JPH067183A - Production of d-glutamic acid - Google Patents

Abstract

PURPOSE:To enable the mass-production of D-glutamic acid useful as a synthetic raw material for pharmaceuticals, agricultural chemicals, etc., at a low cost. CONSTITUTION:D-glutamic acid can be produced by contacting L-glutamic acid with microbial cells, culture liquid or their treated product of a microbial strain belonging to the genus Escherichia and having glutamic acid racemase activity in an aqueous medium to produce DL-glutamic acid, contacting the DL-glutamic acid produced in the aqueous medium with microbial cells, culture liquid or their treated product of a microbial strain having L-glutamic acid decarboxylase activity to decompose the L-glutamic acid in the aqueous medium and collecting the remaining D-glutamic acid from the aqueous medium.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing D-glutamic acid. D-glutamic acid is a substance useful as a synthetic raw material for medicines, agricultural chemicals and the like.

[0002]

2. Description of the Related Art Conventionally, as a method for producing D-glutamic acid, a method in which a hydrolase derived from a microorganism is allowed to act on DL-5-carboxyethylhydantoin (JP-A-55-55)
114291), a method for producing D-glutamic acid by allowing D-amino acid transaminase to act on α-ketoglutaric acid (JP-A-62-205790), and a chemical method for optically resolving DL-glutamic acid by a preferential crystallization method (Japanese Patent Publication No. 31-422, 31-423, 31-297.
2), a method in which a salt of DL-glutamic acid and another optically active compound is prepared and resolved as a diastereomer (Japanese Patent Publication No. 32-5419) and the like are known. However, these methods are not industrially advantageous methods because the raw material substrate is expensive, the manufacturing cost is high, the enzyme system is unstable, the manufacturing process is complicated, and the productivity is low.

Further, by using a microorganism belonging to the genus Lactobacillus (for example, Lactobacillus brevis ATCC8287) having high glutamate racemase activity and glutamate decarboxylase activity, L-glutamic acid was converted to D
-Method for producing glutamic acid (Japanese Patent Application Laid-Open No. 2-5349)
4) is disclosed. However, even though this method has the advantage of efficiently producing D-glutamic acid from L-glutamic acid by a single microorganism, there are problems caused by using a Lactobacillus microorganism. . That is, in general, microorganisms belonging to the genus Lactobacillus have a low yield of cells and it is difficult to prepare a large number of cells, which is an obstacle to improving productivity.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for inexpensively and efficiently producing D-glutamic acid.

[0005]

The inventors of the present invention cloned a gene encoding a glutamate racemase of Lactobacillus brevis ATCC8287 in Escherichia coli, and obtained a wet microorganism of a microorganism carrying the resulting recombinant DNA. Glutamate racemase activity per body was 10 compared to Lactobacillus brevis ATCC8287.
D-glutamic acid, which has been improved by 0 times or more, and which is produced at low cost industrially by fermentation by using the microorganism in combination with an existing microorganism having a high glutamate decarboxylase activity. The present invention has been completed, finding that it can be generated more efficiently.

The present invention will be described in detail below. The present invention relates to a bacterium belonging to the genus Escherichia and having a glutamate racemase activity, a microbial cell, a culture solution or a treated product thereof being contacted with L-glutamic acid in an aqueous medium to produce DL-glutamic acid, and then in the aqueous medium. The produced DL-glutamic acid and L-glutamic acid decarboxylase microbial cells, a culture solution or a treated product thereof are contacted to decompose L-glutamic acid in the aqueous medium, The present invention relates to a method for producing D-glutamic acid, which comprises collecting the remaining D-glutamic acid.

The glutamate racemase and L-glutamate decarboxylase produced by the microorganism of the present invention have different pH regions in which they function. The former is pH 7.5 to 9.5.
, The latter has a pH of 3.5-5.5. Contact with L-glutamic acid in an aqueous medium while keeping the cells, culture solution or treated product of a microorganism belonging to the genus Escherichia and having glutamate racemase activity in an aqueous medium while maintaining the pH at 7.5 to 9.5, preferably pH 8.0 to 9.0. By doing so, L-glutamic acid can be converted to DL-glutamic acid.

The L-glutamic acid used for the contact may be a chemically pure product or a crude product, and may be used in an aqueous medium.
It is used in a concentration range of 10 to 200 g / l, preferably 50 to 200 g / l. The concentration of microbial cells is 0.1 to 50 as wet microbial cells.
g / l, preferably 0.5-10 g / l, 0.1 as enzyme unit
~ 65 U / ml, preferably 0.6 to 13 U / ml. At that time, the bacterial cells may be used as they are, but an organic solvent such as toluene or xylene may be added, or the acetone-treated bacterial cells may be used. When adding an organic solvent such as toluene or xylene, the addition amount is 0.1 to 5.0.
% (V / v) is sufficient. Ammonia water,
An alkali solution such as sodium hydroxide is used, and the temperature during the contact is 25 to 45 ° C, preferably 30 to 40 ° C.
The contact time varies depending on the amount of glutamic acid and the amount of cells used, but is usually 1 to 72 hours.

In the aqueous medium thus obtained,
DL-glutamic acid is present. In the aqueous medium, the produced bacterial cells of a microorganism having DL-glutamic acid and glutamate decarboxylase activity, a culture solution or a treated product thereof is adjusted to pH 3.5 to 5.5, preferably pH 4.0 to
By contacting while maintaining at 5.0, only L-glutamic acid in the aqueous medium can be converted to γ-aminobutyric acid.

The concentration of the bacterial cells used in the contact is 1 to 100 g / l, preferably 1 to 50 g / l, as wet bacterial cells.
0.09-9 U / ml as an enzyme unit, preferably 0.09-4.5 U
/ Ml. At that time, the bacterial cells may be used as they are,
It is also possible to add an organic solvent such as toluene or xylene, or to use acetone-treated cells. When an organic solvent such as toluene or xylene is added, the addition amount may be 0.1 to 5.0% (v / v). The pH is adjusted using an acid solution such as phosphoric acid, hydrochloric acid or sulfuric acid, and the temperature during contact is 25 to 40 ° C. The contact time varies depending on the amount of L-glutamic acid used in the racemization reaction and the amount of cells, but it is usually 1 to 72 hours.

D-glutamic acid and γ-aminobutyric acid are present in the aqueous medium thus obtained. D-glutamic acid can be obtained by removing microbial cells or treated cells from the aqueous medium by means such as centrifugation, and then by direct crystallization method or ion exchange resin treatment. The identification of D-glutamic acid is performed by measurement by high performance liquid chromatography (HPLC) and measurement of specific optical rotation.

Cloning of the glutamate racemase gene of a microorganism belonging to the genus Lactobacillus into Escherichia coli is carried out as follows. The source of glutamate racemase gene may be any microorganism as long as it belongs to the genus Lactobacillus and has glutamate racemase activity. A preferred example is Lactobacillus brevis ATCC8
287 is given. The plasmid vector into which the DNA encoding glutamate racemase is incorporated may be any plasmid vector capable of replicating in Escherichia coli K12 strain. A suitable example is pBR322 (Takara Shuzo).

DN encoding a glutamate racemase
The recombination between the A fragment and the plasmid vector is carried out by treating the chromosomal DNA of a microorganism belonging to the genus Lactobacillus with an appropriate restriction enzyme to obtain a D region containing a glutamate racemase gene region.
An NA fragment was prepared and inserted into a plasmid treated with the same restriction enzyme or a restriction enzyme that produces the same sticky ends, and E. coli was transformed with the recombinant DNA to obtain the desired D
The transformant containing the recombinant DNA containing the NA fragment may be isolated. This series of operations is carried out by a known in vitro recombination technique (Molecular Cloning
ng), T. Maniatis et al. Cold Spring Harbor Laboratory (1982)].

