JPH0530977A - Gene DNA encoding aspartase and use thereof - Google Patents

Abstract

(57) [Summary] [Structure] Brevibacterium flavum MJ-233
A gene DNA encoding aspartase was isolated from the strain and the base sequence of this gene was determined. [Effect] Gene DN encoding this aspartase
Brevibacterium flavum MJ transformed with a plasmid capable of replicative growth in a coryneform bacterium introduced with A
-233 highly produced L-aspartic acid.

Description

Detailed Description of the Invention

[0001]

The present invention relates to aspartase (E
C.4.1.1) -derived gene DNA derived from coryneform bacterium, recombinant plasmid containing the gene DNA, coryneform bacterium transformed with the recombinant plasmid, and coryneform The present invention relates to a method for producing L-aspartic acid using bacteria.

L-aspartic acid is known to exist in proteins as one of the essential amino acids, and is used as a drug or a food additive.

[0003]

2. Description of the Related Art Conventionally, as an industrial production method of L-aspartic acid, fumaric acid and ammonia have been used as starting materials.
Many methods have been proposed for production using a microorganism having aspartase activity [eg, I. Chibata et a.
l., Appl. Microbiol., 27 , 878 (1974);
-29718; see JP-A-60-120983, etc.]. However, these previously proposed L
-There is a limit to improvement in the production method of aspartic acid, and from a new perspective, establishment of a more efficient industrial production method of L-aspartic acid is desired by improving strains by genetic engineering techniques. .

On the other hand, as a gene encoding aspartase, Escherichia coli is used.
Genes (Journal of General Microbiology,
130 , p1271-1278, 1984) and a gene derived from Pseudomonas fluorescens (Journal of Biochemistry, 100 , p697-705,1).
986) is well studied. Among them, aspartase derived from Escherichia coli is known to form a tetramer with a protein molecular weight of 170,000 to 193,000 (Archives of Biochemistry and Biophysics,
147 , p563-570, 1979). However, there has been no previous report on a gene encoding aspartase derived from coryneform bacteria.

[0005]

The object of the present invention is to isolate a gene encoding aspartase derived from coryneform bacterium, introduce the gene into a coryneform bacterium of the same species, and use the coryneform bacterium. It is to efficiently produce L-aspartic acid from a new viewpoint.

[0006]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors have isolated an aspartase gene from a coryneform bacterial chromosome and introduced the gene into an appropriate vector plasmid. As a result, they have found that L-aspartic acid can be efficiently produced by transforming a coryneform bacterium and using the transformed coryneform bacterium, thereby completing the present invention. Thus, according to the present invention, (1) a gene DNA encoding aspartase derived from a coryneform bacterium; (2) a recombinant plasmid having the gene DNA introduced therein; (3) a coryne transformed with the recombinant plasmid. And (4) a method for producing L-aspartic acid from fumaric acid or a salt thereof and ammonia or an ammonium salt using the transformed coryneform bacterium.

The present invention will be described in more detail below.

The "gene DNA encoding aspartase" of the present invention is an enzyme that catalyzes the conversion reaction of fumaric acid and ammonia into L-aspartic acid, that is, aspartase (EC.4.3.1.1). Gene coding for DN
It is A. A large number of microorganisms possess the gene encoding aspartase, but in the present invention, those derived from coryneform bacteria are particularly preferable.

The microorganism serving as a source of a DNA fragment containing a gene encoding aspartase (hereinafter, this may be abbreviated as "A fragment") is not particularly limited as long as it is a coryneform bacterium. However, in general, Brevibacterium flavum MJ-233 (FERM BP-
1497) and its derived strains; Brevibacterium ammoniagenes AT
CC6871, ATCC13745, ATCC13
746; Brevibacterium debaricatum
cterium divaricatum) ATCC14020; Brevibacterium lactofermentum
lactofermentum) ATCC 13869; Corynebacterium glutamicum
ATCC31831 and the like are advantageously used.

An example of the basic procedure for preparing the A fragment from these source microorganisms is as follows.

That is, the A fragment is the above coryneform bacterium,
Brevibacterium flavum, for example.
m flavum) MJ-233 (FERM BP-1497)
It is present on the chromosome of the strain and can be isolated and obtained by the method described below from among the cleaved fragments generated by cleaving this chromosome with an appropriate restriction enzyme.

First, Brevibacterium flavum MJ
Chromosomal DNA is extracted from a culture of strain -233. This chromosomal DNA is partially digested with an appropriate restriction enzyme such as Sau3AI so that the size of the DNA fragment becomes about 20 to 30 kb.

The obtained DNA fragment is a cosmid vector,
For example, insert into pWE15 and insert this cosmid into λDNA i
An Escherichia coli mutant deficient in the aspartase gene (Journal) by transduction using in vitro Packaging Kit
of General Microbiology, 130 , p1271-1278, 1984
Refer to). This E. coli mutant strain is spread on a medium containing L-glutamic acid as a single carbon source.

Cosmid DNA is extracted from the obtained transformant and analyzed with a restriction enzyme to confirm and obtain the inserted A fragment derived from the Brevibacterium flavum MJ-233 strain chromosome.

The A fragment thus obtained has a size of about 2
Since it is as large as 0 to 30 kb and is not practical, it is desirable to specify it to a shorter fragment.

Therefore, the cosmid containing the A fragment obtained above is cleaved with an appropriate restriction enzyme to obtain the resulting DNA.
The fragment is inserted into a vector plasmid that can be replicated in Escherichia coli, and this vector plasmid is introduced into an Escherichia coli mutant strain deficient in the aspartase by a transformation method usually used, for example, the calcium chloride method or the electric pulse method. The E. coli mutant strain is spread on a medium containing L-glutamic acid as a single carbon source.

From the transformant obtained, plasmid DNA is obtained.
The A fragment derived from the chromosome of Brevibacterium flavum MJ-233 strain that has been inserted can be confirmed and obtained by extracting the A.

One of the A fragments thus obtained is
The chromosomal DNA of the Brevibacterium flavum MJ-233 strain can be excised by partial digestion with the restriction enzyme Sau3A1 and further excised with the restriction enzyme EcoRI to give a DNA fragment of about 2.4 kb in size. .

Table 1 below shows the number of recognition sites and the size of the fragment when the DNA fragment containing the gene encoding aspartase of about 2.4 kb was digested with various restriction enzymes.

In the present specification, "the number of recognition sites" by a restriction enzyme means that a DNA fragment or a plasmid is completely decomposed in the presence of a restriction enzyme, and those decomposition products are subjected to 1% agarose according to a method known per se. Gel electrophoresis and 5
% Polyacrylamide gel electrophoresis, and the value determined from the number of separable fragments was adopted.

The "size of the cleaved fragment" and the size of the plasmid are the same as those of Escherichia coli lambda phage (λphage) when agarose gel electrophoresis is used.
Based on the standard line drawn by the migration distance on the same agarose gel of a DNA fragment of known molecular weight obtained by cleaving the DNA of Escherichia coli with a restriction enzyme Hind III, and when using polyacrylamide gel electrophoresis, Escherichia Phi-X 174 phage of coli (φ × 174 phag
Based on the standard line drawn by the migration distance on the same polyacrylamide gel of the DNA fragment of known molecular weight obtained by cleaving the DNA of e) with the restriction enzyme Hae III, the cleaved DNA
Calculate the size of each DNA fragment of the fragment or plasmid. The size of the plasmid is determined by adding the sizes of the respective cut fragments. In addition, in determining the size of each DNA fragment, the size of a fragment of 1 kb or more was 1%.
The results obtained by agarose gel electrophoresis were adopted, and the results obtained by 4% polyacrylamide gel electrophoresis were adopted for fragment sizes of about 0.1 kb to less than 1 kb.

