JPH029384A - Production of l-glutamic acid by fermentation process - Google Patents

Abstract

PURPOSE:To industrially obtain L-glutamic acid at low cost by culture of specific microorganisms tolerant to a specific antibiotic. CONSTITUTION:L-glutamic acid-productive bacteria tolerant to gramicidin, classified as Corynegacterium or Brevibacterium sp. is put to culture in a culture medium to produce and accumulate L-glutamic acid in the resultant cultured product and collect this L-glutamic acid. The above-mentioned mutant tolerant to gramicidin can be obtained by induction from L-glutamic acid-productive bacteria classified as Corynebacterium or Brevibacterium sp., or by imparting bacteria having the above nature with L-glutamic acid productivity.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for producing monoglutamic acid by fermentation. The purpose is to industrially produce L-glutamic acid, which is an important amino acid used as a food additive, at low cost.

Conventional technology Conventionally, for the production of monoglutamic acid by fermentation, wild strains of the genus Corynebacterium and Brevibacterium or mutant strains that require various substances for growth or are resistant to various substances have been used. A method using . In addition, a method using microorganisms that are resistant to antibiotics that inhibit energy metabolism or precursors of ubiquinone biosynthesis and that has the ability to produce L-glutamic acid (Japanese Patent Laid-Open No. 59-1979-1)
15.4995) is known.

Problems to be Solved by the Invention There is a need for an inexpensive method for producing monoglutamic acid, which is not useful as a food additive or the like.

,'J! In order to obtain L-glutamic acid in high yield, the present inventors conducted research on seven bacteria that produce L-glutamic acid. Among the various antibiotic-resistant strains that have been identified, the present invention was completed by discovering that a mutant strain that is particularly resistant to gramicidin produces L-glutamic acid at a high yield.

The present invention will be explained in detail below.

The present invention involves culturing a microorganism belonging to the genus Corynebacterium or the genus Previbacterium, resistant to gramicidin, and capable of producing L-glutamic acid in a medium, producing and accumulating L-glutamic acid in the culture, Provided is a method for producing L-glutamic acid by a fermentation method, which is characterized in that L-glutamic acid is collected from the culture.

As an example of the microorganism used in the present invention, any microorganism that belongs to the genus Corynebacterium or Brevibacterium, has resistance to gramicidin, and has L-glutamic acid producing ability can be used.

This mutant strain can be obtained by deriving from a monoglutamic acid-producing bacterium of the genus Corynebacterium or Brevibacterium, or by imparting L-glutamic acid productivity to a bacterium having the above properties.

For example, L-glutamic acid producing bacteria Corynebacterium
Glutamicum ATCC-13032 is irradiated with X-rays, ultraviolet rays, cobalt, etc., or treated with drugs such as N-methyl-N.
Conventional mutagenesis methods such as contacting with '-nitro-N-nitro-longanidine can be applied as appropriate.

A method for isolating a mutant strain from cells subjected to mutation treatment is to collect a strain that can grow in a solid medium containing an amount of gramicidin in which the parent strain cannot grow.

A specific example of how to obtain a mutant strain is shown below.

Corynebacterium glutamicum ATCC13032
was subjected to the usual mutation treatment with N-methyl-N'-nitro-N-nitroguanidine (200 mg/j', 30°C, 3
0 min), followed by minimal medium containing lO cod/m I of gramicidin [glucose 10 g/β, ammonium chloride 4 g/j! , KH2PO.

Ig#, 2HPO43g/It, Mg5O, 7H2
00,01g/j! , Fe50.・7H,OO,01g
/12. MnSO4'4HzOOlo 1 g/1. Urea 2g/j! , Biotin 50■/I! , agar 20 g/
It.

Apply at pH 7.2). Cultured at 30°C for 2 to 6 days,
Obtain gramicidin-resistant colonies that grow.

Gramicidin manufactured by Sigma (also called Gramicidin D or Gramicidin NF) is used. Fifty strains resistant to gramicidin were harvested, and strains with improved L-glutamic acid production ability than the parent strain were isolated. As a representative strain,
Examples include Corynebacterium glutamicum H-7110.

This strain 1 was deposited as FERM BP-1876 with the Institute of Microbial Technology, Agency of Industrial Science and Technology on May 11, 1986.

The culture medium used in the present invention includes a carbon source and a nitrogen source.

Both synthetic and natural media can be used as long as they contain inorganic substances and other micronutrients required by the strain used.

In other words, carbon sources include glucose, fructose, sucrose, maltose, and mannose.

Carbohydrates such as nlubitol, sugar alcohols, glycerol, 'il powder, starch hydrolysates, molasses, etc. can be used.
In addition, organic acids such as acetic acid, pyruvic acid, lactic acid, fumaric acid, and gluconic acid, and amino acids such as aspartic acid,
Alternatively, lower alcohols such as ethanol can also be used.

Nitrogen sources include various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or amino acids such as aspartic acid,
urea and nitrogen-containing substances such as peptone, meat extracts,
Various products can be used, such as corn steep liquor, casein hydrolyzate, and soybean hydrolyzate.

As the inorganic substances, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate, etc. are used.

