JPH0361438B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-glutamic acid by fermentation. More specifically, L-glutamic acid-producing mutant strains belonging to the genus Corynebacterium or Brevibacterium are cultured in a nutrient medium and the L-glutamic acid produced is collected.
The present invention relates to a method for producing glutamic acid. This mutant strain is endowed with resistance to α-naphthoquinoline (7,8-benzoquinoline). L-glutamic acid is an important amino acid that is useful as a food additive. Therefore, an object of the present invention is to provide an industrially inexpensive method for improving the amino acid. It has been known that bacteria belonging to the genus Corynebacterium or Brevibacterium and having resistance to various substances have the ability to produce significant amounts of L-glutamic acid. The production yields of the known processes are not sufficient from a commercial point of view. Thus, a need exists for a method of producing L-glutamic acid in high yield and at low cost. For this purpose, it has been found that the L-glutamic acid productivity of L-glutamic acid-producing microorganisms belonging to the genus Corynebacterium or Brevibacterium is significantly improved when the microorganisms are given resistance to α-naphthoquinoline. Ta. Conventionally, it has not been known that the productivity of L-glutamic acid can be improved by imparting this property to L-glutamic acid-producing bacteria. According to the present invention, L-glutamic acid is L-glutamic acid belonging to the genus Corynebacterium or Brevibacterium.
- Glutamate-producing mutant strain; this mutant strain has α
- characterized by being resistant to naphthoquinoline; is produced by culturing until L-glutamic acid accumulates in the culture medium and then recovering the L-glutamic acid. The microorganism used in the present invention belongs to Corynebacterium or Brevibacterium and is a transformed strain having the ability to produce L-glutamic acid and has been given resistance to α-naphthoquinoline. Suitable mutant strains can be obtained by mutating microorganisms that already have the ability to produce L-glutamic acid or improved mutant strains thereof to impart this property. Otherwise suitable mutants can be prepared in the opposite way i.e. α-
It can be obtained by imparting L-glutamic acid productivity to bacteria that are resistant to naphthoquinoline. Mutation induction methods include ultraviolet irradiation, X-ray irradiation,
Known techniques such as radiation irradiation, treatment with mutagenic agents, etc. are applied, but N-nitro-
Treatment using N'-methyl-N-nitrosoguanidine (referred to as NTG treatment) is preferably used. The amount of α that makes it impossible for the parent strain to grow mutated bacteria
- Bacteria that can be cultured and grown in a naphthoquinoline-containing medium are selected and then tested for L-glutamic acid productivity. Furthermore, the bacteria used in the present invention may also be endowed with other properties such as auxotrophy and resistance to various substances in combination with the above properties. The above mutation-treated bacteria were cultured in a nutrient medium and L
- A bacterium is selected that has the ability to produce glutamic acid in a better yield than its parent strain. A specific example of a method for obtaining a suitable bacterium is shown below regarding a mutant strain belonging to the genus Corynebacterium. Method Corynebacterium glutamicum
ATCC13032 was treated with NTG using the usual method, and the treated cell suspension was added with 0.5g/dl enzyme extract and 0.7g/dl enzyme extract.
g/dl meat extract, 1g/dl peptone, 0.3g/dl
Cultured on agar medium containing NaCl, 2 g/dl agar, and 100 μg/ml α-naphthoquinoline at 30°C.
Cultivate with A culture test for L-glutamic acid is performed on the colonies produced, and bacteria with excellent productivity are selected. Thus, the L-glutamic acid producing bacterium Corynebacterium glutamicum is resistant to α-naphthoquinoline.
Obtain CQ-306. Similarly, from Brevibacterium lactofermentum ATCC13869 to Brevibacterium
Obtain Lactofermentum BQ-13. These bacteria are from the ARS Culture Collection