[0022]

[Table 1] Table 1 Number of restriction enzyme recognition sites Cleavage fragment size (kb) Ava I 1 1.7, 0.7 Cla I 1 1.3, 1.1 Hind III 2 1.7, 0.35, 0.35 On the other hand, the Brevibacterium flavum MJ-2 described above.
For a DNA fragment of about 2.4 kb in size obtained by excising the chromosomal DNA of 33 with the restriction enzyme EcoRI, the base sequence is the plasmid pUC18 or pUC18.
The dideoxynucleotide enzymatic method using UC19 (dide
oxy chain termination method) (Sanger, F. et al., P
roc. Natl Acad. Sci. USA 74 , 5463, 1977). The gene encoding aspartase determined from the presence of the open reading frame in the nucleotide sequence of the above-mentioned about 2.4 kb DNA fragment thus determined has the following sequence: 526
It is composed of 1578 base pairs encoding the amino acids of: [Sequence] ATG TCT AAG ACG AGC AAC AAG TCT TCA GCA GAC TCA AAG AAT GAC GCA 48 Met Ser Lys Thr Ser Asn Lys Ser Ser Ala Asp Ser Lys Asn Asp Ala 1 5 10 15 AAA GCC GAA GAC ATT GTG AAC GGC GAG AAC CAA ATC GCC ACG AAT GAG 96 Lys Ala Glu Asp Ile Val Asn Gly Glu Asn Gln Ile Ala Thr Asn Glu 20 25 30 TCG CAG TCT TCA GAC AGC GCT GCA GTT TCG GAA CGT GTC GTC GAA CCA 144 Ser Gln Ser Ser Asp Ser Ala Ala Val Ser Glu Arg Val Val Glu Pro 35 40 45 AAA ACC ACG GTT CAG AAA AAG TTC CGA ATC GAA TCG GAT CTG CTT GGT 192 Lys Thr Thr Val Gln Lys Lys Phe Arg Ile Glu Ser Asp Leu Leu Gly 50 55 60 GAA CTT CAG ATC CCA TCC CAC GCA TAT TAC GGC GTG CAC ACC CTT CGT 240 Glu Leu Gln Ile Pro Ser His Ala Tyr Tyr Gly Val His Thr Leu Arg 65 70 75 80 GCG GTG GAC AAC TTC CAA ATC TCA CGA ACC ACC ATC AAC CAC GTC CCA 288 Ala Val Asp Asn Phe Gln Ile Ser Arg Thr Thr Ile Asn His Val Pro 85 90 95 GAT TTC ATT CGC GGC ATG GTC CAG GTG AAA AAG GCC GCA GCT T TA GCA 336 Asp Phe Ile Arg Gly Met Val Gln Val Lys Lys Ala Ala Ala Leu Ala 100 105 110 AAC CGC CGA CTA CAC ACA CTT CCA GCA CAA AAA GCA GAA GCA ATT GTC 384 Asn Arg Arg Leu His Thr Leu Pro Ala Gln Lys Ala Glu Ala Ile Val 115 120 125 TGG GCT TGT GAT CAG ATC CTC ATT GAG GGA CGC TGT ATG GAT CAG TTC 432 Trp Ala Cys Asp Gln Ile Leu Ile Glu Gly Arg Cys Met Asp Gln Phe 130 135 140 CCC ATC GAT GTG TTC CAG GGT GGC GCA GGT ACC TCA CTG AAC ATG AAC 480 Pro Ile Asp Val Phe Gln Gly Gly Ala Gly Thr Ser Leu Asn Met Asn 145 150 155 160 ACC AAC GAA GTT GTT GCC AAC CTT GCA CTT GAG TTC TTA GGC CAT GAA 528 Thr Asn Glu Val Val Ala Asn Leu Ala Leu Glu Phe Leu Gly His Glu 165 170 175 AAG GGC GAG TAC CAC ATC CTG CAC CCC ATG GAT GAT GTG AAC ATG TCC 576 Lys Gly Glu Tyr His Ile Leu His Pro Met Asp Asp Val Asn Met Ser 180 185 190 CAG TCC ACC AAC GAT TCC TAC CCA ACT GGT TTC CGC CTG GGC ATT TAC 624 Gln Ser Thr Asn Asp Ser Tyr Pro Thr Gly Phe Arg Leu Gly Ile Tyr 195 200 205 GCT GGA CTG CAG ACC CTC ATC GCT GAA ATT GAT G AG CTT CAG GTT GCG 672 Ala Gly Leu Gln Thr Leu Ile Ala Glu Ile Asp Glu Leu Gln Val Ala 210 215 220 TTC CGC CAC AAG GGC AAT GAG TTT GTC GAC ATC ATC AAG ATG GGC CGC 720 Phe Arg His Lys Gly Asn Glu Phe Val Asp Ile Ile Lys Met Gly Arg 225 230 235 240 ACC CAG TTG CAG GAT GCT GTT CCC ATG AGC TTG GGC GAA GAG TTC CGA 768 Thr Gln Leu Gln Asp Ala Val Pro Met Ser Leu Gly Glu Glu Phe Arg 245 250 255 GCA TTC GCG CAC AAC CTC GCA GAA GAG CAG ACC GTG CTG CGT GAA GCT 816 Ala Phe Ala His Asn Leu Ala Glu Glu Gln Thr Val Leu Arg Glu Ala 260 265 270 GCC AAC CGT CTC CTC GAG GTC AAC CTT GGT GCA ACC GCA ATC GGT ACT 864 Ala Asn Arg Leu Leu Glu Val Asn Leu Gly Ala Thr Ala Ile Gly Thr 275 280 285 GGT GTG AAC ACT CCA GCA GGC TAC CGC CAC CAG GTT GTC GCT GCT CTG 912 Gly Val Asn Thr Pro Ala Gly Tyr Arg His Gln Val Val Ala Ala Leu 290 295 300 TCT GAG GTC ACC GGA CTG GAA CTA AAG TCC GCA CGT GAT CTC ATT GAG 960 Ser Glu Val Thr Gly Leu Glu Leu Lys Ser Ala Arg Asp Leu Ile Glu 305 310 315 320 GCT ACC TCT GAC ACC GGT GCA T AT GTT CAT GCG CAC TCC GCA ATC AAG 1008 Ala Thr Ser Asp Thr Gly Ala Tyr Val His Ala His Ser Ala Ile Lys 325 330 335 CGT GCA GCC ATG AAA CTG TCC AAG ATC TGT AAC GAT CTA CGT CTG CTG 1056 Arg Ala Ala Met Lys Leu Ser Lys Ile Cys Asn Asp Leu Arg Leu Leu 340 345 350 TCT TCT GGT CCT CGT GCT GGC TTG AAC GAA ATC AAT CTG CCA CCA CGC 1104 Ser Ser Gly Pro Arg Ala Gly Leu Asn Glu Ile Asn Leu Pro Pro Arg 355 360 365 CAG GCT GGT TCC TCC ATC ATG CCA GCC AAG GTC AAC CCA GTG ATC CCA 1152 Gln Ala Gly Ser Ser Ile Met Pro Ala Lys Val Asn Pro Val Ile Pro 370 375 380 GAA GTG GTC AAC CAG GTC TGC TTC AAG GTC TTC GGT AAC GAT CTC ACC 1200 Glu Val Val Asn Gln Val Cys Phe Lys Val Phe Gly Asn Asp Leu Thr 385 390 395 400 GTC ACC ATG GCT GCG GAA GCT GGC CAG TTG CAG CTC AAC GTC ATG GAG 1248 Val Thr Met Ala Ala Glu Ala Gly Gln Leu Gln Leu Asn Val Met Glu 405 410 415 CCA GTC ATT GGC GAA TCC CTC TTC CAG TCA CTG CGC ATC CTG GGC AAT 1296 Pro Val Ile Gly Glu Ser Leu Phe Gln Ser Leu Arg Ile Leu Gly Asn 420 425 430 GCA GCC AA G ACT TTG CGT GAG AAG TGC GTC GTA GGA ATC ACC GCC AAC 1344 Ala Ala Lys Thr Leu Arg Glu Lys Cys Val Val Gly Ile Thr Ala Asn 435 440 445 GCT GAT GTT TGC CGT GCT TAC GTT GAT AAC TCC ATT GGC ATT ATC ACT 1392 Ala Asp Val Cys Arg Ala Tyr Val Asp Asn Ser Ile Gly Ile Ile Thr 450 455 460 TAC CTG AAC CCA TTC CTG GGC CAC GAC ATT GGA GAT CAG ATC GGT AAG 1440 Tyr Leu Asn Pro Phe Leu Gly His Asp Ile Gly Asp Gln Ile Gly Lys 465 470 475 480 GAA GCA GCC GAA ACT GGT CGA CCA GTG CGT GAA CTC ATC CTG GAA AAG 1488 Glu Ala Ala Glu Thr Gly Arg Pro Val Arg Glu Leu Ile Leu Glu Lys 485 490 495 AAG CTC ATG GAT GAA AAG ACG CTC GAG GCA GTC CTA TCC AAG GAG AAC 1536 Lys Leu Met Asp Glu Lys Thr Leu Glu Ala Val Leu Ser Lys Glu Asn 500 505 510 CTC ATG CAC CCA ATG TTC CGC GGA AGG CTC TAC TTG GAG AAC TAA 1581 Leu Met His Pro Met Phe Arg Gly Arg Leu Tyr Leu Glu Asn 515 520 525 A DNA fragment containing a gene encoding the aspartase of the present invention, which comprises the above-mentioned nucleotide sequence, is a natural coli fragment. Not only those isolated from the mold bacterial chromosome DNA,
A commonly used DNA synthesizer, for example, manufactured by Beckman
It may be synthesized using System-1 Plus.

