rhodococcus and the nitrilase activity of the transformant: The resulting plasmid is introduced into a microorganism of the genus Rhodococcus, and its nitrilase activity is determined. </li><li>(6) Deletion plasmids and nitrilase activity: Deletion plasmids are prepared by deleting various regions from the plasmid obtained in step (4) to identify the region essential for expression of the nitrilase structural gene. The plasmids prepared are not necessary to be capable of replicating in E. coli and are sufficient if they include a DNA region capable of replicating in cells of the genus Rhodococcus. </li><li>(7) Nucleotide sequencing: The nucleotide sequence of the region identified in step (6) is determined. </li></ul>

As the above hybrid plasmid vector, mention may be made of pK1, pK2, pK3 and pK4. These plasmids were introduced into R.rhodochrous ATCC 12674 and have been deposited respectively as R.rhodochrous ATCC 12674/pK1 (FERM BP-3728), R.rhodochrous ATCC 12674/pK2 (FERM BP-3729), R.rhodochrous ATCC 12674/pK3 (FERM BP-3730) and R.rhodochrous ATCC 12674/pK4 (FERM BP-3731) with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan (see Japanese Laid-Open Patent Publication No. 68,556/1993).

As the above DNA region capable of replicating in cells of the genus Rhodococcus, mention may be made of those derived from plasmids pRC001, pRC002, pRC003 and pRC004, and these may be the whole of the plasmid or a partial fragment thereof. The above plasmids are derived respectively from the strains R.rhodochrous ATCC 4276, ATCC 14349, ATCC 14348 and IFO 3338 (see Japanese Laid-Open Patent Publication No. 68,556/1993).

Rhodococcuserythropolis SK92 has been deposited as FERM BP-3324 with the Fermentation Research Institute, Agency of Industrial Science and Technology. Plasmid pSK108 containing the nitrilase gene and the regulatory gene has been deposited as transformant JM109/pSK108 (FERM BP-5322) carrying said plasmid pSK108, with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology. The strain SK92 was previously identified as belonging to the genus Rhodococcus on the basis of its bacterial properties (see Japanese Laid-Open Patent Publication No. 280,889/1991). This organism is further identified as Rhodococcuserythropolis on the basis of the following detailed properties: <tables id="tabl0001" num="0001"><img file="EP0719862A2_D0001.tif" /></tables><tables id="tabl0002" num="0002"><img file="EP0719862A2_D0002.tif" /></tables>

EXAMPLES

Hereinafter, the present invention will be illustrated in detail by reference to the following examples which however are not intended to limit the scope of the invention.

