KR20010089979A - 5'-Xanthylic acid producing microorganism - Google Patents

Abstract

PURPOSE: Provided is a mutant microorganism B1045 with superior productability of 5-xanthine acid(XMP) than a parent strain, Corynebacterium ammoniagenes. The5-XMP is an important intermediate of biosynthesis of purine nucleotide and used for the production of 5-guanylic acid(GMP). CONSTITUTION: The mutant microorganism B1045 (KFCC-11138) is prepared by treating Corynebacterium ammoniagenes (KFCC 10743) as a parent strain with mutagens such as UV light, N-methyl-N-nitro-N-nitrosoguanidine(NTG), in usual manner. It has resistance to Bacitracin which inhibits the synthesis of cell wall by diphosphorylating of lipid pyrophosphate, and is thus viable in a medium added with Bacitracin in a concentration of 5mg/l.

Description

5'-Xanthylic acid producing microorganism

The present invention relates to the microorganisms themselves producing 5'-xanthyl acid (XMP). More specifically, the present invention is a variant of Corynebacterium ammoniagenes KFCC 10743, which is a microorganism having baccitracin resistance that inhibits cell wall synthesis by inhibiting dephosphorylation of Lipid pyrophosphate. As relates to a microorganism with improved production capacity of 5'-xanthyl acid as compared to the existing strain.

5'-Xanthyl acid is an intermediate product of the purine nucleotide biosynthetic metabolic system and is an important material for manufacturing 5'-guanylic acid (GMP). The most widely used method of producing 5'-guanylic acid with high taste and high commercial value is microbial fermentation, which produces 5'-xanthyl acid and converts it enzymatically to 5'-guanylic acid. Economically, 5'-xanthyl acid is needed as much as 5'-guanylic acid needs. Conventional methods for preparing 5'-xanthyl acid include chemical synthesis, deamination of 5'-guanylic acid prepared by decomposing ribonucleic acid in yeast, or fermentation, and fermentation includes xanthine (A) as a precursor in fermentation broth. Method of adding Xanthine), preparation by microbial mutant strain, preparation by addition of antibiotics (Japanese Patent No. 42-1477, So44-20390) and preparation by addition of surfactant (Japanese Patent So42-3825, So42- 3838) and the like. Among these, since the direct fermentation method for producing 5'-xanthyl acid by microbial mutant strain is industrially advantageous, the present inventors have improved the traits possessed by the existing Corynebacterium ammonia genes (KFCC 10743) to improve the 5'-k By assigning traits that can produce the highest amount of acid, acidic strains with significantly increased productivity of 5'-xanthyl acid were developed.

Therefore, the present inventors have changed the cell wall synthesis to facilitate the extracellular secretion of microorganisms when the microorganism accumulates intracellular 5'-xanthic acid, thereby producing a large amount of 5'-xanthyl acid. In other words, by imparting resistance to bacitracin that inhibits the dephosphorylation of phospholipids and inhibiting microbial cell wall synthesis, thereby improving cell wall permeability, new strains are given to Corynebacterium ammonia genes KFCC 10743 to retain the conventional strain. The present invention was completed by developing a mutant strain that greatly improved the productivity of 5'-xanthyl acid.

Microorganism B1045 producing 5'-xanthyl acid according to the present invention is irradiated with ultraviolet rays, N-methyl-N'-nitro-N-nitrosoguanidine, based on Corynebacterium ammoniagenes KFCC 10743 as a parent. After treatment according to a conventional method with a mutagenesis agent such as (NTG), it is selected from the mutant strains that can grow in the medium (3) added to the concentration by the bacitracin. At this time, the concentration of bacitracin in the medium used in the experiment was used up to 20 mg / l, and the strain was grown at 5 mg / l of bascitracin, and the strain with improved 5'-xanthyl acid concentration was selected. It was named and deposited with the Korean spawn association (accession number KFCC-11138).

(Note 1) Nutritional medium: Glucose 20g / ℓ, Peptone 10g / ℓ, Yeast extract 10g / ℓ, Sodium chloride 2.5g / ℓ, Urea 3g / ℓ, Adenine 150mg / ℓ, Guanine 150mg / ℓ, pH 7.2

(2) Minimum medium: 20 g / l glucose, 1 g / l potassium phosphate, 1 g / l potassium diphosphate, 2 g / l urea, 3 g / l ammonium sulfate, 1 g / l magnesium sulfate, 100 mg / l calcium chloride , 20 mg / l iron sulfate, 10 mg / l manganese sulfate, 10 mg / l zinc sulfate, 30 μg / l biotin, 0.1 mg / l thiamine salt, 0.8 mg / l copper sulfate, 20 mg / l adenine, 20 mg guanine / l, pH 7.2

(Note 3) Bacitracin-added medium: (Note 2) Medium in which 1 to 10 mg / l of bacitracin was added to the minimum medium.

Biochemical properties of the novel mutant strain B1045 isolated from the present invention are as described in Table 1.

Comparison of Resistance to Bacitracin Bacitracin Concentration (mg / L) 0 0.1 0.2 0.5 1.0 2.0 5.0 10.0 Strain KFCC 10743 +++ +++ ++ + ± - - - B1045 +++ +++ +++ +++ ++ + + - 5 days incubation at 30 ° C. +; Growth,-; Lack of growth

As shown in Table 1, it can be seen that the microorganism B1045 of the present invention is a strain that can be grown even in the addition of bacitracin at a concentration of 5 mg / L.

