FR2764303A1 - Rhodococcus aspartase protein - Google Patents

Abstract

The following are claimed: (1) an aspartase protein having a defined sequence of 477 amino acids given in the specification; (2) DNA encoding the protein of (1); (3) a DNA fragment of about 600 bp that can be amplified with synthetic oligonucleotides having the sequences AACATGAACA CTAACGAGGT and AGGTCGTTGC AGATCTTGGA; (4) a recombinant plasmid containing the DNA of (2) or (3); (5) a host microorganism transformed with the plasmid of (4).

Description

The present invention relates to a protein having activity

  aspartase, a DNA encoding this protein, a recombinant plasmid comprising this DNA, a transformant into which this recombinant plasmid is introduced, a

  process for producing protein and process for producing L- acid

  aspartic by means of this transformant. L-aspartic acid is used in particular in pharmaceutical products and food additives. Recently, L-aspartic acid has also attracted attention as a raw material for surfactants and agents

biodegradable chelators.

  L-aspartic acid is produced mainly by processes

  enzymatic from furmaric acid and ammonia using an aspartase.

  As a gene encoding an aspartase, there is known a gene derived from Escherichia coli [(Journal of General Microbiology, 130, 1271-1278 (1984)] and a gene derived from Brevibacterium flavum MJ-233 (Japanese patent application published before

exam no 5-30 977).

  The object of the present invention is to provide a gene encoding an aspartase which is different from the genes mentioned above and which makes it possible to produce

  L-aspartic acid more effectively.

  Following extensive research to achieve this goal, a protein with aspartase activity and a gene encoding this protein have been isolated from

  bacteria of the genus Rhodococcus, which led to the present invention.

  Thus, the present invention relates to: (1) a protein with aspartase activity which comprises the amino acid sequence represented in SEQ ID NO: 1 or the amino acid sequence represented in SEQ ID NO: 1 comprising a deletion, a substitution or addition of one or more amino acids; (2) DNA encoding the protein; (3) a recombinant plasmid obtained by ligation of DNA with a plasmid vector; (4) a transformant obtained by introduction of the recombinant plasmid into a host microorganism; (5) a process for producing the above protein by culturing the transformant; and (6) a process for producing L-aspartic acid by reacting fumaric acid or one of its salts with ammonia or one of its salts in the presence of

  transforming above or protein above.

  Other characteristics of the invention will appear better in the

  detailed description which follows and refers to the attached drawings, given only

  by way of example, and in which: FIG. 1 is a restriction map of the recombinant plasmid pAR 002. The double line represents the vector pUC 118 and the single line represents the DNA region of the strain EA4 of Rhodococcus. The thick arrow in the DNA region of strain EA4 indicates the position and orientation of the aspartase gene according to the present invention;

  Figure 2 is a restriction map of the recombinant plasmid pAR016.

  The double line represents the region originating from the plasmid pSJ034 and the single line represents the DNA region of the strain EA4. The thick arrow in the DNA region of strain EA4 indicates the position and orientation of the aspartase gene according to the present invention; Figure 3 is a diagram showing the plasmid pSJ034; and

  FIG. 4 shows the stages of construction of the plasmid pSJ034.

  The strain Rhodococcus sp. EA4 (FERM BP-6231) is a specific example of microorganisms capable of producing the protein to

  aspartase activity according to the present invention and a DNA encoding this protein.

  The host microorganism intended to be used in the context of the invention is not particularly limited and mention may be made specifically of the strain EA4 mentioned above as well as Rhodococcus rhodochrous ATCC 12674, ATCC 17041, ATCC 17895 , ATCC 19140 and

ATCC 33258.

  The strain EA4 is deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan, under the access number indicated above and its bacteriological properties are

  described in the Japanese patent application published before examination No. 4- 304 892.

  ATCC 12674, ATCC 17041, ATCC 17895, ATCC 19140 and ATCC

  33,258 are available from the American Type Culture Collection (ATCC).

  The present invention will now be described in more detail.

  detailed by means of the following nonlimiting examples.

Example 1

  (1) Preparation of chromosomal DNA from the strain EA4.

  The strain EA4 was cultivated in 100 ml of MY medium (0.5% of polypeptone, 0.3% of bacteria-yeast extract, 0.3% of bacteria-malt extract, I% of glucose) at 30 C for 72 h while shaking. Then, the cells were harvested and suspended in 4 ml of salt solution-EDTA (0.1 M EDTA, 0.15 M NaCl (pH 8.0)). 8 mg of lysozyme was added to the suspension and shaken at 37 ° C for 1 to 2 h. Then, the resulting suspension was frozen and then 10 ml of Tris-SDS solution (I% SDS, 0.1 M NaCl, 0.1 M Tris-HCl (pH 9.0)) was added thereto. shaking gently. In addition, proteinase K (Merck) was added (final concentration: 0.1 mg / ml) and shaken at 37 ° C for 1 h. An equal volume of TE saturated phenol (TE: 10 mM Tris-HCl, 1 mM EDTA (pH 8.0)) was added to the resulting suspension, stirred and centrifuged. The upper layer was collected and 2 volumes of ethanol added to it. Then we wrapped the DNA around a glass rod. The phenol was removed using 90%, 80%, and 70% ethanol in that order. Then, the DNA was dissolved in 3 ml TE buffer. To this solution was added a solution of ribonuclease A (pretreated at 100 ° C. for 15 min) to obtain a concentration of 10 μg / ml and it was shaken at 37 ° C. for 30 min. In addition, proteinase K was added and shaken at 37 ° C for 30 min, then phenol saturated with TE was added. The resulting solution was centrifuged to separate it into an upper layer and a lower layer. The same operation was repeated twice for the top layer, then an equal volume of a mixed solution of chloroform and isoamyl alcohol (24: 1) was added thereto and the same operation was repeated

