JPH11262398A - Production of l-glutamic acid by fermentation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce the subject compound useful for foods, pharmaceuticals, etc., in high efficiency at a low cost by culturing a microorganism belonging to the genus Alicyclobacillus and capable of producing L-glutamic acid in a medium under aerobic condition and collecting the fermentation product. SOLUTION: A microbial strain belonging to the genus Alicyclobacillus, capable of producing L-glutamic acid and resistant to metabolic antagonist of L-glutamic acid [e.g. Alicyclobacillus acidocaldarius AJ13409 (FERM P-16662)] is cultured in a medium under aerobic condition to effect the production and accumulation of L-glutamic acid in the medium and the product is collected from the medium to obtain the objective L-glutamic acid in high efficiency at a low cost. L-glutamic acid is an amino acid important for foods, pharmaceuticals, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an L-fermentation method.
It relates to a method for producing glutamic acid. L-glutamic acid is
It is an important amino acid for foods and pharmaceuticals.

[0002]

2. Description of the Related Art Conventionally, L-glutamic acid is mainly produced by a fermentation method using so-called coryneform L-glutamic acid-producing bacteria belonging to the genus Brevibacterium, Corynebacterium, Microbacterium or mutants thereof. (Amino Acid Fermentation, Academic Press, 19
5-215, 1986). As a method for producing L-glutamic acid by fermentation using other strains, methods using microorganisms such as Bacillus, Streptomyces, Penicillium (US Pat. No. 3,220,929), Pseudomonas, Arthro A method using microorganisms such as Bacter, Serratia, Candida (US Pat.
563,857) and a method using a mutant strain of Escherichia coli (Japanese Patent Application Laid-Open No. 5-244970).

[0003] Although the productivity of L-glutamic acid has been considerably increased due to the improvement of the breeding and production methods of the microorganisms as described above, in order to respond to a further increase in demand in the future, a more inexpensive and efficient method is required. There is a need for the development of a method for producing L-glutamic acid.

[0004]

An object of the present invention is to find a novel L-glutamic acid-producing bacterium having a high L-glutamic acid-producing ability and to develop an inexpensive and efficient method for producing L-glutamic acid. is there.

[0005]

Means for Solving the Problems In order to solve the above problems, the present inventors have widely searched and studied microorganisms different from conventional microorganisms and having L-glutamic acid producing ability. As a result, they found that a derivative derived from a microorganism belonging to the genus Alicyclobacillus has a high L-glutamic acid-producing ability, and thus completed the present invention.

That is, the present invention is as follows.

(1) belongs to the genus Alicyclobacillus,
-Culturing a microorganism capable of producing glutamic acid in a medium under aerobic conditions to produce and accumulate L-glutamic acid in the medium, and collecting the L-glutamic acid from the medium.
-A method for producing glutamic acid.

(2) In the above method, the microorganism is Alicyclobacillus acidocardarius.

(3) The method as described above, wherein the microorganism is resistant to an L-glutamic acid antimetabolite.

(4) The method as described above, wherein the microorganism is an azaserine-resistant strain derived from Alicyclobacillus acidocardarius.

(5) A strain belonging to Alicyclobacillus acidocardarius which is resistant to L-glutamic acid antimetabolite.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
The microorganism used in the present invention may be any strain that belongs to Alicyclobacillus and accumulates L-glutamic acid.

The following strains belong to the genus Alicyclobacillus, for example. Alicyclobacillus acidcardarius (Alicyclo
bacillus acidocaldarius Alicyclobacillus acidoterrestris
bacillus acidoterrestris Alicyclobacillus cycloheptanikas (Alicyclo)
bacillus cycloheptanicus)

More preferably, the following strains can be mentioned. Alicyclobacillus acidocardarius JCM5260 Alicyclobacillus acidocardarius JCM5261 Alicyclobacillus acidoterrestris ATCC49025 Alicyclobacillus acidoterrestris ATCC49026 Alicyclobacillus acidocardiosa cyclo CC ATCR49027 Ali CC Bacillus cycloheptanicas ATCC 35670 These strains are ATCC (American Type
e CultureCollection) or J
CM (Japan Collectionof Mic
Roganisms Riken).