Example 1

Used strain; Microorganism B1045 of the present invention, KFCC 10743

Species medium; Glucose 30g / l, Peptone 15g / l, Yeast extract 15g / l, Sodium chloride 2.5g / l, Urea 3g / l, Adenine 150mg / l, Guanine 150mg / l, pH 7.2

Fermentation medium; (1) the main medium; Glucose 60g / l, magnesium sulfate 10g / l, iron sulfate 20mg / l, zinc sulfate 10mg / l, manganese sulfate 10mg / l, adenine 30mg / l, guanine 30mg / l, biotin 100µg / l, Copper sulfate 1mg / l, thiamine hydrochloride 5mg / l, calcium chloride 10mg / l, pH 7.2

(2) stellar badge; 10 g / l potassium phosphate, 10 g / l potassium diphosphate, 7 g / l urea, 5 g / l ammonium sulfate

Fermentation method; 5 ml of the seed medium was dispensed into a test tube with a diameter of 18 mm, pressurized and sterilized according to a conventional method, and then inoculated with the strain used, followed by shaking culture at 30 ° C. for 24 hours at 150 rpm to be used as a seed culture solution. In the fermentation broth, the main and stellate broth were sterilized according to the conventional method, and 20 ml and 7 ml were respectively dispensed into a 250 ml shake-type Erlenmeyer flask, which had been autoclaved in advance, and 3 ml of the culture medium was inoculated and incubated for 60 to 70 hours. . The rotation speed was adjusted to 170 rpm and the temperature of 30 degreeC. After completion of the culture, the accumulation amount of 5'-xanthyl acid in the medium was 17.8 g / l for the existing strain KFCC 10743, and the strain B1045 strain according to the present invention was 19.3 g / l with 8 increase (accumulated concentration of 5'-xanthyl acid). Is represented by 5'-sodium xanthate. 7H ₂ O).

Example 2

Used strain; Same as Example 1

Primary seed medium; Same as the seed medium of Example 1

Secondary species; Glucose 60g / l, Potassium phosphate 2g / l, Dipotassium phosphate 2g / l, Magnesium sulfate 1g / l, Iron sulfate 22mg / l, Zinc sulfate 15mg / l, Manganese sulfate 10mg / l, Copper sulfate 1 Mg / l, calcium chloride 100mg / l, biotin 150µg / l, adenine 150mg / l, guanine 150mg / l, thiamine salt 5mg / l, antifoam 0.6ml / l, pH 7.2

Fermentation medium; Glucose 151g / l, Phosphoric acid 32g / l, Potassium hydroxide 25g / l, Adenine 198mg / l, Guanine 119mg / l, Iron sulfate 60mg / l, Zinc sulfate 42mg / l, Manganese sulfate 15mg / l, Copper sulfate 2.4 mg / l, alanine salt 22 mg / l, NCA 7.5 mg / l, biotin 0.4 mg / l, magnesium sulfate 15 g / l, cystine salt 30 mg / l, histidine salt 30 mg / l, calcium chloride 149 mg / l, Thiamine salt 15 mg / l, defoaming agent 0.7 ml / l, CSL 27 ml / l, tuna extract 6 g / l, pH 7.3

Primary seed culture; 50 ml of the primary seed culture medium was dispensed into a 500 ml shaking Erlenmeyer flask, which was autoclaved and cooled at 121 ° C. for 20 minutes, and then inoculated with the used strain, followed by incubation for 24 hours at 30 ° C. at 150 rpm.

Secondary species culture; After dispensing the secondary seed medium into a 5 liter experimental fermenter, each 2 liters, sterilized under pressure at 121 ° C. for 20 minutes, cooled, and inoculated with 50 ml of the culture medium of the primary seed culture medium, while supplying air at 0.5vvm. Shake culture was performed for 24 hours at 900rpm, 33 ℃. The pH of the culture was adjusted to 7.3 with ammonia water.

Fermentation method; The fermentation broth was dispensed by 8 l into a 30-l experimental fermenter, pressurized and sterilized at 121 ° C. for 20 minutes, cooled and inoculated with 1.5 l of the secondary seed culture solution, followed by culturing at 400 rpm and 33 ° C. while supplying air at 1 vvm. When the residual sugar concentration during the culture was less than 1, sterilized glucose was supplied to adjust the total sugar added to the fermentation medium to 30. The pH of the culture was adjusted to 7.3 with ammonia water and incubated for 70 hours. After completion of the culture, the accumulation amount of 5'-xanthyl acid in the medium was 109 g / l for the conventional strain KFCC 10743 strain, and the modified strain B1045 of the present invention was 121 g / l with 11 enhancement (accumulated concentration of 5'-xanthyl acid was 5'- Sodium xanthate, 7H ₂ O).

As described above, the mutant strain B1045 according to the present invention was found to show an increased productivity of about 10 compared to the conventional strain KFCC 10743 in the production of 5'-xanthinic acid.

Therefore, according to the present invention, there is an effect of providing a microorganism which is expected to increase the productivity of 5'- xanthic acid.

Although the present invention has been described in detail only with respect to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the technical scope of the present invention, and such modifications and modifications are within the scope of the appended claims.

Claims (2)

Microorganism B1045, which produces 5'-xanthyl acid as a variant of Corynebacterium ammonia genes (Accession No. KFCC-11138). The method of claim 1, Microorganism B1045 grown at Bacitracin concentration 5 mg / L.