KR100671080B1 - Microorganism producing inosine and process of preparing 5'-inosinic acid using same - Google Patents

Abstract

A novel microorganism producing inosine is provided to be able to produce the inosine with high concentration and high yield through direct fermentation, and show resistance against L-thiazolidine-4-carboxylic acid. The Corynebacterium ammoniagenes CJTP-17 has the resistance against L-thiazolidine-4-carboxylic acid, produces inosine and is deposited as a deposition number of KCCM-10702P. To produce the inosine, the Corynebacterium ammoniagenes CJTP-17 is cultured and then the inosine is isolated from the culture solution.

Description

Microorganism producing inosine and process of preparing 5'-inosinic acid using same}

The present invention relates to a microorganism producing inosine and a method for producing inosine using the same. More specifically, the present invention is a variant of Corynebacterium ammoniagenes CJMPA-122 (KFCC-11136), Corynebacterium ammonia genes which produces inosine at high concentration and high yield by direct fermentation. CJTP-17 (KCCM-10702P) and a method for producing inosine at high concentration and high yield using such microorganisms

Inosine is an important substrate for the chemical synthesis of 5'-inosinic acid, which is spotlighted as a seasoning seasoning, and the synthesis by enzyme transfer reaction. Inosine is an intermediate of the nucleic acid biosynthesis metabolic system and has a physiological significance in the body of animals and plants, and is used in various fields such as food, medicine, and various medical uses. In order to produce such inosine, Bacillus (Agric. Biol. Chem., 46, 2347 (1982); Korean Patent No. 27280) or Corynebacterium ammonia genes (Agric. Biol. Chem., 42, 399 (1978)) and the direct fermentation method using a microorganism, such as pyrolysis of 5'-inosinic acid (Japanese Patent Application No. 43-3320). However, in the case of pyrolysis, a large amount of heat is required to decompose 5′-inosinic acid, so that practicality is a problem, and the production method by direct fermentation has a low titer of inosine production strain, resulting in high production cost and a long time for fermentation. The disadvantage was low productivity.

Therefore, various methods for overcoming the disadvantages of the direct fermentation method and producing inosine at high concentration / high yield have been studied. As one of these methods, development of a strain capable of accumulating high concentration of inosine in a culture solution is required.

Therefore, the present inventors have carried out continuous research to produce inosine at high concentration / high yield by direct fermentation using microorganisms, and as a result, the present invention has been developed by developing microorganisms producing inosine at higher concentration and higher yield by direct fermentation. Was completed.

The present inventors have found that the strain from the above-mentioned microorganism Corynebacterium ammonia genes CJMPA-122 (KFCC-11136), more specifically microorganisms that have given resistance to thiaproline, one of the proline derivatives, by direct fermentation It has been found that inosine can be produced at a higher concentration and higher yield than in the prior art, thereby completing the present invention.

Accordingly, it is an object of the present invention to provide microorganisms which produce inosine in high concentration and high yield by direct fermentation, and which are resistant to thiaproline.

Still another object of the present invention is to provide a method of culturing the microorganisms to directly accumulate inosine in a culture solution at high concentration and high yield.

In one embodiment, the present invention relates to Corynebacterium ammoniagenes CJTP-17 (KCCM-10702P), which is resistant to thiaproline and produces inosine at high concentrations and in high yield.

The microorganism Corynebacterium ammoniagenes CJTP-17 (KCCM-10702P) strain of the present invention is a mutant strain of Corynebacterium ammonia genes CJMPA-122 (KFCC-11136). Corynebacterium ammonia genes CJMPA-122 (KFCC-11136) used as a parent strain in the present invention is a strain producing inosine disclosed in Korean Patent No. 10-0330705.

Therefore, the microorganism according to the present invention shares the following main characteristics and effects of Corynebacterium ammonia genes CJMPA-122 (KFCC-11136). In other words,

i) adenine and biotin requirement properties;

ii) the properties of xanthine and guanine leaky mutants which do not require xanthine and guanine but which promote their growth upon addition thereof;

iii) urease defect properties capable of magnetizing urea;

iv) The cell wall synthesis ability is partially deficient in the cell lysozyme lysozyme having a high susceptibility of 8 ㎍ / ㎖ minimum inhibitory concentration to secrete a large amount of inosine produced in the cell well outside the cell Characteristics;

v) Cell growth is slowed down by the increase in osmotic pressure outside the cells by the high concentration of glucose and various carbon sources added during the culturing process, or by the inosine accumulating in the culture medium later in the culture, thereby inhibiting normal physiological activity of the inosine producing cells. The ability to grow at 3500 μg / ml of dihydroproline (3,4-dihydro-DL-proline) endowed with an osmotic resistance trait, which can prevent inhibition of inosine production;

vi) the ability to grow in 150 µg / ml of azaserrine, an analog of glutamine (L-glutamine), which is essential for purine-based biosynthesis systems;

vii) the ability to grow at 600 μg / ml of guanine analog, mercaptoguanine;

viii) the ability to grow at 1200 μg / ml of the purine analogue of 6-mercaptopurine; And

ix) It has the ability to grow at 300µg / ml of azadenine (8-azaadenine).

