KR100588557B1 - Microorganism producing 5?-inosinic acid and production method of 5?-inosinic acid using the same - Google Patents

Abstract

The present invention is a variant of Corynebacterium ammoniagenes CJHB100 (KCCM-10330), which produces 5'-inosinic acid at high concentration and high yield by direct fermentation method, CJKS101 (KCCM). -10603), and a method for directly accumulating 5'-inosinic acid in a culture medium at high concentration and high yield using such microorganisms. The microorganism according to the present invention is characterized by adenine requirement, xanthine and guanine leak type, urease deficiency, lysozyme high-sensitivity, streptomycin resistance, proline analog resistance, glutamine analog resistance, valine, which are characteristic of CJHB100 (KCCM-10330) strain Leucine and isoleucine analog resistance, tetrahydrofolate analog resistance, as well as alanine analog resistance and tricarboxylic acid cycle pathway inhibitor resistance.

Corynebacterium ammonia genes, inosine acid, alanine analog resistance, tricarboxylic acid cycle pathway inhibitor resistance

Description

Microorganism producing 5'-inosinic acid and production method of 5'-inosinic acid using the same}

The present invention relates to a microorganism producing 5'-inosinic acid and a method of producing 5'-inosine acid using the same. More specifically, the present invention is a variant of Corynebacterium ammoniagenes (former Brevibacterium ) CJHB100 (KCCM-10330), which is more concentrated by direct fermentation than CJHB100 strain. And corynebacterium ammonia genes CJKS101 (KCCM-10603) for producing 5'-inosinic acid in high yield, and a method for producing 5'-inosine acid using the same.

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

Currently, methods for producing 5′-inosine acid by direct fermentation are known, and the most important point is to economically produce high concentration and high yield of 5′-inosine acid.

Applicant has disclosed that valine, which is effective for purine biosynthesis, when fermentation medium is added, as disclosed in Patent Application No. 10-2001-0073897, filed on November 26, 2001 and registered on July 29, 2004, Valine hydroxamate, norvaline, N-methyl-DL-valine, DL-thiaisoleucine, isoleucine hydroloxate (isoleucine), analogs of leucine and isoleucine hydroxamate, leucine hydroxamate and leucine methyl ester, while conferring resistance to hydroxamate, known as one-carbon unit transporters in purine biosynthesis [Escherichia coli and Salmonella . P561-579 (1996)] Microorganisms confer resistance to trimethoprim, pyrimethamine, methotrexate and aminopterin, analogs of tetrahydrofolate (THF) It was confirmed that Corynebacterium ammonia genes CJHB100 (KCCM-10330) can produce 5'-inosinic acid at a higher concentration and higher yield than conventional microorganisms.

Then, the present inventors further tried to develop microorganisms producing 5'-inosine acid at a higher concentration and higher yield, and as a result, the microorganisms producing higher concentrations and higher yields of 5'-inosine acid were developed by direct fermentation. The invention has been completed.

The microorganism of the present invention is a mutant strain from Corynebacterium ammonia genes CJHB100 (KCCM-10330), in which two molecules of alanine (D-alanine) bind during the peptidoglycan biosynthesis process of cell wall components. In order to impart resistance to D-cycloserine (Biochemistry Lehninger second edition p653), an alanine analogue known as a substance that inhibits binding, and smooth the tricarboxylic acid circulation path to shorten cell activity and incubation time Reported as inhibitors of this in order to proceed [Biochem. J, vol; 77, p17 1960] is a microorganism conferring resistance to monofluoroacetic acid. By confirming that these microorganisms can produce 5′-inosinic acid by direct fermentation and fermentation at a higher concentration and higher yield than in the related art, the present invention has been completed.

Accordingly, it is an object of the present invention to provide microorganisms which produce 5′-inosinic acid in high concentration and high yield by direct fermentation, and which are resistant to analogs of alanine and to inhibitors of tricarboxylic acid cycle pathways.

