JPS58141788A - Preparation of l-glutamic acid by fermentation - Google Patents

Abstract

PURPOSE:To prepare L-glutamic acid, in high efficiency, by culturing microorganisms belonging to Brevibacterium genus or Corynebacterium genus. CONSTITUTION:A mutant, belonging to Brevibacterium genus or Corynebacterium genus, resistant to anthracycline antibiotic substance, and capable of producing L-glutamic acid, e.g. Brevibacterium lactofermentum AJ 11776 (FERM- P No. 6339), Corynebacterium glutamicum AJ 11777 (FERM-P No. 6340), etc. is cultured under aerobic conditions at 24-38 deg.C and 6-9pH, and L-glutamic acid is separated from the cultured liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing monoglutamic acid by fermentation. As a result of research to find monoglutamic acid-producing bacteria with higher productivity, the present inventors found that among mutant strains of the genus Brevibacterium or Corynebacterium 1 that are resistant to anthracycline antibiotics. ni, L
- It was discovered that there are many strains with high glutamic acid productivity. The present invention was completed based on this discovery.

That is, the present invention involves culturing a mutant strain that belongs to the genus Brevibacterium or Corynebacterium and is resistant to anthracycline antibiotics and has the ability to produce L-glutamic acid, and the mutant strain is produced and accumulated in the medium. This is a method for producing monoglutamic acid using a fermentation method, which is characterized by collecting monoglutamic acid.

The microorganism used in the present invention belongs to the genus Brevibacterium or Corynebacterium, and is a mutant strain that is resistant to anthracycline antibiotics and has the ability to produce L-glutamic acid. The mutant strain is
After a microorganism of the genus Brevibacterium or Corynebacterium is treated with N-methyl-back-nitroso-N-nitrosoguanidine by a conventional mutation method, an anthracycline is added in such an amount that the parent strain cannot grow. This can be induced by fractionating the bacterial strains that grow on a solid medium containing an antibiotic.

Parent strains used for inducing the mutant strains of the present invention include Brevibacterium sp. and Corynebacterium sp.
It belongs to the so-called coryneform glutamic acid producing bacteria, and specific examples include the following.

Brevibacterium lactofermentum (Brev
ibacterjum lactofermentum
) AT CC13869, Brevibacterium flav
um) AT CC+ 4067, Brevibacterium divaricatum
um) AT CC+4020, Brevibacterium
ium saccharoliticum ) ATCC
14066, Phrinebacterium fistula glutamicum (Co
rynebacterium glutamjcum
) ATCC13032, Corynebacterium φ acetoacillus 1 filler A (Corynebacterium a
cetoacido-philum) ATCC1387
0 Mutant strains resistant to anthracycline antibiotics used in the present invention include 7,819.10-tetrahydro-5,12 such as adriamycin, daunomycin, carminomycin, rubidazone, and aclacinomycin A.
- A mutant strain that is resistant to anthracycline antibiotics such as quinone glycosides in naphthacene, for example,
The following mutant strains are used:

Prepibacterium lactofermentum AJ11
776 (FERM-P 6339), Corynebacterium glutamicum AJ117? ? (FERM-P 63
40) In addition to anthracycline antibiotic resistance, mutations that are known to increase L-glutamic acid productivity, such as ketomalonic acid resistance, monofluoroacetic acid resistance, and adenine resistance, can be added to increase productivity. Stocks can be obtained.

Tables 1 and 2 show the degree of resistance of the mutant strains of the present invention to 7 doriamycin or daunomycin among anthracycline antibiotics.

The degree of resistance of each strain was determined by straining and measuring as follows.
t, ammonium sulfate 0.15 t/dl, KH, PO40, 3f
/dl, K, HPo.

0.1 f/di, MgSO4・7H, 200,
01t/dt.

CaCl, -2H, OO, 1t/di, thiamine hydrochloride lOμt/dls biotin 3 Tll5! /dt%
Na, B, O@IOH, 00, 44w9/di, F
eC1,-6) 1,0 4.85 taps/dl.