Any recipient organism for recombinant DNA may be used as long as it is a microorganism belonging to the genus Escherichia. Generally, since E. coli does not have glutamate racemase activity, L-glutamic acid is used as a single carbon and nitrogen source. It grows well, but it cannot grow with D-glutamic acid or its growth is weak. However, since Escherichia coli having glutamate racemase activity converts D-glutamic acid into L-glutamic acid by glutamate racemase, it can grow well using D-glutamic acid as a single carbon and nitrogen source. Therefore, any microorganism can be used as the recipient of the recombinant DNA as long as it is a microorganism belonging to the genus Escherichia lacking the restriction system, and more preferably, the L-glutamic acid-requiring strain is used.

Such a microorganism can be obtained by subjecting a strain in which the restriction system of the Escherichia coli K12 strain has been deleted by mutation treatment to a parent strain and further performing ordinary mutation treatment. As the mutation induction method, ultraviolet irradiation,
Any of the commonly used mutagenesis treatments such as X-ray irradiation, radiation irradiation and mutagen treatment can be used.

Isolation of the L-glutamic acid-requiring strain from the mutant-treated cells is carried out as follows. That is, the cells subjected to the mutation treatment are applied to an agar nutrient medium (for example, L agar medium) and cultured at 30 ° C. for 1 to 2 days. The emerged colonies are applied to an agar minimal medium (for example, M9 agar medium) and an agar minimal medium containing a trace amount of L-glutamic acid, and cultured at 30 ° C. for 1 to 3 days, and their growth is compared. L-
An L-glutamic acid-requiring strain can be obtained by selecting a colony that grows on an agar minimal medium containing glutamic acid and cannot grow on an agar minimal medium not containing L-glutamic acid.

In general, such a microorganism cannot grow unless L-glutamic acid is present in the medium. On the other hand, in the microorganism having the glutamate racemase activity, the L-glutamic acid causes glutamate racemase to act as L-glutamate. -Has the same effect on the growth of microorganisms as glutamic acid. Therefore, the microorganism having the glutamate racemase activity grows with D-glutamic acid as a required substance. As an example of such a microorganism, Escherichia coli H-TM93 (hereinafter,
H-TM93). H-TM93
Is a microorganism newly isolated by the present inventors from the Escherichia coli K12 strain deficient in the restriction system.

A recombinant DNA containing a DNA encoding a glutamate racemase of a microorganism belonging to the genus Lactobacillus is transformed into a host Escherichia coli L-glutamic acid-requiring strain to obtain D-glutamic acid as a single carbon and nitrogen source or If a transformant that can grow well as a substance is selected, it is highly likely that the transformant is a strain carrying the recombinant DNA. Furthermore, by measuring the glutamate racemase activity of the selected strain, a strain carrying the target recombinant DNA can be obtained.

As such microorganisms, Escherichia
E. coli H-TMGR1 and H-TMGR2. These microorganisms have been deposited as FERM BP-3779 and FERM BP-3780 at the Institute of Microbial Technology, Institute of Industrial Science, as of March 3, 1992.

DN Encoding Glutamate Racemase
The location of the glutamate racemase gene can be further limited by treating the A fragment with various restriction enzymes to prepare small fragments and measuring the glutamate racemase activity of the resulting plasmid containing each small fragment. . The nucleotide sequence of the DNA fragment can be determined by the dideoxy method [Science, 214 , 1205 (1981)] and the like.

The glutamate racemase activity is measured as follows. Phosphate buffer solution (50 mM, containing L-glutamic acid 100 g / l and toluene 2% (v / v)
pH 8.5), add wet bacterial cells to 1 g / l, and add 3
After reacting at 0 ° C. for 2 hours, the reaction solution is kept in boiling water for 10 minutes to stop the reaction. After removing the bacterial cells from the reaction solution by centrifugation, the supernatant is analyzed by HPLC to measure the amount of D-glutamic acid produced.