Cloning of the nitrilase gene from SK92 and the expression thereof in E. coli and Rhodococcus will be further illustrated in Reference Example. (1) Preparation of chromosomal DNA from SK92 The strain SK92 was incubated at 30 °C for 72 hours under shaking in 100 ml MY medium (0.5 % polypeptone, 0.3 % Bacto-yeast extract, 0.3 % Bacto-molt extract). The cells were harvested and the pellet was suspended in 4 ml Saline-EDTA solution (0.1 M EDTA, 0.15 M NaCl, pH 8.0). 8 mg of lysozyme was added to the suspension. The suspension was incubated at 37 °C for 1 to 2 hours under shaking and then frozen. Then, 10 ml of Tris-SDS solution (1 % SDS, 0.1 M NaCl, 0.1 M Tris, pH 9.0) was added to it under gentle shaking, followed by addition of proteinase K (Merk) at a final concentration of 0.1 mg. The mixture was incubated under shaking at 37 °C for 1 hour and then at 60 °C . An equal amount of phenol saturated with TE (TE: 10 mM Tris, 1 mM EDTA, pH 8.0) was added to the mixture, stirred, and centrifuged. A 2-fold excess amount of ethanol was added to the upper layer, and the DNA was recovered using a glass rod. The phenol was removed successively with 90 %, 80 % and 70 % ethanol. Then, the DNA was dissolved in 3 ml TE buffer, and a solution of ribonuclease A (previously treated by heating at 100 °C for 15 min.) was added to it in an amount of 10 µ g/ml. The mixture was incubated at 37 °C for 30 minutes under shaking, followed by addition of proteinase K. The mixture was incubated at 37 °C for 30 minutes under shaking. An equal amount of TE-saturated phenol was added to the mixture, and it was separated by centrifugation into upper and lower layers. The upper layer was subjected twice to the same procedure, followed by the same procedure of extraction with an equal amount of chloroform containing 4 % isoamyl alcohol (these procedures are referred to hereinafter as phenol treatment). Then, a 2-fold excess amount of ethanol was added to the upper layer and the DNA was recovered with a glass rod whereby the chromosomal DNA was obtained. (2) Construction of a DNA library 10 µ l plasmid pSK002 prepared by inserting into vector pUC118 a DNA fragment containing the nitrilase gene from the strain SK92 (see Reference Example) was cleaved at 37 °C for 2 hours with a mixture of 2 µ l of restriction enzyme Sac I, 10µ l of the reaction buffer (10-fold conc.), and 78 µ l of sterilized water, and the digest was electrophoresed on 0.7 % agarose gel to separate an Sal I fragment, 1.1 kb long. Separately, the chromosomal DNA from SK92 obtained in step (1) was digested with Eco RI, electrophoresed on agarose gel and subjected to Southern hybridization where the above 1.1 kb Sac I fragment, labeled using a DIG DNA Labeling Kit (Boehringer Mannheim), was used as the probe (Southern E.M., Mol. Bionl. 98, 503 (1975)) to detect an about 14 kb DNA fragment. A DNA fraction containing the 14 kb fragment hybridized with the probe was cut off from the agarose gel and then inserted into a separately prepared Eco RI-cleaved hybrid plasmid vector pK4 (FERM BP-3731 containing plasmid pRC004 from the genus Rhodococcus and vector pHSG299 from E. coli (see Japanese Laid-Open Patent Publication Nos. 64,589/1993 and 68,566/1993)). The above pK4 fragment used as vector was prepared as follows: 10 µ l of the reaction buffer (10-fold conc.), 77 µ l of sterilized water and 2 µ l of restriction enzyme Eco RI were added to 10 µ l of vector pK4. The mixture was allowed to react at 37 °C for 2 hours, then treated with phenol, precipitated with ethanol, dried, and dissolved in 50 µ l sterilized water. 1 µ l of alkaline phosphatase (Takara Shuzo Co., Ltd.), 10 µ l of the reaction buffer (10-fold conc.) and 39 µ l of sterilized water were added to it. The mixture was allowed to react at 65 °C, treated with phenol, precipitated with ethanol, dried, and dissolved in sterilized water. As described above, 1 µ l of the above DNA fraction containing the 14 kb fragment was inserted into the above Eco RI-cleaved pK4 by overnight reaction at 4°C using a ligation kit (Takara Shuzo Co., Ltd.) to prepare a DNA library. (3) Transformation of E. coli and selection of recombinant DNA E. coli JM109 (available from Takara Shuzo Co., Ltd.) was inoculated into 1 ml of LB medium (1 % Bacto-trypton extract, 0.5 % Bacto-yeast extract, 0.5 % NaCl) and pre-incubated at 37 °C for 5 hours. 100 µ l of the culture was inoculated into 50 ml of SOB medium (2 % Bacto-trypton, 0.5 % Bacto-yeast extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO4, 1 mM MgCl2) and incubated at 18 °C for 20 hours. The cells were recovered by centrifugation, and the pellet was suspended in 13 ml cold TF solution (20 mM PIPES-KOH, pH 6.0, 200 mM KCl, 10 mM CaCl2, 40 mM MnCl2 ), allowed to stand at 0 °C for 10 minutes and centrifuged again. After the supernatant was removed, the E. coli pellet was suspended in 3.2 ml of cold TF solution, followed by addition of 0.22 ml dimethyl sulfoxide. The suspension was allowed to stand at 0 °C for 10 minutes. 10 µ l of the recombinant plasmid (DNA library) prepared in step (2) was added to 200 µ l of the competent cells thus prepared. The mixture was incubated at 0 °C for 30 minutes, then heat-shocked at 42 °C for 30 seconds and cooled at 0 °C for 2 minutes, followed by addition of 0.8 ml of SOC medium (2 % Bacto-trypton, 0.5 % Bacto-yeast extract, 20 mM glucose, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO4, 1 mM MgCl2 ). The mixture was incubated at 37 °C for 60 minutes under shaking. The culture was plated in an amount of 200 µ l per plate on LB agar medium containing 100 µ g/ml ampicillin. The plate was incubated at 37 °C . Selection of transformants carrying the nitrilase gene from the colonies grown on the plate was carried out by colony hybridization in the following manner. The colonies grown on the plate were transferred to a nylon membrane (Biodyne A produced by Nippon Paul) and the microorganisms were lysed. The DNA was fixed on the membrane and then hybridized with the probe (1.1 kb fragment) constructed in step (2), and the colony containing the target recombinant DNA was selected using a DIG Luminescent Detection Kit (Boehringer Mannheim). (4) Preparation of recombinant plasmid The transformant selected in step (3) was incubated at 37 °C overnight in 100 ml of LB medium, and the cells were harvested and washed with sterilized water. 5 ml of solution I (2 mM glucose, 10 mM EDTA, 25mM Tris-HCl buffer, pH 8.0) and 25 mg lysozyme were added to the cells. It was allowed to stand at 0 °C for 30 minutes. 10 ml of solution II (1 N NaOH, 5 % SDS) was added thereto, and the mixture was allowed to stand at 0 °C for 5 minutes. 7.5 ml of solution III (3 M sodium acetate, pH 4.8) was added thereto, and the mixture was allowed to stand at 0 °C for 30 minutes and centrifuged. 50 ml ethanol was added to the supernatant. It was centrifuged again to remove the supernatant. 5 ml of solution IV (10 mM sodium acetate, 50 mM Tris-HCl buffer, pH 8.0) and 2.5 µ l of 10 mg/ml ribonuclease A were added thereto. The mixture was allowed to stand at room temperature for 20 minutes, followed by addition of 12 ml ethanol. It was centrifuged, dried, and dissolved in sterilized water. (5) Transformation of a microorganism of the genus Rhodococcus, and the nitrilase activity of the transformant Rhodococcusrhodochrous ATCC 12674 at the logarithmic growth phase was harvested by centrifugation, washed 3 times with ice-cold sterilized water and suspended in sterilized water. 1 µg of plasmid pSK104 obtained in step (4) was mixed with 10 µ l of the cell suspension, and the mixture was cooled on ice. This mixture of the DNA and the microorganism was introduced into the chamber in a electroporation apparatus CET-200 (Japan Spectroscopic Co., Ltd.), and the sample was pulsed 20 times with a density of electric field of 3.8 kV/cm and a pulse width of 1 ms. The cell suspension thus treated was placed on ice for 10 minutes and heat-shocked at 37 °C for 10 minutes. 500 µ l of MYK medium (0.5 % polypeptone, 0.3 % Bacto-molt extract, 0.3 % Bacto-yeast extract, 0.2 % KH2 PO4, 0.2 % K2 HPO4 (pH 7.0)) was added thereto. The cell suspension was then incubated at 26 °C for 3 hours under shaking. The suspension was plated on an MYK agar plate containing 75 µg/ml kanamycin and incubated at 26 °C for 3 days. The resultant transformant of the genus Rhodococcus was inoculated into 10 ml MYK medium containing 50 µ g/ml kanamycin and pre-incubated at 30 °C for 24 hours. 1 ml of the culture was added to 100 ml of GGP medium (1.5 % glucose, 0.1 % Bacto-yeast extract, 1.0 % sodium glutamate, 0.05 % KH2PO4, 0.05 % K2HPO4, 0.05 % MgSO4 7H2O (pH 7.2)) containing 1.5 % ethylene cyanohydrin (ECH) as inducer and 75 µ g/ml kanamycin. The microorganism was incubated at 30 °C for 48 hours and harvested, and the pellet was suspended in 50 mM phosphate buffer, pH 7.7, and a part of the suspension was allowed to react at 30 °C for 20 minutes in 50 mM phosphate buffer, pH 7.7, containing 100 mM acrylonitrile. The reaction was stopped by addition of 1 N HCl, and the amount of acrylic acid formed in the reaction solution was determined by high performance liquid chromatography (HPLC). The result indicated the formation of 8 mM acrylic acid in the transformant ATCC 12674/pSK104. It was revealed that the gene coding for the regulatory factor necessary for expression of nitrilase is present upstream or downstream of the structural gene of nitrilase. (6) Deletion plasmids and nitrilase activity Because pSK104 was estimated to still contain a number of regions not required for expressing nitrilase, various deletion plasmids were prepared therefrom. Microorganisms transformed with the deletion plasmids were examined for their nitrilase activity (Table 1, Fig. 1). <tables id="tabl0003" num="0003"><table frame="all"><title>Table 1.