In addition, another object of the present invention is to provide a method for culturing the microorganism to directly accumulate 5'-inosinic acid in culture medium at high concentration and high yield.

The present invention relates to a microorganism Corynebacterium ammonia genes CJKS101 (KCCM-10603) which is resistant to analogs of alanine and inhibitors of tricarboxylic acid cycle pathways and produces 5'-inosinic acid in high yield and high concentration.

The present invention also provides a method of culturing Corynebacterium ammonia genes CJKS101 (KCCM-10603) and directly accumulating 5′-inosinic acid at high concentration and high yield in the culture.

Hereinafter, the present invention will be described in more detail.

The microorganism Corynebacterium ammonia genes CJKS101 (KCCM-10603) of the present invention is a mutant strain of Corynebacterium ammonia genes CJHB100 (KCCM-10330). Corynebacterium ammonia genes CJHB100 (KCCM-10330) [see patent application No. 10-2001-0073897] used as a parent strain in the present invention is a variant of brebibacterium ammonia genes ATCC6872 Bacterium ammonia genes CJIP009 (KCCM-10226) (see Korean Patent Publication No. 10-0397321).

Therefore, the microorganism according to the present invention shares the following main characteristics and effects of Corynebacterium ammonia genes CJHB100 (KCCM-10330). In other words,

Iii) properties of xanthine and guanine leaky mutants that are adenine-required and xanthine and guanine non-constitutive but promote growth upon addition of xanthine or guanine;

Ii) urease defect properties capable of magnetizing urea;

Iii) high sensitivity to lysozyme, a cell wall degrading enzyme, and partially deficient in cell wall synthesis ability to secrete large amounts of 5′-inosinic acid produced intracellularly;

Iii) resistant to streptomycin, which can effectively counteract contamination during fermentation;

Viii) The normal physiological activity of microorganisms is inhibited by osmotic pressure outside the cells caused by the accumulation of high concentrations of glucose and other carbon sources in the culture medium, or by the accumulation of 5'-inosinic acid produced in the culture, resulting in slowing of cell growth and consequently In order to prevent the degradation of 5'-inosinic acid production, to play an important role in osmotic pressure control to increase the intracellular concentration of proline (L-proline) that can confer osmotic resistance trait, Endows resistance to osmotic pressure;

Viii) resistance to glutamine analogues essential for the purine biosynthesis system, such as azaserine and DON (6-diazo-5-oxo-L-norleucine);

Iii) analogs of valine, leucine and isoleucine known to promote purine biosynthesis upon addition of fermentation medium, such as valine hydroxamate, norvaline, methylvaline, thiisoleucine, isoleucine hydroxamate, Properties with resistance to leucine hydroxamate and leucine methyl ester;

Iii) it has the property of being resistant to analogs of tetrahydrofolate, known as monocarbon carriers in the purine biosynthesis system, for example, trimethoprim, pyrimetamine, methotrexate and aminopterin.

In addition, Corynebacterium ammonia genes CJKS101 (KCCM-10603) according to the present invention, in addition to the above properties, two molecules of alanine (D-alanine) during the peptidoglycan biosynthesis process of cell wall components As a substance that inhibits the binding of monofluoroacetic acid, to inhibit the alanine analog, D-cycloserine, and to accelerate the tricarboxylic acid cycle in order to shorten cell activity and incubation time By additional resistance to monofluoroacetic acid, 5'-inosinic acid can be directly accumulated in culture at higher concentrations and yields.

As mentioned in the detailed description and examples of the present invention, the nutrient used for the cultivation of microorganisms in the present invention (denoted as' Note 1 '), the minimal medium (denoted as' Note 2'), the species medium ('Note 3 '), flask fermentation medium (denoted as' Note 4'), fermentation broth medium (denoted as' Note 5 '), and fermenter main medium (denoted as' Note 6'), as shown in Table 1, respectively. 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.