Corynebacterium glutamicum CQ-
About 306 NRRL B-15531, Brevibacterium lactofermentum BQ-13
It is given the deposit number NRRL B-15530. As the medium used in the culture step of the invention, either a natural medium or a synthetic medium can be used as long as it contains a carbon source, a nitrogen source, inorganic substances, and small amounts of other nutrients required by the particular mutant strain used. Representative nutritional supplements are shown in the examples below. Suitable carbon sources include carbohydrates such as glucose, sucrose, fructose, maltose, mannose, starch, starch hydrolysates, and molasses, sugar alcohols such as glycerin and sorbitol, and organic acids such as formic acid, acetic acid, butyric acid, fumaric acid, and malic acid. , lower alcohols such as methanol and ethanol, and hydrocarbons. These carbon sources may be used alone or as a mixture in various weight ratios. The entire amount used can be initially fed into the medium or it can be fed intermittently. Ammonia, ammonium chloride, as a nitrogen source
Various inorganic and organic ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium acetate, ammonium nitrate, ammonium phosphate, urea, peptone, polypeptone, meat extract, yeast extract,
Natural nitrogen-containing substances such as corn steep liquor, casein hydrolyzate, soybean meal hydrolyzate, bacterial cells or their hydrolyzate are used. As the inorganic substance, primary potassium phosphate, secondary potassium phosphate, magnesium sulfate, magnesium phosphate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate, etc. are used. If the bacteria used require specific nutrients such as amino bacteria, nucleic acids, vitamins, etc. for growth, the medium must contain appropriate amounts of these substances. In some cases, such nutrient sources are provided by other media components, but then no special provision is required. Cultivation is generally carried out under aerobic conditions at a temperature of 20 to 40°C and a pH of 3 to 9, for example by shaking culture or aerated agitation culture, until a recoverable amount of L-glutamic acid is produced in the culture medium. It is held for days. After completion of the culture, L-glutamic acid is processed by conventional methods such as concentration, adsorption, desorption, salting out, ion exchange resin treatment,
It is recovered from the culture solution by a combination of crystallization, etc. Embodiments of the invention are illustrated by the following examples. Example 1 The inoculum used was Corynebacterium glutamicum ATCC13032, CQ-306, and Brevibacterium lactofermentum ATCC13869.
Four strains of BQ-13 bacteria are used. These include glucose 4g/dl and polypeptone 2g/dl.
g/dl, yeast extract 0.5g/dl, KH ₂ PO ₄ 0.15g/
dl, K ₂ HPO ₄ 0.05g/dl, MgSO ₄・7H ₂ 0 0.05
g/dl, biotin 100μg/ml, urea 0.3g/dl,
Inoculate the seed medium with a pH of 7.2 and culture with shaking at 30°C for 24 hours. Contain 1 ml of the following fermentation medium 20 ml.
Table 1 shows the amount of L-glutamic acid produced and the sugar yield when the cells were inoculated into a 300 ml Erlenmeyer flask and cultured with shaking at 30°C for 3 days. Fermentation medium composition Glucose 10g/dl, Meat Kiss 0.5g/dl, Ammonium sulfate 3g/dl, KH ₂ PO ₄ 0.15g/dl, K ₂ HPO ₄ 0.05
g/dl, MgSO ₄・7H ₂ O 0.05g/dl, thiamine hydrochloride 500μg/, FeSO ₄・7H ₂ O 10mg/,
MnSO ₄・4H ₂ O 10mg/, CuSO ₄・5H ₂ O 1mg/
, urea 0.5g/dl, CaCO ₃ 3g/dl (PH7.2),
Sterilize at 120℃ for 10 minutes.

[Table] Example 2 The same procedure as in Example 1 was carried out except that 10 g/dl of honeydew etc. (in terms of glucose) was used instead of glucose in the fermentation medium and 5 u/ml of penicillin G was added at the start of the culture. Obtain the results shown in Table 2.

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Claims (1)

[Claims] 1. A mutant strain belonging to the genus Corynebacterium or Brevibacterium that is resistant to α-naphthoquinoline and has the ability to produce L-glutamic acid is cultured in a medium, and L-glutamic acid is produced and accumulated in the culture solution. A method for producing L-glutamic acid, which comprises collecting L-glutamic acid.