KR100424847B1 - 5'-inosinic acid producing microorganism and process for production thereof - Google Patents

Abstract

The present invention is a parent strain of the well-known patent strain Brevibacterium ammonia genes CS1019753 (KFCC-10814) and mutated to give azaserine resistance, and then cultured by azacerin resistant mutant Brevibacterium ammonia genes CIAZ109 ( KFCC-11068), and then compared the azaserine resistance of the parent strain and the mutant strain of the present invention, and fermentation culture of the Brevibacterium ammonia genes CIAZ109 of the present invention can produce high concentration, high yield of 5'-inosinic acid. Excellent effect.

Description

5'-inosinic acid producing microorganism and process for production

The present invention relates to a microorganism producing 5'-inosinic acid and a method of producing the same. More specifically, the azaserine resistant strain capable of producing 5'-inosinic acid at high concentration and high yield by direct fermentation culture after granting azaserine resistance to the known patent strain KFCC-10814 and the 5'-inosinic acid using the strain It relates to a production method.

5'-inosinic acid is an intermediate of the nucleic acid biosynthetic metabolic system, which not only has physiological significance in animals and plants, but is also widely used in food, medicine, and medicine. Especially, when used together with sodium glutamate, the synergistic effect is great. It is one of nucleic acid-based seasonings that has been spotlighted as a seasoning seasoning.

Synthesis of 5'-inosinic acid from PRPP (5-phosphoribosyl-α-1-pyrophosphate), a precursor of purine-based nucleic acids, requires two molecules of glutamine, and glutamine synthetase, an enzyme that synthesizes glutamine from sodium glutamate Since glutamine synthetase is highly precisely regulated by amino acids such as glycine, alanine, histidine and CTP, AMP, etc. (Escherichia coli and Salmonella typhimurium, 1987, p302-320), it is possible to supply glutamine in synthesizing 5'-inosinic acid. Is essential. In addition, the produced glutamine is very much used as a precursor of various reactions. In the synthesis of 5'-inosinic acid, PRPP amidotransferase and FGAR (5-phosphoribosyl-N-formylglycinamide) amidotransferase two enzymes (Escherichia coli and Salmonella typhimurium, 1987, p445-473) use glutamine as a substrate. Therefore, in order to make 5'-inosine synthesis more efficient, PRPP amidotransferase and FGAR amidotransferase, both of which are involved in synthesizing 5'-inosic acid, are more sensitive to glutamine than other enzymes. It is very important to increase your affinity.

The present inventors confirmed that the fermentation concentration of 5'-inosinic acid was increased by adding L-glutamine to the 5'-inosinic acid fermentation medium. 5'-inosinic acid was produced by direct fermentation as a result of conferring resistance to azaserrine and DON (6-diazo-5-oxo-L-norleucine), which act as analogues of L-glutamine. Compared to the high concentration, high yield was produced. To date, methods for producing 5'-inosine acid are known and the most important point is to produce 5'-inosine acid economically at high concentration and high yield.

The inventors of the present invention focus on the above-mentioned point as a variant of Brevibacterium ammonia genes (ATCC 6872) Adenine (Leaky Mutant), which produces a conventional 5'-inosinic acid (Leaky Mutant) [Agr, Bio, Chem. , Vol; 47 (5), p1035-1041, 1983, (KY13102, KY13171, KY13184, etc.) or adenine and xanthine or guanine co-required strains require adenine while xanthine or guanine is required. However, by adding it, growth is promoted, urease capable of magnetizing urea is deficient, and cell wall synthesis ability is partially deficient to secrete a large amount of 5'-inosinic acid generated in the cells. It is highly susceptible to Lysozyme, a cell wall degrading enzyme, which is believed to have been made.In addition, when 5'-inosinic acid accumulates in the culture medium due to the high concentration of glucose or other carbon source added during the culture, the osmotic pressure outside the cells increases. Osmotic resistance to prevent normal physiological activity of 5'-inosinic acid-producing cells, slow cell growth, and lower 5'-inosine acid production From a well-known patented strain KFCC-10814, an azaserrine resistant strain, which is an analogue to glutamine, which is essential for purine-based synthesis systems, was selected, and 5'-inosinic acid was used. The present invention was completed by directly accumulating in culture medium in high yield and high concentration.