Furthermore, vitamins necessary for the growth of microorganisms, such as biotin, nicotinamide, pantothenic acid, and thiamine, are also used as necessary, but these substances are provided in the medium along with the other medium compositions mentioned above. If so, there is no need to add it.

Cultivation is performed under aerobic conditions such as shaking culture or deep aeration agitation culture. The culture is maintained at a temperature of 20 to 40°C and a pH in the range of 3 to 9, preferably around neutrality. The pH of the culture medium is adjusted using calcium bromate, an organic or inorganic acid, an alkaline solution, ammonia, a pH reducing solution, or the like. The culture time is usually 1 to 4 days, and L-glutamic acid is produced in the culture solution.

In addition, in order to accumulate L-glutamic acid, as is conventionally known, biotin powder should be kept low, or in the case of a medium with a high biotin content, a surfactant should be used.

Penicillin etc. may be added.

After culturing, to collect L-glutamic acid from the culture solution, solids such as bacterial cells are removed from the culture solution, and a known ion exchange treatment method, acondensation method, and adsorption method are used.

L-glutamic acid can be recovered from the culture solution by using a salting-out method or the like.

Hereinafter, the present invention will be specifically explained with reference to Examples.

Example 1 Corynebacterium glutamicum H-71 as inoculum
10 (FERM BP-1876) strain was used. H-
7110 strain with glucose 40g/I! .

Yeast extract 5g/l, KH2PO41.5g/I! .

K2HPO40,5g/n, MgSO+・7H200
, 5g#, biotin 100g/L urea 3g/I! .

Seed medium with a composition of pH 7.2 containing 20 ml of 25 Qm
The cells were cultured with shaking in a 1-volume Erlenmeyer flask for 24 hours. 1+++l of the obtained seed culture solution was inoculated into a 25 Qml Erlenmeyer flask containing 2 Qml of the following fermentation medium, and incubated at 30°C.

Shaking culture was performed for 24 hours. The production amount of monoglutamic acid at that time was 28.1g/j! Met. Parent strain Δ as control
As a result of culturing in the same manner using TCC-13032, L-
The production amount of glutamic acid is 24.4 g/7! Met.

Additionally, the acquisition of gramicidin-resistant strains and oligomycin, an antibiotic that exhibits other energy metabolism inhibition, were obtained through similar mutational treatments.

Antimycin A, lutamycin 9 palinomycin, and gramicidin S resistant strains M-3482゜M-3
488, M-3490, M-3497, and M-3499 were similarly cultured, and the amount of L-glutamic acid produced was measured. The results are shown in Table 1.

Composition of fermentation medium: Molasses (sugar equivalent) 50g/ffi, NH4H, PO41g
/ R, (NH+)zHP 04 1 g / j
! , (NH4)2 S 042 g/i'
, urea 5 g/l, Mn 504-4H2010vg
/ 1! , Ca CO330g/j! , penicillin G5 un+ts 7ml (pH 6,5)
1 Table: :l'J Jt-Antibiotic Resistance (Concentration) L-Gluta', l-131! ! 2 Oligomycin (0,1
mg/ml)', i-FL3g antimycin A
(0,1mg/ff+l)X: -3490 Lucumycin (0,1zg/if)M-3197 Valinomycin
(0.1mg/ml) M-3499 Gramingin S
(10xr/ml) 1 (-7110 gramicidin (10 ml/ml), T CC-13032 Example 2 mic acid axis/2) 26.3 25.4 25.2 25.3 26.6 24.3 As an inoculum, Corynebacterium glutamicum H-7
110 (FERM BP-1376) strain was used. 39 ml of the seed culture solution obtained by culturing the H-7110 strain in the same seed medium as in Example 1 was added to the fermentation medium 80Q with the following composition.
Incubate the bacteria in a 22-jar fermentor containing ml and aerate.
l VVIII, 1 stirring number 800 rpm.

Culture was performed at pH 7.6 (adjusted with aqueous ammonia).

When the sugar in the medium was consumed and the pH rose, Peri/Lin G was added to 5 units/ml, and then cultured for 43 hours while continuously adding hydrated molasses containing 40% minuta. went. As a control, the parent strain, TCC-1303
Culture was performed in the same manner using two strains. H-7] Glutamic acid i4: production 1 in 10 strains and ATCC-13032 strain
are shown in Table 2.

Composition of fermentation medium: Molasses (iFi equivalent) 30g/l, NH, H2PO.

1.21? /L (NH9)zHP○, 1.2g
/i'.

Mn5O, 4~6H20100g/l.

Cu SO= ・5H2020g/1 (pH7,6
) Table 1 Bacterial Strain L-Glutamic Acid Axis/β) Sugar Yield (%) ΔT CC-13032 Effects of the Invention According to the present invention, L-glutamic acid can be obtained in good yield.

Claims (1)

[Claims] Cultivating a microorganism belonging to the genus Corynebacterium or Brevibacterium, resistant to gramicidin, and capable of producing L-glutamic acid in a medium, producing and accumulating L-glutamic acid in the culture, and collecting the same. A method for producing L-glutamic acid by a fermentation method, characterized by: