JP3100763B2 - Method for producing L-arginine by fermentation - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing L-arginine by a fermentation method. L-arginine is an amino acid useful as a drug, food, and the like.

[0002]

2. Description of the Related Art Conventionally, L-arginine has been produced by a fermentation method or an extraction method from protein. As a method for producing by the fermentation method, a method using a strain resistant to arginine analog [Agricultural & Biological Chemistry (Agricultural & Biological Chemistry)]
emistry), 36 , 1675 (1972), Journal of General and Applied Microbiology (Jou
rnal of General & Applied Microbiology), 19 , 339 (1
973) and JP-B-48-3391], a method using a microorganism belonging to the genus Corynebacterium and having resistance to pyrimidine analogs (JP-B-57-50479), belonging to the genus Corynebacterium and having resistance to cysteine analogs. Method using a microorganism (JP-A-1-257)
No. 486) is known.

[0003] In the prior art, it is known that a strain having a high L-arginine productivity can be obtained by imparting resistance to an analog such as an amino acid or a nucleic acid, but by increasing the degree of resistance to osmotic pressure, It is not known that the productivity of L-arginine increases.

[0004]

There is a need for a method for producing L-arginine in good yield and at low cost.

[0005]

According to the present invention, there is provided a microorganism belonging to the genus Corynebacterium or the genus Brevibacterium, which is capable of growing on a medium having an osmotic pressure in which a parent strain cannot grow, and which has an L-arginine-producing ability. Cultivate in a medium,
The present invention can provide a method for producing L-arginine by a fermentation method, which comprises producing and accumulating L-arginine in a culture and collecting L-arginine from the culture.

Hereinafter, the present invention will be described in detail. As the microorganism used in the present invention, any microorganism can be used as long as it belongs to the genus Corynebacterium or the genus Brevibacterium, can grow on a medium having an osmotic pressure that does not allow the parent strain to grow, and has L-arginine-producing ability. . Such a microorganism belongs to the genus Corynebacterium or the genus Brevibacterium, and a microorganism capable of producing L-arginine is subjected to ordinary mutation treatment to impart the ability to grow on a medium having an osmotic pressure in which the parent strain cannot grow. Can be obtained.

[0007] The parent strain for constructing the mutant strain is Coryneba.
Belonging to the genus Cterium or Brevibacterium, L-
Mutant even in wild strains as long as they have arginine-producing ability
It may be a strain or any. For example, Corynebacterium
・ Glutamicum (Corynebacterium glutamicum)
Nebacterium acetoacidophilum (Corynebacteri
um acetoacidophilum), Corynebacterium lilium
(Corynebacterium lilium), Brevibacterium hula
Bam (Brevibacterium flavum), Brevibacterium
Lactofermentum (Brevibacterium lactofermentu
m), Brevibacterium dibaricatum (Brevibacte
rium divaricatum), Etc.
Specifically, Corynebacterium acetoacidophilum (Cor
ynebacterium acetoacidophilum) H-7117 (FER
MBP-1822). Mutant of interest
Construction and isolation of microorganisms having
-Methyl-N'-nitro-N-nitrosoguanidine treatment
Treatment, UV irradiation, X-ray irradiation, etc.
After that, cultured in a medium having an osmotic pressure that the parent strain can not grow,
What to do by isolating growing colonies
Can be.

In order to obtain the desired mutant strain, it is necessary to adjust the osmotic pressure of the medium to an osmotic pressure at which the parent strain cannot grow. The osmotic pressure is generally proportional to the molarity of the solution, and as the substance for increasing the osmotic pressure of the medium, any substance having high solubility in water can be used, either an inorganic substance or an organic substance. It can also be used as a mixture. For example, salts such as sodium, potassium and ammonium, specifically, sodium chloride, ammonium chloride, ammonium sulfate and the like can be mentioned. Also, a mixed solution containing various inorganic substances and organic substances, such as a culture at the end of fermentation, can be used.