The analytical conditions by HPLC are as follows. HPLC analysis conditions: 1) Column: MCI GEL CRS10W (manufactured by Mitsubishi Kasei Co., Ltd.) 2) Mobile phase: 2 mM CuSO ₄ aqueous solution 3) Column temperature: 35 ° C. 4) Flow rate: 1 ml / min 5) Sample amount: 20 μl 6) Detection : 254 nm ultraviolet absorption In addition, the enzyme activity which produces | generates 1 micromol D-glutamic acid in 1 minute is displayed as 1 unit (U).

The microorganism having glutamate decarboxylase activity may be any microorganism having the activity. Examples of such microorganisms include the following. Escherichia coli 26: Journal of Biological Chemistry (J. Biol. Chem.), 235 , 1649 (1
960) Escherichia coli W: Biochemistry (Biochemi
stry), 9 , 226 (1970) Escherichia coli ATCC11246, ATCC1
3676, ATCC21186 Lactobacillus brevis ATCC8287 It is possible to more efficiently produce D-glutamic acid by using a more active microorganism. For example, Escherichia coli ATCC11246 has 20 times or more glutamate decarboxylase activity per wet cell as compared with Lactobacillus brevis ATCC8287.

As a medium for culturing a microorganism belonging to the genus Escherichia and having a glutamate racemase activity or a microorganism having an L-glutamate decarboxylase activity,
Those containing a carbon source, a nitrogen source, inorganic salts and vitamins can be used. As the carbon source, various kinds of carbohydrates such as glucose, fructose, sucrose, lactose, molasses containing these and starch hydrolysates are used.

As the nitrogen source, ammonium salts of various inorganic acids and organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, amines and other nitrogen-containing compounds, peptone, meat extract, yeast extract, Corn steep liquor, casein hydrolyzate, soybean meal hydrolyzate, various fermented cells and digested products thereof are used. As the inorganic material, potassium dihydrogenphosphate, dipotassium hydrogenphosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used.

The culture temperature is 20 to 42 ° C., preferably 30.
~ 38 ° C. PH during culture is 5.0 to 9.0, preferably 6.0.
Hold to ~ 7.5. The pH is adjusted using an inorganic or organic acid, an alkaline solution, calcium carbonate, ammonia or the like. The culture time is usually 5 to 72 hours.
In each microbial cell obtained by culturing in this manner, glutamate racemase that converts L-glutamic acid into DL-glutamic acid, or L-glutamic acid decarboxylase that decomposes L-glutamic acid into γ-aminobutyric acid, Generated and accumulated. Examples of the present invention will be shown below.

[0027]

【Example】

Example 1. Lactobacillus brevis ATCC 8287 with yeast extract-peptone agar plate (yeast extract 5.5 g /
l, peptone 12.5 g / l, glucose 11.0 g / l, KH ₂
PO ₄ 0.25g / l, K ₂ HPO ₄ 0.25g / l, sodium acetate
_{10g / l, MgSO 4 · 7H} 2 O 0.1g / l, MnSO 4 · 4~6H 2 O 5mg /
1, FeSO ₄ .7H ₂ O 5 mg / l, agar 20 g / l, pH 6.8), and cultured at 37 ° C. for 2 days.

1 platinum loop of the culture bacterium was added to 1 liter of fermentation medium (glucose 20 g / l, corn steep liquor 30 g /
l, sodium acetate 20g / l, KH ₂ PO ₄ 1g / l, K ₂ HPO ₄ 3
_{g / l, MgSO 4 · 7H} 2 O 1g / l, MnSO 4 · 4~6H 2 O 1mg / l, FeSO 4
・ 7H ₂ O 10mg / l, 1ml of trace amount metal solution and 1ml of vitamin mixture solution adjusted to pH 7.0, 120 ℃, 20
The cells were sterilized for 1 minute) and statically cultured at 37 ° C. for 24 hours. The trace metal liquid is (NH ₄ ) ₆ Mo ₇ O ₂₄・ 4H ₂ O 3
_{7mg / l, FeSO 4 · 7H} 2 O 990mg / l, ZnSO 4 · 7H 2 O880mg /
l, CuSO ₄ · 5H ₂ O 393mg / l, MnCl ₂ · 4H ₂ O 72mg / l, and Na ₂ B ₄ O ₇ · 10H ₂ O 88mg / l composition, and a vitamin mixture Is riboflavin 2 g / l, thiamine-HCl 1 g / l, p-aminobenzoic acid 1 g / l, nicotinic acid 1 g / l, calcium pantothenate 1 g / l, pyridoxine 1 g / l, biotin 10 mg / l and folic acid 1 mg / l. A liquid having the composition

The obtained culture solution was put at 4 ° C., 5,000 rpm, 1
After centrifuging for 0 minutes and collecting the cells, the bacterial cells were collected in TE buffer [10 mM
Tris (hydroxymethyl) aminomethane (hereinafter abbreviated as Tris), 1 mM EDTA-Na ₂ , (pH 7.5)] was used to wash and collect the cells, and from the cells, the method of Saito-Miura [Bio-Kimika. Actor (Biochim.Biophys.Acta), 7
2 , 619 (1963)], high molecular chromosomal DNA was isolated. This chromosomal DNA was digested with the restriction enzyme HindIII. That is, the restriction enzyme Hin containing 2 μg of chromosomal DNA
Reaction solution for dIII [Tris 50 mM, MgCl ₂ 10 mM, NaCl
50 mM, (pH 7.5)] 90 μl of 2 units of HindII
I (manufactured by Takara Shuzo Co., Ltd.) was added, the mixture was reacted at 37 ° C. for 60 minutes, and the reaction was stopped by heating at 65 ° C. for 10 minutes.

On the other hand, as a cloning vector,
pBR322 (manufactured by Takara Shuzo) [Gene , 2 , 95
(1975)] was used. H containing 1 μg of this pBR322
2 units of HindIII was added to 90 μl of the reaction solution for indIII, reacted at 37 ° C. for 60 minutes, and then heated at 65 ° C. for 10 minutes to stop the reaction. The two reaction solutions obtained above were mixed, and a 10-fold concentrated T4 DNA ligase buffer [Tris 660 mM, MgCl ₂ 66 mM, dithiothreitol 10
0 mM, (pH 7.6)] 20 μl, 100 mM ATP 2 μl,
Add 350 units of T4 DNA ligase (Takara Shuzo),
The ligation reaction was performed at 12 ° C. for 16 hours. This reaction solution was used for transformation of Escherichia coli H-TM93.

Transformation was carried out by molecular cloning [Molecular Cloning, T. Maniatis et al. Cold.
Spring Harbor Laboratory (1982)]. That is, 1 platinum loop of H-TM93 was inoculated into 100 ml of L medium (10 g of bactotryptone, 5 g of yeast extract, 1 g of glucose and 5 g of sodium chloride in 1 liter of water and adjusted to pH 7.2), and OD _{66 was} inoculated. Incubation was carried out at 37 ° C. until 0 reached 0.2. The culture solution was cooled in ice for 10 minutes and then centrifuged to obtain bacterial cells. Chilled 50 mM calcium chloride-20 mM Tris (pH
The cells were resuspended in 25 ml of 8.0), placed in ice water for 15 minutes, and re-centrifuged. Further, the cells were suspended in 2 ml of 50 mM calcium chloride-20 mM Tris (pH 8.0), and suspended in ice for 18 minutes.
I left it for a while. To 150 μl of the obtained bacterial solution, 50 μl of the above ligase reaction mixture was added and mixed, and the mixture was placed in ice for 10 minutes and then heated at 42 ° C. for 2 minutes. Then, 2 ml of L medium was added and the cells were cultured at 37 ° C. for 2 hours.

