KR100330706B1 - 5'-inosinic acid-producing microorganism and method for preparing 5'-inosinic acid using the same - Google Patents

Abstract

The present invention relates to Corynebacterium ammoniagenes MP377 (KFCC-11141), which magnetizes glucose to produce 5'-inosinic acid. The present invention relates to a high concentration of 5'-inosinic acid in a culture medium using only glucose as a carbon source. Since it can accumulate, 5'-inosinic acid can be manufactured with high concentration and high yield by a more economical method.

Description

5'-inosinic acid-producing microorganism and method for preparing 5'-inosinic acid using the same}

The present invention relates to a microorganism producing 5'-inosinic acid, and more particularly, Corynebacterium (formerly Brevibacterium), which accumulates high concentration of 5'-inosinic acid in a culture solution. ( Brevibacterium )) Variant strain MP377 (KFCC-11141) derived from variant strain KFCC-10814 (Korean Patent No. 127853) of Corynebacterium ammoniagenes ATCC 6872.

5'-inosinic acid is an intermediate in nucleic acid biosynthetic metabolic system and has a physiological significance in animals and plants. It is used in many fields such as food and medicine. Especially, when used in small amounts together with sodium glutamate as a seasoning for food, the taste is increased. It is one of nucleic acid-based seasonings that has been in the spotlight as a savory seasoning, such as increase in effect.

KFCC-10814, a variation of the conventional Corynebacterium ammonia genes ATCC 6872, produces high concentrations of 5'-inosinic acid when fermented using a 1: 1 mixed sugar of glucose and fructose. However, when only glucose is used as the carbon source, the 5'-inosinic acid production capacity of the mutant strain is significantly reduced. In general, the price of glucose used for fermentation as a carbon source is cheaper than the 1: 1 mixed sugar of glucose and fructose obtained by hydrolyzing the raw sugar. Therefore, for more economical production, 5'-inosinic acid is produced in high concentration using only glucose as a carbon source. There is a need to develop microorganisms. There are a number of methods for obtaining such microorganisms, and one of them is using only glucose from a mutant strain that produces a high concentration of 5'-inosinic acid using a 1: 1 mixed sugar of glucose and fructose already obtained by using one of them. There is a method for producing novel mutants that produce high concentrations of 5'-inosinic acid.

Accordingly, an object of the present invention is to produce a high concentration of 5'-inosinic acid when cultured by mixing glucose and fructose as a carbon source, but the use of glucose alone, the magnetization is significantly reduced, especially the 5'-inosinic acid production ability is significantly reduced Mutant strain MP377 which produces high concentration of 5'-inosinic acid while magnetizing glucose by conferring 6-mercaptopurine resistance to the strain KFCC-10814 of bacterium ammonia genes KFCC-10814 -11141), and a method for producing 5'-inosinic acid using the same.

First, the present invention relates to Corynebacterium ammonia genes MP377 (KFCC-11141), which magnetizes glucose to produce 5'-inosinic acid. The microorganism of the present invention is resistant to 6-mercaptopurine of 300 µg / ml or more.

Secondly, the present invention relates to a method of culturing the microorganism to produce 5'-inosinic acid from the culture. In the process according to the invention, preference is given to using glucose as the carbon source.

Hereinafter, the present invention will be described in detail.

