JPH0430275B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Fields] L-glutamine is widely used as a treatment for gastrointestinal ulcers as it promotes the production of glucosamine, which is a component of the mucoprotein that makes up the mucosa of the fire extinguisher, and is also used in amino acid infusions and comprehensive amino acid preparations. It is an important component of The present invention aims to improve the method for producing L-glutamine by fermentation using breeding technology. [Prior Art] Known methods for producing L-glutamine by fermentation include methods using wild strains of the genus Brevibacterium or Corynebacterium and their mutant strains that are resistant to sulfur drugs. [Problems to be Solved] It is an important problem to industrially produce L-glutamine at low cost by improving the fermentation yield and accumulation. [Means for Solving the Problems] The present invention has been made to solve the above-mentioned problems, and is aimed at solving the problems described above.
As a result of research aimed at improving microorganisms capable of producing glutamine and finding strains with even higher fermentation yields, some strains were found to be resistant to purine analogs and/or methionine sulfoxide (hereinafter abbreviated as MSO). They discovered that there is a strain that produces L-glutamine at a higher yield than conventional L-glutamine-producing bacteria, leading to the completion of the present invention. That is, the present invention involves culturing in a liquid medium a microorganism that belongs to the genus Brevibacterium or Corynebacterium and has resistance to purine analogs and/or MSO, and has the ability to produce L-glutamine.
The present invention relates to a method for producing L-glutamine, which comprises collecting L-glutamine produced and accumulated in a culture medium. Purine analogs of the present invention include 8-azaguanine, 8-azaadenine, 8-azaxanthin,
6-mercaptoguanine, 2,6-diaminopurine, 2-fluoroadenine, etc., and strains that are resistant to at least one of these are referred to as purine analog-resistant bacteria in the present invention. The microorganism used in the present invention belongs to the genus Brevibacterium or Corynebacterium and has purine analog resistance and/or MSO resistance,
It is a mutant strain that also has the ability to produce L-glutamine. In order to obtain the mutant strain of the present invention, purine analog-resistant and/or MSO-resistant strains may be induced from the parent strains shown below, sulfur drug-resistant, cobalamin-resistant, vitamin P-resistant, ketomalonic acid-resistant, various amino acid resistant strains, etc. Purine analog resistance and/or
MSO-resistant strains may be induced. The wild strain that serves as the parent strain of this mutant strain is also known as a coryneform L-glutamine producing bacterium such as Brevibacterium or Corynebacterium, and includes, for example, the following. Brevibacterium flavum ATCC 14067 Brevibacterium lactofermentum
ATCC 13869 Brevibacterium deivalicatum
ATCC 14020 Brevibacterium satscaroliticum
ATCC 14066 Corynebacterium glutamicum ATCC 13032 Corynebacterium acetoacidophyllum
ATCC 13870 To obtain the mutant strains of the present invention from these parent strains, conventional mutagenesis methods such as treatment with N-methyl-N'-nitro-N-nitrosoguanidine can be applied. The method for isolating the mutant strain of the present invention from a mutant-treated bacterial fluid is carried out by collecting a strain that grows in a medium containing an analog. The specific method for inducing mutations in the mutant strains shown in the present invention and purine analogs include 8-azaguanine (hereinafter referred to as 8
-Abbreviated as AG. ), and the relationship between the growth rates of strains for these analogs when using MSO is shown below. [Mutation induction method] Brevibacterium flavum ATCC 14067 grown on bouillon agar slant at 30°C for 24 hours
Cells were suspended in M/30 phosphate buffer to determine the cell concentration.
200 μg/ml of N-methyl-N'-nitro-N-nitrosoguanidine was added to a bacterial cell suspension of 10 ⁹ /ml and maintained at 0° C. for 20 minutes. Then, the bacterial cells were collected by centrifugation, and after being well precipitated with M/30 phosphate buffer,
Inoculate the culture medium with the composition shown in Table 1, and store it at 31.5℃ for 2 hours.
Cultured for ~10 days.