The osmotic pressure at which the parent strain cannot grow is, for example, 650 mOSM / kg or more when using ammonium sulfate as a substance for increasing osmotic pressure, or 1000 mOSM / kg or more when using sodium chloride as a substance for increasing osmotic pressure in the case of a minimum agar medium. 500mOSM when using
/ Kg or more (all determined after 5 days of culture). Examples of the mutant strain used in the present invention include Corynebacterium acetoacidophilum ( Corynebacterium acetoacid
ophilum ) H-8492, H- 8493 and H-849
4 is given. Based on the Budapest Treaty, these strains were submitted to the Institute of Microbial Industry and Technology by the Ministry of Industrial Science and Technology of Japan on May 26, 1992, respectively.
BP-3872), Micro-working Kenjo No. 3873 (FERM)
BP-3873), Jiken Kenjo No. 3874 (FER)
MBP-3874).

Hereinafter, a specific method for collecting the strain will be described. The abbreviations in this specification are as follows. ArgHx ^R : arginine hydroxamate resistance 2TA ^R : 2-thiazolealanine resistance [MFA + CBZArg] ^R : N-carbobenzoxyarginine resistance in the presence of monofluoroacetic acid 6AU ^R : 6-azauracil resistance CYS ^R : Cysteine resistance AS ^R : ammonium sulfate resistance NaCl ^R: sodium chloride tolerance AC ^R: ammonium chloride resistant

(1) Method for obtaining H-8492 Corynebacterium acetoacidophilum H-711
7 (hereinafter referred to as ^{H-7117) (ArgHx R,} 2TA
^R , 6AU ^R , [MFA + CBZArg] ^R , CY
S ^R) 30 ° C. at the N- methyl -N'- nitro -N- nitrosoguanidine 250 mg / ml, was treated for 30 minutes, the minimum comprising ammonium sulfate concentration (250 mM) of the processing strain parent showing the growth inhibition Medium agar plate (sucrose
10 g / l, ammonium chloride 1 g / l, urea 2 g / l,
Potassium phosphate 1 g / l, Potassium phosphate 3 g /
1, magnesium chloride dihydrate 0.4 g / l, ferrous sulfate heptahydrate 10 mg / l, manganese sulfate tetrahydrate 10 mg / l
l, zinc sulfate heptahydrate 1mg / l, copper sulfate pentahydrate 1
mg / l, ammonium molybdate tetrahydrate 1mg /
1, biotin 0.05 mg / l, agar 20 g / l, pH 7.2) and cultured at 30 ° C. for 4 to 6 days to obtain a mutant that grows.

[0012] Among the mutant strains thus obtained, L
-As a strain having excellent arginine producing ability, H-8492 (A
^{rgHx R, 2TA R, 6AU R} , [MFA + CBZA
rg] ^R , CYS ^R , AS ^R ).

(2) H-8493 and H-8494
Acquisition method In the acquisition method of the above item (1),
Instead, use sodium chloride (500 mM) or ammonium chloride
Same as (1) except that monium (300 mM) is used
H-8493 (ArgHx^R, 2TA^R, 6A
U^R, [MFA + CBZArg]^R, CYS^R, NaC
l^R) And H-8494 (ArgHx ^R, 2TA^R,
6 AU^R, [MFA + CBZArg]^R, CYS^R, A
C^RGet)

Next, Table 1 shows the growth rates when the parent strain and the mutant strain were cultured at 30 ° C. for 5 days on a minimal medium agar plate containing ammonium sulfate, sodium chloride or ammonium chloride. In these resistant strains, since cross-resistance to various osmotic pressure regulators is observed, it is considered that the strain is not a resistant strain to a specific substance but a strain having an improved degree of osmotic pressure.

[0015]

[Table 1]

In culturing the microorganism used in the method of the present invention, a medium generally used for fermentative production of amino acids is used. That is, a synthetic medium or a natural medium containing a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by microorganisms is appropriately used. As a carbon source, glucose,
Saccharides such as fructose, sucrose, molasses, starch, and starch hydrolysate, organic acids such as acetic acid, fumaric acid, and citric acid, and alcohols such as ethanol, methanol, and glycerol are used.

Examples of the nitrogen source include inorganic salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate; ammonium salts of organic acids such as ammonium fumarate; amines such as ethylamine;
Nitrogen-containing compounds such as urea, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal or its hydrolyzate, fermentation cells such as amino acid fermentation, nucleic acid fermentation, and digestion products thereof Used.

As the inorganic salts, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used. In addition, vitamins such as biotin, thiamine, nicotinic acid and β-alanine, and amino acids such as glutamic acid may be added to the medium as needed.