  extraction (in the following this operation is called "phenol treatment").

  Then two volumes of ethanol were added to the resulting top layer and the DNA was wrapped around a glass rod to obtain a sample

of chromosomal DNA.

  (2) Preparation of a probe. 10 μl of chromosomal DNA obtained in step (1) (1/20 dilution), 10 g of 10 × reaction buffer, 3 g of 50 mM MgCl 2, 2 μl of dNTP 10 mM, 1 p1 (equivalent to 100 pmol) of the oligonucleotide represented in SEQ ID NO: 4 as primer n I and 1 μl of the oligonucleotide represented in SEQ ID NO: 5 as primer n 2 and 1 μl of Taq polymerase (GIBCO BRL) to prepare 100 1l of solution. Each of the above primers was designed based on highly conserved regions among different known aspartases. The resulting solution was subjected to a PCR or PCR reaction (polymerase chain reaction) consisting of 30 cycles each comprising denaturation at 93 C for 30 s to separate the two strands of DNA, hybridization with the two primers at 55 C for 30 s and extension of the primers at 72 C for 2 min. After the end of the reaction, 3 extractions were carried out with chloroform and then precipitated with ethanol to recover the amplified DNA. The DNA was separated by 0.7% agarose gel electrophoresis to obtain a DNA fragment of about 600 bp which is believed to encode part of the aspartase gene of the strain EA4. This DNA fragment was labeled with the kit

  DNA labeling DIG (Boehringer Mannheim) to prepare a probe.

  (3) Preparation of a DNA bank At 200 μl of chromosomal DNA of the strain EA4, 40 μl of 10x restriction enzyme buffer, 145 g of sterilized water and 15 μl of restriction enzyme were added. BamHI and reacted at 37 C for 6 h. Then, an ethanol precipitation was carried out to recover the DNA which was subjected to an agarose gel electrophoresis. A DNA fragment of about 8 kb was taken from the gel which was recovered with DNA PREP (DIA IATRON). This DNA fragment was inserted into the BamHI site of the E. coli pUC 118 vector using a

  ligation kit (Takara Shuzo) to prepare a recombinant DNA library.

  The fragment of pUC118 used in the ligation was prepared in the following manner. To 10 µl of pUC118 was added 10 µl of 10x restriction enzyme buffer, 77 µl of sterilized water and 2 µl of BamHI restriction enzyme and reacted at 37 ° C for 2 h. Then a phenol treatment and an ethanol precipitation were carried out. The resulting DNA was dried and then dissolved in 50 g of sterilized water. To this solution, 1 µl of alkaline phosphatase, 10 µl of 10x buffer and 39 µl of sterilized water were added and reacted at 65 C. Then a phenol treatment and ethanol precipitation were carried out . The resulting DNA was dried and

  it was dissolved in sterilized water.

  (4) Preparation of transformants and selection of the recombinant DNA The strain of E. coli JMI09 was inoculated with 1 ml of LB medium (1% of bacterotryptone, 0.5% of bacteria-yeast extract, 0.5 % NaCI) and cultured at 37 C for 5 h. 100 µl of the resulting culture was added to 50 ml of SOB medium (2% bacto-tryptone, 0.5% bacto-yeast extract, 10 mM NaCI, 2.5 mM KCI, 1 mM MgSO4, MgCI2 1 mM) and cultured at 18 C for 20 h. The cells were then harvested by centrifugation, 13 ml of cold TF solution (PIPES-KOH 20 mM (pH 6.0), KCI 200 mM, CaCI, 10 mM, MnCI2 40 mM) were added to the cells. allowed to stand at 0 C for 10 min and centrifuged again. After removing the supernatant, the precipitated E. coli was suspended in 3.2 ml of cold TF solution. 0.22 ml of dimethyl sulfoxide was added to the resulting suspension and allowed to stand at 0 C for min. To 200 μl of the competent cells thus prepared, 10 μl of the solution containing the recombinant plasmid prepared in step (3) above was added (ie DNA bank) and left to stand. at 0 C for 30 min. Then a thermal shock was applied to the mixture at 42 C for 30 s and the mixture was cooled to 0 C for 2 min. Then, 0.8 ml SOC medium (2% bacto-tryptone, 0.5% bacto-yeast extract, 20 mM glucose, 10 mM NaCl, 2.5 mM KCI, 1 mM MgSO4) was added. , MgC12 1 mM. Cells were grown at 37 ° C for 60 min with shaking. The resulting culture was plated out in 200 μl aliquots on agar LB medium containing 100 gg / ml ampicillin and cultured at 37 C. A transformant having the aspartase gene was selected by in situ hybridization. among the transforming colonies cultivated on the agar medium. Briefly, the transforming colonies cultivated on the agar medium were transferred to a nylon membrane (BIODYNE A; Nihon Pall Limited), then the cells were lysed and the DNA fixed. Next, the membrane was treated with the probe (approximately 600 kb fragment) prepared in step (2) above. Colonies containing the recombinant DNA of interest were selected using the kit

  DIG luminescent detection system (Boehringer Mannheim).