Further, as described above, the DNA fragment of the present invention obtained from the chromosomal DNA of Brevihacterium flavum MJ-233 is one of the nucleotide sequences unless it substantially impairs the function of encoding aspartase. Part of the base may be replaced with another base or deleted, or a new base may be inserted, or a part of the base sequence may be rearranged. Often, any of these derivatives is included in the DNA fragment containing the gene encoding the aspartase of the present invention.

The size detailed above is about 2.4 kb D
A map of the cleavage points of the NA fragment by the restriction enzyme is shown in FIG.

The DNA fragment (A fragment) containing the gene encoding the aspartase of the present invention is introduced into a plasmid vector containing at least a gene that controls the replication / proliferation function of the plasmid in the coryneform bacterium to obtain a coryneform bacterium. It is possible to obtain a recombinant plasmid capable of high expression of aspartase.

The promoter for expressing the gene encoding the aspartase of the present invention may be the promoter of the gene itself possessed by the coryneform bacterium, or may be used for initiating the transcription of the aspartase gene. Any promoter may be used as long as it is a nucleotide sequence derived from a prokaryote.

The A fragment of the present invention can be introduced,
Examples of the plasmid vector containing at least a gene controlling the replication / proliferation function in a coryneform bacterium include, for example, Japanese Patent Application No.
-4212 plasmid pCRY30;
Plasmid pC described in JP-A-2-276575
RY21, pCRY2KE, pCRY2KX, pCRY
3K7, pCRY3KE and pCRY3KX;
Plasmids pCRY2 and pCRY3 described in JP-A-191686; pAM330 described in JP-A-58-67679; pHM1519 described in JP-A-58-77895; pAJ655 described in JP-A-58-192900. , PAJ611 and pAJ1844;
PCG1 described in JP-A-57-134500; pCG2 described in JP-A-58-35197;
No. 183799, pCG4 and pCG11
Etc. can be mentioned.

Among them, the plasmid vector used in the coryneform bacterium host vector system has a gene that controls the replication / proliferation function of the plasmid in the coryneform bacterium and a gene that controls the plasmid stabilizing function in the coryneform bacterium. Are preferred, for example plasmids pCRY30, pCR
Y21, pCRY2KE, pCRY2KE, pCRY2
KX, pCRY3K7, pCRY3KE and pCRY3
KX or the like is preferably used.

As a method for preparing the above-mentioned plasmid vector pCRY30, Brevibacterium stationis IFO12144 (FE
Plasmid pBY503 (see JP-A-1-95785 for details of this plasmid) DNA was extracted from RMBP-2515) and contained a gene responsible for the replication-proliferation function of a plasmid having a size of about 4.0 kb with a restriction enzyme XhoI. A DNA fragment is cut out, and a DNA fragment containing a gene having a stabilizing function of a plasmid having a size of about 2.1 kb with restriction enzymes EcoRI and KpnI is cut out. Both of these fragments were used as plasmid pHSG298 (Takara Shuzo).
The plasmid vector pCRY30 can be prepared by incorporating it into the EcoRI, KpnI and SalI sites of E. coli.

Next, the introduction of the A fragment of the present invention into the above plasmid vector is carried out by, for example, cleaving a restriction enzyme site existing at only one site in the plasmid vector with the restriction enzyme,
If necessary, the A fragment and the cleaved plasmid vector may be treated with S1 nuclease to make it blunt-ended, or in the presence of an appropriate adapter DNA.
It can be performed by ligating with ligase treatment.

The introduction of the A fragment of the present invention into the plasmid pCRY30 is carried out by using the plasmid pCRY30 with the restriction enzyme Eco.
It can be carried out by cleaving with RI and ligating the DNA fragment (A fragment) containing the gene encoding the aspartase thereto with DNA ligase.

The plasmid pC constructed in this way
The recombinant plasmid in which the A fragment of the present invention having a size of about 2.4 kb was introduced into RY30 is one of the recombinant plasmids which can be preferably used for the production of L-aspartic acid, and the present inventors This is plasmid pCRY30-A
It was named spB. Plasmid pCRY30-AspB
The details of the method for creating are described in Example 4 below.

A map of restriction enzyme cleavage points of this plasmid pCRY30-AspB is shown in FIG. A plasmid capable of replicative growth in a coryneform bacterium containing an aspartase gene constructed in this manner is introduced into a host microorganism, and a culture of the microorganism is used to stably and efficiently produce L-aspartic acid. Is possible.

Host microorganisms which can be transformed with the plasmid according to the present invention include coryneform bacteria such as Brevibacterium flavum MJ-233 (FERM BP-1).
497), Brevibacterium flavum MJ-233.
-AB-41 (FERM BP-1498), Brevibacterium flavum MJ-233-ABT-11 (F
ERM BP-1500), Brevibacterium flavum MJ-233-ABD-21 (FERM BP-1)
499) and the like.

The above-mentioned FERM BP-1498 strain is an ethanol-assimilating microorganism to which DL-α-aminobutyric acid resistance has been positively imparted by using the FERM BP-1497 strain as a parent strain (Japanese Patent Publication Sho-59-59). 28398 gazette column 3-4 reference). In addition, FERM BP-1500
Strain is a highly active mutant of L-α-aminobutyric acid transaminase using the strain of FERM BP-1497 as a parent strain (see JP-A-62-51998). Furthermore, the strain of FERM BP-1499 is FERMBP-
It is a highly active mutant strain of D-α-aminobutyric acid deaminase using the strain 1497 as a parent strain (JP-A-61-177993).
(See the official gazette).

In addition to these microorganisms, Brevibacterium ammoniagenes
ATCC 6871, ATCC 13745, ATCC
13746; Brevibacterium debaricatum (Brevi
bacterium divaricatum) ATCC14020; Brevibacterium lactofer
mentum) ATCC 13869; Corynebacterium glutamicum ATCC31
831 etc. can also be used as a host microorganism.

When a strain derived from Brevibacterium flavum MJ-233 is used as a host, the plasmid pBY502 possessed by this strain (Japanese Patent Laid-Open No. 63-3678).
(See Japanese Patent Publication No. 7), transformation may be difficult.
It is desirable to remove BY502. As a method for removing such a plasmid pBY502, for example,
By repeating subculture, it is possible to spontaneously delete or artificially remove [B
act. Rev. 36 p. 361-405 (1972)]. An example of the method for artificially removing the plasmid pBY502 is as follows.

Host Brevibacterium flavum MJ-
Approximately 10 cells per ml in a medium containing acridine orange (concentration: 0.2 to 50 μg / ml) or ethidium bromide (concentration: 0.2 to 50 μg / ml) at a concentration that incompletely inhibits the growth of 233. Inoculate so that
Incubate at about 35 ° C. for about 24 hours while incompletely inhibiting the growth. After diluting the culture solution, the culture solution is applied to an agar medium and cultured at about 35 ° C for about 2 days. Each of the emerged colonies is independently subjected to a plasmid extraction operation to select a strain from which the plasmid pBY502 has been removed. By this operation, plasmid p
A strain derived from Brevibacterium flavum MJ-233 from which BY502 is removed is obtained.

As a method for transforming the above-mentioned plasmid into the strain derived from Brevibacterium flavum MJ-233 thus obtained, as is known for Escherichia coli and Erwinia carotovora [Calvin,
NM and Hanawalt, PC, Journal of Bacteriolog
y, 170 , 2796 (1988); Ito, K., Nishida,
T. and Izaki. K., Agricultural and Biological Chem
istry, 52 , 293 (1988)], pulsed wave energization to DNA recipient bacteria [Satoh, Y. et al., Journal of In
dustrial Microbiology, 5 , 159 (1990)] and the like.