The culture is carried out under aerobic conditions such as shaking culture or deep aeration stirring culture at 20 to 40 ° C., preferably 25 to 38 ° C.
Performed at a temperature of ° C. The pH of the medium is maintained at 5 to 9, preferably near neutrality. PH adjustment of the culture medium is calcium carbonate, inorganic or organic acid, alkaline solution, ammonia,
This is performed using a pH buffer or the like. The culture period is usually 1 to 7 days, and L-arginine is produced and accumulated in the culture.

After completion of the culture, precipitates such as bacterial cells are removed from the culture solution, and L-arginine is removed from the culture solution by using ion exchange treatment, concentration, salting out, isoelectric point precipitation and the like. Can be recovered. Hereinafter, examples of the present invention will be described.

[0021]

【Example】

Example 1 Corynebacterium acetoacidophilum H-711
7, H-8492, H-8493 and H-8494 were each converted into 50 g / l glucose, 10 g / l peptone,
Yeast extract 10 g / l, ammonium sulfate 5 g / l, potassium monophosphate 5 g / l, magnesium sulfate dihydrate
30 g of seed medium (pH 7.2) consisting of 0.5 g / l, biotin 0.05 mg / l and sodium chloride 5 g / l
C., and cultured for 24 hours. After inoculating 2 ml of the obtained various culture solutions into a 300 ml Erlenmeyer flask containing 20 ml of a production medium described below, the cells were cultured at 30 ° C. for 72 hours with shaking. Composition of Production Medium Molasses 80 g / l (in terms of glucose), potassium monophosphate 0.5 g / l, potassium diphosphate 0.5 g / l, ammonium sulfate 45 g / l, urea 2 g / l, calcium carbonate 3
Table 2 shows the amount of L-arginine produced in the 0 g / l (pH 7.2) culture solution.

1 liter of the filtrate obtained by removing cells and other insolubles from the culture solution of H-8492 was passed through a column of a strongly acidic ion exchange resin (Dowex 50X8 (Na type), manufactured by Dow Chemical Co.), and subjected to a conventional method. L-arginine was separated, purified, concentrated and crystallized according to the procedure described above to obtain 18.8 g of L-arginine crystals.

[0023]

[Table 2]

[0024]

According to the present invention, L-arginine can be obtained in good yield.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12P 13/10 BIOSIS (DIALOG) WPI (DIALOG)

Claims (5)

(57) [Claims] An L-A by a fermentation method comprising the following steps :
Method for producing luginin. (A) Corynebacterium or Brevibacterium
Microorganisms selected from microorganisms belonging to the genus
Culture in a medium containing the substance
Osmotic pressure that does not allow colony formation or cell division
(B) Determined by treating the microorganism used in (a) with a mutagen
Have the osmotic pressure determined in (a)
A medium capable of growing on a medium and having the ability to produce L-arginine.
Select a different strain (c) Culture the mutant strain selected in (b) in a medium,
L-arginine is produced and accumulated in the nutrient, and
Collect L-arginine 2. The method of claim 1 wherein the microorganism is Corynebacterium aceto A Sid Fi Lamb (Corynebacterium acetoacidophilu
m), Corynebacterium glutamicum
rium glutamicum), Corynebacterium lilium (C
orynebacteriumlilium), Brebbacterium flava
(Corynebacterium flavum), Brevibacterium
Lactofermentum (Corynebacterium lactoferment)
um), Brevibacterium dibaricatum (Coryneba)
The method according to claim 1, which is a microorganism selected from the group consisting of Cterium divaricatum) . 3. The mutant strain is Corynebacterium acetoacidophilum H-.
8492 (FERM BP-3872), H-8493 (FERM BP-3873) or H-84
The production method according to claim 1 , wherein the production method is 94 (FERM BP-3874). 4. A corynebacterium acetoacidophile.
Medium containing a substance that increases the osmotic pressure of ram microorganisms
And the microorganisms form colonies in the medium.
Determines the osmotic pressure at which cell division is not possible and mutates the microorganism
Among the microorganisms obtained by treatment with the agent
Have the ability to produce L-arginine.
A microorganism obtained by selecting a microorganism. 5. The method according to claim 5, wherein the microorganism is Corynebacterium acetoacetate.
Acidophilum (Corynebacterium acetoacidophilum)
H-8492 (FERM BP-3872), H-8493 (FERM BP-3873) or H-
The microorganism according to claim 4, which is 8494 (FERM BP-3874).