KR20010089981A - 5'-Xanthylic acid producing microorganism - Google Patents

Abstract

PURPOSE: A microorganism TP400-35 (KFCC-11140) producing 5-xanthylic acid(XMP) is provided which is a mutant strain of Corynebacterium ammoniagenes (KFCC 10743) having thioprolin resistance and reinforced osmoresistance, thereby having superior productivity of 5-xanthylic acid than conventional strains. CONSTITUTION: A microorganism TP400-35 (KFCC-11140) producing 5-xanthylic acid(XMP) is obtained by treating Corynebacterium ammoniagenes (KFCC 10743) with mutagens such as, N-methyl-N-nitro-N-nitrosoguanidine(NTG) or UV rays, to have thioprolin resistance and osmoresistance. Therefore, it has improved productivity of 5-xanthylic acid. 5-xanthylic acid is an intermediate of biosynthesis of purine nucleotide and used as a source in manufacturing 5-guanylic acid.

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 special microorganism having resistance to thioprolin, which has enhanced osmotic resistance, thereby increasing 5'-xanthyl acid as compared to existing strains. The present invention relates to an improved microorganism.

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 increased the osmotic resistance because microorganisms are fermented in high concentrations of 5'-xanthyl acid and sugars, thereby producing a large amount of 5'-xanthyl acid. Thioproline, one of proline derivatives, was used in the present invention as a substance that enhances osmotic resistance (Lett. Appl. Microbiol., 222 , pp. 202-205, 1996). In other words, by giving resistance to thioproline, which enhances osmotic resistance, in order to increase resistance to high concentrations of extracellular sugar or 5'-xanthyl acid, 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 which greatly improved the productivity of 5'-xanthyl acid.

Microorganism TP400-35 producing 5'-xanthyl acid according to the present invention is irradiated with ultraviolet rays, N-methyl-N'-nitro-N-nitro, based on Corynebacterium ammoniagenes KFCC 10743. After treatment with a mutagenesis agent such as soguanidine (NTG) according to a conventional method, it is selected from among the mutants that can grow in the medium (3) added with thioproline by concentration. At this time, the thioproline concentration in the medium used in the experiment was used up to 500 mg / ℓ, grown at a thioproline concentration 400 mg / ℓ, strains with improved 5'-xanthyl acid concentration were selected, and the strain was TP400-35 It was named and deposited in the Korean spawn association (accession number KFCC-11140).

(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) Thioproline-added medium: (Note 2) Medium in which 100-500 mg / l of thioprol was added to the minimum medium.

Biochemical properties of the novel mutant strain TP400-35 isolated from the present invention are as described in Table 1.

Comparison of resistance to thioproline Thioproline concentration (mg / L) 0 100 200 250 300 350 400 500 Strain KFCC 10743 +++ +++ + - - - - - TP400-35 +++ +++ +++ +++ ++ + + - 5 days incubation at 30 ° C. +; Growth,-; Lack of growth

As shown in Table 1, it can be seen that the microorganism TP400-35 of the present invention is a strain capable of growing even in an azide salt addition medium having a concentration of 400 mg / L.

Example 1

Used strain; Microorganism TP400-35 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 18 mm diameter test tube, pressurized and sterilized according to a conventional method, and then inoculated with the used strain, followed by shaking culture at 30 ° C. for 25 hours at 150 rpm to use 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 21.0 g / l for the existing strain KFCC 10743, and the strain TP400-35 strain according to the present invention was 22.3 g / l with an increase of 6.2 (5'-xanthyl acid). accumulation levels were represented by 5'-sodium and xanthan tilsan 7H ₂ O).

Example 2

Used strain; Same as Example 1

Primary seed medium; Same as the primary 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 110 g / l for the conventional strain KFCC 10743 strain, and the modified strain TP400-35 of the present invention was 125 g / l with an improved 14 (accumulated concentration of 5'-xanthyl acid was 5 '-Sodium xanthate. 7 H ₂ O).

As described above, the mutant strain TP400-35 according to the present invention was found to exhibit 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 TP400-35, which produces 5'-xanthyl acid as a variant of Corynebacterium ammonia genes (Accession No. KFCC-11140). The method of claim 1, Microorganism TP400-35 grown at thioproline concentration 400 mg / l.