JPH0644871B2 - Fermentation method for producing L-leucine - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing L-leucine by a fermentation method.

L-leucine has various uses in a wide range of fields such as pharmaceuticals and foods.

2. Description of the Related Art Conventionally, L-leucine has been produced by a fermentation method or a decomposition extraction method from a protein. As a method for producing by fermentation, a method using a valine-requiring mutant strain (Japanese Patent Publication No. 38-
4395), a method using a mutant strain belonging to the genus Corynebacterium and having S- (2-aminoethyl) -L-cysteine resistance (Japanese Patent Publication No. 51-37347), phenylalanine, isoleucine, valine of Corynebacterium glutamicum or A method using a methionine-requiring mutant (Japanese Patent Publication No. 54-36233), a method using a 2-thiazolealanine-resistant mutant belonging to the genus Brevibacterium or Corynebacterium (Japanese Patent Publication No. 48-2427).
3), a method of allowing L-valine to be present in the medium when culturing a microorganism capable of producing L-leucine (JP-A-56-1)
27095) and the like are known.

Problems to be Solved by the Invention Development of a method for industrially inexpensively producing L-leucine, which has various uses in a wide range of fields such as pharmaceuticals, foods or raw materials thereof, has been desired.

Means for Solving Problems The present inventors have conducted extensive studies on L-leucine-producing bacteria by a fermentation method in order to efficiently and inexpensively obtain L-leucine. As a result, they have found that the productivity of L-leucine-producing bacteria is stabilized and improved by imparting valine analog resistance, and completed the present invention.

The present invention will be described in detail below.

The present invention relates to a coryneform glutamic acid-producing bacterium, culturing in a medium a microorganism having resistance to a valine analog and having L-leucine-producing ability, and producing and accumulating L-leucine in the culture. There is provided a method for producing L-leucine by a fermentation method, which comprises collecting L-leucine.

The valine analog as used in the present invention refers to one that inhibits the growth of coryneform glutamic acid-producing bacteria, but this inhibition is released by the presence of valine. For example,
Examples include α-aminobutyric acid, β-hydroxyvaline, valine hydroxamate and norvaline.

As a parent strain for obtaining the mutant strain of the present invention, Corynebacterium glutamicum ATCC13032, Corynebacterium acetoacidophilum ATCC1383
7, Corynebacterium Lilium ATCC1599
0, Brevibacterium flavum ATCC 1406
7, Brevibacterium lactofermentum ATC
C13869, Brevibacterium divalicatum A
TCC14020 etc. are mentioned.

Coryneform glutamic acid-producing bacteria or other microorganisms are L
-To have leucine-producing ability, 5 ', 5', 5'-
It is known that resistance to leucine analogs such as trifluoroleucine and azaleucine, and drugs such as S- (2-aminoethyl) -L-cysteine and 2-thiazolealanine may be imparted. However, many of these drug resistant strains have unstable L-leucine producing ability. The present inventors have found that imparting valine analog resistance to these stabilizes and improves the L-leucine-producing ability.

Induction of the mutant strain in the present invention is carried out by an ordinary mutation induction method, for example, ultraviolet irradiation or chemical treatment with a drug.
Specifically, the above parent strain was treated with N-methyl-N'-nitro-N-
Treatment with nitrosoguanidine (250 μg / ml) at 30 ° C. for 30 minutes may be performed to obtain a mutant strain that grows on a minimal medium agar plate containing a valine analog at a concentration at which the parent strain shows growth inhibition, eg, α-aminobutyric acid 20 mg / ml. . By culturing the resistant mutant strain thus obtained and examining the L-leucine-producing ability, a mutant strain in which the L-leucine-producing ability is stabilized and improved can be isolated.

Examples of the parent strain and valine analog resistant strains derived from the parent strain are shown below.

Parent strain Corynebacterium glutamicum CU-25 (FERM P-1834): AEC ^R resistant strain H-4669 (FERM BP-1311): AEC ^R , AB
A ^R parent strain Corynebacterium aceto A Sid Fi lamb H-
4667 (FERM BP-1309): TFL ^R (derived from Corynebacterium acetoacidophilum ATCC13837) resistant strain H-4668 (FERM BP-1310): TFL ^R , A
BA ^R AEC ^R is S- (2-aminoethyl) -L- cysteine resistance, ABA ^R is α-aminobutyric acid-resistant, TFL ^R 5 ',
It shows resistance to 5 ', 5'-trifluoroleucine.