The strain Corynebacterium ammonia genes MP377 of the present invention is a variant of the conventional Corynebacterium ammonia genes ATCC 6872, i) adenine (leaky mutant) that produces 5'-inosinic acid (leaky mutant) [ Agric . Biol. Chem. , Vol. 47, No. 5, p1035-1041, 1983 (KY13102, KY13171, KY13184, etc.) or, unlike adenine and xanthine or guanine co-required strains, require adenine, while not requiring xanthine or guanine However, by adding them, growth is promoted, ii) urease capable of magnetizing urea is depleted, and iii) cell wall synthesis to secrete large amounts of 5'-inosinic acid produced in cells well outside the cell. It has a high sensitivity to lysozyme, a cell wall degrading enzyme that is considered to be partially deficient, and i) adds a high concentration of glucose and fructose or other sugars as a carbon source to the culture process, When 5'-inosinic acid accumulates at a high concentration in the culture medium, the osmotic pressure outside the cells increases, thereby inhibiting normal physiological activity of 5'-inosinic acid producing cells. The osmolality resistant trait is provided to prevent the slowing and lowering of 5'-inosinic acid production. I) Purine base as part of releasing the control by purine bases in 5'-inosine acid biosynthesis. It has a resistance trait to 6-mercaptopurine, which is a kind of analog. In particular, it is possible to use only glucose as a carbon source by imparting resistance to 6-mercaptopurine, and by aerobic culture in a nitrogen source-limited nutrient medium, 5'-inosinic acid is directly accumulated in high concentration and high yield. . The strain was deposited on January 29, 2000 with the Korean spawn association, which was granted accession number KFCC-11141.

Referring to the method for producing a strain according to the invention in more detail as follows.

First of all, Corynebacterium ammonia genes ATCC 6872 is a parent strain, and it has the ability to produce 5'-inosinic acid by X-ray, ultraviolet irradiation, etc., and requires adenine for growth, and it promotes growth in the minimum medium containing adenine and guanine (Note 4). Get CS101. At this time, mutant strains are separated using a nutrient medium (Note 1), a minimum medium (Note 2), and a minimum addition medium (Notes 3 and 4) as the mutant strain separation medium. Next, CS101 was used as a parent strain, and treated with chemical mutagen such as X-ray, ultraviolet ray or N-methyl-N-nitro-N-nitrosoguanine, diethylsulfide, ethylamine, and then plated on the medium (Note 1). Colony is purely separated. CS1019 having higher lysozyme susceptibility than CS101 is then selected using medium supplemented with lysozyme by concentration. Next, after mutant CS1019 was treated with the above-described organic mutants, pure strain was isolated from the medium (Note 1), and mutant CS10197 lacking urease was selected from the Christensenurea test medium and the medium (Note 7). Variant CS10197 was again used in the same manner using 3,4-dihydroproline higher than CS10197 using 3,4-dehydro-DL-proline-concentrated medium (Note 6). Mutant strain CS101975 with resistance is obtained. At this time, most of the mutants can not grow because the concentration of 3,4-dihydroproline is high, and the strains with high osmotic resistance can be grown, so the mutants that can be grown at 3500㎍ / ㎖ by gradually increasing the concentration of 3,4-dihydroproline Get The mutant strains thus obtained were centrifuged to plate 3,4-dihydroproline in a medium (Note 6) containing 3500 µg / ml to purely separate the resulting colonies to obtain KFCC-10814.

In the present invention, the mutant strain CSJ6 having higher 6-mercaptopurine resistance than KFCC-10814 was prepared using the medium (Note 8) in which the mutant strain KFCC-10814 was added to the 6-mercaptopurine concentration by using the same method again. Get At this time, most of the mutants were not able to grow due to the high concentration of 6-mercaptopurine, MP377 by pure separation of colonies produced by smearing the medium (Note 8) containing 6-mercaptopurine at a concentration of 300 ㎍ / ㎖ It was named.

(Note 1) nutrient medium; 1% peptone, 1% gravy, 0.25% sodium chloride, 1% yeast extract, 2% agar, pH 7.2

(Note 2) Minimum Medium; Glucose 2%, Sodium sulfate 0.3%, Potassium phosphate 0.1%, Dipotassium phosphate 0.3%, 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 pantothenate 10mg / l, thiamine hydrochloride 5mg / l, biotin 30µg / l, urea 0.2%, pH 7.3

(Note 3) adenine minimal addition medium; (Note 2) A medium in which adenine was added in a concentration range of 10 to 500 mg / l.