A method for culturing a coryneform bacterium having an aspartase-producing ability obtained by transformation by the above method, for example, a strain derived from Brevibacterium flavum MJ-233 will be described below.

Cultivation can be carried out in an ordinary nutrient medium containing a carbon source, a nitrogen source, an inorganic salt, etc. Examples of the carbon source include glucose, ethanol, methanol, molasses, etc.
As the nitrogen source, for example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea and the like are used alone or in combination. Further, as the inorganic salt, for example, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like are used. In addition, nutrients such as various vitamins such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid, biotin and the like can be added to the medium.

Culturing can be carried out usually under aerobic conditions such as aeration and stirring, at a temperature of about 20 to 40 ° C., preferably 25 to 35 ° C. PH during culture is 5-10,
It can be preferably around 7 to 8, and the pH during culture
Can be adjusted by adding an acid or an alkali.

The carbon source concentration at the start of culture is preferably 1
-5% by volume, more preferably 2-3% by volume. The culture period can be usually 1 to 7 days, and the optimum period is 3 days.

The cells can be collected from the thus obtained culture, washed with water or an appropriate buffer, and used in the L-aspartic acid production reaction.

In the L-aspartic acid production reaction,
These bacterial cells can be used as they are, or can be used as a disrupted bacterial cell product subjected to ultrasonic treatment or the like, or can be immobilized on a suitable carrier before use. More preferably, a treated product obtained by subjecting the microbial cell or its crushed product or immobilized product to heat treatment in the presence of L-aspartic acid and ammonium ion at a temperature of about 40 to 60 ° C. in an alkaline region of pH can be used. .

In the present specification, the crushed products, immobilized products, heat-treated products and the like of the bacterial cells as described above are collectively referred to as "treated bacterial cells".

According to the present invention, therefore, there is provided a method for producing L-aspartic acid, which comprises reacting fumaric acid or a salt thereof with ammonia or an ammonium salt in the presence of the above-mentioned cultured bacterial cell or treated bacterial cell. To be done.

The enzymatic reaction between fumaric acid or its salt and ammonia or ammonium salt is about 0 in an aqueous medium.
It can be carried out in the range of -60 ° C, but it is preferably carried out in the range of 20-50 ° C in consideration of the stability of aspartase. The molar ratio of fumaric acid or its salt to ammonia or ammonium salt is usually 1: 1 to
A range of 1: 5 is suitable.

[0049]

The present invention has been described above, but the present invention will be described in more detail with reference to the following examples.

Example 1 Asbestos derived from Brevibacterium flavum MJ-233
A DNA fragment containing a gene encoding partase (A fragment)
(A) Cloning (A) Extraction of total DNA of Brevibacterium flavum MJ-233 Semi-synthetic medium A medium [composition: urea 2 g, (NH ₄ ) ₂ SO ₄
7g, K ₂ HPO ₄ 0.5g, KH ₂ PO ₄ 0.5g, Mg
_{SO 4 0.5g, FeSO 4 · 7H} 2 O 6mg, MnSO 4
4-6H ₂ O 6mg, yeast extract 2.5g, casamino acid 5
g, biotin 200 μg, thiamine hydrochloride 200 μg, glucose 20 g, distilled water 11] 11, and Brevibacterium flavum MJ-233 (FERM BP-149).
7) was cultured until the late logarithmic growth phase and the bacterial cells were collected. 10m containing the obtained bacterial cells at a concentration of 10mg / ml containing lysozyme
M NaCl-20 mM Tris buffer (pH 8.0)-
It was suspended in 15 ml of 1 mM EDTA-2Na solution. Next, proteinase K was added so that the final concentration was 100 μg / ml, and the mixture was incubated at 37 ° C. for 1 hour. Further, sodium dodecyl sulfate was added so that the final concentration was 0.5%, and the mixture was kept at 50 ° C. for 6 hours for lysis. In this lysate,
After adding an equal volume of phenol / chloroform solution and shaking gently at room temperature for 10 minutes, centrifuge the whole volume (5,
(000 × g, 20 minutes, 10 to 12 ° C.), and the supernatant fraction was collected, sodium acetate was added to the mixture at 0.3 M, and then twice the amount of ethanol was gently added. The DNA existing between the aqueous layer and the ethanol layer was scattered with a glass rod, washed with 70% ethanol, and then air-dried. 10mM Tris buffer (pH 7.5) -1m to the obtained DNA
5 ml of M EDTA.2Na solution was added, and the mixture was allowed to stand at 4 ° C. overnight and used for the subsequent experiments.

(B) Creation of Recombinant Brevibacterium flavum MJ obtained in the above (A)
90 μl of the total DNA solution of S-233 with the restriction enzyme Sau3A
I 1 unit was used to carry out reaction at 37 ° C. for 20 minutes for partial decomposition. Cosmid pWE15 (manufactured by Stradagene) was digested with the restriction enzyme BamHI to the partially digested DNA,
Dephosphorylated mixture was mixed, 50 mM Tris buffer (pH 7.6), 10 mM dithiothreitol, 1 m
Each component of MATP, 10 mM MgCl ₂ and 1 unit of T4 DNA ligase was added (concentration of each component is the final concentration), reacted at 4 ° C. for 15 hours to bind.

(C) Selection of cosmid containing gene encoding aspartase The aspartase-deficient Escherichia coli mutant strain used for the above-mentioned gene selection was Escherichia coli K-.
12JRG1114 (aspA23) [inside parentheses indicates aspartase genotype (Genotype). For details and obtaining method of this strain, see Journal of Gen.
eral Microbiology, 130 , 1271-1278 (1
984)]].

Using the cosmid mixture obtained in the above (B),
A selective medium [K ₂ HPO ₄ which was transduced with the Escherichia coli JRG1174 strain and contained 50 mg of ampicillin.
7g, KH ₂ PO ₄ 2g, (NH ₄ ) ₂ SO ₄ 1g, Mg
SO ₄ .7H ₂ O 0.1 g, L-glutamic acid sodium salt 30 mM and agar 16 g were dissolved in distilled water 11]. For transduction, λ sold by Takara Shuzo
It was performed using the DNA in vitro Packaging Kit. The strain grown on the medium was subjected to liquid culture by a conventional method, cosmid DNA was extracted from the culture solution, the cosmid was cleaved with a restriction enzyme, and the cosmid pWE15 was examined for length 8. In addition to the 8 kb DNA fragment, a DNA fragment of about 30 kb in length was observed. This cosmid was named pWE15-Asp.

(D) Plasmid pHSG3 of DNA fragment (A, fragment) containing a gene encoding aspartase
Subcloning into 99 The DNA insert fragment contained in the cosmid pWE15-Asp obtained in (C) above is as large as about 30 kb and is not practical, so in order to miniaturize only the necessary portion of the obtained fragment, DNA containing a gene encoding aspartase into plasmid pHSG399 (commercially available from Takara Shuzo)
The fragment was subcloned as follows.

Cosmid pWE15-obtained in the above item (C)
Asp digested with restriction enzyme EcoRI and plasmid pHSG399 digested with restriction enzyme EcoRI were mixed, and 50 mM Tris buffer (pH 7.6), 1
0 mM dithiothreitol, 1 mM ATP, 10 mM
Each component of 1 unit of MgCl ₂ and T ₄ DNA ligase was added (the concentration of each component is the final concentration), and the mixture was reacted at 12 ° C. for 15 hours to be bound.

Using the obtained plasmid mixture, the calcium chloride method (Journal of Molecular Biology, 53 , 15) was used.
9, 1970) by Escherichia coli K-12JR
G1114 and (aspA23) strain was transformed, selection medium containing chloramphenicol 50mg [K ₂ HPO ₄ 7
g, KH ₂ PO ₄ 2g, (NH ₄ ) ₂ SO ₄ 1g, MgS
_{O 4 · 7H 2 O 0.1g,} L- sodium glutamate 3
0 mM and 16 g of agar were dissolved in 1 liter of distilled water].

The strain grown on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture solution, the plasmid was cleaved with a restriction enzyme and examined by agarose gel electrophoresis to find that the length of plasmid pHSG399 was long. In addition to the 2.2 kb DNA fragment, an inserted DNA fragment of about 2.4 kb in length was observed. The number of restriction enzyme recognition sites and the size of the cleaved fragment of a DNA fragment of about 2.4 kb in length when cleaved with various restrictions were as shown in Table 1 above. A map of restriction enzyme cleavage points of this DNA fragment is shown in FIG.