This bacterial solution was centrifuged, the resulting bacterial cells were washed twice with physiological saline, and then M9 agar medium containing 100 μg / ml of ampicillin and 250 μg / ml of D-glutamic acid [Na ₂ HPO ₄
6 g, KH ₂ PO ₄ 3 g, NaCl 0.5 g, NH ₄ Cl 1 g, MgSO ₄
7H ₂ O 0.5 g, glucose 2 g, calcium chloride 0.
01 g and 0.1 mg of thiamine hydrochloride in 1 liter of water and adjusted to pH 7.2 (1 liter of a medium (M9 medium) to which 20 g of agar was added) were applied and cultured at 30 ° C. for 5 days. When colonies grown on M9 agar medium containing D-glutamic acid were picked up, cultured in L medium and then collected, the glutamate racemase activity was examined, and the strain was 100 times or more that of Lactobacillus brevis ATCC 8287. It was found to have glutamate racemase activity. The results are shown in Table 1.

[0033]

[Table 1]

Plasmid DNA was isolated from this transformant and analyzed by restriction enzyme digestion and agarose gel electrophoresis. As a result, the obtained plasmid was 7.1 kilobase (k).
b), at the HindIII breakpoint of pBR322, at the DN of Lactobacillus brevis ATCC 8287
It had a HindIII fragment of about 2.7 kb from A. This plasmid was named pGAR1. Similarly, when other transformants were examined, it was found that there was a plasmid in which the HindIII fragment of about 2.7 kb inserted in pGAR1 was in the opposite direction to pGAR1. This plasmid was named pGAR2. Plasmid pGAR
The restriction enzyme maps of 1 and pGAR2 are shown in FIGS. 1 and 2.

The recombinant strain prepared by introducing the plasmid pGAR1 into Escherichia coli H-TM93 was transformed into H-TMGR1 and
The plasmid pGAR2 was transformed into Escherichia coli H-TM9.
The recombinant strains introduced in 3 were each named H-TMGR2. Recombinant strain H-TM carrying plasmid pGAR1
GR1 was inoculated into 300 ml of L medium containing 1 platinum loop and 100 μg / ml of ampicillin, and cultured with shaking at 30 ° C. for 18 hours. After culturing, the cells were collected and L-in a 1-liter Erlenmeyer flask.
To 500 ml of an aqueous solution containing 100 g / l of glutamic acid and 2% (v / v) of toluene (adjusted to pH 8.5 with sodium hydroxide), 3 g / l of H-TMGR1 as wet bacterial cells
And gently shaken at 30 ° C. for 5 hours.
As a result, D-glutamic acid was 49.7 g / l in the aqueous medium.
Was produced and the amount of remaining L-glutamic acid was 49.8 g / l.

The pH of the obtained aqueous medium containing DL-glutamic acid was adjusted to 4.2 with 6N hydrochloric acid, and 500 ml of the aqueous medium in a 1-liter Erlenmeyer flask was charged with Escherichia coli ATCC 11246 having L-glutamic acid decarboxylase activity. Add the cultured cells of No. 3 as wet cells to a concentration of 5 g / l, and add 30
The mixture was gently shaken at 10 ° C for 10 hours. Escherichia coli
Culturing of ATCC 11246 was performed according to the culturing method of H-TMGR1 except that L medium not containing ampicillin was used. As a result, L-glutamic acid in the aqueous medium was quantitatively converted to γ-aminobutyric acid, and 48.1 g / l of D-glutamic acid remained in the aqueous medium. In addition, L-glutamic acid was not detected.

After taking 100 ml of the obtained aqueous medium and removing the bacterial cells by centrifugation, the pH of the supernatant was adjusted to 3.2 with 6N hydrochloric acid and then concentrated to obtain 4.2 g of D-glutamic acid crystals. .
The specific rotation of the obtained D-glutamic acid is [α] ²⁰ _D =
It was −31.8 ° (c = 10, 2N HCl).

Example 2. To locate the glutamate racemase gene in the approximately 2.7 kb HindIII fragment inserted into pGAR1 and pGAR2, the restriction enzyme cleavage site present on this insert was used to
Various small pieces were prepared. These fragments and plasmids treated with restriction enzymes to generate sticky ends
The vector pUC18 or pUC19 was ligated and introduced into Escherichia coli TM93, and the glutamate racemase activity of the recombinant was measured.