  (5) Preparation of a recombinant plasmid The transformant selected in step (4) above was cultured in ml of LB medium at 37 ° C. overnight. The cells were harvested and washed with sterilized water. 5 ml of solution I (2 mM glucose, 10 mM EDTA, 25 mM Tris-HCl (pH 8.0)) and 25 mg of lysozyme were added to the cells and left to stand at 0 C for 30 min. In addition, 10 ml of solution II (1 N NaOH, 5% SDS) was added and allowed to stand at 0 C for 5 min. Then, 7.5 ml of solution III (3M sodium acetate (pH 4.8)) was added and allowed to stand at 0 C for 30 min. The resulting mixture was centrifuged, 50 ml of ethanol was added to the supernatant and centrifuged again. Then the supernatant was removed and 5 ml of solution IV (mM sodium acetate, 50 mM Tris-HCl (pH 8.0)) and 2.5 1 l of ribonuclease A solution (10 mg / ml) were added to the precipitate. and allowed to stand at room temperature for 20 min. 12 ml of ethanol was added to the resulting mixture and the plasmid was collected by centrifugation, rinsed with 70% ethanol, dried and dissolved in 0.4 ml of sterilized water. After treating with phenol, the plasmid was recovered by ethanol precipitation, dried and dissolved in 0.4 ml of sterilized water. The recombinant plasmid thus obtained was called pAR002. This plasmid has been deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan as transforming E. coli JMI09 / pAR002 under the access number FERM

BP-6233.

  (6) Preparation of a restriction map and determination of the region

with which the probe hybridizes.

  A restriction map was prepared by digestion of the plasmid pAR002 obtained in step (5) above with several restriction enzymes (FIG. 1). In addition, pAR002 was digested with restriction enzymes KpnI, BglII, PstI, SalI, etc., and subjected to agarose gel electrophoresis. Then a Southern hybridization was carried out to determine a

fragment to which the probe hybridizes.

  (7) Determination of the nucleotide sequence The nucleotide sequences were determined for the regions flanking the region specified in step (6) above and for this region itself with a Pharmacia ALF II fluorescence sequencer. The nucleotide sequence represented in SEQ ID NO: 3 was thus obtained. In this sequence, an open reading frame was found which has the amino acid sequence indicated in SEQ ID NO: 1. By making a comparison using the NBRF (National Biomedical Research) amino acid sequence database. Foundation), it was found that the gene has 50 to 60% homology with known aspartases at the amino acid sequence level and that the polypeptide also has significant homology in regions of great homology among the known aspartases. It is therefore considered that this polypeptide constitutes an aspartase. The nucleotide sequence of this open reading frame is shown in

SEQ ID NO: 2.

Example 2

  (1) Preparation of the plasmid pAR0 16 10 μl of pAR002 (1/20 dilution) were mixed, 10 μl of 10 × reaction buffer, 3 μl of 50 mM MgCl 2, 2 μl of 10 mM dNTP, 1 μl (equivalent to 100 pmol) of the oligonucleotide represented in SEQ ID NO: 6 as primer n 3 and 1 μl of the oligonucleotide represented in SEQ ID NO: 7 as primer n 4, and 1 g of Taq polymerase (GIBCO BRL) to prepare 100 μl of solution. The primers were designed so that a fragment containing the open reading frame of EA4 aspartase was amplified. The solution thus prepared was subjected to PCR or PCR of 30 cycles, each cycle consisting of denaturation at 93 C for 30 s, hybridization at 55 C for 30 s and extension at 72 C for 2 min. After the end of the reaction, 3 extractions were carried out with

  chloroform and then ethanol precipitation to recover the amplified DNA.

  Then, the recovered DNA was digested with the restriction enzymes XbaI and Sse8387I, and then separated by 0.7% agarose gel electrophoresis. A 1.2 kb band was recovered from the gel which was inserted into the XbaI-Sse8387I site of the plasmid pSJ034 (FIG. 3) having a strong promoter activity for the Rhodococcus bacteria, to thus prepare the recombinant plasmid pAR016 (figure 2). Plasmid pSJ034 was prepared from plasmid pSJ023 (described in Japanese patent application No. 9-65616) according to the steps indicated in FIG. 4. Plasmid pSJ023 was deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan as transforming R. rhodochrous ATCC 12674 / pSJ023 under the access number

FERM BP-6232.

