JPS61274691A - Production of l-glutamic acid - Google Patents

Abstract

PURPOSE:To produce a large amount of L-glutamic acid inexpensively, by cultivating a specific mold belonging to the genus Corynebacterium in a mold. CONSTITUTION:A cell of Corynebacterium glutamicum B-15 is suspended in a buffer solution, and varied by ultraviolet light irradiation, to give a variant (e.g., Corynebacterium glutamicum G-28) which grows in a minimum medium comprising glucose as a carbon source but has no ability or extremely reduced ability to grow using L-glutamine or L-glutamic acid as the only carbon source. Then, the variant is aerobically cultivated in a medium consisting of a carbon source (e.g., glucose), a nitrogen source (e.g., peptone), an inorganic salt (e.g., NaCl), a growth factor, etc. at 5-9pH at 24-37 deg.C for 2-7, and L-glutamic acid is accumulated in the culture mixture. Then, L-glutamic acid is isolated by in exchange resin method, etc.

Description

[Detailed description of the invention] Industrial applications The present invention relates to a method for producing L-glutamic acid.

BACKGROUND OF THE INVENTION Conventionally, various methods have been known for producing L-glutamic acid using microorganisms, but the method related to the present invention uses L-glutamic acid belonging to the genus Brevibacterium as the sole carbon source. A method using unavailable mutant strains is known (Japanese Unexamined Patent Publication No. 57-138395).

Problems to be Solved by the Invention In recent years, the demand for L-glutamic acid has increased, and improvements in methods for increasing the productivity of L-glutamic acid are always desired.

Means for Solving the Problems The present inventors have discovered that L-glutamic acid producing bacteria belonging to the genus Corynebacterium lack the ability to grow using L-glutamine or L-glutamic acid as the sole carbon source (incomplete). The inventors have discovered that L-glutamic acid can be obtained in good yield by using a bacterial strain containing deletions (including deletions), and have completed the present invention.

The present invention will be explained in detail below.

The present invention involves culturing in a medium a microorganism that belongs to the genus Corynebacterium and has the ability to produce L-glutamic acid, and has lost or significantly reduced the ability to grow using L-glutamine or L-glutamic acid as the sole carbon source, L-glutamic acid is produced by a fermentation method characterized by accumulating L-glutamic acid in a culture and collecting the accumulated L-glutamic acid from the culture.
Provided is a method for producing glutamic acid.

The microorganisms used in the present invention are microorganisms that belong to the genus Corynebacterium, have the ability to produce L-glutamic acid, and have lost or significantly reduced the ability to grow using L-glutamine or L-glutamic acid as the sole carbon source. Any one can be used.

That is, the microorganism of the present invention can grow in a minimal medium containing glucose as the sole carbon source, but does not grow or grows extremely slowly on a minimal medium containing L-glutamine or L-glutamic acid as the sole carbon source.

The microorganism used in the present invention is a microorganism that has the ability to produce L-glutamic acid and is subjected to mutation treatment to induce a mutant strain that lacks or significantly reduces the ability to grow using L-glutamine or L-glutamic acid as the sole carbon source. You can get it by doing

As the mutation treatment, a conventional mutation treatment method such as ultraviolet irradiation or chemical treatment with N-methyl-N'-nitro-N-nitroguanidine (NTG), nitrous acid, etc. is employed. Mutant strains can also be induced by other genetic techniques (eg, genetic recombination, transduction, cell fusion, etc.). Furthermore, in addition to the above-mentioned properties, the microorganism of the present invention has other properties (for example, various nutritional auxotrophies).

(drug resistance, drug sensitivity, drug dependence, changes in assimilation of various sugars or organic acids, etc.).

Specific examples of microorganisms used in the present invention include Corynebacterium glutamicum G-28 and G- 37 are mentioned.

Next, a specific example of operation for obtaining such microorganisms will be explained.

Operation example Corynebacterium glutamicum B-15 (hereinafter referred to as B
-15) cells were suspended in 0.1N Tris-maleate buffer (pH 6.0) at a concentration of 108 cells/ml. To this, N-methyl-N'-nitroN-
Nitroguanidine was added to a final concentration of 0.2 μg/ml and left at room temperature for 30 minutes.
．． KH2F0゜0, 15 g7dl, K2HPO40
,05g/dl, NaC10,01g/a, Mg5O
<・7820 0.05g/di, CaC1z・2
H2Olx/ml, Pe5o4・7 H2O10J
1g/m1. MnC1*” 4 Ha○7g/ml
, Chiami7 ・HCI O, lx/m1. Ammonium sulfate 0,1
5 g/a, Hitfon 30x/J, Agar 1.5
g/di (adjusted to pH 7.2 with Na011)] and statically cultured at 30°C for 3 days.