In general, in order to obtain a microorganism having an amino acid-producing ability, a technique of obtaining a strain resistant to an antimetabolite of the target amino acid is used. Such a technique can also be applied when acquiring a microorganism having the ability to produce L-glutamic acid belonging to the genus Alicyclobacillus.

The L-glutamic acid antimetabolite as referred to in the present invention means to inhibit the growth of bacteria belonging to the genus Alicyclobacillus,
It is a substance whose growth inhibition is restored by the addition of L-glutamic acid. Alternatively, it is a substance that has an action of suppressing the expression of an enzyme involved in the L-glutamic acid biosynthesis system or an action of inhibiting the activity of the enzyme, and the suppression or inhibition is restored by the addition of L-glutamic acid.

Examples of the compound having a growth inhibitory effect by antagonizing L-glutamic acid as described above include, for example, azaserine, 4-phloroglutamic acid, α-methylglutamic acid, N-methylglutamic acid and the like. In addition to these compounds, L-glutamic acid antimetabolites can be selected as follows. Soft agar containing bacterial cells of the genus Alicyclobacillus is overlaid on a plate medium, an appropriate amount of a candidate compound is placed thereon, and an appropriate amount of L-glutamic acid is placed at a position different from that position. The compound which was cultured as it was and formed an inhibition circle around the placed compound, and the compound whose growth was recovered around L-glutamic acid was L-glutamic acid.
It is determined to be an analog that antagonizes glutamate.

The strain resistant to L-glutamic acid antimetabolite is obtained by irradiating with X-rays or ultraviolet rays, or by using N-methyl-N'-nitro-N-nitrosoguanidine (hereinafter referred to as "N
G "), a method of treating with a mutagen such as ethyl methanesulfonic acid to introduce a mutation into the parent strain, and then growing on an agar medium containing an L-glutamic acid antimetabolite at such a concentration that the parent strain cannot grow. A possible strain may be collected.

The concentration of the L-glutamic acid antimetabolite at which the parent strain cannot grow can be determined by inoculating a medium containing the L-glutamic acid antimetabolite at various concentrations with bacteria of the genus Alicyclobacillus and examining the presence or absence of growth. can do. Next, the thus determined concentration, preferably agar medium containing L-glutamic acid antimetabolite at the minimum concentration that cannot grow, is spread on the mutated Alicyclobacillus bacterium, and a strain that forms a colony is selected. Good. The selection may be performed once or plural times. In addition, selection of a strain resistant to an L-glutamic acid antimetabolite may be performed for one type of L-glutamic acid antimetabolite, or may be performed for a plurality of L-glutamic acid antimetabolites.

In the present invention, the genus Alicyclobacillus belonging to the genus Alicyclobacillus, which is resistant to an antimetabolite of L-glutamic acid, is specifically the minimum concentration which inhibits the growth of a wild strain determined as described above or higher. Mutant strains capable of forming colonies on a glucose minimal medium containing the L-glutamic acid antimetabolite. More specifically, a mutant strain capable of forming a colony on a glucose minimal medium containing 0.5 g / L of azaserine is exemplified.

Specific examples of the mutant strains resistant to L-glutamic acid antimetabolite obtained as described above include Alicyclobacillus acidocardarius AJ13409 resistant to azaserine. Alicyclobacillus acidocardarius AJ13409 has been deposited on Feb. 24, 1998 with the Ministry of International Trade and Industry's Institute of Industrial Science and Technology, under the accession number FERM P-16662.

