JP2578474B2 - Method for producing L-glutamic acid - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing L-glutamic acid. Therefore, the present invention is useful in the industrial fields of food and medicine.

2. Description of the Related Art Conventionally, various methods have been known as methods for producing L-glutamic acid using coryneform glutamate-producing bacteria.
The present invention relates to a method using a mutant strain of the genus Brevibacterium having reduced isocitrate lyase activity (Japanese Patent Application Laid-Open No. 56-92795), or a method of growing using L-glutamine or L-glutamic acid as a sole carbon source. Using mutant strains that lack or have significantly reduced ability to
57-138395).

Problems to be Solved by the Invention In recent years, the demand for L-glutamic acid has been increasing, and to improve the productivity of L-glutamic acid, an improvement in the method for producing L-glutamic acid has always been desired.

Means for Solving the Problems The present inventors belonged to coryneform glutamic acid producing bacteria, and
-A mutant in which ketoglutarate dehydrogenase activity was significantly reduced, a mutant in which both activities of isocitrate lyase and α-ketoglutarate dehydrogenase were significantly reduced, and the mutant further imparted resistance to a proline analog The present inventors have found that the productivity of L-glutamic acid is improved by using the mutant strain, and completed the present invention.

 Hereinafter, the present invention will be described in detail.

The present invention belongs to coryneform glutamic acid producing bacteria, α-
Ketoglutarate dehydrogenase activity, or both isocitrate lyase activity and α-ketoglutarate dehydrogenase activity are significantly reduced, and a microorganism having L-glutamic acid-producing ability, or further imparting resistance to the proline analog to the microorganism The cultured microorganism is cultured in a medium, L-glutamic acid is produced and accumulated in the culture, and L-glutamic acid is produced from the culture.
Provided is a method for producing L-glutamic acid, comprising collecting glutamic acid. In the present specification, the coryneform glutamate-producing bacteria refers to a group of glutamate-producing bacteria belonging to the genera Corynebacterium, Brevibacterium, and Microbacterium (“Fermentation and Industry”, No. 40).
Vol. 102, 1982).

As the microorganism used in the present invention, a microorganism belonging to coryneform glutamate-producing bacteria, α-ketoglutarate dehydrogenase activity, or a microorganism in which both isocitrate lyase activity and α-ketoglutarate dehydrogenase activity are significantly reduced,
Alternatively, any microorganism can be used as long as the microorganism further imparts resistance to a proline analog. Examples of proline analogs include 3,4-dehydroproline, 4-thioproline, azetidine carboxylic acid, and the like.
Further, these microorganisms may have other properties in addition to these properties, such as various auxotrophy, drug resistance, drug sensitivity, drug dependency, and the like.

Such a microorganism can be obtained by a conventional method from a group of cells which have been subjected to a conventional mutation treatment, for example, ultraviolet irradiation or chemical treatment such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or nitrous acid. Obtainable. Microorganisms having such properties can also be induced by other genetic techniques, such as gene recombination, transduction, cell fusion and the like.

Mutants with reduced isocitrate lyase activity can be easily separated from mutants that assimilate glucose but do not assimilate acetic acid. Furthermore, the mutant strain in which the α-ketoglutarate dehydrogenase activity is reduced in addition to the decrease in the isocitrate lyase activity is subjected to a mutation treatment using the above-described mutant strain in which the isocitrate lyase activity is reduced as a parent strain. However, citric acid can be easily separated from mutant strains that do not assimilate.

Furthermore, a mutant having resistance to a proline analog can be easily separated from a mutant that can grow on a minimal agar medium containing a proline analog at a concentration at which the parent strain cannot grow, by performing a mutation treatment using the above mutant as a parent strain. .

Hereinafter, a specific method for inducing Corynebacterium glutamicum G-41 (a mutant lacking isocitrate lyase activity-deficient α-ketoglutarate dehydrogenase activity), which is a representative example of the strain used in the present invention, will be described below. Is described.

Corynebacterium glutamicum B-15 (FERM 7982, hereinafter referred to as B-15) is 10 ⁸ cells / ml in cell 0.1N Tris maleate buffer in (pH 6.0) As above. To this, NTG was added to a final concentration of 0.2 mg / ml, left at room temperature for 30 minutes, and then allowed to stand at room temperature for a minimum agar medium using glucose as a sole carbon source (glucose 0.5 g / dl, KH ₂ PO ₄
0.15 g / dl, K ₂ HPO ₄ 0.05 g / dl, NaCl 0.01 g / dl, MgSO ₄・ 7H ₂ O0.
_{05g / dl, CaCl 2 · 2H} 2 O1μg / ml, FeSO 4 · 7H 2 O10μg / ml, MnCl
₂ · 4H _{2 O7μg} / ml, thiamine _{· HCl0.1μg / ml, (NH 4} ) 2 SO
₄ 0.15 g / dl, Bition 30 μg / dl, Agar 1.5 g / dl (NaOH pH 7.
2)), and incubated at 30 ° C. for 3 days.

