KR100402320B1 - A microorganism Corynebacterium ammoniagenes NV4-82-9 being capable of producing 5'-xanthylic acid in higher yield and a producing method of 5'-xanthylic acid by using the same - Google Patents

Abstract

The present invention is directed to UV irradiation, N-methyl-N`-nitro-N, based on Corynebacterium ammoniagenes KFCC-10743, which produces 5'-Xanthylic acid (XMP). Norvalin, an analogue to valine, which affects the biosynthesis of 5'-xanthyl acid by modifying the traits of the parent strain by treating a mutant inducer such as nitrosoguanidine (NTG) according to a conventional method ( norvaline) to screen the resistant strain, and as a result, the present invention relates to a microorganism that directly accumulates 5'-xanthyl acid in a culture medium with high yield and high concentration, and a method of producing 5'-xanthyl acid using the same.

Description

A microorganism Corynebacterium ammoniagenes NV4-82-9 being capable of producing microorganism Corynebacterium ammonia genes V4-82-9 producing 5'-xanthyl acid in high yield 5'-xanthylic acid in higher yield and a producing method of 5'-xanthylic acid by using the same}

The present invention relates to a microorganism producing 5'-xanthylic acid (XMP) and a method for producing 5'-xanthyl acid using the same, and more particularly, Corynebacterium ammonia genes KFCC-10743 is a mutant strain, which is a special microorganism having resistance to norvaline and relates to a microorganism having improved production capacity of 5'-xanthyl acid compared to the existing strain, and a 5'-xanthyl acid production method using the same.

5'-Xanthyl acid is an intermediate product of the Purine nucleotide biosynthetic metabolic system and is an important material for the preparation of 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, or a method for deamination of 5'-guanylic acid prepared by decomposing ribonucleic acid in yeast, and fermentation, for example, xanthine as a precursor in fermentation broth. Method of adding and producing microbial mutants, method of adding antibiotics (Japanese Patent No. 42-1477, cow 44-20390) and method of adding surfactant (Japanese Patent No. 42-3825, Small 42-3838) Etc. are known. Among these, since the method of directly producing fermentation of 5'-xanthyl acid by microbial mutants is industrially advantageous, the present inventors have improved the traits owned by the existing Corynebacterium ammonia genes (KFCC10743) to make xantyl acid (XMP). By giving these maximal traits, mutant strains with significantly increased productivity of xantyl acid (XMP) were developed.

The inventors of the present invention first have a certain effect on the production of 5'-xanthyl acid by direct fermentation of various amino acids for the purpose of increasing the 5'-xanthic acid production ability of Corynebacterium ammoniagenes KFCC-10743. The experiment was carried out to see if.

Experimental results show that 0.1-5 g / L of L-valine, L-glutamine, and L-serine, respectively, were added to 5'-xanthyl acid fermentation broth to increase the fermentation concentration of 5'-xanthyl acid. Confirmed. The proper supply of L-valine, which has the most remarkable effect, is of great importance in the production of 5'-xanthyl acid and confers resistance to norvaline, which acts as an analog of L-valine. Thus, the present invention was completed by discovering that strains resistant to norvaline can produce 5'-xanthyl acid in high concentration and high yield by the direct fermentation method.

Next, the microorganism isolation and acquisition method of the present invention will be described in more detail.

The microorganism NV4-82-9 of the present invention is directed to Corynebacterium ammoniagenes KFCC-10743, and is irradiated with ultraviolet rays, such as N-methyl-N`-nitro-N-nitrosoguanidine (NTG). After treatment with a mutagenic agent according to a conventional method, it was selected from among the mutants that can grow in medium (NOTE 3) to which novalin is added by concentration. At this time, the concentration of norvaline in the medium used in the experiment was used up to 8 g / l, strains with improved 5'- xanthyl acid concentration grown at 4 g / l in the norvalin concentration was selected, this strain was NV4-82-9 It was deposited as Accession No. KFCC-11248 as of December 15, 2000 to the Korean spawn association in Hongje-dong, Seodaemun-gu, Seoul, for the general sale to third parties.

(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.

(Note 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 thiamate, copper sulfate 0.8 mg / l, adenine 20 mg / l, guanine 20 mg / l, pH 7.2.

(Note 3) Norvalin added medium: (Note 2) Medium to which novalin 2-8 g / l was added to the minimum medium.

The biochemical properties of the novel mutant strain NV4-82-9 isolated from the present invention are as described in Table 1, and the microorganism of the present invention can be seen that the strain can be grown even in the addition of 4g / l norvalin medium.

The characteristics of the microorganism of the present invention are as described in Table 1 below.

Comparison of resistance to norvaline Norvalin concentration (g / l) 0 0.5 One 2 4 6 8 KFCC-10743 +++ +++ ++ + - - - NV4-82-9 +++ +++ +++ +++ ++ + -

(Note) +: growth; -Not grown, incubated for 5 days at 30 ° C.

Example 1

Strains used: Microorganisms NV4-82-9, KFCC-10743 of the present invention.

Species medium: glucose 30 g / l, peptone 15 g / l, yeast extract 15 g / l, sodium chloride 2.5 g / l, urea 3 g / l, adenine 150 mg / l, guanine 150 mg / l, pH 7.2.