In order to produce L-glutamic acid using a microorganism belonging to the genus Alicyclobacillus and capable of producing L-glutamic acid, a carbon source, a nitrogen source, inorganic salts, and, if necessary, organic compounds such as amino acids and vitamins. It can be performed by a conventional method using a normal nutrient medium containing micronutrients. Either a synthetic medium or a natural medium can be used. The carbon source and nitrogen source used in the medium may be those available for the strain to be cultured.

As the carbon source, sugars such as glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolyzate, molasses and the like are used. In addition, organic acids such as acetic acid, citric acid and the like can be used alone or in addition. It is used in combination with a carbon source.

As the nitrogen source, ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate and ammonium acetate, nitrates and the like are used.

As the organic trace nutrients, amino acids, vitamins, fatty acids, nucleic acids, and peptones, casamino acids, yeast extracts, soybean protein decomposed products containing these, and the like are used. When a sex mutant is used, it is necessary to supplement the required nutrients.

As the inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like are used.
As the culture method, aeration culture is performed while controlling the fermentation temperature to 30 to 70 ° C and the pH to 2 to 7. If the pH falls below this range during the culture, neutralize with an alkali such as ammonia gas. Thus, by culturing for about 10 hours to 4 days, a remarkable amount of L-glutamic acid is accumulated in the culture solution.

After the completion of the culture, the L-glutamic acid accumulated in the culture solution may be isolated according to a known method. For example, it can be isolated by a method of removing the cells from the culture and then concentrating and crystallization, or by ion exchange chromatography.

[0028]

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

(1) Search for L-glutamic acid antimetabolite of Alicyclobacillus acidocardarius Alicyclobacillus acidocardarius JCM52
60 strains were transformed into a BAM (JCM) liquid medium (glucose 0. 1).
5 g / L, ammonium sulfate 0.1 g / L, magnesium sulfate heptahydrate 0.25 g / L, potassium dihydrogen phosphate 0.3 g / L, calcium chloride dihydrate 0.125 g
/ L, yeast extract 0.5 g / L, pH 3-4) 5
The cells were cultured overnight at 0 ° C., washed with a 50 mM potassium phosphate buffer (pH 4.5), and used as indicator bacteria.

Glucose minimal medium (Glu) kept at 50 ° C.
Lucose 0.5 g / L, ammonium sulfate 0.1 g / L
L, magnesium sulfate heptahydrate 0.25 g / L, phosphoric acid
Potassium dihydrogen 0.3 g / L, calcium chloride dihydrate
0.125 g / L, pH 4) soft agar plate (agar concentration
0.8%) to the agar with normal agar concentration
Overlaid on a course minimal medium plate. At this time, the bacterial concentration
About 10⁶Individual cells / cm ^TwoIndicator bacteria addition amount
Was adjusted.

Various compounds (azaserine, 4-phloroglutamic acid, DL-α-methylglutamic acid, N-methylglutamic acid, etc.) are placed on the plate thus prepared, one cup for each ear, and about 3.5 cm away from the position.
Glutamic acid was likewise placed on one full earpick. The compound which was cultured at 50 ° C. for 1 to 3 days as it was and formed an inhibitory circle around the placed compound and the growth of the bacteria was restored around L-glutamic acid was determined to be an L-glutamic acid antimetabolite. did. As a result of examining growth inhibition and antagonism with L-glutamic acid for various compounds, it was found that azaserine was found to be Aricyclobacillus acidocardarius JCM.
5260 strain was found to act as an L-glutamic acid antimetabolite.