CuSO4・5Hg O1, 95m9/dl −,
(NH4)6 Mo7 ot4” 4H200,185
m"j/dl, ZnSO4-7H, 04489/di
.

MnC1g @4)120 0.36 N9/d
Into a 20 m (1500-volume flask) a sterilized medium containing adriamycin in the amount shown in Table 1 or daunomycin in the amount shown in Table 2, a natural medium (Pep) 7 lt/di, Yeast extract 7.1f
l/di.

NaC10.5t/dl, pH7.0) 24 in slant
The cultured bacteria were suspended in sterile water, inoculated, and cultured with shaking in a flask for 24 hours. The growth rate of each strain was 562 n.
The turbidity was measured at m, and the relative growth rate is shown in each table.

The method of producing and accumulating L-glutamic acid using these strains is the same as that used for culturing conventional glutamic acid-producing microorganisms. Sugar juice from cane and sugar beet containing scallions, carbohydrates such as blackstrap molasses and starch saccharification liquid, acetic acid, ethyl alcohol, etc. are used, and nitrogen sources include, for example, ammonium salts, aqueous ammonia, ammonia gas, Urea etc. are used. Other inorganic ions such as phosphates and magnesium salts are added to the medium as necessary. Further, micronutrients such as thiamine and biotin are used as appropriate. Furthermore, in a medium containing excess biotin,
Biotin/action control substances such as polyoxyethylene sorbitan monopalmitate and penicillin are added to the culture medium in a conventional manner.

Cultivation is best carried out under aerobic conditions, with a culture temperature of 24~24°C.
It is recommended to control the pH at 6 to 9 during 38C1 culture.
For the adjustment, inorganic or organic acidic or alkaline substances, as well as urea, calcium carbonate, ammonia gas, etc. can be used.

Collection of monoglutamic acid from the fermentation liquid is carried out by appropriately combining the ion exchange resin method and other known methods.

Examples will be described below.

Example 1 1 ot/dl using gain molasses as reducing sugar.

KH, Po, 0.1 f/di (p) (7,0)
Prepare a medium with the composition of
The mixture was dispensed into a 00ml shaking flask and sterilized by heating with 115U for 10 minutes. Each of the bacterial strains shown in Table 3 was inoculated into this medium, and cultured at 31.5 C with shaking. In addition, in order to maintain the culture solution at pH 6.5 to 9.0 during culturing,
017di of urea water was added as appropriate. Six hours after the start of culture, polyoxyethylene sorbitan monopalmitate was added at a concentration of 4 t/di, and the fermentation was completed in 30 hours. The yield of L-glutamic acid accumulated in the fermentation liquid relative to sugar was determined. The terminology is shown in Table 3.

Example 2 Ammonium acetate 1. ot/di, sodium acetate 1.
0t/dlt, KH2PO40,2f/d
i, MgSO4-7H20o, o8t/dll, Fe
SO4-? H202vxti/dl.

k'1nSO4・4H202mq/1ies Soybean protein hydrolyzate as bag nitrogen 36mg/di, thiamine hydrochloride 2θ1z y /de, biotia 0.06 pt
/dt to pH 8 with J″: potassium solution.
, 0, and 30 ml of it was dispensed into a 600 ml shaking flask, and the mixture was heat sterilized at 115 C for 10 minutes. This medium was inoculated with the bacterial strains shown in Table 4, and cultured with shaking at 31.5C. After the start of the culture, when the residual acetic acid reached 0.1 t/dl or less, a previously prepared ammonium acetate solution was added twice at 1.4 tldi each, and the fermentation was completed in 40 hours. Table 4 shows the yield of 1 gram of retamic acid to acetic acid accumulated in the fermentation solution.

Claims (1)

[Claims] Brevibacterium or Corynebacterium genus, anthracycline antibiotic Wr: resistant;
and culturing a mutant strain having the ability to produce L-glutamic acid 1-
1. A fermentation method for producing monoglutamic acid, which is characterized in that monoglutamic acid produced and accumulated in a medium is collected.