Among these recombinants, HindIII-
PGA was added to the recombinant having about 1.4 kb of EcoRI fragment.
Recombinant H-TMGR having R1 or pGAR2
Since the glutamate racemase activity equivalent to that of 1 or H-TMGR2 was observed, it was proved that the glutamate racemase gene was located on the HindIII to EcoRI fragment. Approximately 1.4 kb of this HindIII-EcoRI fragment
As a result of determining the nucleotide sequence by the dideoxy method, the sequence shown in SEQ ID NO: 1 was the only open reading frame of 150b or more. This open reading frame is HindIII ~ E
HindIII to EcoRI on the approximately 1.4 kb fragment of coRI
Exists in the direction.

Further, about HindIII to EcoRI
Approximately 0.7 kb of HindIII to PstI from the 1.4 kb fragment (23 in the open reading frame of SEQ ID NO: 1)
The recombinant lacking the (2b upstream portion) did not show glutamate racemase activity at all, and thus it was proved that the open reading frame shown in SEQ ID NO: 1 was the glutamate racemase structural gene.

[0041]

INDUSTRIAL APPLICABILITY According to the present invention, D-glutamic acid, which is important as a synthetic raw material for pharmaceuticals and agricultural chemicals, can be efficiently produced from L-glutamic acid which is industrially inexpensive and produced in large quantities.

[0042]

[Sequence listing] SEQ ID NO: 1 Sequence length: 831 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Lactobacillus brevis Strain name: ATCC 8287 Sequence ATG CAA AAT GAT CCG ATT GGT CTA ATG GAT TCA GGG GTC GGT GGT TTG 48 Met Gln Asn Asp Pro Ile Gly Leu Met Asp Ser Gly Val Gly Gly Leu 1 5 10 15 ACC GTC TTA AAG GAA GTT CAA CGG TTG TTG CCC ACT GAA AAT ACA GTA 96 Thr Val Leu Lys Glu Val Gln Arg Leu Leu Pro Thr Glu Asn Thr Val 20 25 30 TTT CTG GGC GAT CAG GCG CGT TTG CCA TAT GGA CCG CGC TCG GTG GCT 144 Phe Leu Gly Asp Gln Ala Arg Leu Pro Tyr Gly Pro Arg Ser Val Ala 35 40 45 GAG GTC ACA ATG TTT ACC AAA CAA ATT GCG CAA TTT TTA CGC CAA CAA 192 Glu Val Thr Met Phe Thr Lys Gln Ile Ala Gln Phe Leu Arg Gln Gln 50 55 60 GCA AGA ATC AAA GCG TTA GTG ATT GCG TGT AAC ACG GCA ACT GCA GCG 240 Ala Arg Ile Lys Ala Leu Val Ile Ala Cys Asn Thr Ala Thr Ala Ala 65 70 75 80 GCT CTA ACA ACG ATG CAG CAA ACA TTG CCC ATT CCG GTA ATC GGC GTT 288 Ala Leu Thr Thr Met Gln Gln Thr Leu Pro Ile Pro Val Ile Gly Val 85 90 95 ATT GCA CCG GGT GCG CAG GCC GCG GTG CAG ACA ACC CGT AAT CAC CGA 336 Ile Ala Pro Gly Ala Gln Ala Ala Val Gln Thr Thr Arg Asn His Arg 100 105 110 ATT GGG GTG ATT GCA ACG GCC GGA ACG GTT AAG AGT GAC CAG TAT CGG 384 Ile Gly Val Ile Ala Thr Ala Gly Thr Val Lys Ser Asp Gln Tyr Arg 115 120 125 CGA GAT ATT TTA GCA GCA GCA CCT AAT AGT CAA ATT TTT AGT GTG GCT 432 Arg Asp Ile Leu Ala Ala Ala Pro Asn Ser Gln Ile Phe Ser Val Ala 130 135 140 TGT CCG GAA ATG GTG ACG CTG GCA GAG CAA AAT GAC TTA ACG ACC ACG 480 Cys Pro Glu Met Val Thr Leu Ala Glu Gln Asn Asp Leu Thr Thr Thr 145 150 155 160 CAT GCC CAG TCC GTA GTA GCG GCA AAC TTG GCG TCA CTT ATG GAT AAG 528 Ser Gln Val Thr Leu Val Asp Pro Gly Leu Ala Thr Ala Glu Gln Thr 165 170 175 AAA ATT GAT ACG TTG GTG ATG GGC TGT ACG CAT TTT CCA TTA TTG CGA 576 Val Ala Ile Leu His Ala Gln Ser Val Val Ala Ala Asn Leu Ala Ser 180 185 190 AGC GCT ATT CAG CAC GCT GTG GGT TCA CAG GTG ACC TTG GTG GAT CC C 624 Leu Met Asp Lys Lys Ile Asp Thr Leu Val Met Gly Cys Thr His Phe 195 200 205 GGC TTA GCA ACT GCG GAA CAA ACA GTG GCT ATC CTA AAG ACG CGG GGG 672 Pro Leu Leu Arg Ser Ala Ile Gln His Ala Val Gly Lys Thr Arg Gly 210 215 220 TTA CTC AAT TCT GCC ACG ACA CGT GGG ACG GCT CAA TTC TTT ACC ACG 720 Leu Leu Asn Ser Ala Thr Thr Arg Gly Thr Ala Gln Phe Phe Thr Thr 225 230 235 240 GGT GAG ACA GAC CAG TTT GAT ACG TTG GCT AGT CAG TGG TTG GAT CAG 768 Gly Glu Thr Asp Gln Phe Asp Thr Leu Ala Ser Gln Trp Leu Asp Gln 245 250 255 CAA CCA ACG CCA GCG AAG CAC GTG GCG ATT GCG CAG CTG ACA ACT CCG 816 Gln Pro Thr Pro Ala Lys His Val Ala Ile Ala Gln Leu Thr Thr Pro 260 265 270 ATG GAG GTT AAC TAA 831 Met Glu Val Asn 275