The plasmid obtained above was cleaved with various restriction enzymes, and the size of the cleaved fragment was measured. The results are shown in Table 2 below.

[0059]

Table 2 Plasmid pHSG399-Asp Restriction number of restriction enzyme recognition sites Size of cleavage fragment (kb) AvaI 2 3.6, 1.0 ClaI 1 4.6 EcoRI 2 2.4, 2.2 The above restriction enzymes The plasmid characterized by
It was named SG399-Asp.

Based on the above, a DNA fragment (Ec) having a size of about 2.4 kb containing a gene encoding aspartase is obtained.
was obtained.

Example 2 Determination of Nucleotide Sequence of Gene Encoding Aspartase The DNA fragment of about 2.4 kb containing the gene encoding aspartase obtained in item (D) of Example 1 was analyzed. Nucleotide sequence of plasmid pUC18 or pU
The dideoxynucleotide enzymatic method (dideox) using C19
y chain termination method) (Sanger, F. et al., Pro
c. Nat. Acad. Sci. USA 74 , 5463, 197
According to the strategy diagram shown in FIG. From the presence of the open reading frame in the base sequence,
The gene encoding aspartase encodes 526 amino acids having the nucleotide sequence shown in the sequence listing below 1
It was found to consist of 578 base pairs.

Example 3 Plasmid vector pC which is stable and replicates in coryneform bacteria
Construction of RY30 (A) Preparation of plasmid pBY503 Plasmid pBY503 was prepared from Brevibacterium statinis IFO12144 (FERM BP-251).
The plasmid was isolated from 5) and has a molecular weight of about 10 megadalton, and was prepared as described in JP-A-1-95785. Semi-synthetic medium A medium [urea 2 g, (NH
₄ ) ₂ SO ₄ 7 g, K ₂ HPO ₄ 0.5 g, KH ₂ PO ₄ 0.
_{5g, MgSO 4 0.5g, FeSO 4} · 7H 2 O 6m
g, MnSO ₄ .4-6H ₂ O 6 mg, yeast extract 2.5
g, casamino acid 5 g, biotin 200 μg, thiamine hydrochloride 200 μg, glucose 20 g and distilled water 1 l] 1 l
And Brevibacterium statiornis IFO121
44 was cultured until the late logarithmic growth phase, and the bacterial cells were collected. A buffer containing lysozyme at a concentration of 10 mg / ml [25 mM tris (hydroxymethyl) aminomethane, 10 mM EDTA, 50 mM glucose] 20 m
It was suspended in 1 and reacted at 37 ° C. for 1 hour. Alkali-SDS solution [0.2N NaOH, 1% (w / v)]
SDS] 40 ml was added, mixed gently and allowed to stand at room temperature for 15 minutes. Next, a potassium acetate solution [5M potassium acetate-60 ml, acetic acid 11.5 m] was added to the reaction solution.
1, mixed solution of distilled water 28.5 ml] 30 ml,
After mixing well, the mixture was allowed to stand in ice water for 15 minutes.

The total amount of the lysate was transferred to a centrifuge tube and centrifuged at 4 ° C. for 10 minutes at 15,000 × g to obtain a supernatant.

To this, an equal amount of phenol-chloroform solution (phenol: chloroform = 1: 1 mixture) was added and suspended, then transferred to a centrifuge tube, and 15,000 at room temperature for 5 minutes.
The water layer was collected by centrifugation at x g. To the aqueous layer, twice the amount of ethanol was added, and the mixture was allowed to stand at -20 ° C for 1 hour and then centrifuged at 4 ° C for 10 minutes at 15,000 xg to recover the precipitate.

After drying the precipitate under reduced pressure, TE buffer [Tris 1
0 mM, EDTA 1 mM; adjusted to pH 8.0 with HCl] dissolved in 2 ml. Cesium chloride solution [5]
170 g of cesium chloride in 100 ml of double concentration TE buffer
15 ml and 10 mg / ml ethidium bromide solution (1 ml) were added to give a density of 1.392 g /
adjusted to ml. This solution is stored at 12 ° C for 42 hours, 11
Centrifugation at 6,000 xg was performed.

The plasmid pBY503 is found as a lower band in the centrifuge tube by UV irradiation. By pulling this band from the side of the centrifuge tube with a syringe,
A fraction containing the plasmid pBY503 was obtained.

Next, this fraction solution was treated with an equal amount of isoamyl alcohol four times to extract and remove ethidium bromide, and thereafter, it was dialyzed in a TE buffer solution. A 3M sodium acetate solution was added to the final concentration of 30 mM to the dialysate containing the plasmid pBY503 thus obtained, and then double the amount of ethanol was added and the mixture was allowed to stand at -20 ° C for 1 hour. This solution is centrifuged at 15,000 × g to obtain D
NA was precipitated to obtain 50 μg of plasmid pBY503.

(B) Plasmid vector-pCRY30
The plasmid pHSG298 (Takara Shuzo) 0.5 μg was reacted with the restriction enzyme Sa1I (5 units) at 37 ° C. for 1 hour,
The plasmid DNA was completely degraded.

Plasmid pBY prepared in the above (A)
Restriction enzyme XhoI (1 unit) to 2 μg of 503 at 37 ° C
For 30 minutes to partially decompose the plasmid DNA.

Both plasmid DNA digests were mixed,
After heat treatment at 65 ° C. for 10 minutes to inactivate the restriction enzyme, the final concentration of each component in the inactivating solution was 5
0 mM Tris buffer pH 7.6, 10 mM MgCl ₂ ,
10 mM dithiothreitol, 1 mM ATP and T4
Strengthen each component to 1 unit of DNA ligase,
It was kept warm at 6 ° C for 15 hours. Using this solution, Escherichia coli JM109 competent cells (Takara Shuzo) were transformed.

Transformed strain is 30 μg / ml (final concentration)
Kanamycin, 100 μg / ml (final concentration) IP
TG (isoipropyl-β-D-thiogalactopyranoside) 100 μg / ml (final concentration) of X-gal (5-
L medium containing bromo-4-chloro-3-indolyl-β-galactopyranoside) (10 g tryptone, 5 g yeast extract, 5 g NaCl and 1 l pure water, pH 7.2)
After culturing at 37 ° C. for 24 hours, it was obtained as a growing strain.
Of these growing strains, those that grew in white colonies were selected, and the respective plasmids were subjected to the alkali-SDS method [T. Maniatis E. F. Fritsch, J .; Sambrook,
"Molecular cloning" (1982) p90-91].

As a result, S of plasmid pHSG298
About 4.0k derived from the plasmid pBY503 at the alI site
Plasmid pHSG298-or in which the fragment of b was inserted
i was obtained.

Then, using the same method, the plasmid pBY503DNA obtained in the above (A) was digested with the restriction enzyme Kpn.
A DNA fragment of about 2.1 kb obtained by treatment with I and EcoRI was cloned into the above-mentioned plasmid pHSG298-ori at KpnI and EciRI sites to prepare a plasmid vector-pCRY30.

Example 4 Construction of plasmid pCRY30-AspB and introduction into coryneform bacterium Plasmid pHSG39 obtained in section (D) of Example 1
5 unit of 9-Asp 5 μg of restriction enzyme EcoRI,
A mixture obtained by reacting at 37 ° C. for 1 hour and digested with 301 μg of the plasmid pCRY obtained in the section (B) of Example 3 and digested at 37 ° C. for 1 hour was mixed, and mixed with 50 mM Tris buffer. Liquid (pH
7.6) 10 mM dithiothreitol, 1 mM AT
Each component of P, 10 mM MgCl ₂ and 1 unit of T4 DNA ligase was added (the concentration of each component is the final concentration), and the mixture was reacted at 12 ° C. for 15 hours to be bound. Using this plasmid, Escherichia coli K was prepared according to the method described above.
-12JRG1114 the (aspA ₂₃₎ strain was transformed,
Selection medium [K ₂ HP containing 50 μg / ml of kanamycin
O ₄ 7 g, KH ₂ PO ₄ 2 g, (NH ₄ ) ₂ SO ₄ 1 g, M
gSO ₄ .7H ₂ O 1 g, L-glutamic acid + sodium 30 mM and agar 16 g were dissolved in distilled water 1 l].