Fermentation medium

① 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 5 mg / l, calcium chloride 10 mg / l, pH 7.2.

② starch broth: 10g / l potassium phosphate, 10g / l potassium phosphate, 7g / l urea, 5g / l ammonium sulfate.

Fermentation method

5 ml of the seed medium was dispensed into a 18 mm diameter test tube, autoclaved and sterilized according to a general method, followed by inoculation of the used strain, followed by shaking culture at 30 ° C. for 18 hours at 180 rpm to be used as a seed culture solution. In the fermentation broth, the main medium and the starlet medium were respectively sterilized by autoclaving according to the conventional method, and 29 ml and 10 ml were respectively dispensed into a 500 ml shake-type Erlenmeyer flask pre-sterilized, and 1 ml of the culture medium was inoculated and then incubated for 90 hours. The rotation speed was adjusted to 200 rpm and temperature 30 degreeC. After completion of the culture, the accumulation amount of 5'-xanthyl acid in the medium was 22.1 g / l for the existing strain KFCC-10743, and the variant strain NV4-82-9 of the present invention was 24.3 g / l improved by 10.4% (of 5'-xanthyl acid). accumulation levels are expressed as 5'xanthan tilsan sodium · 7H ₂ O).

Example 2

Strain used: same as Example 1.

Primary seed medium: the same as the primary seed medium of Example 1.

Secondary species: glucose 60 g / l, potassium dibasic phosphate 2g / l, potassium dibasic phosphate 2g / l, magnesium sulfate 1g / l, iron sulfate 22mg / l, zinc sulfate 15mg / l, manganese sulfate 10mg / l, Copper sulfate 1mg / l, calcium chloride 100mg / l, biotin 150ug / 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 2.4mg / l, alanine salt 22mg / l, NCA 7.5mg / l, biotin 0.4mg / l, magnesium sulfate 15g / l, cystine salt 30mg / l, histidine salt 30mg / l, calcium chloride 149mg / l, thiamine salt 15mg / l, Antifoam 0.7ml / l, CSL 27ml / l, Tuna Extract 6g / l, pH 7.3.

Primary seed culture: 50 ml of the primary seed culture medium was dispensed into a 500 ml Erlenmeyer flask for shaking, cooled by autoclaving at 121 ° C. for 20 minutes, inoculated with the used strain, and incubated with shaking at 30 ° C. for 24 hours at 180 rpm.

Secondary seed culture: Dispense the second seed medium into 5 liter experimental fermenter, 2 liters each, pressurize and sterilize at 121 ℃ for 10 minutes, cool, inoculate 50ml of the primary seed culture solution, and inflate 0.5vvm of air. Incubated at 900rpm, 31 ° C for 24 hours while feeding. The pH of the culture was adjusted to 7.3 with ammonia water.

Fermentation method

The fermentation broth was dispensed by 8 liters into a 30-liter experimental fermenter, pressurized and sterilized at 121 ° C. for 20 minutes, cooled and inoculated with 1.5 liters of the secondary seed culture solution, followed by incubation at 400 rpm and 33 ° C. while supplying air at 1vvm. However, when the residual sugar concentration during the culture was 1% or less, 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 90 hours. After culture completion, the accumulation amount of 5'-xanthyl acid in the medium was 121 g / l for the KFCC-10743 strain and the mutant strain NV4-82-9 of the present invention was 136 g / l, which was about 12.5% higher than the conventional strain (5'- Accumulated concentration of xanthyl acid was expressed as 5'-sodium xanthate 占 7H ₂ O).

The present invention relates to a microorganism producing 5'-xanthylic acid (XMP) and a method for producing 5'-xanthyl acid using the same. More specifically, Corynebacterium ammoniagenes KFCC-10743 is a special microorganism having a resistance to norvaline as a mutant KFCC-10743, and a microorganism having improved production capacity of 5'-xanthyl acid compared to the existing strain and The present invention relates to a method of producing 5'-xanthyl acid, by modifying a trait owned by the existing Corynebacterium ammonia genes KFCC-10743 and assigning a trait that can produce the maximum amount of xanthyl acid (XMP). XMP) has developed mutant strains with significantly increased productivity.

Claims (3)

Corynebacterium ammonia genes strain NV4-82-9 (KFCC-11248), which produces 5′-xanthyl acid (XMP) and is resistant to norvaline. The Corynebacterium ammonia genes strain NV4-82-9 (KFCC-11248) according to claim 1, characterized in that it is viable in the presence of trace amounts to 6 g / l of norvaline. Microbial Corynebacterium ammonia genes strain NV4-82-9 (FCC-11248) according to claim 1 was incubated for 24 hours at 30 ° C., 180 rpm, pH 7.3 in a primary seed medium, and then in a secondary seed medium. After activating by shaking culture for 24 hours at 31 ℃, 900rpm, pH 7.3, and shaking culture for 90 hours at 33 ℃, 400rpm in a fermentation medium, if the residual sugar concentration in the culture medium is less than 1%, added glucose added to the culture medium Method for producing 5'-xanthyl acid, characterized in that the total sugar content is added to be 30% culture.