Mutation of microorganisms includes X-rays, ultraviolet rays, chemical mutation inducing agents (e.g., N-methyl-N-nitro-N-nitrosoguanine, diethylsulfate, ethylamine, etc.). In order to do this, Corynebacterium ammonia genes CJHB100 (KCCM-10330) was treated with N-methyl-N-nitro-N-nitrosoguanine. Thereafter, this was appropriately diluted in a medium containing 1.7% agar and alanine analogs in different concentrations in a minimum medium (Note 2), respectively, and then smeared to obtain colonies.

Each colony obtained was incubated in a nutrient medium (Note 1), incubated for 24 hours in a seed medium (Note 3), and then cultured for 5 to 6 days in a fermentation medium (Note 4), and 5 'accumulated in the fermentation broth. Microorganisms with good inosinic acid production were selected for alanine analogs (CJSB7). Subsequently, colonies were obtained by appropriate dilution in medium containing tricarboxylic acid cycle pathway inhibitors, each of which was resistant to CJSB7, to which the resistance was given.

Each colony obtained was incubated in a nutrient medium (Note 1), incubated for 24 hours in a seed medium (Note 3), and then cultured for 5 to 6 days in a fermentation medium (Note 4). The strains with the highest yield of 5'-inosinic acid accumulated in were selected.

These strains were named Corynebacterium ammonia genes CJKS101, and under the Treaty of Budapest, the Korean Culture Center of Microorganisms (KCCM) in Hongdae 1-dong, Seodaemun-gu, Seoul on October 27, 2004 Deposited as -10603.

Biochemical properties of representative analogs of the novel variant strain CJKS101 (KCCM-10603) selected according to the present invention are listed in Table 2 in comparison to Corynebacterium ammoniagenes ATCC6872.

The production method of 5'-inosinic acid used in the present invention is as follows.

Corynebacterium ammonia genes CJKS101 (KCCM-10603) according to the present invention is incubated in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins and the like while controlling the temperature, pH and the like under aerobic conditions.

As the carbon source, carbohydrates such as glucose, fructose, sterilized pretreated molasses (ie, molasses converted to reducing sugars) and the like can be used. As nitrogen sources, various inorganic nitrogen sources such as ammonia, ammonium chloride and ammonium sulfate and peptone, NZ-amine, meat Organic nitrogen sources such as extracts, yeast extracts, corn steep liquors, casein hydrolysates, fish or degradation products thereof, skim soybean 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 20 to 40 ° C. The pH of the medium is preferably maintained near neutral during incubation, the incubation lasted for 5-6 days, and the 5'-inosinic acid accumulated by direct fermentation was analyzed according to the conventional method.

Hereinafter, the present invention will be described in detail with reference to Examples. However, it does not mean that the scope of the present invention is limited by the embodiment in any form.

<Example>

Example 1 Selection of Cycloserine Resistant CJSB7

Variants according to the present invention were suspended at 10 ⁷ to 10 ⁸ cells / ml in phosphate buffer (pH 7.0) or citrate buffer (pH 5.5) with Corynebacterium ammonia genes CJHB100 (KCCM-10330) as parent. -Methyl-N-nitro-N-nitrosoguanine was added to a final concentration of 10-50 μg / ml and treated at 32 ° C. for 20-40 minutes to induce mutations, then washed twice with 0.85% saline. Then, after diluting appropriately in a culture medium containing 50 μg / ml and 100 μg / ml of cycloserine in a minimum medium (Note 2) containing 1.7% agar, smears were obtained to obtain colonies. Each colony was cultured in a nutrient medium (Note 1), incubated for 24 hours in a seed medium (Note 3), and then cultured for 3 to 4 days in a fermentation medium (Note 4). This best CJSB7 could be selected.

Table 3 shows the concentration of resistance to the cycloserine of the microorganism and parent CJHB100 (KCCM-10330) according to the present invention.