In addition, Corynebacterium ammonia genes CJTP-17 (KCCM-10702P) according to the present invention in addition to the above properties, in addition to the resistance to thiaproline (thiaproline) inosine in a high concentration and high yield in the culture medium Can be accumulated directly.

As mentioned in the detailed description and examples of the present invention, the nutrient medium (denoted as' Note 1 '), the minimal medium (denoted as' Note 2'), the seed medium (' Note 3 '), flask fermentation medium (marked as' Note 4'), fermenter seed medium (marked as' Note 5 '), and fermenter main medium (marked as' Note 6'), respectively, as shown in Table 1 It means a medium consisting of the composition. However, the medium composition is not limited to those shown in Table 1, and any culture medium suitable for the culture of Corynebacterium ammonia genes can be used.

In order to obtain the microorganism according to the present invention, the normal physiological activity of the microorganism is inhibited by the osmotic pressure caused by the high concentration of glucose in the culture medium and other various carbon sources or the osmotic pressure caused by the accumulation of the inosine produced during the culture. In order to prevent the slowing of growth and consequently the decrease of inosine production, a similar role of proline is used to increase the intracellular concentration of proline (L-proline) that can play an important role in the control of osmotic pressure and confer osmotic resistance traits. The traits of existing strains were modified to enhance properties not affected by osmotic pressure by increasing resistance to chain thiaproline, and among these strains, mutants with new traits with improved inosine productivity were finally selected.

As a specific embodiment for obtaining the microorganism according to the present invention, Corynebacterium ammonia genes CJMPA-122 (KFCC-11136) is X-ray, ultraviolet or N-methyl-N-nitro-N-nitrosoguanine (N- Chemical mutagenic agents such as methyl-N-nitro-N-nitrosoguanine, diethylsulfide and ethylamine were treated. The treated strain was then diluted appropriately in a medium in which thiaproline concentration was added to 50 to 300 µg / ml in a minimum medium (Note 2), followed by spreading to obtain colonies. Each colony obtained according to the thiaproline concentration was cultured in nutrient medium (Note 1), incubated in seed medium (Note 3) for 24 hours, and then cultured in fermentation medium (Note 4) for 5 to 6 days, and accumulated in fermentation broth. The strains with the highest yield of inosine production were selected.

These strains were named Corynebacterium ammoniagenes CJTP-17, and they were assigned to the Korean Culture Center of Microorganisms (KCCM) in Hongje 1-dong, Seodaemun-gu, Seoul under the Budapest Treaty. Deposited on day 11 with accession no. KCCM-10702P.

Biochemical properties for the new variant strain CJTP-17 (KCCM-10702P) selected according to the present invention are listed in Table 2 compared to Corynebacterium ammoniagenes CJMPA-122 (KFCC-11136).

In another aspect, the present invention relates to a method of culturing Corynebacterium ammonia genes CJTP-17 (KCCM-10702P), and accumulating inosine at high concentration and high yield in the culture.

According to the present invention, the Corynebacterium ammonia genes CJTP-17 (KCCM-10702P) strain is cultured in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins, etc. while controlling the temperature, pH, etc. under aerobic conditions do.

Carbohydrates such as glucose, sterilized pretreated molasses (ie, converted molasses to reduced sugar) may be used as the carbon source, and various inorganic nitrogen sources such as ammonia, ammonium chloride, and ammonium sulfate and peptone, NZ-amine, and meat may be used as the nitrogen source. Organic nitrogen sources such as extracts, yeast extracts, corn steep liquors, casein hydrolysates, fish or degradation products thereof, skim soy cakes or degradation products thereof can be used.

As the inorganic compound, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like may be used. In addition, vitamins and nutrient-containing bases may be added as necessary.

The culturing is carried out under aerobic conditions, for example by shaking culture or aeration stirred culture, preferably at a temperature of 28 to 36 ° C. The pH of the medium is preferably maintained near neutral during the culture. Incubation was continued for 5-6 days, and inosine accumulated by direct fermentation was analyzed by conventional methods using HPLC.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated concretely based on an Example. However, it does not mean that the scope of the present invention is limited by the embodiment in any form.