  (2) Transformation of Rhodococcus bacteria and aspartase activity of the resulting transformant. Rhodococcus rhodochrous ATCC 12674 strain cells were harvested in the log growth phase, washed 3 times with sterilized water and ice-cold and suspended in sterilized water. 10 µl of this cell suspension was mixed with 1.1 of plasmid pAR016 and cooled in ice. This mixture of DNA and cells was placed in a cuvette. Then electric pulses were applied to it at 2.0 kV and 200 n with a Gene Pulser device (Bio Rad). The liquid thus treated was allowed to stand for 10 min by cooling it in ice and then a thermal shock was applied to it at 37 ° C. for 10 min. Then, 500 g of MYK medium was added (0.5% of polypeptone, 0.3% of bacteria-yeast extract, 0.3% of bacteria-malt extract, 0.2% of K2HPO4, 0 , 2% KH, PO 4) and allowed to stand at 30 ° C for 5 h. Then the resulting mixture was applied to agar MYK medium containing 50 kig / ml

  of kanamycin and cultured at 30 ° C for 3 days.

  The transformant thus prepared was inoculated with Rhodococcus bacteria in ml of MYK medium (containing 50 gg / ml of kanamycin) and cultured at 30 ° C. for 72 h in the form of preculture. 1% of the preculture was inoculated into 10 ml of MYK medium (containing 50 μg / ml of kanamycin) and cultured at 30 ° C. for 96 h. Then the cells were harvested by centrifugation, washed with 100 mM phosphate buffer (pH 8.0) and suspended in a small amount of buffer. This cell suspension was sonicated with ice cooling and centrifuged to obtain a crude enzyme solution on which aspartase activity was determined. As

  control, the strain ATCC 12674 cultivated without the addition of kanamycin was used.

  The aspartase activity was determined as described below.

  Briefly, 1.0 M ammonium fumarate / 1 mM magnesium chloride (pH 8.8) was contacted with the crude enzyme solution. Then aspartic acid produced at 30 ° C. for 60 min was determined quantitatively by HPLC using an ODS column (hydrophilic adsorbent column with octadecylsilyl groups (ODS). It was found that the aspartase activity was increased

  in the transformant containing the plasmid.

  L-aspartic acid produced (mM) ATCC 12674 / pAR016 322

ATCC 12674 0.1

Example 3

  (1) The plasmid pAR016 was introduced into the strains ATCC 17895 and ATCC 19140 in the same manner as in Example 2 to prepare transformants of Rhodococcus bacteria. Each of these transformants was inoculated with 10 ml of MYK medium (containing 50, tg / ml of kanamycin) and cultured at 30 ° C. for 72 h in the form of preculture. 1% of the preculture was inoculated with 100 ml of GGPK medium (1.5% of glucose, I% of sodium glutamate, 0.1% of bacteria-yeast extract, 0.5% of K2HPO4, 0, 5% KH2PO4, 0.5% MgSO4, 7H20, pH 7.2 (containing 50 gg / ml kanamycin) and cultured at C for 96 h. The cells were harvested by centrifugation, washed with 100 mM phosphate buffer (pH 8.0) and suspended in a small amount of this buffer. Aspartic acid production reaction was carried out using the resulting cell suspension. Briefly, added the cell suspension to 100 ml of 1.0 M ammonium fumarate / 1 mM magnesium chloride (pH 8.8) and the mixture was stirred at 30 ° C. for 20 h.

  used strain ATCC 12674 cultivated without the addition of kanamycin.

  The aspartase activity was determined by quantitatively determining the aspartic acid produced by the above reaction (at 30 ° C for 20 h) by HPLC using an ODS column. It was thus found that the activity of aspartase

  was increased in transformants containing the plasmid.

  L-aspartic acid produced (mM) ATCC 17895 / pAR016 990

ATCC 17895 5

ATCC 19140 / pAR016 985

ATCC 19140 8

  Thus, the present invention provides a gene encoding a protein with aspartase activity. Using a transforming microorganism with multiple copies of this gene it is possible to prepare L-aspartic acid

  effectively from fumaric acid and ammonia.

  Sequence list (2) Information for SEQ ID NO: 1: (i) Characteristics of the sequence: (A) Length: 477 amino acids (B) Type: amino acid (C) Topology: linear (ii) Type of molecule: protein (vi) Initial source: (A) Organism: Rhodococcus (B) Strain: EA4

  (xi) Description of the sequence: SEQ ID NO: 1:

  Phr Met Arg lie Glu His Asp Leu Leu Gly Asp Arg Glu Val Pro 3s 10 15 lu Ala Tyr Tyr Gly lie His Thr Leu Arg Ala Leu Glu Asn Phe