[Table] Among the 50 8-AG resistant strains grown on agar medium, Brevibacterium flavum AJ 12210 (FERM
-P 8123) was obtained. On the other hand, 8-
By using a medium containing 0.2 g/dl of MSO instead of AG, 50 MSO-resistant strains were obtained, and Brevibacterium flavum AJ 12211 was selected as a strain with high L-glutamine production ability.
(FERM-P 8124) was obtained. Brevibacterium flavum AJ 12210 (FERM-P 8123)
further mutated under similar mutation conditions.
By inoculating and culturing the above medium containing 0.2 g/dl of MSO, 40 strains showing resistance to 8-AG and MSO were obtained, and among them, Brevibacterium was selected as a strain with improved L-glutamine production ability. Um・
Flavum AJ 12212 (FERM-P 8125) was obtained. Corynebacterium glutamicum ATCC
When using 13032 as the parent strain, Corynebacterium glutamicum AJ 12213 (FERM P-
8126), and Corynebacterium spp. as an MSO-resistant strain.
Glutamicum AJ 12214 (FERM P-8127),
8-Corynebacterium glutamicum AJ 12215 as a strain showing resistance to AG and MSO at the same time
(FERM P-8128) was obtained. The degree of analog resistance of the thus obtained mutant strain was compared with that of the parent strain. Glucose 0.5g/dl, urea 0.2g/dl, ammonium sulfate
0.15g/dl, KH ₂ PO ₄ 0.3g/dl, K ₂ HPO ₄ 0.1
g/dl, MgSO ₄・7H ₂ O0.01g/dl, CaCl ₂・
2H ₂ O 0.1mg/dl, biotin 100μg/, thiamine hydrochloride 100μg/, FeSO ₄・7H ₂ O 0.002g/
dl, MnSO ₄・7H ₂ O0.002g/dl and the amounts of analogs shown in Tables 2 to 4, and PH
Natural medium (peptone 1
g/dl, yeast extract 1g/dl, NaCl 0.5g/dl,
PH7.0) Bacterial cells cultured on a slant for 24 hours were suspended in sterilized water and inoculated, cultured for 24 hours, and the degree of growth was measured by turbidity. These results are summarized in the second to fourth
Shown in the table.

【table】

【table】

[Effect]

The medium used for culturing such mutant strains is a normal medium containing a carbon source, a nitrogen source, inorganic ions, substances that should satisfy the above-mentioned requirements, and other organic micronutrients such as vitamins as necessary. . Suitable carbon sources include carbohydrates such as glycol and sucrose, alcohols such as ethanol or methanol, and organic acids such as acetic acid and fumaric acid. As nitrogen sources, aqueous ammonia, ammonia gas, and ammonium salts are suitable. As inorganic ions, potassium ions, sodium ions, magnesium ions, phosphate ions, and others are appropriately added to the medium as necessary. It is preferable to culture under aerobic conditions, and the culture medium should be
More favorable results can be obtained by controlling the pH to 4 to 8 and the temperature to 25 to 37°C. Thus, if the culture is continued for 1 to 7 days, a significant amount of L will be present in the medium.
- Glutamine is produced and accumulated. L- from the culture solution
Glutamine can be collected using a conventional method such as using an ion exchange resin. This will be explained below using examples. Example 1 300 ml of an aqueous culture medium having the composition shown in Table 5 was dispensed into a small glass jar fermentor, sterilized by a conventional method, and then grown on a bouillon slant at 30°C for 24 hours. Various strains shown in Table 6 were inoculated. Then, while maintaining its pH at 6.5 with ammonia gas, it was heated to 31.5°C at 1200 rpm and aeration at 1/4 volume per minute.
Cultured for hours. Table 5 Glucose 10% Ammonium sulfate 1% Potassium phosphate 0.25% Magnesium sulfate 0.04% Ferrous sulfate 0.001% Thiamine hydrochloride 350γ/ Biotin 5γ/ Ajiru (registered trademark) (PH7.0) 0.5ml/dl Fermentation Table 6 shows the amount of L-glutamine produced and accumulated in the finished solution.

【table】

[Table] Brevibacterium flavum AJ 12212
Fermentation finished liquid 1 using (FERM-P 8125)
The bacterial cells were removed by centrifugation to obtain a supernatant, from which L-glutamine was separated and purified using a conventional method using an ion exchange resin.
-21.5 g of glutamine crystals were obtained.

Claims (1)

[Claims] 1. A microorganism belonging to the genus Brevibacterium or Corynebacterium that has purine analog resistance and/or methionine sulfoxide resistance and has the ability to produce L-glutamine is cultured in a liquid medium, and the L produced and accumulated in the medium is cultured. - A method for producing L-glutamine, which comprises collecting glutamine.