KR920005749B1 - Method for producing l-arginine and new microorganism - Google Patents

Abstract

A L-arginine is prepd. by fermenting corynebacterium glutamicum MWEX-46 (KCTC 8485P) under aerobic condition at 25-40 deg.C and pH 5.5-8.0 for 1-5 days in the medium contg. 10 % glucose, 4.0 % ammonium sulfate, 0.3 % urea, 0.3 % KH2PO4, 0.1 % K2HPO4, 0.025 % MgSO4 7H2O and 50 μg/l biotin, and collecting from the fermented broth. The microorganism with argininehydroxamate tolerance and L-alanine requirement produces increased yields of L- arginine. The L-arginine is used as a feed additive, food additive or medicine.

Description

New strain Corynebacterium glutamicum MWEX-46 and preparation method of L-arginine using the same

The present invention relates to a novel microorganism belonging to the genus Corynebacterium and a method for producing L-arginine using the same.

L-arginine is a kind of natural amino acid that is widely used in medicine, food, and other animal feeds. It is used in liver function accelerator, brain disorder treatment, male infertility treatment, synthetic amino acid preparation, amino acid sap, etc. It is a substance that is spotlighted as a main raw material.

Conventionally known methods for producing L-arginine by microbial fermentation include a method of culturing Brevibacterium flavo strains lacking L-arginine diimase (Patent Office Publication No. 81-897), genus Brevibacterium or Cory A method of culturing strains that impart acetic acid requirement to a conventionally known L-arginine producing strain in Nebacterium (Japanese Patent Application Laid-Open No. 57-163487), and Bres having 2-thiazoalanine resistance and L-lysine requirement There is known a method of culturing microorganisms belonging to the genus Nonbacterium or Corynebacterium (Japanese Patent Publication No. 60-83593).

The present inventors have studied for the industrial production of L-arginine by fermentation method. As a strain belonging to Corynebacterium glutamicum, L-arginine production ability and resistance to arginine hydroxamate and at the same time by L-alanine it was confirmed that the invention: (May 22, 1900 deposited with the Korea Institute of Science and technology KCTC 8485P) ^* and this new strain is accumulated in the medium to large amounts of L- arginine promotes growth of new strains MWEX-46.

The microorganism used in the present invention is based on the wild strain (ATCC 13032) of L-glutamic acid producing bacteria belonging to Corynebacterium glutamicum and UV irradiation or NTG (N-methyl-N'-nitro-N-nitroso guanidine) Strain MWE-1010 having a production capacity of L-arginine obtained using a conventional mutagenic treatment method as described above was used as a parent strain. After giving arginine hydroxamate resistance to this parent strain, strain MWEX-46 (KCTC 8485P) ^* of the present invention which promoted growth by addition of L-alanine was isolated.

It is not clear about the mechanism by which the strain of the present invention accumulates a large amount of L-arginine in the medium layer, but L-acetylglutamate kinase activity, which is an enzyme in the L-arginine biosynthesis step, was enhanced compared to the parent strain and the final product L- Not only does not inhibit or suppress feedback by arginine, but also inhibits the production of L-alanine, thereby maintaining the balance of L-aspartic acid and L-citrulline for L-arginine biosynthesis, thereby producing L-arginine. It is assumed that this is promoted.

When the present invention is described in more detail, the strain MWE-1010 secreted L-arginine in the medium and the L-arginine degrading ability was secreted among strains whose growth was inhibited by the addition of arginine hydroxyxamate. The parent strain was further mutated to select strains resistant to arginine hydroxyxamate 2 mg / ml, and then resistant strain MWE-4125 strain obtained by increasing the production of L-arginine was obtained by culturing the flask. The resistant mutants obtained in this way were not suitable for industrial use because L-citrulline was by-produced in the culture medium.