</title><tgroup cols="3" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col3" align="center">Deletion plasmids and formation of acrylic acid</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col3" align="center">amount of formed acrylic acid (mM)</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col3" align="center">inducer (ECH)</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">absent</entry><entry namest="col3" nameend="col3" align="center">present</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="right">1) pSK102</entry><entry namest="col2" nameend="col2" align="char" char=".">0</entry><entry namest="col3" nameend="col3" align="char" char=".">0</entry></row><row><entry namest="col1" nameend="col1" align="right">2) pSK104</entry><entry namest="col2" nameend="col2" align="char" char=".">0.77</entry><entry namest="col3" nameend="col3" align="char" char=".">8.00</entry></row><row><entry namest="col1" nameend="col1" align="right">3) pSK105</entry><entry namest="col2" nameend="col2" align="char" char=".">0</entry><entry namest="col3" nameend="col3" align="char" char=".">1.71</entry></row><row><entry namest="col1" nameend="col1" align="right">4) pSK123</entry><entry namest="col2" nameend="col2" align="char" char=".">0</entry><entry namest="col3" nameend="col3" align="char" char=".">0</entry></row><row><entry namest="col1" nameend="col1" align="right">5) pSK124</entry><entry namest="col2" nameend="col2" align="char" char=".">0</entry><entry namest="col3" nameend="col3" align="char" char=".">0</entry></row><row><entry namest="col1" nameend="col1" align="right">6) pSK106</entry><entry namest="col2" nameend="col2" align="char" char=".">1.14</entry><entry namest="col3" nameend="col3" align="char" char=".">6.38</entry></row><row><entry namest="col1" nameend="col1" align="right">7) pSK107</entry><entry namest="col2" nameend="col2" align="char" char=".">0</entry><entry namest="col3" nameend="col3" align="char" char=".">3.40</entry></row><row><entry namest="col1" nameend="col1" align="right">8) pSK125</entry><entry namest="col2" nameend="col2" align="char" char=".">0</entry><entry namest="col3" nameend="col3" align="char" char=".">0</entry></row><row><entry namest="col1" nameend="col1" align="right">9) pSK126</entry><entry namest="col2" nameend="col2" align="char" char=".">0</entry><entry namest="col3" nameend="col3" align="char" char=".">0</entry></row><row><entry namest="col1" nameend="col1" align="right">10) pSK127</entry><entry namest="col2" nameend="col2" align="char" char=".">0</entry><entry namest="col3" nameend="col3" align="char" char=".">0</entry></row><row><entry namest="col1" nameend="col1" align="right">11) pSK109</entry><entry namest="col2" nameend="col2" align="char" char=".">0</entry><entry namest="col3" nameend="col3" align="char" char=".">0</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="right">12) pSK108</entry><entry namest="col2" nameend="col2" align="char" char=".">0</entry><entry namest="col3" nameend="col3" align="char" char=".">8.05</entry></row></tbody></tgroup></table></tables> As is evident from the table, ATCC12674/pSK108 (6.2 kb HindIII-EcoRV fragment) (FIG. 2) is of high nitrilase activity. Additional deletion plasmids were constructed and examined for the gene coding for the regulatory factor. The result revealed that the gene is located within a far upstream region (about 3 kb BamHI-EcoRV fragment) from the structural gene of nitrilase. (7) Nucleotide sequencing The gene coding for the regulatory factor essential for expression of nitrilase, revealed in step (6), was sequenced using Fluorescence Sequencer ALFII (Pharmacia). The sequence analysis revealed the nucleotide sequence of SEQ ID No: 5, and the presence of 2 open reading frames coding respectively for the amino acid sequences of SEQ ID Nos: 1 and 2 was found. Comparison with Amino Acid Sequence Data Base NBRF (National Biomedical Research Foundation) suggested that the regulatory factor belongs to a family of two-component regulator. The nucleotide sequences of these open reading frames are shown in SEQ ID Nos: 3 and 4.

