JPS6066990A - Production of l-glutamic acid - Google Patents

Abstract

PURPOSE:To obtain the titled compound as an amino acid important for food additives industrially at a low cost, by cultivating a special variant strain belonging to the genus Corynebacterium or Brevibacterium in a culture medium. CONSTITUTION:A variant strain, e.g. Corynebacterium glutamicum CQ-306 (NRRL B-15531) or Brevibacterium lactofermentum BQ-13 (NRRL B-15530), belonging to the genus Corynebacterium or Brevibacterium, having the tolerance to alpha-naphthoquinone, and having the ability to produce L-glutamic acid is cultivated in a culture medium at 20-40 deg.C and 3-9pH preferably under aerobic conditions for 1-4 days to give the aimed compound.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-glutamic acid by fermentation. More specifically, the present invention relates to a method for producing L-glutamic acid by culturing an L-glutamic acid-producing mutant strain belonging to the genus Corynebacterium or Brevibacterium in a nutrient medium and collecting the produced L-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 monoglutamic acid.

The production yields of the known processes are not sufficient from a commercial point of view. Thus, a need exists for a process for producing L-glutamic acid in high yield and at low cost.

For this purpose, the monoglutamic acid productivity of a monoglutamic acid-producing microorganism belonging to the genus Corynebacterium or Brevibacterium is significantly improved when the microorganism is endowed with resistance to α-naph-1 quinoline. was discovered. Conventionally, the productivity of monoglutamic acid was L-
It is not known that imparting these properties to glutamic acid-producing bacteria can improve performance.

According to the present invention, L-glutamic acid is produced by a 1°-glutamic acid-producing mutant strain belonging to the genus Corynebacterium or Brevibacterium; this mutant strain is characterized by being resistant to α-naph I and quinoline. is produced by culturing until I,-glutamic acid accumulates in the culture medium, and then collecting l,-glutamic acid.

The microorganism used in the present invention belongs to Corynebacterium or Brevibacterium and is a transformed strain that has been endowed with resistance to α-naphthoquinoline and has the ability to produce glutamic acid. 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, preferred mutant strains can be obtained by the reverse method, namely α- It can be obtained by imparting L-glutamic acid productivity to bacteria that are resistant to naphthoquinoline.

Mutation induction methods include ultraviolet irradiation and X-ray irradiation.

Known techniques such as radiation irradiation, treatment with a mutagenic agent, etc. can be applied, but treatment using N-nitro-N'-medyru-N-nitrosoguanidine (referred to as NTG treatment) is preferably used.

The mutated bacteria are cultured in a medium containing α-naphthoquinoline in an amount that the parent strain cannot grow, and bacteria that can grow 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 mutagenized bacteria are cultured in a nutrient medium, and those that have the ability to produce monoglutamic acid at a higher yield than the parent strain are selected. A specific example of a method for obtaining a suitable bacterium is shown below regarding a mutant strain belonging to the genus Corynebacterium.

Corynebacterium glutamicum 8”I’ CC13
032 was treated with N'rG in the usual manner, and the treated cell suspension was given at 0.5 g/d! Enzyme extract, 0.7g, /
di meat extract, 1g/dI peptone, 0.3'g/di
NaCn, 2g7dl agar, and I 00 p g/
Cultured on an agar medium containing mlα-naphthoquinoline,
Incubate at 30°C.

A culture test for L-glutamic acid is performed on the colonies produced, and bacteria with excellent productivity are selected.

Thus, the α-naphthoquinoline-resistant L-butamic acid producing bacterium Corynebacterium glutamicum CQ, -306
get.

Similarly, Brevibacterium lactofermentum BQ-13 is obtained from Brevibacterium lactofermentum ATCC 13869.

These bacteria are ARS Coltura Co11ec.
tionResearch+ FementaLion
Brevibacterium glutamicum CQ-306 was internationally deposited at the Laboratory on July 21, 1982 under the Budaves 1 Treaty, and the NRRL
B-15531, Brevibacterium lactofermentum B Q-13 NRRL B-1553
A zero value and an 1L number are given.

Carbon source as a medium used in the culture stage of the invention.

A natural medium if it contains a nitrogen source, minerals as well as small amounts of other nutrients required by the particular mutant strain used.

Any synthetic medium may be used. Representative nutritional supplements are shown in the examples below.

