JPH0362396B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a method for producing L-ornithine by fermentation. Conventional technology Conventionally, methods for producing L-ornithine include a method using an arginine or citrulline auxotroph of the genus Corynebacterium (Japanese Patent Publication No. 18439/1983), a method using an arginine or citrulline auxotroph of the genus Brevibacterium, Method to use (Special Public Interest Publication 43-
8712) is known. Problems to be Solved by the Invention In the conventional methods, the yield of L-ornithine is still not satisfactory. Always excellent L-
There is a need for a method for producing ornithine. Means for Solving the Problems According to the present invention, a significant amount of L- Ornithine can be produced. The microorganisms used in the present invention belong to the genus Corynebacterium, and include 2-thiazolylalanine,
Microorganisms that are resistant to sulfaguazine or 2-fluoropyruvate and have the ability to produce L-ornithine can be induced by mutant strains induced by ultraviolet irradiation or chemical treatment, cell fusion methods, or gene scanning methods. Any recombinant strain etc. can be used. Specifically, Coryhnebacterium glutamicum H-3795
(2-Thiazolylalanine resistance)
656) (hereinafter referred to as H-3795), Corynebacterium daltamicum H-3796 (sulfaguanidine resistance) (Feikoken Joyori No. 657) (hereinafter referred to as H-
3796), Corynebacterium dactamicum H-3797 (resistant to sulfaguanidine and 2-thiazolylalanine) (Feikoken Article No. 658)
(hereinafter referred to as H-3797), Corynebacterium
Daltamicum H-3798 (resistant to sulfaguanidine and 2-fluoropyruvate)
No. 659) (hereinafter referred to as H-3798). Next, the method for collecting the mutant strain of the present invention and the growth of the strain in various drug-added media will be explained. (1) Method for collecting mutant strains Yeast bouillon slant (meat extract 0.5
g/dl, yeast extract 0.5g/dl, peptone 1
g/dl, salt 0.25g/dl, agar 2g/dl, PH
Corynebacterium glutamicum ATCC13232 (hereinafter referred to as
ATCC13232) was suspended in trismalate buffer (PH6.0) to a bacterial cell concentration of 10 ⁸ to 10 ⁹ /ml. Then, N-methyl-
After adding N'-nitro-N-nitrosoguanidine to a final concentration of 250 μg/ml,
Leave it for a minute. Thereafter, the bacterial cells were collected by centrifugation, washed with trismalate (PH6.0) in a buffer solution, and then the bacterial cells were collected with 100 μg of sulfaguanidine/
30°C, 5-7 ml of plate culture shown in Table 1.
Cultured for 1 day.

[Table] A mutant strain H-3796 with improved L-ornithine productivity was obtained from the colonies that appeared on the flat agar plate. Furthermore, H-3795, H-3797 and H-3795, H-3797 and H-
Got 3798.

[Table] (2) Growth of drug-resistant strains in drug-containing medium H-3795 and H- obtained by method (1) above
3796, H-3797, H-3798 and ATCC13232 were inoculated into yeast bouillon slant and incubated at 30℃ for 24 hours.
Cultured for hours. Then, the culture solution was suspended in physiological saline. The suspension was treated with sulfaguanidine, 2-thiazolylalanine or 2-thiazolylalanine at the concentrations shown in Table 3.
The cells were inoculated onto a plate medium containing fluoropyruvate shown in Table 1 (10 ⁸ cells/plate) and cultured at 30°C for 48 hours. The results are shown in Table 3.