In order to prevent L-citrulline by-products of resistant strains, a single colony was isolated and mutated. As a result, the strain MWEX-46 (KCTC 8485P) ^* , which promotes growth by the addition of L-alanine, was introduced into the medium. The present invention was completed by not only accumulating high concentrations but also byproducts of L-citrulline.

In the present invention, for example, a method of separation of mutant strains, the cells cultured at 30 ° C. for 24 hours in a complete slope medium were collected by centrifugation or filtration, and then washed twice with 0.05M TM (Tris-Malate) buffer (pH 6.2). The cell was diluted to 10 ⁸ -10 ⁹ / ml with the same buffer, and then NTG was added to 250μg / ml and treated for 30 minutes at 30 ℃. After the NTG treatment was completed, the cells were collected by centrifugation or filtration, washed twice with physiological saline, and then plated in a medium to which arginine hydroxamate was added, and then cultured at 30 ° C. for 3-5 days to isolate mutants.

The composition of the complete medium and the minimum medium used in the present invention is as follows.

(Note 1). Complete medium: glucose 1.0%, broth 0.5%, enzyme extract 0.5%, peptone 1.0%, salt 0.25%, agar 1.5-2.0%, pH7.2.

(Note 2). Minimum medium: glucose 0.5%, ammonium sulfate 0.15%, urea 0.15%, potassium monophosphate 0.1%, dipotassium phosphate 0.3%, magnesium sulfate monohydrate 0.01%, calcium chloride dihydrate 1mg / ℓ, biotin 100㎍ / ℓ , Chiamine Hydrochloride 100㎍ / ℓ, Trace Urea ^* 1mg / ℓ, Agar 1-2%

^* Trace elements: 2 cups of sulfur wire 270mg / ℓ, zinc sulfate 10mg / ℓ, ferrous chloride 870mg / ℓ, manganese chloride 7.2mg / ℓ, sodium borate 88mg / ℓ, ammonium molybdate 40mg / ℓ

The result of comparing the strain of the present invention with the parent strain MWE-1010 and the resistant strain MWE-4125 is as follows.

TABLE 1

TABLE 2

In the present invention, the L-arginine analysis method in the culture solution is a variation of the Sakaguchi method, 0.5ml of the diluted solution so that the L-arginine concentration in the culture medium is 50-100μg / ml mixed with 2.5ml 0.43N caustic soda solution and A solution ( 1.0 ml of a solution containing 5.0 g of 8-hydroxyquinoline and 2.5 ml of a 1% aqueous solution of diacecyl) in 100 ml of n-propanol was added, mixed, and reacted at 25 ° C. for 1 hour, and the absorbance at 520 nm was measured. Separately, the standard curve of the absorbance value obtained using the L-arginine standard and the absorbance value obtained using the culture solution were compared to calculate the L-arginine concentration in the culture solution.

In the method for producing L-arginine using the microorganism of the present invention, L-arginine accumulated in the medium was recovered by culturing in a medium containing a carbon source, nitrogen source, minerals and other nutrients used in normal fermentation.

As the main carbon source used in the present invention, sugars such as glucose, sugar, starch hydrolyzate, and fructose can be used alone or in combination. As a nitrogen source, ammonium sulfate, urea, ammonia gas, peptone, gravy, yeast extract, CSL, Casein hydrolyzate, defatted soybean meal hydrolyzate and the like. Potassium phosphate, magnesium sulfate, salt, ferrous sulfate, and manganese sulfate may be used as the inorganic substance, and as a nutrient, an appropriate amount of a nutrient required for microbial growth may be added to the medium.

The culture is incubated for 1-5 days in the range of 25 ℃ -40 ℃ pH5.5-8.0 under normal aerobic conditions. The pH control during the cultivation is organic or inorganic alkaline substance urea, ammonia gas, ammonia water, calcium carbonate, etc. Use As a method for recovering L-arginine from the culture completion solution, a conventional method using an ion exchange resin or the like is used.

The present invention will be described in detail with reference to the following Examples.