Suitable carbon sources include carbohydrates such as glucose, sucrose, fructose, maltose, mannose, #flour, starch hydrolyzate, and molasses, sugar alcohols such as glycerin and sorbitol, formic acid, acetic acid, butyric acid, fumaric acid, malic acid, etc. Organic acids, lower alcohols such as methanol and ethanol, hydrocarbons, etc. are used. These carbon sources may be used alone or as a mixture in various ffI 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 nitrate, fIe, ammonium, ammonium phosphate, urea, peptone, polypeptone, meat extract, 6-ge mother extract 5, corn steep liquor, casein hydrolyzate .

Natural nitrogen-containing substances such as soybean meal hydrolyzate, bacterial cells or their hydrolyzate are used.

Inorganic substances include primary potassium phosphate, secondary potassium phosphate,
Magnesium sulfate 3 Magnesium phosphate.

Sodium chloride, ferrous sulfate, manganese sulfate dicalcium carbonate, etc. are used.

If the bacteria used require specific nutrients such as amino acids, 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, pH 3 to 9, e.g. shaking) 8 nutrient or aerated agitation 1 ↑ Until a recoverable amount of L-glutamic acid is produced in the culture solution by culturing. It will be held for ~4 days.

After completion of the culture, I2-glutamic acid is processed by conventional methods such as concentration, vA, desorption, salting out, and treatment with ion exchange resin.

Crystallization etc. are recovered from the culture solution using a combination cell.

Embodiments of the invention are illustrated by the following examples.

Example 1 As the inoculum, Corynebacterium glutamicum AT
CC13032, CQ-306+'ji, Brevibacterium lactofer-mentum 8T (:C13
Four strains, 869 bacteria and BQ-13 bacteria, are used.

These are glucose 4g/lU, polypeptone 2a/
di, yeast extract 0.5g/d1. KH□po.

0.15 g/J, K2HPO40.05 g/dl.

M B S Oa ・71L 0 0.05 g/d
l-biotin 100μg/ml, urea 0.3g/d1.
The cells are inoculated into a seed medium with a composition of pH 7.2 and cultured with shaking at 30°C for 24 hours. 1 ml of this was inoculated into a 300 ml Erlenmeyer flask containing 20 ml of the following Hage medium, and the mixture was incubated at 30°C for 3 days.
The amount of monoglutamic acid produced and the yield relative to sugar when cultured with shaking for days are shown in Table 1.

Fermentation medium composition Glucose 10g/d1. Meat Kiss 0.5g/d1゜Ammonium Sulfate 3g/d1. KH2PO40,1,5g/d! .

Kz IIP Os O,05g/d! , Mg SO
a ・711200.05g/d! , thiamine hydrochloride 500μg/LF esOa ・7Hz 0 10mg
/(1, MnSO4・4tlzO10ne/l, Cu5
On・5Hz01 tvr/1, urea 0.5g/
d/, CaCO53g/d1 (pH 7,2), 120°C
, lQmin, sterilization.

Table 1 Example 2 Using blackstrap molasses (reduced glucose) Jog/dI instead of glucose in the fermentation medium), culture b! j ”!I'
1. Addition of Penicillin G 5u/Miriritsutoru.
It was carried out in the same manner as in Example 1, and the results are shown in Table 2.
7ru.

Table 2 Patent Applicant (+02) l<4Wa Hakko Kogyo Co., Ltd.] Procedural Amendment 1, Indication of Matter A'1 DB 58 Hourly Wage Application No. 174319 2, Title of Invention Method for producing L-glutamic acid 3. What is the relationship between the person filing the amendment and the case? Patent applicant postal code: 100 Address: 6-1 Hitotecho-chome, Chiyoda-ku, Tokyo Name:
(102) Kyowa Hakko Kogyo Co., Ltd. 4, subject of amendment 1111s R'l Detailed explanation column 5 of the invention? lH Correct contents 1) Correct 1 Co1turej to 1Cu1tureJ in the 7th line directly below the 4th line of the specification.

2) 4 lines directly below the 4th line of the same book, ``Predobacterium glutamicum'' is changed to ``1 Corynebacterium glutamicum''nTd:'1J.

Claims (1)

[Claims] Cultivating 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 in a medium, producing and accumulating L-glutamic acid in medium II, A method for producing monoglutamic acid, which comprises collecting this.