The colony-grown cells are then transferred to the minimal agar medium described above and a minimal agar medium containing acetic acid as the sole carbon source (a medium in which glucose is removed from the above glucose medium and 0.5 g / dl acetic acid is added). Transcription was performed by the replica method, and static culture was performed at 30 ° C. for 3 days. As a result, a number of mutant strains that grew on the minimal medium using glucose as a carbon source but did not grow on the minimal medium using acetic acid as a carbon source were obtained.

These mutants were transformed into a liquid medium using glucose and acetic acid as carbon sources [glucose 3%, ammonium acetate 2%,
(NH ₄ ) ₂ SO ₄ 0.1%, K ₂ HPO ₄ 0.2%, MgSO ₄ .7H ₂ O 0.05%, biotin 500 μg /, thiamine / HCl 500 μg /, calcium pantothenate 500 μg /, nicotinamide 500 μg /
, CaCO ₃ 2%, (pH 7.2)] and aerobically cultured in a 300 ml Erlenmeyer flask containing 50 ml under shaking conditions of 210 rpm. The obtained strains were collected, and the strain was crushed by an ultrasonic cell crusher, and centrifuged at 12,000 rpm by a centrifuge. The isocyanate lyase activity in the obtained supernatant was measured by Method in Enzymology (Method
s in Enzymology), vol. 13, p. 163 (1969).
−40 was selected.

G-40 thus obtained was treated with NTG in the same manner as described above, smeared on a minimal medium containing glucose as a carbon source, and the grown colonies were separated from the above-mentioned minimal agar medium containing glucose as a carbon source and citric acid. Smear on both the minimal agar medium as the carbon source (the above-mentioned minimal agar medium with glucose as the carbon source and remove the glucose and add 0.5 g / dl of sodium citrate). A number of mutants that did not grow on the medium were isolated. In the same manner as in the above-mentioned measurement of isocitrate lyase activity, these mutant strains were cultured in liquid culture, collected, and disrupted to obtain a cell extract, and each of them was α-ketoglutarate dehydrogenase. The activity was determined by Agricultural and Biological Chemistry (Agric. Biol. Chem.) Vol. 44 (19
As a result of measurement according to the method described in (80), strain G-41 was selected as a strain having reduced α-ketoglutarate dehydrogenase activity.

The G-41 strain obtained as described above, in which both the isocitrate lyase activity and the α-ketoglutarate dehydrogenase activity were significantly reduced, was identified as Corynebacterium glutamicum G-41 (FERM BP-1652). January 1, 1988
It was deposited with the Institute of Microbial Industry and Technology (MIC) on April 4.

Further, the Corynebacterium glutamicum G-41 strain obtained above was used as a parent strain, and a proline analog (3,4
-Dehydroproline),
The induction was performed as follows.

Bouillon agar G-41 strain grown overnight at slant medium cells (FERM BP-1652), so that 10 ⁹ / ml 1/1
Suspend in 5M Tris-maleate buffer, add NTG to 250
It was added to a concentration of μg / ml and left at room temperature for 30 minutes. Collect cells by centrifugation and use 1 / 15M of same volume as above.
Resuspend in Tris-maleate buffer and add 0.1 ml
Minimal agar medium containing 0 mg / dl of 3,4-dehydroproline [glucose 0.5 g / dl, (NH ₄ ) ₂ SO ₄ 0.15 g / dl, KH ₂ PO ₄ 0.15
_{g / dl, K 2 HPO 4} 0.05g / dl, NaCl4.6g / dl, MgSO 4 · 7H 2 O0.05g / d
_{l, CaCl 2 · 2H 2 O1μg} / ml, FeSO 4 · 7H 2 O10μg / ml, MnCl 2 · 4H
₂ O7μg / ml, Thiamine / HCl 0.1μg / ml, Vithion 30μg /
, Agar 1.5 g / dl (adjusted to pH 7.2 with NaOH)] and incubated at 30 ° C for 3 days. On the surface of the medium to which 3,4-dehydroproline was added, 3,4-dehydroproline-resistant strains grew as about 250 colonies. On the other hand, in the control medium without 3,4-dehydroproline, the bacteria grew on the entire surface of the medium. One hundred strains of the grown 3,4-dehydroproline resistant strains were picked and subjected to an L-glutamic acid production test according to the method of Example 1 described later. As a result, G-42 was identified as a bacterium having excellent L-glutamic acid productivity. The strain was selected.

The G-42 strain having resistance to 3,4-dehydroproline obtained as described above was Corynebacterium
Glutamicum G-42 (FERM BP-1845)
Deposited with the Japan Institute of Fabrication on May 11, 2008.