[0043]

[Brief description of drawings]

FIG. 1 shows Lactobacillus brevis ATCC 8287.
1 shows a restriction enzyme map of a plasmid pGAR1 containing a gene encoding a derived glutamate racemase.

FIG. 2 shows Lactobacillus brevis ATCC 8287.
1 shows a restriction enzyme map of a plasmid pGAR2 containing a gene encoding a derived glutamate racemase.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location C12P 41/00 A 8931-4B // C12N 1/20 7236-4B (C12N 1/21 C12R 1: 19) (C12N 15/61 C12R 1:24) (C12P 41/00 C12R 1:19) (C12N 1/20 C12R 1:19)

Claims (4)

[Claims] 1. A microbial cell belonging to the genus Escherichia and having a glutamate racemase activity, a culture solution or a treated product thereof is contacted with L-glutamic acid in an aqueous medium to produce DL-glutamic acid, and then the aqueous medium. The DL-glutamic acid produced therein is contacted with a microbial cell having a L-glutamic acid decarboxylase activity, a culture solution or a treated product thereof to decompose L-glutamic acid in the aqueous medium, and the aqueous medium A method for producing D-glutamic acid, which comprises collecting residual D-glutamic acid from 2. When producing D-glutamic acid from L-glutamic acid, the aqueous medium is kept at pH 7.5 to 9.5 to racemize L-glutamic acid into DL-glutamic acid,
The method according to claim 1, which comprises subsequently decomposing L-glutamic acid while maintaining the pH at 3.5 to 5.5. 3. A DNA encoding the glutamate racemase polypeptide shown in SEQ ID NO: 1. 4. A microorganism belonging to the genus Escherichia into which a recombinant DNA containing a DNA encoding a glutamate racemase of a microorganism belonging to the genus Lactobacillus is incorporated.