Reference Example

<ul id="ul0002" list-style="none" compact="compact"><li>(1) Preparation of the chromosomal DNA from the strain SK92 The chromosomal DNA from SK92 was prepared in the same manner as in Example, step (1). </li><li>(2) Preparation of a probe and construction of a DNA library Polymerase chain reaction was carried out using 100 µ l solution containing 10 µ l of DNA as substrate (diluted 20-fold), 10 µ l of the reaction buffer (10-fold conc.), 4 µ l of 5 mM dNTP, 5 µ l (500 pmol) each of 5'-AACTGCTGGGA(AG)CACTTCCA-3' as primer #1 (20 nucleotides corresponding to the amino acid sequence NCWEHFQ) and 5'-GA(AG)TA(AG) TG(AG)CC(CG)AC(ACTG)GG(AG)TC-3' as primer #2 (20 nucleotides corresponding to the amino acid sequence DPVGHYS), and 1 µ l of Tth DNA polymerase (Toyo Boseki). The above 2 primers were prepared on the basis of amino acid sequences having high homologies with known various nitrilases. The reaction involved 50 cycles each consisting of the incubation of the sample at 93 °C for 30 seconds (denaturation step), 45 °C for 30 seconds (annealing step) and 72 °C for 2 minutes (elongation step). A 410 bp DNA fragment coding for the nitrilase from SK92 was obtained from the reaction solution. This DNA fragment was labeled as probe using a DIG DNA Labeling Kit (Boehringer Mannheim). 10 µ l of the reaction buffer (10-fold conc.), 37 µ l of sterilized water and 3µ l of restriction enzyme Sal I were added to 50 µ l of the chromosomal DNA from SK92. The mixture was allowed to react at 37 °C for 2 hours, then precipitated with ethanol and electrophoresed on agarose gel. A DNA fragment, about 1.1 kb, was recovered using DNA PREP (DIA-IATRON). The DNA fragment was inserted into the Sal I site of E. coli vector pUC118 using a ligation kit (Takara Shuzo Co., Ltd.) whereby a recombinant DNA library was prepared. The above pUC118 fragment was prepared in the following manner. 10 µ l of the reaction buffer (10-fold conc.), 77 µ l of sterilized water and 2 µ l of restriction enzyme Sal I were added to 10 µ l of pUC118. The mixture was allowed to react at 37 °C for 2 hours, then treated with phenol, precipitated with ethanol, dried, and dissolved in 50 µ l of sterilized water. 1 µ l of alkaline phosphatase (Takara Shuzo Co., Ltd.), 10 µ l of the reaction buffer (10-fold conc.) and 39 µ l of sterilized water were added thereto. The sample solution was allowed to react at 65 °C, treated with phenol, precipitated with ethanol, dried, and dissolved in sterilized water. </li><li>(3) Transformation of E. coli and selection of recombinant DNA Competent cells of E. coli JM109 were prepared in the same manner as in Example, step (3). 10 µ l solution (DNA library) containing the recombinant plasmid prepared in step (2) was added to 200 µ l of the competent cells. The cells were allowed to stand at 0 °C for 30 minutes, then heat-shocked at 42 °C for 30 seconds and cooled at 0 °C for 2 minutes. 0.8 ml of SOC medium was added thereto, and the cells were incubated at 37 °C for 60 minutes under shaking. The culture was plated in an amount of 200 µ l per plate onto LB agar medium containing 100 µ g/ml ampicillin, followed by incubation at 37 °C. Selection of a transformant carrying the nitrilase gene from the colonies grown on the agar medium was carried out by colony hybridization in the following manner. The transformants grown on the agar medium were transferred to a nylon membrane (Biodaine A produced by Paul Co., Ltd.) and they were lysed to fix DNA. The DNA was treated with the probe (410 bp fragment) prepared in step (2), and the colony containing the target recombinant DNA was selected using a DIG Luminescent Detection Kit (Boehringer Mannheim). </li><li>(4) Construction of recombinant plasmids and preparation of a restriction enzyme map The transformant selected in step (3) was treated in the same manner as in Example, step (4). The recombinant plasmid pSK002 thus obtained was cleaved with several restriction enzymes to prepare a restriction enzyme map. </li><li>(5) Production of nitrilase by transformed E. coli and conversion of a nitrile into an acid The JM109/pSK002 strain was inoculated into 1 ml of 2×YT medium (1.6 % Bacto-trypton, 1.0 % Bacto-yeast extract, 0.5 % NaCl) containing 50 µ g/ml ampicillin and incubated at 37 °C for 8 hours. 1 ml of the culture was inoculated into 100 ml of 2×YT medium containing 50 µ g/ml ampicillin and 1 mM IPTG, followed by incubation at 37 °C for 14 hours. After harvested, the microorganisms were suspended in 50 mM phosphate buffer, pH 7.7, and a part of the suspension was allowed to react at 30 °C for 20 minutes in 50 mM phosphate buffer, pH 7.7, containing 100 mM acrylonitrile. The reaction was stopped by addition of 1 N HCl, and the amount of acrylic acid formed in the reaction solution was determined by HPLC. In the control test, the strain JM109 before transformation was used. The result indicates that while no acrylic acid was detected in the host JM109, the formation of 18 mM acrylic acid was found in the transformant JM109/pSK002. </li><li>(6) Introduction of the DNA fragment containing the nitrilase gene into a hybrid plasmid vector A DNA fragment (5.8 kb BgIII-HindIII fragment) containing the nitrilase structual gene and a region speculated to contain its promoter were cloned into hybrid plasmid vector pK4 whereby plasmid pSK 120 was constructed. </li><li>(7) Transformation of a microorganism of the genus Rhodococcus and the nitrilase activity of the transformant Rhodococcusrhodochrous ATCC 12674 at the logarithmic growth phase was harvested by centrifugation, washed 3 times with ice-cold sterilized water, and suspended in sterilized water. 10µ g cell suspension was mixed with 1 µ g of plasmid pSK120 obtained in step (6), and the mixture was then cooled on ice. This mixture of the DNA and the microorganism was introduced into the chamber in a gene-introducing unit CET-200 (Nippon Bunko) where the sample was pulsed 20 times with a density of electric field of 3.8 kV/cm and a pulse width of 1ms. The cell suspension thus treated was placed on ice for 10 minutes and heat-shocked at 37 °C for 10 minutes. 500 µ l of MYK medium was added to the suspension and the mixture was then incubated at 26 °C for 3 hours under shaking. The culture was plated onto MYK agar medium containing 75 µg/ml kanamycin and incubated at 26 °C for 3 days. The thus obtained transformant of the genus Rhodococcus was inoculated into 10 ml MYK medium containing 50 µ g/ml kanamycin and pre-incubated at 30 °C for 24 hours. 1 ml of the culture was added to 100 ml of GGP medium containing 75 µ g/ml kanamycin. 1.5 % ECH was added thereto as inducer. The transformant was incubated at 30 °C for 48 hours. After recovered, the cells were suspended in 50 mM phosphate buffer, pH 7.7, and their nitrilase activity was examined in the same manner as in step (5). No activity was found in it. A number of references are cited herein, the disclosures of which are incorporated in their entireties by reference herein. </li></ul>