The strain grown on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture solution, the plasmid was cleaved with a restriction enzyme and examined by agarose gel electrophoresis. As a result, plasmid pCRY30 was obtained. In addition to the 8.6 kb long DNA fragment, an inserted DNA fragment of 2.4 kb in size was observed.

Plasmid DNA prepared as described above
Was transformed into a coryneform bacterium.

Transformation was carried out as follows using the electric pulse method.

Brevibacterium flavum MJ-23
3 (FERM BP-1497) plasmid pBY50
The 2 removed strain was cultivated in 100 ml of the medium A until the initial logarithmic growth, penicillin G was added to 1 unit / ml, and the mixture was further cultivated with shaking for 2 hours, and the cells were collected by centrifugation to collect the cells. 20 ml pulse solution (272 mM
Sucrose, 7 mM KH ₂ PO ₄ , 1 mM MgCl ₂ ; p
It was washed with H7.4). Further, the cells were collected by centrifugation, suspended in 5 ml of the pulse solution, and 0.75 ml of the cells were mixed with 50 μl of the plasmid DNA solution obtained above and left standing in water for 20 minutes. Using Gene Pulser (manufactured by Bio-Rad), 2500 V, 25 μ
After setting to FD and applying a pulse, the plate was allowed to stand in ice for 20 minutes. The whole amount was transferred to 3 ml of the A medium and cultured at 30 ° C. for 1 hour, then inoculated into the A agar medium containing 15 μg / ml (final concentration) of kanamycin, and cultured at 30 ° C. for 2 to 3 days. From the emerged kanamycin resistant strain, the above-mentioned Example 3 was selected.
A plasmid was obtained using the method described in section (A). This plasmid was cleaved with various restriction enzymes and the size of the cleaved fragment was measured. The results are shown in Table 3 below.

[0079]

Table 3 Plasmid pCRY30-AspB Number of restriction enzyme recognition sites Size of cleaved fragment (kb) EcoRI 2 8.6, 2.4 BamH1 1 11.0 The plasmid characterized by the above restriction enzymes was pCR.
It was named Y30-AspB. This plasmid pCRY3
A restriction map of 0-AspB is shown in FIG.

Brevibacterium flavum MJ transformed with the plasmid pCRY30-AspB.
Brevibacterium flavum MJ-233-AspB transformed with -233-AspB was submitted to the Institute of Microbial Technology, Institute of Industrial Technology, 1-3-1 Higashi, Tsukuba City, Ibaraki Prefecture on May 9, 1991. : Deposited as Micromachine Lab. No. 12228 (FERM P-12228).

Example 5 Stability of Plasmid pCRY30-AspB 100 ml of the above-mentioned A medium was dispensed into a 500 ml Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes, and the transformant Brevibacterium obtained in Example 4 was obtained. Umm flavum M
After inoculating J-233-AspB and shaking culture at 30 ° C. for 24 hours, 100 ml of A medium prepared in the same manner
Was dispensed into a 500 ml Erlenmeyer flask and heated at 120 ° C for 15
The cells sterilized for 1 minute were subcultured at a rate of 50 cells per 1 ml, and shake culture was carried out at 30 ° C. for 24 hours. Next, the cells were collected by centrifugation and washed, and then a fixed amount was spread on a plate medium prepared using A medium supplemented with kanamycin at a rate of 15 μg / ml and A medium without supplement, at 30 ° C. After culturing for 1 day, the number of growing colonies was counted.

As a result, it was confirmed that the same number of colonies grew in the kanamycin-added medium and the kanamycin-free medium, and that all the A medium-grown colonies grew in the kanamycin-added medium, that is, the high stability of the plasmid.

Example 6 Production of L-Aspartic Acid 100 ml of the above-mentioned A medium was dispensed into a 500 ml Erlenmeyer flask and sterilized (pH 7.0 after sterilization), and then Brevibacterium fravum MJ-2.
33-AspB was inoculated and glucose was aseptically added at 5 g / l.
Was added so that the concentration became, and shake culture was performed at 33 ° C. for 2 days.

Next, the main culture medium (glucose 5%, ammonium sulfate 2.3%, KH ₂ PO ₄ 0.05%, K ₂ HP
_{O 4 0.05%, MgSO 4 ·} 7H 2 O0.05%, FeS
_{O 4 · 7H 2 O20ppm, MnSO} 4 · nH 2 O20pp
m, biotin 200 μg / l, thiamine / HCl 10
0 μg / l, casamino acid 0.3%, yeast extract 0.3%)
1000 ml of the above was placed in a 2 l aeration and stirring tank, sterilized (120 ° C., 20 minutes), 20 ml of the culture was added, and the number of revolutions was 1000 rpm, the aeration rate was 1 vvm, the temperature was 33 ° C., and the pH was 7.6. Culturing was carried out for an hour.

After completion of the culture, these culture solutions were centrifuged (4000 rpm, 15 minutes), and the collected cells were suspended in distilled water to give OD (optical density, absorbance at wavelength of 610 nm) value of 50. A body suspension was prepared, and the cell suspension was used as a test solution.

For the production of L-aspartic acid, 50 ml of the reaction solution shown in Table 4 below was reacted at 45 ° C. for 5 hours, and the reaction completed solution was centrifuged (4000 rpm, 15 minutes).
The amount of aspartic acid produced in the supernatant was determined by a microorganism quantification method using Leuconostoc mesenteroides ATCC 8042.

The results are shown in Table 5 as relative values with the production amount by the FERM BP-1497 strain being 1.

[0088]

[Table 4 fumarate 5g MgSO ₄ · 7H ₂ O 0.1g polyoxyethylene (20) sorbitan monolaurate 0.05ml ammonia (28% strength) 14 ml test solution 10ml total amount 50 ml (pH 9.4)

[0089]

[Table 5] As is clear from the results shown in Table 5, by using the microorganism of the present invention, L-aspartic acid could be efficiently produced from fumaric acid or its salt and ammonia or ammonium salt.

[0090]

INDUSTRIAL APPLICABILITY The novel gene DNA of the present invention is a gene DNA encoding aspartase derived from coryneform bacteria.
Therefore, it becomes possible to efficiently produce L-aspartic acid from fumaric acid and ammonia using a coryneform bacterium into which the plasmid of the present invention containing the gene DNA has been introduced.

[0091]