(Note 4) adenine, guanine minimal addition medium; Medium in which adenine and guanine (or xanthine are added at a concentration of 10 to 500 mg / l, respectively) in a minimum medium.

(Note 5) lysozyme addition medium; (Note 1) A medium added by concentration up to 1 to 100 µg / L in lysozyme in the medium.

(Note 6) 3,4-dihydroproline addition medium; (Note 1) A medium added by concentration up to 100 to 5000 µg / L in 3,4-dihydroproline in the medium.

(7) Christensenurea test medium; 1.0 g of peptone, 1.0 g of glucose, 5.0 g of sodium chloride, 2.0 g of potassium phosphate, 0.012 g of phenol red, 20 g of agar, 20 g of urea, 1 l of distilled water

(Note 8) 6-mercaptopurine addition medium; (Note 1) A medium to which 300 µg / ml of 6-mercaptopurine was added.

Biochemical properties of the novel mutant strain MP377 obtained separately in the present invention are as described in Table 1, Table 2 and Table 3.

characteristic ATCC 6872 (parent) KFCC-10617 KFCC-10814 MP377 (KFCC-11141) Adenine Composition Demand Demand Demand Guanine (Xanthine) Composition Leak type Leak type Leak type Biotin Demand Demand Demand Demand Lysozyme (lowest inhibitory concentration) 80 µg / ml 15 µg / ml 8 µg / ml 8 µg / ml 3,4 dihydroproline resistance 1000 µg / ml 2000 µg / ml 3500 µg / ml 3500 µg / ml 6-mercaptopurine resistance 20 µg / ml 50 µg / ml 100 µg / ml 300 µg / ml

Test strain name Badge color Urease ATCC 6872 (Protein stock, urease strain) Red + E. coli (standard strain lacking urease) yellow - KFCC-10814 yellow - MP377 (KFCC-11141) yellow -

Intracellular L-proline Concentration Strain Concentration ATCC 6872 (parent) - KFCC-10617 (conventional strain) 0.91 KFCC-10814 1.77 MP377 (KFCC-11141) 1.83

As described above, strain MP377 of the present invention is an adenine-required, guanine-leak type mutant having 5'-inosine acid and 5'-inosine acid production capacity directly in fermentation broth, and secreting a specific metabolite out of the cell wall. This increased sensitivity to lysozyme, and when a high concentration of carbon source is present in the culture medium or a large amount of 5'-inosinic acid is produced during the culture process, normal physiological activity inhibition by the osmotic pressure generated by the high concentration solute outside the cells To prevent this, 6-mercapto has a high resistance to 3,4-dihydroproline, which can increase the concentration of a specific solute proline (L-proline) inside the cell to exclude the effects of osmotic pressure It is a mutant strain that has the characteristics of purin resistance and increased ability to produce 5'-inosinic acid through glucose magnetization.

5'-inosinic acid was separated and purified according to the conventional method by accumulating 5'-inosinic acid accumulated in aerobic culture in a 5 L fermenter for 3 to 4 days while maintaining the temperature at 25 to 35 ° C and pH 6.0 to 8.0 as described in the following Examples. To obtain.

EXAMPLE

Hereinafter, the present invention will be described in more detail with reference to Examples, which are only intended to help understanding of the present invention, but the scope of the present invention is not limited thereto.

Example 1

Use strain: MP377 (KFCC-11141)

Species medium: glucose 5%, peptone 0.5%, broth 0.5%, yeast extract 1%, sodium chloride 0.25%, adenine 100 mg / l, guanine 100 mg / l, pH 7.6

Fermentation medium: 1.5% potassium phosphate, 2.5% potassium diphosphate, 1% ammonium chloride, 1% magnesium sulfate, 0.01% calcium chloride, 10mg / l iron sulfate, 10mg / l manganese sulfate, 10mg / l zinc sulfate, corn 20% dipping filtrate, biotin 30㎍ / ℓ, thiamine hydrochloride 5mg / ℓ, adenine 100mg / ℓ, guanine 100mg / ℓ, glucose and molasses are mixed and added to reducing sugar 6% (glucose 5.6%, molasses 0.4%) It was.