(2) Acquisition of an azaserine-resistant strain derived from Alicyclobacillus acidocardarius Alicyclobacillus acidocardarius JCM52
60 strains were transformed into a BAM (JCM) liquid medium (glucose 0. 1).
5 g / L, ammonium sulfate 0.1 g / L, magnesium sulfate heptahydrate 0.25 g / L, potassium dihydrogen phosphate 0.3 g / L, calcium chloride dihydrate 0.125 g
/ L, yeast extract 0.5g / L, pH3-4) 50m
at 50 ° C. overnight, and the cells were collected by centrifugation. The cells were suspended in a 50 mM potassium phosphate buffer (pH 5), and the cells were collected by repeating the operation of collecting cells twice, and then washed with the same buffer 8 containing 2 g / L of NG.
The suspension was suspended in 50 ml at 50 ° C. for 60 minutes, and then collected by centrifugation. The cells were suspended in the same buffer and collected by centrifugation. After repeating the same operation twice to wash the cells, 40 ml of BAM (JCM) liquid medium was added, and the cells were shake-cultured at 50 ° C. for 12 hours to fix the mutation. After collection, the cells were spread on a minimal glucose medium plate containing 0.5 g / L of azaserine, and cultured at 50 ° C. for 24 hours.

A colony which appeared on a glucose minimal medium containing 0.5 g / L of azaserine was picked up, a single colony was separated on a medium having the same composition, and an azaserine-resistant strain AJ1 was isolated.
3409 was isolated. When the parent strain JCM5260 strain was applied to a glucose minimal medium containing 0.5 g / L of azaserine, it could not grow at all, and no colony formation was observed.

(3) Culture of L-glutamic acid producing strain and production of L-glutamic acid Azaserine-resistant strain AJ134 isolated as described above
09 and its parent strain, JCM5260, were
(JCM) After culturing on an agar medium for 24 hours, 120
BAM2 medium (glucose)
40 g / L, ammonium sulfate 5 g / L, magnesium sulfate heptahydrate 0.5 g / L, potassium dihydrogen phosphate 2
g / L, calcium chloride dihydrate 0.25 g / L, ferrous sulfate heptahydrate 0.02 g / L, manganese sulfate tetrahydrate
0.02 g / L, zinc sulfate dihydrate 0.72 mg / L,
Copper sulfate pentahydrate 0.64mg / L, cobalt chloride hexahydrate
0.72 mg / L, boric acid 0.4 mg / L, sodium molybdate dihydrate 1.2 mg / L, yeast extract
0.5 g / L, calcium carbonate 5 g / L, pH2.
5) Inoculation was carried out in a 500 ml shaking Sakaguchi flask into which 25 ml was injected, followed by shaking culture at 50 ° C. or 60 ° C. until the sugar was depleted, and the results shown in Table 1 were obtained.

[0035]

Table 1 L-glutamic acid accumulation 蓄積 L-glutamic acid accumulation (mg / L) ５０ 50 ℃ 60 ℃ Ｊ JCM5260 8 13 AJ13409 564 483

JCM5260 hardly accumulated L-glutamic acid, while the azaserine-resistant strain AJ
13409 accumulated significant amounts of L-glutamic acid. AJ
Many Azaserine-resistant strains other than the 13409 strain were isolated and evaluated by the method shown in (3).
As in the case of No. 9, a large number of strains which accumulated a significant amount of L-glutamic acid were found.

[0037]

Since the microorganism used in the present invention has a high L-glutamic acid-producing ability, a higher productivity can be imparted by using a conventionally known breeding technique for coryneform L-glutamic acid-producing bacteria. It is expected that study of culture conditions and the like will lead to the development of an inexpensive and efficient method for producing L-glutamic acid.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:01)

Claims (5)

[Claims] 1. A microorganism belonging to the genus Alicyclobacillus and capable of producing L-glutamic acid is cultured in a medium under aerobic conditions to produce and accumulate L-glutamic acid in the medium, which is collected from the medium. A process for producing L-glutamic acid. 2. The method for producing L-glutamic acid according to claim 1, wherein the microorganism is Alicyclobacillus acidocardarius. 3. The method for producing L-glutamic acid according to claim 1, wherein the microorganism is resistant to an L-glutamic acid antimetabolite. 4. The method for producing L-glutamic acid according to claim 2, wherein the microorganism is an azaserine-resistant strain derived from Alicyclobacillus acidocardarius. 5. A strain belonging to Alicyclobacillus acidocardarius, which is resistant to an antimetabolite of L-glutamic acid.