Valine analog (α-aminobutyric acid) 20 mg
Medium agar plate containing 10 ml / ml (glucose 10 g /,
Ammonium chloride 1g /, urea 2g / KH ₂ PO ₄
1 g /, K ₂ HPO ₄ 3 g /, MgCl ₂ · 2H ₂ O
_{0.4g /, FeSO 4 · 7H 2} O 10mg /,
_{MnSO 4 · 4H 2 O 10mg /} , ZnSO 4 · 7H 2
O 1 mg /, CuSO ₄ 5H ₂ O 1 mg /, (NH
_{4) 6 Mo 7 O 24 ·} 4H 2 O 1mg /, biotin 50μg
Table 1 shows the degree of growth when cultured for 24 hours on agar / agar 20 g /, pH 7.2).

The above-mentioned parent strains and valine analog-resistant mutants derived from them have been deposited at the Institute of Microbiology, Institute of Industrial Technology (MICRO) on March 10, 1987.

For culturing the bacterium used in the method of the present invention, a medium generally used for fermentative production of amino acids is used. In other words, carbon sources, nitrogen sources, inorganic substances that can be assimilated by bacteria,
A synthetic medium or a natural medium containing a growth factor or the like is appropriately used.

As the carbon source, saccharides such as glucose, fructose, sucrose, sugar condensate, starch hydrolysate, various organic acids such as acetic acid, fumaric acid and citric acid, and alcohols such as ethanol, glycerol and annose can be used.

As a nitrogen source, various inorganic salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, ammonium salts of organic acids, amines, other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, casein Hydrolysis,
Soybean meal hydrolyzate, various fermented bacterial cells and digested products thereof are used.

As the inorganic substance, potassium dibasic phosphate, dibasic potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used.

The culture is carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is 20 to 40 ° C, preferably 25
It is in the range of to 38 ° C. The pH of the medium is in the range of 5-9,
It is preferably kept near neutral. The pH of the medium is adjusted with calcium carbonate, an inorganic or organic acid, an alkaline solution, ammonia, a pH buffer solution or the like.

The culture period is usually 1 to 7 days, and L-leucine is produced and accumulated in the culture. After the culture is completed, the precipitates such as bacterial cells are removed from the culture solution, and L-leucine is recovered from the culture solution by using the ion exchange treatment method, the concentration method, the salt folding method, the isoelectric point precipitation method, and the like in combination. You can

Examples of the present invention will be shown below.

Example 1. Corynebacterium glutamicum CU-25 (FER
MP-1834) and H-4669 (FERM B
P-1311) was grown on a storage agar medium having the following composition, and then 1 platinum loop was added to 20 ml of production medium having the following composition.
Inoculate (300 ml Erlenmeyer flask) and incubate at 30 ℃ 72
Culture was performed with shaking for an hour. 10.2 mg / each in the culture broth
ml, 13.5 mg / ml L-leucine was produced and accumulated.

The H-4469 culture-finished solution was centrifuged, and the resulting supernatant 1 was used as a strongly acidic cation exchange resin DIAION SK +.
After passing through a column packed with 1B (manufactured by Mitsubishi Kasei Co., Ltd.), it was eluted with 0.2N aqueous ammonia, and the fraction containing L-leucine was treated with activated carbon. The treatment liquid was adjusted to pH 6.0 and then concentrated to obtain 12.2 g of crude L-leucine crystals.

Example 2. Corynebacterium acetoacidophilum H-466
7 (FERM BP-1309), H-4668 (FE
When RM BP-1310) was cultured in the same manner as in Example 1, L-leucine was produced and accumulated as shown in Table 2.

Effects of the Invention According to the present invention, L-leucine can be obtained in good yield.

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Claims (1)

[Claims] 1. A bacterium belonging to the genus Corynebacterium or Brevibacterium, which is resistant to valine analogues and L-.
A method for producing L-leucine by a fermentation method, which comprises culturing a microorganism having leucine-producing ability in a medium, producing and accumulating L-leucine in the culture, and collecting L-leucine from the culture.