Example 2: Selection of monofluoroacetic acid resistant strain CJKS101

The selected cycloserine resistant strain CJSB7 is suspended as a parent strain at 10 ⁷ to 10 ⁸ cells / ml of phosphate buffer (pH 7.0), and the final concentration of N-methyl-N-nitro-N-nitrosoguanine is 10 to 50 µg / add to ml and treat for 20-40 minutes at 32 ° C. to induce mutations, wash twice with 0.85% saline and then monofluoroacetic acid in minimal medium containing 1.7% agar (Note 2) After appropriate dilution in a medium containing 75 μg / ml and 100 μg / ml, respectively, they were plated to obtain colonies. Each colony was cultured in a nutrient medium (Note 1), incubated for 24 hours in a seed medium (Note 3), and then cultured for 3 to 4 days in a fermentation medium (Note 4) to finally accumulate in the fermentation broth. CJKS101 was selected with the highest yield of inosine acid.

Table 4 shows the concentration of resistance to monofluoroacetic acid of the microorganism and parent CJSB7 according to the present invention.

Example 3: Erlenmeyer flask fermentation titer experiment

Use strain: Corynebacterium ammonia genes CJKS101

Species medium: see note 3 in table 1

Flask Fermentation Medium: See Note 4 in Table 1

Fermentation method: 3 ml of the seed medium was dispensed into a 18 mm diameter test tube, inoculated with the strain used after autoclaving according to a conventional method, and shake-cultured at 30 ° C. for 24 hours to use as a seed culture solution. 27 ml of fermentation medium was dispensed into a 500 ml shake flask for 3 minutes, sterilized at 120 ° C. for 10 minutes, inoculated with 3 ml of the seed culture solution, and then cultured 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, the accumulation amount of 5'-inosinic acid in the medium was 20.4 g / l. This was better than the result of incubating the parent strain CJHB100 under the same conditions (19.7 g / l).

Example 4 Fermentation Titer Experiment in 5-L Fermenter

Used strain: CJKS101

Species medium: same as Example 3

Fermenter Seed Medium: See Note 5 in Table 1.

Fermenter Main Medium: See Note 6 in Table 1

Fermentation method: 50 ml of the seed medium was added to a 500 ml shake flask.

After dispensing and autoclaving according to the conventional method, inoculated strains were used, followed by shaking culture at 30 ° C. for 24 hours to use as a seed culture. 1000 ml of fermented crude medium was placed in a 2.5 L-fermentation tank and autoclaved at 15 ° C. for 15 minutes to inoculate 50 ml of the seed culture, followed by incubation for 1-2 days. The number of revolutions was adjusted to 900 times per minute, temperature 28-34 ° C., pH 7.2.

In addition, 1250 ml of the fermenter broth medium was placed in a 5 L-fermentation tank and sterilized under pressure at 120 ° C. for 15 minutes to inoculate 250 ml of the fermenter broth broth. Then, when the reducing sugar in the culture became 2%, the primary, secondary, tertiary, Fourth additional sugar was mixed with fructose, glucose and molasses and added to be 32% as the final reducing sugar, followed by incubation for 5 to 6 days. Rotation speed was adjusted to 900 times per minute, temperature 30 ℃, pH 7.2. At this time, the accumulation amount of 5'-inosinic acid in the medium was 80.3 g / l. This was better than the result of incubating the parent strain CJHB100 under the same conditions (71.9 g / l).

As described above, when 5'-inosinic acid is produced by using Corynebacterium ammonia genes CJKS101 (KCCM-10603) according to the present invention, 5'-inosinic acid is economically higher by direct fermentation and It can be produced in high yield.

Claims (2)

Production of 5'-inosinic acid of Accession No. KCCM-10603 Corynebacterium ammoniagenes CJKS101. A method of culturing the microorganism according to claim 1 and producing 5'-inosinic acid from the culture solution.