[Table]: Very good growth +: Good growth -: No growth The medium used in the present invention may be a natural medium or an artificial medium as long as it contains carbon sources, nitrogen sources, inorganic substances, and other growth-promoting substances. Any medium may be used.
Carbon sources include glucose, fructose, sucrose, maltose, starch hydrolyzate,
Fruit juice, blackstrap molasses, organic acids, alcohol, etc. are used. As the nitrogen source, ammonia, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium nitrate, ammonium carbonate, ammonium hydroxide, urea, yeast extract, corn steep liquor, peptone, etc. are used. As the inorganic substance, potassium phosphate, dipotassium phosphate, magnesium sulfate, etc., ferrous sulfate, manganese sulfate, zinc sulfate, etc. are used. As growth promoting substances, amino acids and vitamins such as pantothenic acid, nicotinic acid, and thiamine hydrochloride are used. In addition, the microorganism used is L-citrulline or L-citrulline.
- If the medium has auxotrophy for arginine, 100 to 300 mg/L-arginine may be present in the medium. Culture is performed under aerobic conditions such as aeration and stirring, culture temperature is 24-40℃, pH is 5.5-8.5, and culture days are 1-40℃.
It's been 4 days. Urea, calcium carbonate, aqueous ammonia, ammonia gas, magnesium phosphate, etc. are used to adjust the pH. L-ornithine can be collected from the fermentation liquid using a conventional method.
For example, an ion exchange resin method, a concentration method, an organic solvent addition method, etc. are used. Example 1 H-3796 was used as an inoculum. The strain was cultured on bouillon slant at 30°C for 24 hours. One loopful of the culture was mixed with 70 g of glucose/
, ammonium sulfate 30g/, KH ₂ PO ₄ , 0.5
g/, MgSO ₄ 0.125g/, FeSO ₄・7H ₂ O0.02
g/, MnSO ₄・7H ₂ O0.02g/, ZnSO ₄・
7H ₂ O 0.01g/, Biotin 100γ/, Thiamine Hydrochloride 1mg/, Corn Steep Liquor 10
g/, urea 3g/, L-alkinine 250mg/
, butyl citrate 3ml/, CaCO ₃ 30g/
The cells were inoculated into a 250 ml Erlenmeyer flask containing 20 ml of a medium having the following composition, and cultured at 30°C for 48 hours. The amount of L-ornithine accumulated in the culture solution was 30.6 g/(in terms of hydrochloride). Next, the bacterial cells were removed from the culture solution 1, and the resulting solution was poured into an Amberlite (H ⁺ type) (manufactured by Rohm and Haus Co., Ltd.) column and washed with water. The adsorbed material was then eluted with 5% aqueous ammonia. The L-fornithine fraction was collected and concentrated under reduced pressure. Hydrochloric acid in an equimolar amount as L-ornithine was added to the obtained residue. The resulting crude crystals were recrystallized from aqueous methanol to obtain 27.5 g of L-ornithine hydrochloride. As a target, ATCC13232 instead of H-3796
L-ornithine was prepared in the same manner as above except for using
26.8 g/(in terms of hydrochloride) was obtained. Example 2 The same procedure as in Example 1 was carried out except that the strains shown in Table 4 were used instead of H-3796 as the inoculum. The results are shown in Table 4.

[Table] Example 3 H-3796 was used as the inoculum. The bacterial strain was cultured on a bouillon slant at 30°C for 24 hours. Add 1 platinum loop of the culture to 70g of blackstrap molasses/
(gluose equivalent), ammonium sulfate 30g/,
KH ₂ PO ₄ 0.5/, MgSO ₄ 0.125g/, Urea 3
g/, corn steep liquor 10g/, thiamine hydrochloride 150γ/, butyl citrate 3
ml/, L-Arginine 250mg/, CaCO ₃ 30
250 ml containing 20 ml of medium with a composition of
It was inoculated into an Erlenmeyer flask and cultured at 30°C for 48 hours.
27.5 g/(hydrochloride equivalent) of L-ornithine was accumulated in the culture solution. As a control, ATCC13232 instead of H-3796
L-ornithine was prepared in the same manner as above except for using
22.1 g/(in terms of hydrochloride) was obtained. Effects of the Invention A significant amount of L-ornithine can be obtained by the method of the present invention.

Claims (1)

[Claims] 1. A microorganism belonging to the genus Corynebacterium that is resistant to 2-thiazolylalanine, sulfaguanidine, or 2-fluoropyruvate and capable of producing L-ornithine is cultured in a medium, and L-ornithine is added to the culture solution. - A method for producing L-ornithine by a fermentation method, which comprises producing and accumulating ornithine and collecting the produced and accumulated L-ornithine.