JP2886550B2 - Method for producing 5'-inosinic acid by fermentation method - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing 5′-inosic acid by a fermentation method. 5'-Inosinic acid is widely used as a seasoning and the like, and is a useful substance in the field of food industry.

2. Description of the Related Art Conventionally, as a method for producing 5′-inosinic acid by fermentation, microorganisms belonging to the genus Corynebacterium are used in a concentration of 0.1 to 0.01.
% Of L-proline [Applied Microbiology (Applied Microbiology) 16 9
81-987 (1968)], belonging to the genus Corynebacterium as a method for directly producing 5'-inosic acid from sugars by fermentation.
A method of obtaining and cultivating a strain having adenine requirement and not affected by sterilization by pressurization of a fermentation medium to produce and accumulate 5'-inosinic acid in the medium (Japanese Patent Publication No. 58-46319); Belongs to, having an adenine requirement, culturing a mutant strain having resistance to adenine or rifampicin,
A method of producing and accumulating 5'-inosinic acid in a medium (Japanese Patent Application Laid-Open No. 56-160999) is known.

In addition, a microorganism having 5'-inosinic acid
A method for culturing in a medium containing 5.05.0% L-proline and / or an analog thereof has been disclosed by the present applicant. (Japanese Patent Application No. 1-55835) In the present specification, the strain conventionally referred to as Brevibacterium ammoniagenes is changed to Corynebacterium ammoniagenes and described. The name of this strain has been changed by the International Journal of Systematic Bacteriology
nal of Systematic Bacteriology, 442-443, October, 198
7 years).

An object of the present invention is to provide a method for industrially and efficiently producing 5'-inosic acid, which is widely used as a seasoning, by a direct fermentation method.

Means for Solving the Problems According to the present invention, it belongs to the genus Corynebacterium,
Resistant to proline antagonists such as 3,4-dehydro-D, L-proline, L-azetidine-2-carboxylic acid and L-thiazolidine-4-carboxylic acid, and 5 '
By culturing a microorganism capable of producing inosinic acid in a medium, 5'-inosic acid can be produced and accumulated in the culture, and 5'-inosic acid can be collected from the culture.

 Hereinafter, the present invention will be described in detail.

The microorganism to be used belongs to the genus Corynebacterium, has resistance to L-proline antagonists, and has 5'-
Any microorganism can be used as long as it is capable of producing inosinic acid. A microorganism having resistance to an L-proline antagonist is
It refers to a mutant strain that can grow on a medium containing an L-proline antagonist at a concentration at which the parent strain shows growth inhibition.

In the present invention, the term "L-proline antagonist" means to inhibit the growth of a 5'-inosinic acid-producing bacterium belonging to the genus Corynebacterium when an L-proline antagonist is added to a minimal medium agar plate and cultured. However, it refers to a compound whose inhibition is released by the presence of L-proline. Examples of L-proline antagonists include 3,4-
Dehydro-D, L-proline, L-azetidine-2-carboxylic acid, L-thiazolidine-4-carboxylic acid and the like. An appropriate amount of the L-proline antagonist added to the medium is 1 to 15 g /.

The mutant strain as described above imparts L-proline antagonist resistance to a 5'-inosinic acid-producing bacterium belonging to the genus Corynebacterium or has resistance to an L-proline antagonist belonging to the genus Corynebacterium. It can be obtained by imparting 5'-inosinic acid-producing ability to a strain.

The mutant strain of the present invention comprises Corynebacterium Ammoniagenes KY13184 (FER
MP-3790) (hereinafter referred to as KY13184 strain) as a parent strain.
It can be obtained by subjecting this to a usual mutation treatment such as N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet light, X-ray.

Hereinafter, a specific method for obtaining the mutant strain used in the present invention will be described.

Using the KY13184 strain as a parent strain, the strain was N-methyl-
N'-nitro-N-nitrosoguanidine 100 μg / ml
After treatment at 30 ° C for 30 minutes, the concentration at which the parent strain shows growth inhibition (3 mg /
ml) of a minimal medium agar plate containing 3,4-dehydro-D, L-proline (glucose 20 g /, ammonium chloride 2 g /, K
H ₂ PO ₄ 0.1g /, K ₂ HPO ₄ 0.3g /, MgCl ₂・ 2H ₂ O 0.3g /
_{, FeSO 4 · 7H 2 O 10mg} /, MnSO 4 · 4~6H 2 O 1mg /, Z
_{nSO 4 · 7H 2 O 1mg /} , CuSO 4 · 5H 2 O 0.2mg /, CaCl 2 · 2H
₂ O 10 mg /, L-cysteine 40 mg /, L-asparagine 0.5 g /, thiamine hydrochloride 10 mg /, calcium pantothenate 20 mg /, biotin 60 μg /, nicotinic acid 3 g /
, Adenine 20mg /, guanine 20mg /, agar 20g /,
pH 7.2). After culturing at 30 ° C. for 7 to 10 days, among the mutant strains that grew, a strain having a higher ability to produce 5′-inosinic acid than the parent strain was selected. One of the most excellent strains is Corynebacterium ammoniagenes H-
7464 (hereinafter referred to as strain H-7644).

Also, instead of 3,4-dehydro-D, L-proline, L-
The same method as described above was performed except that azetidine 2-carboxylic acid was used. Among the obtained strains, one strain having particularly excellent productivity was Corynebacterium ammoniagenes H-7465 (hereinafter referred to as H-7465 strain). ).