SEQUENCE LISTING

<ul id="ul0003" list-style="none"><li>SEQ ID No: 1 LENGTH: 244 TYPE: amino acid TOPOLOGY: linear MOLECULAR TYPE: protein ORIGINAL SOURCE ORGANISM: Rhodococcuserythropolis STRAIN: SK92 SEQUENCE:<img file="EP0719862A2_D0003.tif" /><img file="EP0719862A2_D0004.tif" /></li><li>SEQ ID No: 2 LENGTH: 534 TYPE: amino acid TOPOLOGY: linear MOLECULAR TYPE: protein ORIGINAL SOURCE ORGANISM: Rhodococcuserythropolis STRAIN: SK92 SEQUENCE:<img file="EP0719862A2_D0005.tif" /><img file="EP0719862A2_D0006.tif" /><img file="EP0719862A2_D0007.tif" /><img file="EP0719862A2_D0008.tif" /></li><li>SEQ ID No: 3 LENGTH: 735 TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear ORIGINAL SOURCE ORGANISM: Rhodococcuserythropolis STRAIN: SK92 SEQUENCE:<img file="EP0719862A2_D0009.tif" /><img file="EP0719862A2_D0010.tif" /></li><li>SEQ ID No: 4 LENGTH: 1605 TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear ORIGINAL SOURCE ORGANISM: Rhodococcuserythropolis STRAIN: SK92 SEQUENCE:<img file="EP0719862A2_D0011.tif" /><img file="EP0719862A2_D0012.tif" /><img file="EP0719862A2_D0013.tif" /></li><li>SEQ ID No: 5 LENGTH: 2336 TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear ORIGINAL SOURCE ORGANISM: Rhodococcuserythropolis STRAIN: SK92 SEQUENCE:<img file="EP0719862A2_D0014.tif" /><img file="EP0719862A2_D0015.tif" /><img file="EP0719862A2_D0016.tif" /><img file="EP0719862A2_D0017.tif" /><img file="EP0719862A2_D0018.tif" /></li></ul>