An object of the present invention is to impart azaserrine resistance, a glutamine analogue, to a well-known patented strain Brevibacterium ammonia genes CS1019753 (KFCC-10814), and to culture a high concentration and high yield of 5'-inosinic acid by direct fermentation. To provide a selection of azaserine resistant strains that can be directly accumulated in the air. Another object of the present invention is to provide a method for producing 5'-inosinic acid at high concentration and high yield by directly fermenting the strain.

The object of the present invention is to isolate the azaserine resistant strain showing azaserine resistance by mutating the parent strain of the known patent strain Brevibacterium ammonia genes CS1019753 (KFCC-10814), and then the known strain strain and parent strain known patent After comparing the azaserine-resistant concentrations of strain KFCC-10814, the selected azaserine-resistant strains of the present invention were fermented and cultured to evaluate the production efficiency of 5'-inosinic acid.

The present invention is treated with a chemically modified organic agent in the parent strain Brevibacterium ammonia genes CS1019753 and then cultured in an azaserine-added medium to grow the colonies in a nutrient medium to produce 5'-inosinic acid in high yield. Separating and screening the novel azaserine resistant strain Brevibacterium ammonia genes CIAZ109; Comparing and measuring streptomycin resistance concentrations of Brevibacterium ammonia genes CS1019753 and the novel azaserine resistant strain Brevibacterium ammoniagenes CIAZ109 of the present invention; Inoculating a novel azaserine resistant strain Brevibacterium ammonia genes CIAZ109 of the present invention in a seed medium and further culturing the fermentation medium to produce 5'-inosine acid; The novel azaserine resistant strain Brevibacterium ammonia genes CIAZ109 of the present invention is prepared by culturing different components from the above medium and fermentation medium to produce 5'-inosinic acid.

Variation strain CS1019753 of Brevibacterium ammonia genes (ATCC 6872) used as a parent strain in the present invention does not require xanthine or guanine, but growth is promoted by addition, and urease capable of magnetizing urea is lacking. It is highly susceptible to Lysozyme, a cell wall degrading enzyme that is thought to be partially deficient in cell wall synthesis ability to secrete large amounts of 5'-inosinic acid produced intracellularly. When 5'-inosinic acid accumulates in the culture medium due to glucose and various carbon sources or late in culture, osmotic pressure outside the cells increases, which inhibits normal physiological activity of 5'-inosinic acid producing cells, slowing cell growth and producing 5'-inosinic acid. The domestic patent No. 127853 strain is given an osmotic resistance trait that can prevent the degradation.

Hereinafter, specific examples of the present invention will be described in detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited to these Examples and Experimental Examples.

Example 1 Separation of Azaserine Resistant Strain Brevibacterium Ammonia Genes CIAZ109

Variant of the present invention was suspended at 107-108 cells / mL in phosphate buffer (pH7.0) or citrate buffer (pH 5.5) with Brevibacterium ammonia genes CS1019753 (KFCC-10814) as parent strain. N-nitro-N-nitrosoguanine was added to a final concentration of 10-50 ㎍ / mL and treated for 20-40 minutes at room temperature or 32 ° C to induce mutations, and then washed twice with 0.85% saline solution. Colonies were obtained by appropriate dilution in azaserine-added medium containing 50 μg / mL, 75 μg / mL, and 100 μg / mL, respectively. Each colony was cultured in a nutrient medium, incubated for 24 hours in a seed medium, and then incubated for 3-4 days in a fermentation medium, and CIAZ109 was selected for the best production of 5'-inosinic acid accumulated in the fermentation broth.

