JP3006929B2 - Production method of L-valine by fermentation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing L-valine. L-valine is mainly used for pharmaceuticals such as nutrient infusions and general amino acid preparations, but is also a useful amino acid that has uses as a seasoning and feed.

[0002]

2. Description of the Related Art Conventionally, a method for producing L-valine using a so-called coryneform glutamate-producing bacterium belonging to the genus Corynebacterium or the genus Brevibacterium includes:
A method using a microorganism having resistance to D, L-aminobutyric acid (JP-A-63-160592), a method using a microorganism having resistance to thiazolealanine (Japanese Patent Publication No. 52-116), a method using a microorganism having resistance to aminoethylcysteine Method used
58-2678).

[0003]

In recent years, the demand for L-valine has been increasing, and there is a need for an improved method for producing L-valine more efficiently.

[0004]

DISCLOSURE OF THE INVENTION The present invention relates to a genus of Corynebacterium or Brevibacterium, which is a medium having acetic acid as a sole carbon source, having resistance to L-valine, and having glucose as a sole medium. Culturing a microorganism having an ability to produce L-valine that is sensitive to a pyruvate analog in a medium as a carbon source, producing and accumulating L-valine in the culture, and collecting L-valine from the culture And a process for producing L-valine. Hereinafter, the present invention will be described in detail.

The microorganism used in the present invention is a microorganism belonging to the genus Corynebacterium or Brevibacterium, which is known as a coryneform glutamic acid-producing bacterium, has L-valine-producing ability, and has an ability to produce acetic acid. The medium as the sole carbon source shows resistance to L-valine,
In a medium using glucose as a sole carbon source, any medium may be used as long as it is a strain sensitive to pyruvate analogs. Examples of coryneform glutamate-producing bacteria used as a parent strain for inducing a microorganism having such properties include the following strains.

Corynebacterium glutamicum ATCC13032 Corynebacterium acetoacid film ATCC13870 Corynebacterium herculis ATCC13868 Corynebacterium lillium ATCC15990 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 Brevibacterium divaribacrem ATCC140 Bacterium thiogenitalis ATCC19240 These strains are inhibited from growing by L-valine on a medium containing acetic acid as a carbon source.

[0007] The microorganism of the present invention utilizes this property to induce a mutant which has become insensitive to L-valine in a medium containing L-valine at a concentration at which the parent strain is sensitive, using acetic acid as a sole carbon source. Alternatively, the mutant strain can be obtained by selecting a mutant strain which is sensitive to pyruvate analog at a concentration at which the parent strain does not show sensitivity, using glucose as a sole carbon source. Conversely, after inducing mutants that are sensitive to pyruvate analogs at concentrations in which the parent strain is insensitive to glucose as the sole carbon source, the parent strain is sensitive to acetate as the sole carbon source from those mutants. Can be also obtained by selecting an L-valine-insensitive mutant strain in a medium containing L-valine at a concentration of

Specifically, the parent strain is irradiated with ultraviolet light or N-methyl-N'-nitro-N-nitrosoguanidine (hereinafter referred to as N
After treatment with a commonly used mutation treatment method such as chemical treatment such as TG) and nitrous acid, a large colony growing on a medium containing L-valine using acetic acid as a sole carbon source is collected. Alternatively, the microorganism of the present invention can be obtained by collecting a colony sensitive to the pyruvate analog in a medium containing glucose as the sole carbon source and selecting a colony having the other trait. Examples of the pyruvate analog according to the present invention include β-fluoropyruvic acid, β-bromopyruvic acid, β-chloropyruvic acid, β-cyclohexylpyruvic acid, β-mercaptopyruvic acid, β-imidazolylpyruvic acid, trimethylpyruvic acid, β-hydroxypyruvic acid, α-ketobutyric acid and the like.

The microorganism of the present invention is characterized by exhibiting L-valine resistance in a medium containing acetic acid as a sole carbon source and being sensitive to a pyruvate analog in a medium containing glucose as a sole carbon source. , In addition to this trait,
Characters such as auxotrophy of amino acids known to confer L-valine productivity and amino acid analog resistance may be shared. These multiple mutant strains can be obtained by sequentially giving each mutation or by crossing via a protoplast fusion between strains having each mutation individually.

The production of L-valine by the microorganism of the present invention can be carried out by a usual culture method. As the medium to be used, either a synthetic medium or a natural medium can be used as long as it contains a carbon source, a nitrogen source, an inorganic substance and a trace amount of nutrients required by the used strain. As the carbon source, carbohydrates such as glucose, glycerol, fructose, sucrose, maltose, mannose, starch, starch hydrolysate, molasses, and various organic acids such as polyalcohol, pyruvic acid, fumaric acid, lactic acid, and acetic acid can be used. Further, depending on the assimilation of microorganisms, hydrocarbons, alcohols and the like are also used.

Examples of the nitrogen source include ammonia or various inorganic and organic ammonium salts such as ammonium chloride, ammonium sulfate, ammonium carbonate, and ammonium acetate, urea and other nitrogen-containing substances, peptone, NZ-amine, meat extract, yeast extract, corn and corn. Various types such as steep liquor, casein hydrolyzate, fish meal or its digest can be used.

Further, as inorganic substances, potassium monohydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate,
Use ammonium chloride, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate and calcium carbonate. The culture is performed under aerobic conditions such as shaking culture or aeration culture. The culture temperature is generally 20 to 40.
C is preferred. It is desirable to maintain the pH of the medium around neutral. The culture period is usually 1 to 5 days.