Fermentation method: 3 ml of the seed medium was dispensed into a 18 mm diameter test tube, autoclaved and sterilized according to a conventional method, followed by inoculation of the strain used, followed by shaking culture at 30 ° C. for 24 hours. 40 ml of the fermentation broth was dispensed into a 500 ml shake flask, and sterilized under pressure at 120 ° C. for 10 minutes to incubate 3 ml of the culture medium and incubated for 5 days. Rotation speed was adjusted to 200 times per minute, temperature 30 ℃, pH 7.0. The accumulation amount of 5'-inosinic acid in the medium was 17.6 g / l.

Example 2

Use strain: MP377 (KFCC-11141)

Species medium: glucose 5%, peptone 0.5%, broth 0.5%, yeast extract 1%, sodium chloride 0.25%, adenine 100 mg / l, guanine 100 mg / l, pH 7.6

Fermentation medium: 1.5% potassium phosphate, 2.5% potassium diphosphate, 1% ammonium chloride, 1% magnesium sulfate, 0.01% calcium chloride, 10mg / l iron sulfate, 10mg / l manganese sulfate, 10mg / l zinc sulfate, corn Dipping Filtrate 20%, Biotin 30㎍ / ℓ, Thiamine Hydrochloride 5mg / ℓ, Adenine 100mg / ℓ, Guanine 100mg / ℓ, Fructose, Glucose and Molasses are mixed, 6% as reducing sugar (Fructose 2.8%, Glucose 2.8%, Molasses 0.4 %) Was used.

Fermentation method: 3 ml of the seed medium was dispensed into a 18 mm diameter test tube, autoclaved and sterilized according to a conventional method, followed by inoculation of the strain used, followed by shaking culture at 30 ° C. for 24 hours. 40 ml of the fermentation broth was dispensed into a 500 ml shake flask, sterilized under pressure at 120 ° C. for 10 minutes, and 3 ml of the culture medium was incubated for 5 days. Rotation speed was adjusted to 200 times per minute, temperature 30 ℃, pH 7.0. The accumulation amount of 5'-inosinic acid in the medium was 15.5 g / l.

Comparative Example 1

Strain used: KFCC-10814

Species medium: glucose 5%, peptone 0.5%, broth 0.5%, yeast extract 1%, sodium chloride 0.25%, adenine 100 mg / l, guanine 100 mg / l, pH 7.6

Fermentation medium: 1.5% potassium phosphate, 2.5% potassium diphosphate, 1% ammonium chloride, 1% magnesium sulfate, 0.01% calcium chloride, 10mg / l iron sulfate, 10mg / l manganese sulfate, 10mg / l zinc sulfate, corn 20% dipping filtrate, biotin 30㎍ / ℓ, thiamine hydrochloride 5mg / ℓ, adenine 100mg / ℓ, guanine 100mg / ℓ, glucose and molasses are mixed and added to reducing sugar 6% (glucose 5.6%, molasses 0.4%) It was.

Fermentation method: 3 ml of the seed medium was dispensed into a 18 mm diameter test tube, autoclaved and sterilized according to a conventional method, followed by inoculation of the strain used, followed by shaking culture at 30 ° C. for 24 hours. 40 ml of the fermentation broth was dispensed into a 500 ml shake flask, sterilized under pressure at 120 ° C. for 10 minutes, and 3 ml of the culture medium was incubated for 5 days. Rotation speed was adjusted to 200 times per minute, temperature 30 ℃, pH 7.0. The accumulated amount of 5'-inosinic acid in the medium was 7.2 g / lL. As a result of measuring the residual glucose using the glucose analyzer, it could be seen that the glucose could not be decomposed 100% with 1.6% remaining glucose.