Next, the cells grown in colonies were placed on the above-mentioned minimal agar medium and on a minimal agar medium containing L-glutamine as the sole carbon source (glucose was removed from the above-mentioned glucose medium, and 0%
．． As a result of transferring to a medium (medium supplemented with 5x/J L-glutamine) using the replica method and statically culturing at 30°C for 3 days,
Although it grows on a minimal medium with glucose as the carbon source, L
- We obtained many mutant strains that either do not grow on a minimal medium containing glutamine as a carbon source or grow significantly slower than the parent strain B-15. These mutant strains were further grown in a minimal medium containing glucose as a carbon source, a minimal medium containing L-glutamine as a carbon source, and a minimal medium containing L-glutamic acid as a carbon source (from the above-mentioned minimal medium containing glucose as a carbon source to Excluding sodium L-glutamate 0.
5g/d1 supplemented medium).
It grows well on a minimal medium with glucose as the carbon source, but it does not grow or grows poorly compared to the parent strain on both minimal medium with L-glutamine as the carbon source and minimal medium with L-glutamic acid as the carbon source. We isolated 40 significantly slower mutant strains. All of these mutant strains were subjected to an L-glutamic acid fermentation test (same method as in Example 1), and G.
Both strains -28 and G-37 were selected.

G-28 has been deposited with the Institute of Microbial Technology (Feikoken) of the Agency of Industrial Science and Technology on May 2, 1985, and its deposit number is FERM P-g2b7.

The results of growth tests of these strains on minimal agar media containing various carbon sources are shown in Table 1.

Table 1 G-28++++-- G-37++++ --B-15 (parent strain) +++++ ++ ++ Cells were spread on a minimum agar medium containing various carbon sources, and incubated at 30°C for 4 hours.
It shows the growth rate when statically cultured for days.

− No growth, ++: good growth, ++++: extremely good growth.

The composition of the basal medium is shown in the text.

In the present invention, as a medium for culturing microorganisms, a nutrient medium or a synthetic medium containing a carbon source, a nitrogen source, inorganic salts, growth factors, etc. is used.

Carbon sources include glucose and fructose.

Carbohydrates such as sucrose, molasses, starch, starch hydrolyzate, and fruit juice, and alcohols such as ethanol, methanol, and propatool can be used.

Nitrogen sources include ammonium sulfate, ammonium nitrate, ammonium hydrochloride, ammonium phosphate, ammonium acetate, ammonia, and amines.

Peptone, meat extract, yeast extract, corn steep
Liquor, casein hydrolyzate, various fermented microbial cells, and digested products thereof can be used.

Inorganic salts include potassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, and manganese sulfate.

Calcium carbonate etc. can be used.

When using mutant strains that exhibit auxotrophy, the auxotrophic substances are added as standard products or natural products containing them.

Cultivation is performed under aerobic conditions such as aeration and shaking culture. The culture temperature is 24-37°C, and the number of culture days is 2-7 days. The pH of the culture solution is maintained in the range of 5-9. p) To adjust I, urea, calcium carbonate, ammonia gas, aqueous ammonia, magnesium phosphate, etc. are used as a neutralizing agent.

After completion of the culture, known methods such as ion exchange resin method and solvent extraction method are used to isolate L-glutamic acid from the culture solution.

The present invention will be specifically illustrated by giving examples below.

Example 1゜ Corynebacterium glutamicum G-2 as inoculum
8 and G-37 were used.

These mutants were treated with 40 g/l of glucose, 10 g/l of peptone, 5 g/l of meat extract, and 5 g/l of yeast extract.
l, KH2PO41g/l, 2HPO41g/j
! , Mg50m・? H200,5g/LFeSO4'
7820 20mg/CMn5On・4H2020■/
l, urea 5 g/l, (NH4)zsO45
A 300 II Erlenmeyer flask containing 20 ml of a seed medium (p)I7.2) consisting of g/l was inoculated and incubated at 30°C for 2 hours.
It was cultured for 4 hours. 7 ml of this culture solution was added to fermentation medium [glucose 77 g/l, urea 7.7 g/1, (NH
4) 2so4 3 g/1.

KH, PO41.5g/l, 2HP0.1.5g/l
l.

M n S 04 ・4 Hzo 30mg/1
, Piotine 77z/1. Calcium pantothenate 770
■/1. Nicotinamide T70/l, Thiamine H
Cl 300g/l, pH 7, containing 2113ml 3
00ml Erlenmeyer flask, and then inoculated with penicillin G.
Solution (1[111) was added to the final concentration of 5 U/ml, and cultured with shaking at 34°C. During culture, the pH of the culture solution
The cells were cultured for 30 hours while adding 3.5 ml of sterilized 10% urea solution to maintain the pH between 6.5 and 8.0. As a control, the parent strain Day-15 was cultured at the same time under the same conditions. The amount of L-glutamic acid accumulated by each strain was as shown in Table 2.

G-2826,0 G-3728,2 Effects of the Invention According to the method of the present invention, the fermentation production of L-glutamic acid can be significantly improved, so that L-glutamic acid can be supplied in large quantities at low cost.

Claims (2)

[Claims] (1) A microorganism that belongs to the genus Corynebacterium and has the ability to produce L-glutamic acid, and has lost or significantly reduced the ability to grow using L-glutamine or L-glutamic acid as the sole carbon source, is cultured in a medium. A method for producing L-glutamic acid by a fermentation method, which comprises accumulating L-glutamic acid in a culture and collecting the accumulated L-glutamic acid from the culture. (2) The method according to claim 1, wherein the microorganism belongs to Corynebacterium glutamicum.