[0013] A method for collecting L-valine from a culture solution is as follows.
After completion of the culturing, the cells are removed by a known method such as a method of removing the cells and concentrating and crystallization, a treatment with activated carbon or a treatment with an ion exchange resin. Hereinafter, the present invention will be described specifically with reference to Examples.

[0014]

【Example】

Example 1 Cells cultured at 30 ° C. for 16 hours in a complete medium containing 20 g of Corynebacterium glutamicum ATCC13032 (a medium containing 20 g of powdered broth and 5 g of yeast extract in 1 liter of water and adjusted to pH 7.2) were collected. After washing with a 05M Tris-maleate buffer (pH 6.0), the cells were suspended in the same buffer so that the concentration of the cells was about 10 ⁹ cells / ml, and NTG was added to the final concentration of 500 mg / l. The mixture was maintained at 30 ° C. for 20 minutes to perform mutation treatment. After washing the mutated cells with the same buffer, the cells were applied to a plate agar medium containing 0.5 g / l of L-valine in a minimal medium having the composition shown in Table 1.

[0015]

[Table 1]

After culturing at 30 ° C. for 2 to 4 days, of the colonies growing on the plate medium, large colonies were picked. Among these strains, the growth was slow on a minimal agar medium containing 5 g / l of glucose as a carbon source instead of sodium acetate in Table 1 and 6 mg / l of β-fluoropyruvic acid (hereinafter abbreviated as βFP). The strain was selected. Among these mutants, Corynebacterium glutamicum AV1 was obtained as a strain having a high L-valine productivity.

This strain has been deposited as FERM BP-3006 on July 12, 1990 with the Institute of Microbial Industry and Technology (MIC) of the National Institute of Advanced Industrial Science and Technology. Table 2 shows the sensitivity of parent strain ATCC13032 and mutant strain AV1 to L-valine and βFP. The L-valine sensitivity was measured using the minimum agar medium shown in Table 1 containing 0.5 g / l L-valine,
The FP sensitivity was determined by applying both strains to a minimal agar medium containing 6 mg / l of βFP and using 5 g / l of glucose as a carbon source instead of sodium acetate, and growing at 30 ° C for 2 days.

Example 2 Brevibacterium lactofermentum ATCC1386
9 was cultured in complete medium at 30 ° C. for 16 hours, and the cells were collected.
After washing with a 0.05 M Tris-maleate buffer (pH 6.0), the cells were suspended in the same buffer to a concentration of about 10 ⁹ cells / ml. And kept at 30 ° C. for 20 minutes to perform mutation treatment. After washing the mutated cells with the same buffer, the cells were added to a minimal medium having the composition shown in Table 1 with 0.5 g / L-valine.
1 on a plate agar medium.

After culturing at 30 ° C. for 2 to 4 days, a large colony among the colonies growing on the plate medium was picked. Among these strains, 5 g / l of glucose was used as a carbon source instead of sodium acetate shown in Table 1, and βFP
Slow growing strains were selected on a minimal agar medium containing 6 mg / l. Among such mutant strains, Brevibacterium lactofermentum AV11 was obtained as a strain having a high L-valine productivity.

This strain has been deposited on July 12, 1990 with MICK as FERM BP-3007. Table 2 shows the sensitivity of parent strain ATCC13869 and mutant strain AV11 to L-valine and βFP. L-valine sensitivity and βFP sensitivity tests were performed in the same manner as in Example 1.

[0021]

[Table 2]

Example 3 The mutant strain of Corynebacterium strains obtained in Examples 1 and 2
Glutamicum AV1 (FERM BP-3006), Brevibacterium lactofermentum AV11 (FERMBP-3007)
And their parent strains in 3 ml of seed medium (glucose 1%,
A test tube containing 2% powdered bouillon, 0.5% yeast extract, pH 7.2) was inoculated and cultured with shaking at 30 ° C. for 24 hours.
1 ml of this seed culture is mixed with 30 ml of the following fermentation medium.
A 0 ml Erlenmeyer flask was inoculated and cultured with shaking at 30 ° C. for 48 hours. After the culture, the culture filtrate was subjected to paper chromatography, and after ninhydrin was developed, colorimetric determination was performed and L-
The amount of valine produced was measured.

The results are shown in Table 3. Composition of fermentation medium: glucose 8%, KH ₂ PO ₄ 0.1
%, Magnesium sulfate 0.1%, ammonium sulfate 5%,
Urea 0.2%, thiamine hydrochloride 120 μg / l, biotin 180 μg / l, FeSO ₄ .7H ₂ O 12 mg / l,
_{MnSO 4 · 4~6H 2 O 12mg /} l, corn steep liquor 1%, calcium carbonate 2% (pH 7.2)

[0024]

[Table 3]

[0025]

According to the present invention, L-valine can be produced industrially at low cost.

Claims (1)

(57) [Claims] 1. A medium belonging to the genus Corynebacterium or Brevibacterium, which is resistant to L-valine in a medium containing acetic acid as a sole carbon source, and a pyruvate analog in a medium containing glucose as a sole carbon source. Microorganisms which are susceptible to L-valine and have the ability to produce L-valine are cultured in a medium, L-valine is produced and accumulated in the culture, and L-valine is collected from the culture. -Method for producing valine.