Comparative Example 2

Strain used: KFCC-10814

Species medium: glucose 5%, peptone 0.5%, broth 0.5%, yeast extract 1%, sodium chloride 0.25%, adenine 100 mg / l, guanine 100 mg / l, pH 7.6

Fermentation medium: 1.5% potassium phosphate, 2.5% potassium diphosphate, 1% ammonium chloride, 1% magnesium sulfate, 0.01% calcium chloride, 10mg / l iron sulfate, 10mg / l manganese sulfate, 10mg / l zinc sulfate, corn Dipping Filtrate 20%, Biotin 30㎍ / ℓ, Thiamine Hydrochloride 5mg / ℓ, Adenine 100mg / ℓ, Guanine 100mg / ℓ, Fructose, Glucose and Molasses are mixed, 6% as reducing sugar (Fructose 2.8%, Glucose 2.8%, Molasses 0.4 %) Was used.

Fermentation method: 3 ml of the seed medium was dispensed into a 18 mm diameter test tube, autoclaved and sterilized according to a conventional method, followed by inoculation of the strain used, followed by shaking culture at 30 ° C. for 24 hours. 40 ml of the fermentation broth was dispensed into a 500 ml shake flask, and sterilized under pressure at 120 ° C. for 10 minutes to incubate 3 ml of the culture medium and incubated for 5 days. Rotation speed was adjusted to 200 times per minute, temperature 30 ℃, pH 7.0. The accumulation amount of 5'-inosinic acid in the medium was 15.2 g / l.

Example 3

Use strain: MP377 (KFCC-11141)

Species Medium: same as Example 1

Fermentation medium: 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 steep filtrate 20%, biotin 30µg / ml , 5 mg / l thiamine hydrochloride, 200 mg / l adenine, 100 mg / l guanine, glucose and molasses were mixed and used to add 18% (glucose 16.8%, molasses 1.2%) as total reducing sugar.

Fermentation method: 40 ml of the seed medium was dispensed into a 500 ml shake culture Erlenmeyer flask and autoclaved according to the conventional method, followed by inoculation of the used strain and shaking culture at 30 ° C. for 24 hours to use as a seed culture solution. 1600 ml of fermentation broth was inserted into a 5 L fermenter, autoclaved at 120 ° C. for 15 minutes, and 200 ml of the culture medium was incubated for 3 to 4 days. The rotation speed was adjusted to 900 times per minute, at a temperature of 28 to 34 ° C. and pH 7.0. In addition, when the sugar became 2% at the time of fermentation, the primary, secondary and tertiary additional sugars were added to be 8% as the final reducing sugar by mixing glucose and molasses, respectively. The accumulation amount of 5'-inosinic acid in the medium was 66.4 g / l.

Example 4

Use strain: MP377 (KFCC-11141)

Species Medium: same as Example 1

Fermentation medium: 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 steep filtrate 20%, biotin 30µg / ml Thiamine hydrochloride 5mg / l, adenine 200mg / l, guanine 100mg / l, fructose, glucose and molasses were mixed and used to add 18% (8.4% fructose, 8.4% glucose, 1.2% molasses) as the total reducing sugar.

Fermentation method: 40 ml of the seed medium was dispensed into a 500 ml shake culture Erlenmeyer flask and autoclaved according to the conventional method, followed by inoculation of the used strain and shaking culture at 30 ° C. for 24 hours to use as a seed culture solution. 1600 ml of fermentation broth was inserted into a 5 L fermenter, autoclaved at 120 ° C. for 15 minutes, and 200 ml of the culture medium was incubated for 3 to 4 days. The rotation speed was adjusted to 900 times per minute, 28 to 34 ° C., pH 7.0. In addition, when the sugar became 2% during fermentation, the primary, secondary and tertiary additional sugars were added so that 8% of the final reducing sugars were mixed with glucose and molasses. The accumulated amount of 5'-inosinic acid in the medium was 58.8 g / l.