[Sequence listing] SEQ ID NO: 1 Array length: 1581 Sequence type: Nucleic acid Number of chains: double-stranded Topology: linear Sequence Type: Genomic DNA origin Organism name: Brevibacterium flavum Share Name: MJ-233 Sequence features Characteristic symbol: peptide Location: 1-1581 Method by which the characteristics were determined: P Array ATG TCT AAG ACG AGC AAC AAG TCT TCA GCA GAC TCA AAG AAT GAC GCA 48 Met Ser Lys Thr Ser Asn Lys Ser Ser Ala Asp Ser Lys Asn Asp Ala   1 5 10 15 AAA GCC GAA GAC ATT GTG AAC GGC GAG AAC CAA ATC GCC ACG AAT GAG 96 Lys Ala Glu Asp Ile Val Asn Gly Glu Asn Gln Ile Ala Thr Asn Glu              20 25 30 TCG CAG TCT TCA GAC AGC GCT GCA GTT TCG GAA CGT GTC GTC GAA CCA 144 Ser Gln Ser Ser Asp Ser Ala Ala Val Ser Glu Arg Val Val Glu Pro          35 40 45 AAA ACC ACG GTT CAG AAA AAG TTC CGA ATC GAA TCG GAT CTG CTT GGT 192 Lys Thr Thr Val Gln Lys Lys Phe Arg Ile Glu Ser Asp Leu Leu Gly      50 55 60 GAA CTT CAG ATC CCA TCC CAC GCA TAT TAC GGC GTG CAC ACC CTT CGT 240 Glu Leu Gln Ile Pro Ser His Ala Tyr Tyr Gly Val His Thr Leu Arg  65 70 75 80 GCG GTG GAC AAC TTC CAA ATC TCA CGA ACC ACC ATC AAC CAC GTC CCA 288 Ala Val Asp Asn Phe Gln Ile Ser Arg Thr Thr Ile Asn His Val Pro                  85 90 95 GAT TTC ATT CGC GGC ATG GTC CAG GTG AAA AAG GCC GCA GCT TTA GCA 336 Asp Phe Ile Arg Gly Met Val Gln Val Lys Lys Ala Ala Ala Leu Ala             100 105 110 AAC CGC CGA CTA CAC ACA CTT CCA GCA CAA AAA GCA GAA GCA ATT GTC 384 Asn Arg Arg Leu His Thr Leu Pro Ala Gln Lys Ala Glu Ala Ile Val         115 120 125 TGG GCT TGT GAT CAG ATC CTC ATT GAG GGA CGC TGT ATG GAT CAG TTC 432 Trp Ala Cys Asp Gln Ile Leu Ile Glu Gly Arg Cys Met Asp Gln Phe     130 135 140 CCC ATC GAT GTG TTC CAG GGT GGC GCA GGT ACC TCA CTG AAC ATG AAC 480 Pro Ile Asp Val Phe Gln Gly Gly Ala Gly Thr Ser Leu Asn Met Asn 145 150 155 160 ACC AAC GAA GTT GTT GCC AAC CTT GCA CTT GAG TTC TTA GGC CAT GAA 528 Thr Asn Glu Val Val Ala Asn Leu Ala Leu Glu Phe Leu Gly His Glu                 165 170 175 AAG GGC GAG TAC CAC ATC CTG CAC CCC ATG GAT GAT GTG AAC ATG TCC 576 Lys Gly Glu Tyr His Ile Leu His Pro Met Asp Asp Val Asn Met Ser             180 185 190 CAG TCC ACC AAC GAT TCC TAC CCA ACT GGT TTC CGC CTG GGC ATT TAC 624 Gln Ser Thr Asn Asp Ser Tyr Pro Thr Gly Phe Arg Leu Gly Ile Tyr         195 200 205 GCT GGA CTG CAG ACC CTC ATC GCT GAA ATT GAT GAG CTT CAG GTT GCG 672 Ala Gly Leu Gln Thr Leu Ile Ala Glu Ile Asp Glu Leu Gln Val Ala     210 215 220 TTC CGC CAC AAG GGC AAT GAG TTT GTC GAC ATC ATC AAG ATG GGC CGC 720 Phe Arg His Lys Gly Asn Glu Phe Val Asp Ile Ile Lys Met Gly Arg 225 230 235 240 ACC CAG TTG CAG GAT GCT GTT CCC ATG AGC TTG GGC GAA GAG TTC CGA 768 Thr Gln Leu Gln Asp Ala Val Pro Met Ser Leu Gly Glu Glu Phe Arg                 245 250 255 GCA TTC GCG CAC AAC CTC GCA GAA GAG CAG ACC GTG CTG CGT GAA GCT 816 Ala Phe Ala His Asn Leu Ala Glu Glu Gln Thr Val Leu Arg Glu Ala             260 265 270 GCC AAC CGT CTC CTC GAG GTC AAC CTT GGT GCA ACC GCA ATC GGT ACT 864 Ala Asn Arg Leu Leu Glu Val Asn Leu Gly Ala Thr Ala Ile Gly Thr         275 280 285 GGT GTG AAC ACT CCA GCA GGC TAC CGC CAC CAG GTT GTC GCT GCT CTG 912 Gly Val Asn Thr Pro Ala Gly Tyr Arg His Gln Val Val Ala Ala Leu     290 295 300 TCT GAG GTC ACC GGA CTG GAA CTA AAG TCC GCA CGT GAT CTC ATT GAG 960 Ser Glu Val Thr Gly Leu Glu Leu Lys Ser Ala Arg Asp Leu Ile Glu 305 310 315 320 GCT ACC TCT GAC ACC GGT GCA TAT GTT CAT GCG CAC TCC GCA ATC AAG 1008 Ala Thr Ser Asp Thr Gly Ala Tyr Val His Ala His Ser Ala Ile Lys                 325 330 335 CGT GCA GCC ATG AAA CTG TCC AAG ATC TGT AAC GAT CTA CGT CTG CTG 1056 Arg Ala Ala Met Lys Leu Ser Lys Ile Cys Asn Asp Leu Arg Leu Leu             340 345 350 TCT TCT GGT CCT CGT GCT GGC TTG AAC GAA ATC AAT CTG CCA CCA CGC 1104 Ser Ser Gly Pro Arg Ala Gly Leu Asn Glu Ile Asn Leu Pro Pro Arg         355 360 365 CAG GCT GGT TCC TCC ATC ATG CCA GCC AAG GTC AAC CCA GTG ATC CCA 1152 Gln Ala Gly Ser Ser Ile Met Pro Ala Lys Val Asn Pro Val Ile Pro     370 375 380 GAA GTG GTC AAC CAG GTC TGC TTC AAG GTC TTC GGT AAC GAT CTC ACC 1200 Glu Val Val Asn Gln Val Cys Phe Lys Val Phe Gly Asn Asp Leu Thr 385 390 395 400 GTC ACC ATG GCT GCG GAA GCT GGC CAG TTG CAG CTC AAC GTC ATG GAG 1248 Val Thr Met Ala Ala Glu Ala Gly Gln Leu Gln Leu Asn Val Met Glu                 405 410 415 CCA GTC ATT GGC GAA TCC CTC TTC CAG TCA CTG CGC ATC CTG GGC AAT 1296 Pro Val Ile Gly Glu Ser Leu Phe Gln Ser Leu Arg Ile Leu Gly Asn             420 425 430 GCA GCC AAG ACT TTG CGT GAG AAG TGC GTC GTA GGA ATC ACC GCC AAC 1344 Ala Ala Lys Thr Leu Arg Glu Lys Cys Val Val Gly Ile Thr Ala Asn         435 440 445 GCT GAT GTT TGC CGT GCT TAC GTT GAT AAC TCC ATT GGC ATT ATC ACT 1392 Ala Asp Val Cys Arg Ala Tyr Val Asp Asn Ser Ile Gly Ile Ile Thr     450 455 460 TAC CTG AAC CCA TTC CTG GGC CAC GAC ATT GGA GAT CAG ATC GGT AAG 1440 Tyr Leu Asn Pro Phe Leu Gly His Asp Ile Gly Asp Gln Ile Gly Lys 465 470 475 480 GAA GCA GCC GAA ACT GGT CGA CCA GTG CGT GAA CTC ATC CTG GAA AAG 1488 Glu Ala Ala Glu Thr Gly Arg Pro Val Arg Glu Leu Ile Leu Glu Lys                 485 490 495 AAG CTC ATG GAT GAA AAG ACG CTC GAG GCA GTC CTA TCC AAG GAG AAC 1536 Lys Leu Met Asp Glu Lys Thr Leu Glu Ala Val Leu Ser Lys Glu Asn             500 505 510 CTC ATG CAC CCA ATG TTC CGC GGA AGG CTC TAC TTG GAG AAC TAA 1581 Leu Met His Pro Met Phe Arg Gly Arg Leu Tyr Leu Glu Asn         515 520 525

[Brief description of drawings]

1 is a restriction map of a DNA fragment containing a gene encoding an aspartase of the present invention, which is digested with a restriction enzyme.

FIG. 2 is a strategy diagram for determining the nucleotide sequence of a DNA fragment of the present invention having a size of about 2.4 kb.