Example 1 Screening of Thiaproline Resistant CJTP-17 Strains

Corynebacterium ammonia genes CJMPA-122 (KFCC-11136) was suspended in phosphate buffer (pH 7.0) or citrate buffer (pH 5.5) at 10 ^{7-10 8} cells / ml, followed by N-methyl-N- Nitro-N-nitrosoguanine was added to a final concentration of 10-50 μg / ml and treated at room temperature or 30-32 ° C. for 40-90 minutes to induce mutations. The strains obtained after centrifugation were washed three times with 0.85% saline, and then 50 μg / ml, 100 μg / ml, 150 μg / ml, 200 μg / ml, 250 μg / ml and 300 μg of thiaproline, respectively. After appropriate dilution and spreading in a minimum medium (Note 2) added to the / ml concentration, colonies were grown while incubating for 4 to 5 days at a temperature of 30 to 34 ℃. In the present invention, the CJTP-17 strain was found to be a viable bacterium even in the addition of 200µg / ml thiaproline. Table 3 shows the degree of resistance to thiaproline between the strain CJMPA-122 (KFCC-11136) and the new strain CJTP-17 (KCCM-10702P), the strain of the microorganism according to the present invention.

Example 2: Erlenmeyer flask fermentation titer experiment

3 ml of seed medium (Table 1, Note 3) was dispensed into a 18 mm diameter test tube and autoclaved at 120 ° C. for 10 minutes, followed by strain Corynebacterium ammonia genes CJMPA-122 (KFCC-11136) and 200 μg / ml thiaproline. Each of several strains resistant to the strain was inoculated and shaken at 30 ° C. for 24 hours to use as a culture medium. 27 ml of the fermentation broth was dispensed into a 500 ml shake flask for 3 minutes, autoclaved at 120 ° C. for 10 minutes, and then inoculated with 3 ml of the seed culture solution, followed by incubation for 5 to 6 days. The rotation speed was adjusted to 200 times per minute at a temperature of 32 ° C. and pH 7.2. As a result of culture, inosine accumulation in the medium was 9.5 g / L for the parent strain CJMPA-122 (KFCC-11136), and the production of one of the resistant strains was higher than that of the parent strain. After culturing this strain in a nutrient medium (Note 1), diluting to an appropriate concentration in a solid medium, pure separation to obtain a single cell, dozens of colonies were cultured in a seed medium (Note 3) for 24 hours After incubation for 4 to 5 days in fermentation broth 4, CJTP-17 producing 10.5 g / L of inosine, which had the highest amount of inosine accumulated in the fermentation broth, could be selected. As a result, the strain showed a 10% increase in productivity relative to the parent strain.

Example 3 Fermentation Titer Test in 5-L Fermenter

50 ml of seed medium (Note 3) was dispensed into a 500 ml shake flask for 10 minutes, and autoclaved at 120 ° C for 10 minutes, followed by Corynebacterium ammonia genes CJMPA-122 (KFCC-11136) strain and CJTP-17 strain. Each was inoculated and shaken at 30 ° C. for 24 hours to use as a culture medium. Fermenter seed medium (Note 5) was placed in a 2.5L-fermentation tank and autoclaved at 120 ° C. for 15 minutes, inoculated with 50 ml of seed culture solution, and then incubated for 1-2 days. Rotation speed was adjusted to 900 times per minute, temperature 28 to 34 ℃, pH 7.2. In addition, 1250 ml of the fermenter main medium (Note 6) was placed in a 5 L-fermentation tank and autoclaved at 15 ° C. for 15 minutes, followed by incubation with 250 ml of the fermenter broth culture solution, when the residual reducing sugar in the culture became 2%. According to the production method of this medium (Note 6), additional sugar made by mixing fructose, glucose, and molasses is added as 32% of the final reducing sugar, respectively. Incubate 5-6 days with repeated additions. Rotation speed was adjusted to 900 times per minute, temperature 30 ℃, pH 7.2. As a result of fermentation, the culture of parent strain CJMPA-122 (KFCC-11136) produced 34.5 g / L of inosine, and the culture of CJTP-17 strain produced 38.5 g / L of inosine, yielding about 12% productivity. An improvement was shown.

When producing inosine using the Corynebacterium ammonia genes CJTP-17 (KCCM-10702P) strain according to the present invention, it can be produced in high concentration and high yield compared to the existing strain by direct fermentation method. In addition, the inosine acid can be economically produced at a higher concentration / high yield than the direct fermentation method through the enzyme transfer reaction from the inosine produced by the strain.

Claims (2)

Corynebacterium ammonia genes CJTP-17 (KCCM-10702P) that is resistant to thiaproline and produces inosine. A method of culturing the microorganism of claim 1 and producing inosine from the culture solution.