The novel variant strain Brevibacterium ammonia genes CIAZ109 (KFCC-11068) isolated from the present invention was deposited on 12 December 1998 by the Korean spawn association, the biochemical properties of which are listed in Table 1. As shown.

Biochemical Properties of Microbial Strains of the Invention characteristic CS1019753 (KFCC-10814) CIAZ109 (KFCC-11068) Adenine Demand Demand Guanine (Xanthine) Leak type Leak type Biotin Demand Demand Lysozyme susceptibility (lowest growth inhibitory concentration) 8 µg / mL 8 µg / mL 3,4-dihydroproline resistance 83500 µg / mL 3500 µg / mL Azaserine Resistance 25 µg / mL 100 µg / mL

The nutrient medium used in this example was composed of 1% peptone, 1% juicy, 0.25% sodium chloride, 1% yeast extract, 2% agar, and adjusted to pH 7.2. The minimum medium was 2% glucose, 0.3% sodium sulfate, and phosphoric acid. 0.1% first potassium, 0.3% dipotassium phosphate, magnesium sulfate 0.3%, calcium chloride 10mg / L, iron sulfate 10mg / L, zinc sulfate 1mg / L, manganese sulfate 1mg / L, L-cysteine 20mg / L, calcium plate Totenate 10mg / L, thiamine hydrochloride 5mg / L, biotin 30g / L, adenine 20mg / L, guanine 20mg / L, agar 2% and used to adjust to pH7.3. In addition, azaserine-added medium used a medium in which 25, 50, 75,100 mg / L of azaserine was added to the minimum medium, and seed medium was 5% glucose, 0.5% peptone, 0.5% broth, 1% yeast extract, and 0.25% sodium chloride. %, Adenine 100mg / L, guanine 100mg / L was used to adjust the pH to 7.6 was used. Fermentation medium is 1.5% potassium phosphate, 2.5% potassium diphosphate, 1% ammonium chloride, magnesium sulfate 1%, calcium chloride 0.01%, iron sulfate 10mg / L, manganese sulfate 10mg / L, zinc sulfate 10mg / L, Corn immersion filtrate 20%, biotin 30ug / L, thiamine hydrochloric acid 5mg / L, adenine 100mg / L, guanine 100mg / L, fructose, glucose and molasses were mixed and used to add 6% as reducing sugar.

Experimental Example 1 Determination of Azaserine Tolerance in Mutant and Parent strains

In this experimental example, the resistance of the strain to azaserine was measured while culturing Brevibacterium ammonia genes CS1019753 (KFCC 10814) used as the parent strain in Example 1 in the minimal medium used in Example 1. As a result, the conventional patent strain CS1019753 (KFCC-10814), which was used as a parent strain, grew up to 25 µg / mL of azaserine and did not grow at all, but the present invention variant strain Brevibacterium ammonia genes CIAZ109 (KFCC-11068) described above. As shown in Example 1, growth was also observed at 100 µg / mL. This is summarized in Table 2.

Azaserine Tolerance characteristic CS1019753 (KFCC-10814) CIAZ109 (KFCC-11068) Azaserine Tolerance 25 µg / mL 100 µg / mL