Example 1

Strain medium (pH 7.2) containing broth 0.5%, yeast extract 0.5%, peptone 1.0%, salt 0.25%, and agar 2.0% was inoculated with the strain MWEX-46 of the present invention and incubated at 28 ° C for 24 hours, followed by seed medium. Was inoculated. Seed medium was prepared by using a medium containing 5% glucose, 1.0% yeast extract, 0.3% urea, 0.3% C3L, 0.5% peptone, 1.0% salt, 0.25% biotin, and 50 μg / ml biotin to pH 7.2. After the adjustment, 50 ml was dispensed into a 500 ml shake flask and sterilized at 121 ° C. for 20 minutes. Platinum was inoculated in the slope medium and then incubated for 24 hours at 30 ℃ inoculated in fermentation medium. The fermentation broth is a 5N-caustic medium containing 10% glucose, 4.0% ammonium sulfate, 0.3% urea, 0.1% potassium monophosphate, 0.1% dibasic potassium phosphate, 0.025% magnesium sulfate hexahydrate, and 50 μg / l biotin. After adjusting to pH 7.2 with a soda solution, 30 ml was dispensed into 500 ml shake flasks, autoclaved at 121 ° C. for 20 minutes, and separately sterilized calcium carbonate was added to 3.0%. 1 ml from the seed medium was inoculated into the fermentation medium and then reciprocated shaking culture at 30 ℃. In the same manner, the parent strain and the mutant strain were cultured to investigate the production of L-arginine in the culture, and the results as shown in Table 3 were obtained.

TABLE 3

Example 2

After culturing in the same manner as in Example 1, 1 ml of the seed culture solution was inoculated in a secondary seed medium and incubated at 30 ° C. for 24 hours. The secondary seed medium was 5.0% of molasses (in terms of glucose), 4% of defatted soybean hydrolyzate, 0.5% of CSL, 0.05% of potassium monophosphate, 0.05% of potassium diphosphate, and 0.025% of magnesium sulfate. Ferrous 7 hydrochloride 10 mg / l, manganese sulfate 4 hydrochloride 10 mg / l, bio-value 50μg / l, chlorine hydrochloride 100μg / l, medium containing 0.3% urea was adjusted to pH 7.2 with 5N-caustic soda solution The solution was dispensed into a 100 ml shake flask and sterilized at 121 ° C. for 20 minutes. As fermentation medium, waste molasses (in terms of glucose) 15.0%, ammonium sulfate 1.0%, potassium monophosphate 0.1%, dibasic potassium phosphate 0.1%, magnesium sulfate.7 hydrochloride 0.05%, biotin 100mg / l, chiamin hydrochloride 100mg / ℓ, solubilized 2 liter of medium into a 5 L fermenter, sterilized at 121 ° C. for 20 minutes, adjusted to pH 7.0 with ammonia, inoculated with secondary culture and incubated at 30 ° C. at a stirring rate of 500 rpm and 1 vm. As a result of culturing the parent strain and resistant strains by the same method to examine the amount of L-arginine produced, the results shown in Table 4 were obtained.

TABLE 4

The fermentation broth cultured by the above method was centrifuged to remove the cells, the supernatant was passed through a strong acidic ion exchange resin, and the resin was eluted with 2N-caustic soda solution after washing with water. The eluate was decolorized and concentrated to obtain L-arginine crystals, which were compared with the standard product. The result was pure L-arginine, and the recovery yield was 91.5%.

Claims (3)

Corynebacterium glutamicum MWEX-46, which has the ability to produce L-arginine, is resistant to arginine hydroxamate, and is promoted by L-alanine. A method for producing L-arginine, which is obtained by culturing Corynebacterium glutamicum MWEX-46 in a nutrient medium. The method of claim 2, wherein the culturing is carried out for 1 to 5 days under aerobic conditions in the range of 25 ℃ to 40 ℃, pH5.5 ~ 8.0 for the production of L-arginine.