FIG. 3 is a restriction enzyme map of the plasmid pCRY30-AspB of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location (C12N 15/60 C12R 1:13) (C12N 1/20 C12R 1:13) (C12P 13/20 (72) Inventor Hideaki Yukawa 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Sanryo Yuka Co., Ltd. Tsukuba Research Institute

Claims (8)

[Claims] 1. An aspartase (E derived from coryneform bacterium)
A gene DNA encoding C.4.3.1.1). 2. The gene DNA according to claim 1, wherein the coryneform bacterium is Brevibacterium flavum MJ-233. 3. A gene DN encoding (EC.4.3.1.1) aspartase represented by the following DNA base sequence:
A. ATGTCTAAGA CGAGCAACAA GTCTTCAGCA GACTCAAAGA ATGACGCAAA AGCCGAAGAC 60 ATTGTGAACG GCGAGAACCA AATCGCCACG AATGAGTCGC AGTCTTCAGA CAGCGCTGCA 120 GTTTCGGAAC GTGTCGTCGA ACCAAAAACC ACGGTTCAGA AAAAGTTCCG AATCGAATCG 180 GATCTGCTTG GTGAACTTCA GATCCCATCC CACGCATATT ACGGCGTGCA CACCCTTCGT 240 GCGGTGGACA ACTTCCAAAT CTCACGAACC ACCATCAACC ACGTCCCAGA TTTCATTCGC 300 GGCATGGTCC AGGTGAAAAA GGCCGCAGCT TTAGCAAACC GCCGACTACA CACACTTCCA 360 GCACAAAAAG CAGAAGCAAT TGTCTGGGCT TGTGATCAGA TCCTCATTGA GGGACGCTGT 420 ATGGATCAGT TCCCCATCGA TGTGTTCCAG GGTGGCGCAG GTACCTCACT GAACATGAAC 480 ACCAACGAAG TTGTTGCCAA CCTTGCACTT GAGTTCTTAG GCCATGAAAA GGGCGAGTAC 540 CACATCCTGC ACCCCATGGA TGATGTGAAC ATGTCCCAGT CCACCAACGA TTCCTACCCA 600 ACTGGTTTCC GCCTGGGCAT TTACGCTGGA CTGCAGACCC TCATCGCTGA AATTGATGAG 660 CTTCAGGTTG CGTTCCGCCA CAAGGGCAAT GAGTTTGTCG ACATCATCAA GATGGGCCGC 720 ACCCAGTTGC AGGATGCTGT TCCCATGAGC TTGGGCGAAG AGTTCCGAGC ATTCGCGCAC 780 AACCTCGCAG AAGAGCAGAC CGTGCTGCGT GAAGCTGCCA ACCGTCTCCT CGAGGTCAAC 840 CTTGGTGCAA CCGCAATCGG TACTGGTGTG AACACTCCAG CAGGCTACCG CCACCAGGTT 900 GTCGCTGCTC TGTCTGAGGT CACCGGACTG GAACTAAAGT CCGCACGTGA TCTCATTGAG 960 GCTACCTCTG ACACCGGTGC ATATGTTCAT GCGCACTCCG CAATCAAGCG TGCAGCCATG 1120 AAACTGTCCA AGATCTGTAA CGATCTACGT CTGCTGTCTT CTGGTCCTCG TGCTGGCTTG 1180 AACGAAATCA ATCTGCCACC ACGCCAGGCT GGTTCCTCCA TCATGCCAGC CAAGGTCAAC 1240 CCAGTGATCC CAGAAGTGGT CAACCAGGTC TGCTTCAAGG TCTTCGGTAA CGATCTCACC 1300 GTCACCATGG CTGCGGAAGC TGGCCAGTTG CAGCTCAACG TCATGGAGCC AGTCATTGGC 1360 GAATCCCTCT TCCAGTCACT GCGCATCCTG GGCAATGCAG CCAAGACTTT GCGTGAGAAG 1420 TGCGTCGTAG GAATCACCGC CAACGCTGAT GTTTGCCGTG CTTACGTTGA TAACTCCATT 1480 GGCATTATCA CTTACCTGAA CCCATTCCTG GGCCACGACA TTGGAGATCA GATCGGTAAG 1540 GAAGCAGCCG AAACTGGTCG ACCAGTGCGT GAACTCATCC TGGAAAAGAA GCTCATGGAT 1600 GAAAAGACGC TCGAGGCAGT CCTATCCAAG GAGAACCTCA TGCACCCAAT GTTCCGCGGA 1660 AGGCTCTACT TGGAGAACTA A 1681 // 4. A gene DNA encoding aspartase (EC.4.3.1.1) represented by the following amino acid sequence. Met Ser Lys Thr Ser Asn Lys Ser Ser Ala Asp Ser Lys Asn Asp Ala 1 5 10 15 Lys Ala Glu Asp Ile Val Asn Gly Glu Asn Gln Ile Ala Thr Asn Glu 20 25 30 Ser Gln Ser Ser Asp Ser Ala Ala Val Ser Glu Arg Val Val Glu Pro 35 40 45 Lys Thr Thr Val Gln Lys Lys Phe Arg Ile Glu Ser Asp Leu Leu Gly 50 55 60 Glu Leu Gln Ile Pro Ser His Ala Tyr Tyr Gly Val His Thr Leu Arg 65 70 75 80 Ala Val Asp Asn Phe Gln Ile Ser Arg Thr Thr Ile Asn His Val Pro 85 90 95 Asp Phe Ile Arg Gly Met Val Gln Val Lys Lys Ala Ala Ala Leu Ala 100 105 110 Asn Arg Arg Leu His Thr Leu Pro Ala Gln Lys Ala Glu Ala Ile Val 115 120 125 Trp Ala Cys Asp Gln Ile Leu Ile Glu Gly Arg Cys Met Asp Gln Phe 130 135 140 Pro Ile Asp Val Phe Gln Gly Gly Ala Gly Thr Ser Leu Asn Met Asn 145 150 155 160 Thr Asn Glu Val Val Ala Asn Leu Ala Leu Glu Phe Leu Gly His Glu 165 170 175 Lys Gly Glu Tyr His Ile Leu His Pro Met Asp Asp Val Asn Met Ser 180 185 190 Gln Ser Thr Asn Asp Ser Tyr Pro Thr Gly Phe Arg Leu Gly Ile Tyr 195 200 205 Ala Gly Leu Gln Thr Le u Ile Ala Glu Ile Asp Glu Leu Gln Val Ala 210 215 220 Phe Arg His Lys Gly Asn Glu Phe Val Asp Ile Ile Lys Met Gly Arg 225 230 235 240 Thr Gln Leu Gln Asp Ala Val Pro Met Ser Leu Gly Glu Glu Phe Arg 245 250 255 Ala Phe Ala His Asn Leu Ala Glu Glu Gln Thr Val Leu Arg Glu Ala 260 265 270 Ala Asn Arg Leu Leu Glu Val Asn Leu Gly Ala Thr Ala Ile Gly Thr 275 280 285 Gly Val Asn Thr Pro Ala Gly Tyr Arg His Gln Val Val Ala Ala Leu 290 295 300 Ser Glu Val Thr Gly Leu Glu Leu Lys Ser Ala Arg Asp Leu Ile Glu 305 310 315 320 Ala Thr Ser Asp Thr Gly Ala Tyr Val His Ala His Ser Ala Ile Lys 325 330 335 Arg Ala Ala Met Lys Leu Ser Lys Ile Cys Asn Asp Leu Arg Leu Leu 340 345 350 Ser Ser Gly Pro Arg Ala Gly Leu Asn Glu Ile Asn Leu Pro Pro Arg 355 360 365 Gln Ala Gly Ser Ser Ile Met Pro Ala Lys Val Asn Pro Val Ile Pro 370 375 380 Glu Val Val Asn Gln Val Cys Phe Lys Val Phe Gly Asn Asp Leu Thr 385 390 395 400 Val Thr Met Ala Ala Glu Ala Gly Gln Leu Gln Leu Asn Val Met Glu 405 410 415 Pro Val Ile Gly Glu Se r Leu Phe Gln Ser Leu Arg Ile Leu Gly Asn 420 425 430 Ala Ala Lys Thr Leu Arg Glu Lys Cys Val Val Gly Ile Thr Ala Asn 435 440 445 Ala Asp Val Cys Arg Ala Tyr Val Asp Asn Ser Ile Gly Ile Ile Thr 450 455 460 Tyr Leu Asn Pro Phe Leu Gly His Asp Ile Gly Asp Gln Ile Gly Lys 465 470 475 480 Glu Ala Ala Glu Thr Gly Arg Pro Val Arg Glu Leu Ile Leu Glu Lys 485 490 495 Lys Leu Met Asp Glu Lys Thr Leu Glu Ala Val Leu Ser Lys Glu Asn 500 505 510 Leu Met His Pro Met Phe Arg Gly Arg Leu Tyr Leu Glu Asn 515 520 525 5. A recombinant plasmid into which the gene DNA according to any one of claims 1 to 4 has been introduced. 6. A recombinant plasmid having the gene DNA according to any one of claims 1 to 4 and a DNA containing a gene that controls a replication / proliferation function in a coryneform bacterium. 7. A coryneform bacterium transformed with the recombinant plasmid according to claim 6. 8. L-aspartic acid, characterized in that fumaric acid or a salt thereof is reacted with ammonia or ammonium salt in the presence of the cultured bacterial cells or treated bacterial cells of the coryneform bacterium of claim 7. Manufacturing method.