25 30

  ! the Thr Gly Ile Pro Leu Ser Val His Pro Asp Met Val Ser Ala

40 45

  Ala Ala Val Lys Gln Ala Ala Ala Arg Ala Asn Ala Asp Leu Gly

55 60

  eu Ser Pro Glu His Ala Ala Ala lie Glu Gln Ala Cys Lys Glu

75 80

  rg Ala Gly Arg Phe Thr Asp Gln Phe Val Val Asp Val lIe Gin

90 95

  Gly Gly Ala Gly Thr Ser Ser Asn Met Asn Ala Asn Glu Val Val Ala

105 110

  Asn Arg Ala Leu Glu Ile Leu Gly Gly Glu Arg Gly Gin Tyr Gln Phe

120 125

  Leu His Pro Leu Glu His Val Asn Met Ser Gln Ser Thr Asn Asp Val

135 140

  Tyr Pro Thr Ala lie Lys lie Gly Leu Gln GIY Ala Val Thr Arg Leu

150 155 160

  Arg Gly Ala Met Asp Glu Leu Ala GIY Ala Phe Ala Glu Lys Ala Ala

170 175

  Glu Phe Ser His Val Leu Lys Val Gly Arg Thr Gln Leu Gln Asp Ala

185 190

  Val Pro Met Thr Leu Gly Gln Glu Ile Val Thr Phe Ala Val Met Ile

200 205

  Lys Glu Asp Ser Gln Arg Leu Glu Glu Ala Ala Arg Leu Ile Ser Glu

210 215 220

  le Asn Leu Gly Gly Thr Ala binds Gly Thr Gly Leu Asn Ala His Pro

225 230 235 240

  Glu Tyr Ala Arg Arg Val Arg Glu His Leu Val Ser Ile Thr Gly Leu

245 250 255

  Asp le Ser Thr Ala Ser Asp Leu Ile Glu Ala Thr Gln Asp Val GIY

260 265 270

  Ala Phe Val Gin Leu Ser Gly Val Leu Lys Arg Thr Ala Val Lys Leu

275 280 285

  Ser Lys lie Cys Asn Asp Leu Arg Leu Leu Ser Ser Gly Pro Arg Ala

290 295 300

  Gly Leu Gly Glu [le Asn Leu Pro Ala Val Gain Ala Gly Ser Ser Ile

305 310 315 320

  Met Pro Gly Lys Val Asn Pro Val Ile Pro Glu Val Val Asn Gin Ile

325 330 335

  Ala Tyr Arg Val Val GlY Asn AsP Leu Thr Ile Thr Met Ala Ala Glu

340 345 350

  Ala Gly Gln Leu Gin Leu Asn Ala Phe Glu Pro Val lie Ala His Ser

355 360 365

  Leu Phe Glu Thr Ala Glu Leu Leu Thr Arg Gly Cys Thr Val Leu Arg

370 375 380

  Glu Arg Cys Val lie Gly Ile Thr Ala Asn Val Glu His Leu Glu Arg

385 390 395 400

  Thr Val Ala Ala Ser Ile Gly Val Val Thr Ala Leu Asn Pro Tyr lie

405 410 415

  Gly Tyr Thr Ala Ala Thr Glu Leu Ala Ala Asp Ala Leu Ala Ser Gly

420 425 430

  Arg Thr Val Val Glu Leu Val Leu Glu Arg Gly Leu Leu Gly Lys Glu

435 440 445

  AsP Leu Asp Ala Ile Met Glu Pro Ala Asn Leu Ala Arg Thr Ala Ile

450 455 460

  Val Ser A; a Pro Asp Phe Asp Pro Leu Val Pro Ser Thr

465 470 475

  (2) Information for SEQ ID NO: 2: (i) Characteristics of the sequence: (A) Length: 1434 base pairs (B) Type: nucleic acid (C) Configuration of the strands: double (D) Topology: linear ( ii) Type of molecule: DNA (genomics) (vi) Initial source: (A) Organism: Rhodococcus (B) Strain: EA4 (ix) Characteristics (A) Name / key: CDS (B) Position: 1..1431

  (xi) Description of the sequence: SEQ ID NO: 2:

  ATG ACG ATG CGC ATC GAG CAC GAC CTG CTC GGT GAT CGC GAA GTG CCT 48

  Met Thr Met Arg Ile Glu His Asp Leu Leu Gly Asp Arg Glu Val Pro

1 5 10 15

  GCG GAG GCC TAC TAC GGC ATC CAC ACG CTG AGA GCA CTG GAG AAC TTC 96

  Ala Glu Ala Tyr Tyr Gly Ile His Thr Leu Arg Ala Leu Glu Asn Phe

25 30

  CCG ATC ACC GGT ATT CCT CTC TCG GTC CAC CCCG GAC ATG GTA TCG GCG 144

  Pro Ile Thr GIy le Pro Leu Ser Val His Pro Asp Met Val Ser Ala

35 40 45

  TTG GCT GCG GTC AAG CAG GCC GCT GCT CGG GCG AAC GCC GAC CTC GGA 192

  Leu Ala Ala Val Lys Gln Ala Ala Ala Arg Ala Asn Ala Asp Leu Gly

55 60

  ATC CTC TCG CCC GAG CAT GCA GCG GCG ATC GAG CAA GCT TGC AAG GAG 240

  lie Leu Ser Pro Glu His Ala Ala Ala Ile Glu Gln Ala Cys Lys Glu

70 75 80

  ATT CGT GCG GGC AGG TTC ACC GAC CAG TTC GTC GTC GAC GTC ATT CAG 288

  lie Arg Ala GIY Arg Phc Thr Asp Gln Phe Val Val Asp Val le Gin

90 95

  GGT GGT GCC GGC ACG TCA TCG AAC ATG AAC GCC AAC GAA GTG GTC GCG 336

  Gly Gly Ala Gly Thr Ser Ser Asn Met Asn Ala Asn Glu Val Val Ala

105 110

  AAC CGC GCG CTC GAA ATT CTC GGC GGC GCAA CGG GGG CAG TAC CAA TTC 384

  Asn Arg Ala Leu Glu le Leu Gly Gly Glu Arg Gly Gln Tyr Gln Phe

120 125

  CTC CAT CCG CTC GAA CAC GTC AAC ATG AGT CAG AGC ACC AAC GAC GTC 432

  Leu His Pro Leu Glu His Val Asa Met Ser Gin Ser Thr Asn Asp Val

135 140

  TAC CCC ACC GCG ATC AAA ATC GGC TTG CAA GGT GCC GTC ACC AGG CTT 480

  Tyr Pro Thr Ala Ile Lys Tle Gly Leu Gln Gly Ala Val Thr Arg Leu i50 155 160

  CGA GGT GCCG ATG GAC GAA CTT GCA GGC GCA TTC GCG GAG AAA GCA GCC 528

  Arg Gly Ala Met Asp Glu Leu Ala Gly Ala Phe Ala Glu Lys Ala Ala

170 175

  GAG TTC TCC CAC GTG CTC AAG GTC GGG CGG ACT CAG TTG CAA GAC GCG 576

  Glu Phe Ser His Val Leu Lys Val Gly Arg Thr Gln Leu Gln Asp Ala i8C 185 190

  GTC CCG ATG ACT CTC GGT CAA GAG ATT GTC ACG TTC GCC GTG ATG ATC 624

  Val Pro Met Thr Leu Gly Gln Glu lie Val Thr Phe Ala Val Met Ile

200 205

  AAG GAG GAC TCT CAG AGA CTr GAA GAG GCC GCT CGG TTG ATC TCG GAG 672 Lys Glu Asp Ser Gin Arg Leu Glu Glu Ala Ala Arg Leu lie Ser Glu

210 215 220

  ATC MJC CTG GGA GGG ACG GCG ATA GGT ACC GGT CTC AAT GCG CAC CCG 720

  Ile Asn Leu Gly Gly Thr Ala lie Gly Thr Gly Leu Asn Ala His Pro

225 230 235 240

  GAG TAC GCC AGA CGT GTG CGT GAG CAT CTG GTG TCG ATC ACC GGT CTC 768

  Glu Tyr Ala Arg Arg Val Arg Glu His Leu Val Ser lie Thr Gly Leu

245 250 255

  GAC ATC TCG ACG GCA TCG GAT CTC ATC GAA GCA ACC CAG GAC GTC GGG 816

  Asp bind Ser Thr Ala Ser Asp Leu le Glu Ala Thr Gin Asp Val Gly

260 265 270

  GCC TTC GTT CAG CTG TCG GGA GTG CTC AAG CGT ACC GCG GTC AAA CTG 864

  Ala Phe Val Gin Leu Ser GIy Val Leu Lys Arg Thr Ala Val Lys Leu

275 280 285

  TCC AAG ATC TGC AAC GAT CTG AGA TTG CTG TCC TCA GGC CCA CGA GCA 912

  Ser Lys lie Cys Asn Asp Leu Arg Leu Leu Ser Ser Gly Pro Arg Ala

290 295 300

  GGT CTG GGT GAG ATC AAT TTA CCT GCG GTG CAG GCA GGT TCG TCG ATC 960

  Gly Leu Gly Glu lie Asa Leu Pro Ala Val Gln Ala Gly Ser Ser Ile

305 310 315 320

  ATG CCC GGA AAG GTC AAT CCG GTC ATT CCG GAA GTG GTC AAT CAG ATT 1008

  Met Pro Gly Lys Val Asn Pro Val lie Pro Glu Val Val Asn Gin Ile

325 330 335

  GCC TAC CGG GTG GTG GGA AAC GAT CTG ACC ATC ACC ATG GCT GCC GAG 1056

  Ala Tyr Arg Val Val Gly Asn Asp Leu Thr lie Thr Met Ala Ala Glu

340 345 350

  GCG GGT CAA CTG CAG CTC.AAC GCA TTC GAA CCG GTC ATT GCA CAC AGC 1104

  Ala Gly Gln Leu Gin Leu Asn Ala Phe Glu Pro Val Ile Ala His Ser

355 360 365

  TTG TTC GAG ACT GCA GAA CTC CTC ACG CGG GGC TGC ACT GTG TTG CGC 1152 Leu Phe Glu Thr Ala Glu Leu Leu Thr Arg Gly Cys Thr Val Leu Arg