Example 2: Erlenmeyer flask fermentation titer experiment

Using the present invention Brevibacterium ammonia genes CIAZ109 (KFCC011068) mutant strains glucose 5%, peptone 0.5%, juicy 0.5%, yeast extract 1%, sodium chloride 0.25%, adenine 100mg / L, guanine 100mg / L ( 3 mL of the seed medium containing PH7.6) was dispensed into an 18 mm diameter test tube and inoculated with the test strain after autoclaving, followed by shaking culture at 30 ° C. for 24 hours. Potassium phosphate 1.5%, dipotassium phosphate 2.5%, ammonium chloride 1%, magnesium sulfate 1%, calcium chloride 0.01%, iron sulfate 10mg / L, manganese sulfate 10mg / L, zinc sulfate 10mg / L, corn steep filtrate 40 mL of fermentation broth prepared by mixing 20%, biotin 30ug / L, thiamine hydrochloride 5mg / L, adenine 100mg / L, guanine 100mg / L, fructose, glucose and molasses in 6% of reducing sugar was added to a 500mL shaking Erlenmeyer flask. After dispensing and autoclaving at 121 ° C. for 10 minutes, 3 mL of the culture medium was inoculated and incubated for 3-4 days. The rotational speed was adjusted to 200 times per minute, temperature 30 ℃, pH 7.0. As a result, the accumulation amount of 5'-inosinic acid in the medium was 17.1 g / L.

Example 3: Fermentation Titer Test in 5 L Fermenter

Brevibacterium ammonia genes CIAZ109 mutant strain of the present invention as a test strain, using the same seed medium as in Example 1, 40mL of the seed medium solution in a 500mL shaking Erlenmeyer flask and autoclave sterilization and inoculated the test strain 30 Shake incubation for 24 hours at ℃. Phosphoric Acid 2%, Potassium Hydroxide 2%, Magnesium Sulfate 1%, Calcium Chloride 0.01%, Iron Sulfate 10mg / mL, Manganese Sulfate 10mg / mL, Zinc Sulfate 10mg / mL, Corn Soak Filtrate 20%, Biotin 30µg / mL, Thiamine Hydrochloride 5,600 ml of adenine 200 mg / l, guanine 100 mg / l, fructose, glucose, and molasses were added to a total reducing sugar of 18%. 1600 ml of fermentation broth was added to a 5 L-test fermenter and autoclaved at 121 ° C. for 15 minutes. 200 mL of the culture was inoculated and incubated for 3-4 days. It was adjusted to 900 revolutions per minute, temperature 28-34 ° C., pH 7.0. In addition, when the sugar became 2% during fermentation, primary, secondary and tertiary additional sugars were added such that fructose, glucose and molasses were mixed to 8% as the final reducing sugar. As a result, the accumulation amount of 5'-inosinic acid in the final fermentation broth was 65.1g / L.

As described in detail through the above examples and experimental examples, the present invention mutated the known strain Brevibacterium ammonia genes CS1019753 (KFCC-10814) to a glutamine analogue of azaserrine (azaserine) resistant strain Brevibacterium ammonia Genes CIAZ109 (KFCC-11068) has the effect of screening, and the present invention is a very useful invention in the fermentation industry because it has an excellent effect of producing a high concentration, high yield of 5'-inosinic acid by direct fermentation method.

Claims (2)

Belonging to Brevibacterium ( Brevibacterium ), adenine and biotin are required, guanine or xanthine leaky strain, high susceptibility to lysozyme, 3.4-dihydroproline concentration can be grown above 3500㎍ / mL, It is also resistant to azaserine 100 ㎍ / mL, microbial Brevibacterium ammonia genes CIAZ109 (KFCC-11068) capable of producing 5'-inosinic acid in the fermentation broth by direct fermentation. 5'-inosinic acid by the direct fermentation method, characterized in that incubated for 3 to 4 days at 200 ~ 900 times per minute, temperature 28 ~ 34 ℃ conditions in a fermentation medium (pH 7.0) using the microorganism of claim 1 Production method. Here, the fermentation broth is 2% phosphoric acid, potassium hydroxide 2%, magnesium sulfate 1%, calcium chloride 0.01%, iron sulfate 10mg / mL, manganese sulfate 10mg / mL, zinc sulfate 10mg / mL, corn steep filtrate 20%, biotin μg / mL, thiamine hydrochloride 5 mg / L, adenine 100 mg / L, guanine 100 mg / L, fructose, glucose and molasses mixture 18% (total reducing sugar value).