Comparative Example 3

Use strain: KFCC-10814

Species Medium: same as Example 1

Fermentation medium: 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 steep filtrate 20%, biotin 30µg / ml , 5 mg / l thiamine hydrochloride, 200 mg / l adenine, 100 mg / l guanine, glucose and molasses were mixed and used to add 18% (glucose 16.8%, molasses 1.2%) as total reducing sugar.

Fermentation method: 40 ml of the seed medium was dispensed into a 500 ml shake culture Erlenmeyer flask and autoclaved according to the conventional method, followed by inoculation of the used strain and shaking culture at 30 ° C. for 24 hours to use as a seed culture solution. 1600 ml of fermentation broth was inserted into a 5 L fermenter, autoclaved at 120 ° C. for 15 minutes, and 200 ml of the culture medium was incubated for 3 to 4 days. The rotation speed was adjusted to 900 times per minute, at a temperature of 28 to 34 ° C. and pH 7.0. In addition, when the sugar became 2% at the time of fermentation, the primary, secondary and tertiary additional sugars were added to be 8% as the final reducing sugar by mixing glucose and molasses, respectively. The accumulation amount of 5'-inosinic acid in the medium was 44.7 g / l.

Comparative Example 4

Use strain: KFCC-10814

Species Medium: same as Example 1

Fermentation medium: 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 steep filtrate 20%, biotin 30µg / ml Thiamine hydrochloride 5mg / l, adenine 200mg / l, guanine 100mg / l, fructose, glucose and molasses were mixed and used to add 18% (8.4% fructose, 8.4% glucose, 1.2% molasses) as the total reducing sugar.

Fermentation method: 40 ml of the seed medium was dispensed into a 500 ml shake culture conical flask and autoclaved according to the conventional method, followed by inoculation of the used strain and shaking culture at 30 ° C. for 24 hours to use as a seed culture solution. 1600 ml of fermentation broth was inserted into a 5 L fermenter, autoclaved at 120 ° C. for 15 minutes, and 200 ml of the culture medium was incubated for 3 to 4 days. The rotation speed was adjusted to 900 times per minute, at a temperature of 28 to 34 ° C. and pH 7.0. In addition, when the sugar became 2% at the time of fermentation, the primary, secondary and tertiary additional sugars were added to be 8% as the final reducing sugar by mixing glucose and molasses, respectively. The accumulation amount of 5'-inosinic acid in the medium was 58.4 g / l.

As described above, by imparting 6-mercaptopurine resistance to Corynebacterium ammonia genes KFCC-10814, which produces a high concentration of 5'-inosinic acid using a 1: 1 mixed sugar of glucose and fructose as a carbon source, Using only glucose as a carbon source, it was easy to obtain a mutant strain that produces a high concentration of 5'-inosinic acid. Since the price of glucose is lower than that of 1: 1 mixed sugar of glucose and fructose of raw sugar hydrolyzate as a carbon source in conventional fermentation, it is possible to produce 5'-inosinic acid in a more economical manner by using the mutants obtained by the above method. We have established a foundation for this.

Corynebacterium ammonia genes MP377 according to the present invention can accumulate 5'-inosinic acid in a high concentration in the culture medium using only glucose as a carbon source, it is possible to produce 5'-inosinic acid at high concentration and high yield in a more economic way Can be.

Claims (4)

Corynebacterium ammoniagenes MP377 (KFCC-11141), which magnetizes glucose to produce 5'-inosinic acid. The Corynebacterium ammonia genes MP377 according to claim 1, which is resistant to at least 300 µg / ml 6-mercaptopurine. A method for producing 5'-inosinic acid from the culture by culturing the microorganism according to claim 1 or 2. 4. The method of claim 3, wherein glucose is used as the carbon source.