370 375 380

  GAG CGA TGC GTG ATC GGC ATT ACC GCC AAC GTC GAG CAT CTC GAA CGA 1200

  Glu Arg Cys Val lle Gly Ile Thr Ala Asn Val Glu His Leu Glu Arg

385 390 395 400

  ACG GTC GCG GCT TCG ATC GGT GTC GTG ACC GCCG CTC AAT CCC TAC ATC 1248

  Thr Val Ala Ala Ser lie Gly Val Val Thr Ala Leu Asn Pro Tyr ile

405 410 415

  GGC TAC ACA GCC GCG ACG GAA TTG GCG GCG GAC GCG CTT GCT TCC GGT 1296

  Gly Tyr Thr Ala Ala Thr Glu Leu Ala Ala Asp Ala Leu Ala Ser Gly

4120 425 430

  CGT ACC GTC GTG GAG TTG GTG CTC GAA CGC GGG TTG TTC GC AAG GAG 1344

  Arg Thr Val Val Glu Leu Val Leu Glu Arg Gly Leu Leu Gly Lys Glu

435 440 445

  GAT TTG GAC GCG ATC ATG GAG CCT GCG AAT TTG GCA CGA ACT GCG ATC 1392

  Asp Leu Asp Ala Ile Met Glu Pro Ala Asn Leu Ala Arg Thr Ala lIe

450 455 460

  GTG TCG GCT CCC GAT: TC GAT CCA CTC GTC CCG TCG ACC TGA 1434

  Val Ser Ala Pro Asp Phe Asp Pro Leu Val Pro Ser Thr

465 470 475

  (2) Information for SEQ ID NO: 3: (i) Characteristics of the sequence: (A) Length: 1912 base pairs (B) Type: nucleic acid (C) Configuration of the strands: double (D) Topology: linear ( ii) Type of molecule: DNA (genomics) (vi) Initial source: (A) Organism: Rhodococcus (B) Strain: EA4

  (xi) Description of the sequence: SEQ ID NO: 3:

  TTCGAjAGCCA CGCCCGTTCA GGTGACCTGC GTGCGGCGAA GTTCGGTGGA GCCGAACTAC 60

  CGTGGGGCGA GATCCCGGTG GCGGCAAACC TCAAATGACG ATGCGCATCG AGCACGACCT 120

  GCTCGGTGAT CGCGAAGTGC CTGCGGAGGC CTACTACGGC ATCCACACGC TGAGAGCACT 180

  GGAGAACTTC CCGATCACCG GTATTCCTCT CTCGGTCCAC CCCGACATGG TATCGGCGTT 240

  GGCTGCGGTC AAGCAGGCCG CTGCTCGGGC GAACGCCGAC CTCGGAATCC TCTCGCCCGA 300

  GCATGCAGCG GCGATCGAGC AAGCTTGCAA GGAGATTCGT GCGGGCAGGT TCACCGACCA 360

  GTTCGTCGTC GACGTCATTC AGGGTGGTGC CGGCACGTCA TCGAACATGA ACGCCAACGA 420

  AGTGGTCGCG AACCGCGCGC TCGAAATTCT CGGCGGCGAA CGGGGGCAGT ACCAATTCCT 480

* CCATCCGCTC GAACACGTCA ACATGAGTCA GAGCACCAAC GACGTCTACC CCACCGCGAT 540

  CAAAATCGGC TTGCAAGGTG CCGTCACCAG GCTTCGAGGT GCGATGGACG AACTTGCAGG 600

  CGCATTCGCG GAGAAAGCAG CCGAGTTCTC CCACGTGCTC AAGGTCGGGC GGACTCAGTT 660

  GCAAGACGCG GTCCCGATGA CTCTCGGTCA AGAGATTGTC ACGTTCGCCG TGATGATCAA 720

  GGAGGACTCT CAGAGACTGG AAGAGGCCGC TCGGTTGATC TCGGAGATCA ACCTGGGAGG 780

  GACGGCGATA GGTACCGGTC TCAATGCGCA CCCGGAGTAC GCCAGACGTG TGCGTGAGCA 840

  TCTGGTGTCG ATCACCGGTC TCGACATCTC GACGGCATCG GATCTCATCG AAGCAACCCA 900

  GGACGTCGGG GCCTTCGTTC AGCTGTCGGG AGTGCTCAAG CGTACCGCGG TCAAACTGTC 960

  CAAGATCTGC AACCATCTGA GATTGCTGTC CTCAGGCCCA CGAGCAGGTC TGGGTGAGAT 1020

  CAATTTACCT GCGGTGCAGG CAGGTTCGTC GATCATGCCC GGAAAGGTCA ATCCGGTCAT 1080

  TCCGGAAGTG GTCAATCAGA TTGCCTACCG GGTGGTGGGA AACGATCTGA CCATCACCAT 1140

  GGCTGCCGAG GCGGGTCAAC TGCAGCTCAA CGCATTCGAA CCGGTCATTG CACACAGCTT 1200

  GTTCGAGACT GCACAACTCC TCACGCGGGG CTGCACTGTG TTGCGCGAGC GATGCGTGAT 1260

  CGGCATTACC GCCAACGTCG AGCATCTCGA ACGAACGGTC GCGGCTTCGA TCGGTGTCGT 1320

  GACCGCGCTC AATCCCTACA TCGGCTACAC AGCCGCGACG GAATTGGCGG CGGACGCGCT 1380

  TGCTTCCGGT CGTACCGTCG TGGAGTTGGT GCTCGAACGC GGGTTGTTGG GCAAGGAGGA 1440

  TTTGGACGCG ATCATGGAGC CTGCGAATTT GGCACGAACT GCGATCGTGT CGGCTCCCGA 1500

  TTTCGATCCA CTCGTCCCGT CGACCTGACC AGGTCTTCAG GGCGGTTGAC ACCGCACATG 1560

  TGGGCGGTAT CGTTTAGTGA TACGTATAAC TACTTGCAGT CCTGTTGTGT CAGGATTGAC 1620

  AGAAGCTGAG TCGGCACGGT TCAAGGACGT ACCGCGCCAA GCCAGCCAGG AGACGGAAAC 1680

  CATGTTCGAA AAGACACGAA TGGACAGTCA TCCCCCCTGT AAGAGCAACT ACGACGTCAT 1740

  CGTCGTGGGA AGCGGAATGG GGGGTGGAAC GATGGCATAC GCTCTCMAAGG ATTCCGGCCT 1800

  CGATGTGCTC TTGATCGAGA GGGGAGACTT CCTTCCACAG GAACGTCAGA ATTGGGACCC 1860

  CCGAGCGGTG TTCGAGAAAG ATCGGTACAA GAACGCCGA AAATGGGTCG AC 1912

  (2) Information for SEQ ID NO: 4: (i) Characteristics of the sequence: (A) Length: 20 base pairs (B) Type: nucleic acid (C) Configuration of the strands: simple (D) Topology: linear ( ii) Type of molecule: other nucleic acid

  (A) Description: / desc = "synthetic DNA"

  (xi) Description of the sequence: SEQ ID NO: 4:

AACATGAACACTAACGAGGT 20

  (2) Information for SEQ ID NO: 5: (i) Characteristics of the sequence (A) Length: 20 base pairs (B) Type: nucleic acid (C) Configuration of the strands: simple (C) Topology: linear (ii ) Type of molecule: other nucleic acid

  (A) Description: / desc = "synthetic DNA"

  (xi) Description of the sequence: SEQ ID NO: 5:

AGGTCGTTGC AGATCTTGGA 20

  (2) Information for SEQ ID NO: 6: (i) Characteristics of the sequence: (A) Length: 33 base pairs (B) Type: nucleic acid (C) Configuration of the strands: simple (D) Topology: linear ( ii) Type of molecule: other nucleic acid

  (A) Description: / desc = "synthetic DNA"

  (xi) Description of the sequence: SEQ ID NO: 6:

  AATCTAGAAT GACGATGCGC ATCGAGCACG ACC 33

  (2) Information for SEQ ID NO: 7: (i) Characteristics of the sequence: (A) Length: 27 base pairs (B) Type: nucleic acid (C) Configuration of the strands: simple (D) Topology: linear ( ii) Type of molecule: other nucleic acid

  (A) Description: / desc = "synthetic DNA"

  (xi) Description of the sequence: SEQ ID NO: 7:

CTCTAGACCT GCGTACGGCG AAGTTCG 27

Claims (12)

  1. Protein characterized in that it has aspartase activity and has the amino acid sequence indicated in SEQ ID NO: 1 or the amino acid sequence indicated in SEQ ID NO: 1 having a deletion, a substitution or an addition of one or more amino acids. 2. DNA characterized in that it codes for the protein according to the claim 1.   3. DNA according to claim 2, characterized in that it has the sequence   nucleotide indicated in SEQ ID NO: 2.   4. DNA according to any one of claims 2 and 3, characterized   in that it is derived from Rhodococcus bacteria.   5. DNA according to claim 4, characterized in that the bacteria   Rhodococcus are part of the Rhodococcus sp. EA4.   6. DNA fragment of approximately 600 bp characterized in that it is amplifiable with the synthetic DNAs represented in SEQ ID NO: 4 and 5 respectively. 7. Recombinant plasmid characterized in that it is obtained by ligation of   DNA according to any one of claims 2 to 6 with a vector   plasmid. 8. Recombinant plasmid characterized in that it is obtained by ligation   with a plasmid DNA vector according to any one of claims   2 to 6 and a region of DNA capable of replication and proliferation in Rhodococcus bacteria.   9. Transformer characterized in that it is obtained by introduction of the   recombinant plasmid according to any one of claims 7 and 8 in a host microorganism.   10. Transformant according to claim 9 characterized in that the   Host microorganism is a Rhodococcus bacteria.   11. A method of producing the protein according to claim 1 characterized in that it comprises the culture of the transformant according to any one of claims 9 and 10.   12. A method of producing L-aspartic acid characterized in that it comprises the reaction of fumaric acid or one of its salts with ammonia or one of its salts in the presence of the transformant according to l any of   claims 9 and 10 or the protein according to claim 1.