As a medium for culturing the above microorganisms, a nutrient medium or a synthetic medium containing a carbon source, a nitrogen source, inorganic salts, growth factors and the like is used.

As the carbon source, carbohydrates such as glucose, fructose, sucrose, molasses, starch, starch hydrolysate, fruit juice, and alcohols such as ethanol, methanol, and propanol can be used.

Nitrogen sources include ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, ammonium acetate, urea, ammonium, amines, peptone,
Meat extract, yeast extract, corn steep liquor, casein hydrolyzate, various fermented cells and digests thereof can be used.

As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate and the like can be used.

When a mutant showing auxotrophy is used, nutrients can be added as a standard or a natural product containing it.

As the culture conditions, under aerobic conditions such as aeration and stirring,
The culture temperature is 24-37 ° C, and the culture days are 2-7 days. The pH of the culture is maintained in the range of 5-9. Urea, calcium carbonate, ammonia gas, aqueous ammonia,
Magnesium phosphate, ammonium carbonate and the like are used.

As a method for isolating L-glutamic acid from the culture solution after completion of the culture, a known method such as an ion exchange resin method and a solvent extraction method is used.

 Hereinafter, the present invention will be specifically described with reference to examples.

Example 1 Corynebacterium glutamicum G-41 as inoculum
(FERM BP-1652) was used. G-41 strain was replaced with glucose 4
0g /, Peptone 10g /, Meat extract 5g /, Yeast extract 5g
_{/, KH 2 PO 4 1g /} , K 2 HPO 4 1g /, MgSO 4 · 7H 2 O0.5g /, FeS
_{O 4 · 7H 2 O20mg /,} MnSO 4 · 4H 2 O20mg /, urea 5 g /, (N
H ₄₎ 30 containing ₂ SO ₄ 5 g / consisting seed medium (pH 7.2) 20 ml
A 0 ml Erlenmeyer flask was inoculated and cultured at 30 ° C. for 24 hours.
7 ml of this seed culture was added to a production medium [glucose 77 g /, urea
7.7 g /, (NH ₄ ) ₂ SO ₄ 3 g /, KH ₂ PO ₄ 1.5 g /, K ₂ HPO ₄ 1.5 g
_{/, MnSO 4 · 4H 2 O30mg} /, biotin 77μg /, calcium pantothenate 770μg /, nicotinamide 770Myug /
, Thiamine / HCl 300 μg / (pH 7.2)]
The mixture was inoculated into a 00 ml Erlenmeyer flask, penicillin G was further added to a final concentration of 5 units / ml, and cultured with shaking at 34 ° C. During the culture, maintain the pH of the culture solution at 6.5 to 8.0,
The cells were cultured for 30 hours while adding 0.5 ml of a sterilized 10% urea solution. After completion of the culture, the amount of L-glutamic acid produced in the culture solution was measured. As a result, 27.2 mg / ml of L-glutamic acid was accumulated in the culture solution. As a control, the parent strain B-15 was cultured in the same manner as described above. As a result, 24.3 mg / ml of L-glutamic acid was produced.

Example 2 30 baffle plates containing 20 ml of a production medium having the following composition
G-4 obtained in the same manner as in Example 1 was placed in a 0 ml Erlenmeyer flask.
One strain or 1 ml of a seed culture of the G-42 (FERM BP-1875) strain was inoculated, respectively, and shake-cultured at 30 ° C. for 40 hours under shaking conditions of 210 rpm. As a result, the G-41 strain was 10.2 mg / ml. L-glutamic acid and the G-42 strain accumulated 49 mg / ml L-glutamic acid in the culture broth, respectively. Parent strain B- used as a control
In the case of the 15 strains and the G-40 strain, the accumulated amount of L-glutamic acid was 0.1 mg / ml or less.

Composition of production medium: molasses 100 g / (glucose conversion), (NH ₄ ) ₂ SO ₄ 20 g /, KH ₂ PO ₄ 0.5 g /, MgSO ₄ .7H ₂ O
0.5 g /, urea _{3g /, FeSO 4 · 7H 2} O10mg /, thiamine · H
Cl2.5 mg /, Bition 500 μg /, CaCO ₃ 30 g / pH 7.2 Effects of the Invention According to the present invention, L-glutamic acid can be produced in good yield and at low cost.

Claims (3)

(57) [Claims] The present invention relates to a coryneform glutamic acid producing bacterium,
Ketoglutarate dehydrogenase activity is significantly reduced and L
-Culturing a microorganism capable of producing glutamic acid in a medium,
L-glutamic acid is produced and accumulated in a culture, and L-glutamic acid is collected from the culture.
A method for producing glutamic acid. 2. The method according to claim 1, wherein said microorganism is a microorganism further reduced in isocitrate lyase activity. 3. The method according to claim 1, wherein the microorganism further has resistance to a proline analog.