Based on the Budapest Treaty, the H-7746 strain and the H-7465 strain were each sent to the Institute of Microbial Industry and Technology by the Ministry of Industrial Science and Technology of Japan on May 18, 1989.
425) and No. 2426 (FERM BP-2426).

Strain KY13184, strain H-7644 and strain H-7465 were plated on a minimal medium agar plate containing an L-proline antagonist, respectively.
Table 1 shows the degree of growth when cultured at 10 ° C for 10 days.

In culturing the microorganism used in the present invention, a medium generally used for fermentative production of nucleic acids is used. Any medium such as a synthetic medium or a natural medium can be used as long as the medium contains a carbon source, a nitrogen source, inorganic salts, growth factors, and the like that can be assimilated by the microorganism.

Examples of the carbon source include hydrolysates such as glucose, fructose, sucrose, molasses, and starch, various organic acids such as acetic acid, fumaric acid, and citric acid, and alcohols such as ethanol and glycerol.

Examples of the nitrogen source include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, various inorganic salts such as 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, various fermentation cells such as amino acid fermentation, nucleic acid fermentation, and digestion thereof Objects are used.

As the inorganic substance, 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, biotin, thiamine, nicotinic acid, β-
Vitamins such as analine and amino acids such as glutamic acid may be added.

Cultivation is performed under aerobic conditions such as shaking and deep stirring at a temperature of 20 ° C.
The reaction is carried out at 4040 ° C., preferably 25-38 ° C., while maintaining the pH at 5-9, preferably near neutrality, and is usually completed in 2-7 days. The pH of the medium is adjusted with calcium carbonate, an inorganic or organic acid, an alkaline solution, ammonia, a pH buffer and the like.

After completion of the culture, remove precipitates such as cells from the culture solution,
By using an ion exchange treatment method, an adsorption method, an extraction method, a precipitation method, or the like, 5′-inosinic acid can be collected from the culture solution.

 Hereinafter, examples of the present invention will be described.

Example 1 300 ml of a seed medium having the following composition in a two-volume Erlenmeyer flask each containing the KY13184 strain, the H-7746 strain and the H-7465 strain
And cultured at 30 ° C. for 24 hours.

Seed medium composition: glucose 20 g /, peptone 10 g /, meat extract 10 g /, yeast extract 5 g /, NaCl 3 g /, biotin 20 μg /, adenine 100 mg /, guanine 100 mg / (pH
7.2, adjusted with NaOH or H ₂ SO ₄ , sterilized by autoclaving at 120 ° C. for 10 minutes) Inoculate 300 ml of the obtained three seed cultures into a production medium 3 having the following composition in a 5-volume culture tank. At 30 ° C
The cells were cultured for 96 hours (at a rotation speed of 600 rpm and an aeration rate of 30 / min). The pH during the culture was adjusted to about 6.8 with aqueous ammonia.

Production medium composition: glucose 150 g /, KH ₂ PO ₄ 10 g /,
K ₂ HPO ₄ 10 g /, MgSO ₄・ 7H ₂ O 10 g /, CaCl ₂・ 2H ₂ O 0.1
_{g /, ZnSO 4 · 7H 2} O 5mg /, FeSO 4 · 7H 2 O 20mg /, MnC
_{l 2 · 4H 2 O 5mg /} , biotin 30 [mu] m /, calcium pantothenate 10 mg /, vitamin B ₁ 5mg /, nicotinic acid 5mg /
, Adenine 100 mg /, guanine 100 mg /, corn steep liquor 20 g /, urea 2 g / (boiled separately pressurized steam sterilization 120 ° C., 5 minutes) (pH 7.2, adjusted with NaOH or H ₂ SO _4, 1
Pressurized steam sterilization at 20 ° C for 10 minutes) As a result, KY13184 strain, H
The amounts of 5'-inosinic acid produced in the culture solutions of the -7464 strain and the H-7465 strain were 26.3 g /, 28.7 g / and 28.9 g /, respectively.
Met.

The supernatant 1 obtained by removing the cells from the culture termination solution of the H-7644 strain was adjusted to pH 1.4 with hydrochloric acid, and Diaion SK # 1 was used.
Immediately after passing through a resin tower (H type, manufactured by Mitsubishi Kasei Corporation), distilled water was passed through to obtain an eluate. Thereafter, the resin tower was washed with water, and the fraction containing 5'-inosic acid in the running water at the initial stage of washing was combined with the eluate already obtained, and adjusted to pH 7.2 with sodium hydroxide. This was concentrated under reduced pressure using a rotary evaporator to obtain 20.1 g of crude crystals of sodium 5'-inosinate.

Effects of the Invention According to the present invention, 5'-inosic acid can be produced industrially at low cost and efficiently.

Claims (2)

(57) [Claims] 1. A microorganism belonging to the genus Corynebacterium, resistant to L-proline antagonists and capable of producing 5'-inosic acid, is cultured in a medium, and 5'-inosic acid is added to the culture. A method for producing 5'-inosinic acid by fermentation, comprising producing and accumulating 5'-inosinic acid from the culture. 2. The L-proline antagonist is a substance selected from the group consisting of 3,4-dehydro-D, L-proline, L-azetidine-2-carboxylic acid and L-thiazolidine-4-carboxylic acid. A method for producing 5'-inosinic acid by the fermentation method according to claim (1).