Contents7

Every citation, both ways

Citations
Document	Relation	Office	Cited during
US5830693A	Cited by	United States of America	Search report
EP0759474A2	Cited by	European Patent Office (EPO)	Search report
US6849442B1	Cited by	United States of America	Search report
EP0759474A3	Cited by	European Patent Office (EPO)	Search report
US6869783B1	Cited by	United States of America	Applicant
EP0502476A2	Cites	European Patent Office (EPO)	Search report

13 members in 7 offices

This recordMember, by publication datePriority claim

Priority claims5

Priority claims
Document	Office	Kind	Date
33765294	Japan	–	1994-12-28
33765294	Japan	A	1994-12-28
33765294	Japan	A	1994-12-28
33765294	–	–	–
JP19940337652	–	–	–

Members13

Family members, oldest first
Document	Office	Kind
EP0719862A2This record	European Patent Office (EPO)	A2	1996-07-03
JPH08173169A	Japan	A	1996-07-09
KR960023057A	Republic of Korea	A	1996-07-18
CN1133341A	China	A	1996-10-16
US5602014A	United States of America	A	1997-02-11
EP0719862A3	European Patent Office (EPO)	A3	1998-02-25
TW387895B	Taiwan Province of China	B	2000-04-21
JP3154633B2	Japan	B2	2001-04-09
EP0719862B1	European Patent Office (EPO)	B1	2001-08-16
DE69522192D1	Germany	D1	2001-09-20
CN1080306C	China	C	2002-03-06
DE69522192T2	Germany	T2	2002-04-18
KR100358532B1	Republic of Korea	B1	2003-03-28

30 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
2016-01-29	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2016-01-20	Patent expired after termination of 20 yearsExpiredPE20	PE20	GB
2015-12-31	Patent ceasedCeasedPL	PL	CH
2015-12-27	Expiry of rightR071	R071	DE
2015-04-30	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2015-02-27	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2015-01-30	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2015-01-30	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2002-08-07	No opposition filedOpposition26N	26N	EP
2002-06-21	No opposition filed within time limitOppositionORIGINAL CODE: 0009261PLBE	PLBE	EP
2002-06-21	Information on the status of an ep patent application or granted ep patentGrantedSTATUS: NO OPPOSITION FILED WITHIN TIME LIMITSTAA	STAA	EP
2002-01-11	Fr: translation filedET	ET	EP
2002-01-01	European patent in force as of 2002-01-01IF02	IF02	GB
2001-11-30	New agentNV	NV	CH
2001-09-20	Corresponds to:REF	REF	EP
2001-08-31	European patent takes effect as a national patent in ch/liEP	EP	CH
2001-08-16	Designated contracting statesAK	AK	EP
2001-06-29	(expected) grantORIGINAL CODE: 0009210GRAA	GRAA	EP
2001-05-03	Despatch of communication of intention to grant a patentORIGINAL CODE: EPIDOS IGRAGRAH	GRAH	EP
2001-04-25	Party data changed (applicant data changed or rights of an application transferred)RAP1	RAP1	EP
2001-02-14	Despatch of communication of intention to grantORIGINAL CODE: EPIDOS AGRAGRAG	GRAG	EP
2001-02-14	Despatch of communication of intention to grant a patentORIGINAL CODE: EPIDOS IGRAGRAH	GRAH	EP
2000-10-23	Despatch of communication of intention to grantORIGINAL CODE: EPIDOS AGRAGRAG	GRAG	EP
2000-04-12	First examination report despatched17Q	17Q	EP
1999-05-06	Party data changed (applicant data changed or rights of an application transferred)RAP1	RAP1	EP
1998-09-02	Request for examination filed17P	17P	EP
1998-02-25	Designated contracting statesAK	AK	EP
1998-01-09	Search report despatchedORIGINAL CODE: 0009013PUAL	PUAL	EP
1996-07-03	Designated contracting statesAK	AK	EP
1996-05-17	Public reference made under article 153(3) epc to a published international application that has entered the european phaseORIGINAL CODE: 0009012PUAI	PUAI	EP