KR20010100223A - 5'-Inosinic acid-producing microorganism - Google Patents

Abstract

The present invention relates to a microorganism producing 5'-inosinic acid, which is capable of growing in a high concentration of azetidine carboxylic acid (L-azetidine-2-carboxylic acid), and resistant to osmotic pressure. The present invention relates to Corynebacterium ammonia genes, which directly accumulate 5'-inosinic acid in a high yield, high concentration in a culture medium.

Description

5'-Inosinic acid-producing microorganism}

The present invention relates to a mutant strain of a microorganism producing 5'-inosinic acid, more specifically Corynebacterium ammoniagenes ( formerly known as Brevibacterium ammonia genes) strain (ATCC 6872).

The microorganism of the present invention is adenine (Leaky Mutant), which produces a conventional 5'-inosinic acid (Leaky Mutant) [ Agr. Bio. Chem ., Vol; 47 (5), p.1035-1041, 1983, (KY13102, KY13171, KY13184, etc.) or adenine and xanthine or guanine co-required strains, whereas adenine is required while xanthine Or do not require guanine, but by adding them, growth is promoted, urease (magnifying urease) that can magnetize urea (Urea) is lacking, and cell wall synthesis ability is partially missing, high in Lysozyme (lysozyme) cell wall degrading enzyme Sensitization allows not only to secrete large amounts of 5'-inosinic acid produced intracellularly well, but also to give an osmotic resistance trait, thereby allowing high concentrations of glucose and various carbon sources added during the cultivation process, Even though 5'-inosinic acid accumulates in the culture medium and the osmotic pressure outside the cells increases, the cell culture resulted from the inhibition of normal physiological activity of 5'-inosinic acid producing cells. It was developed from a microorganism strain (KFCC-10814) (Korean Patent No. 128553) that can prevent the slowing of and lowering of 5'-inosinic acid production, and has been developed for proline (L-proline), which plays an important role in osmotic pressure control. In order to screen for L-azetidine-2-carboxylic acid resistant strains, which are analogues, they were treated with various mutants to increase the concentrations inside the cells against the high concentration of solutes accumulated outside the cells. In comparison, by effectively eliminating the effects of osmotic pressure, 5'-inosinic acid can be directly accumulated in the culture medium in high yield and high concentration.

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 used in various fields such as food, medicine, and various medical uses. 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, several methods for producing 5'-inosine acid using direct fermentation are known. The most important point is to economically produce high concentration and high yield of 5'-inosine acid.

The present inventors conducted a thorough study by selecting resistant strains against various proline analogues (L-proline analogues). As a result, strains conferring resistance to azetidine carboxylic acid (L-azetidine-2-carboxylic acid) were identified by direct fermentation. -It has been found that inosinic acid can be produced at a higher concentration and higher yield than the conventional one, and thus, the present invention has been completed.

Accordingly, an object of the present invention is an azetidine carboxyl which is a proline analogue, which is selected from known strains (KFCC-10814) developed by our company and produces 5'-inosinic acid in high yield and high concentration by direct fermentation method than the known technique. To provide a strain of microorganisms resistant to acid (L-azetidine-2-carboxylic acid).

Most bacteria accumulate organic solutes called potassium ions and osmolytes by increasing the osmotic pressure inside the cells to prevent osmotic dehydration under osmotic pressure outside the cells. Osmolite is known to proline (L-proline), glutamate, sugar (Sugar), N-methylated amino acid derivatives (L-proline) is the osmotic pressure control As an important factor of, the activity of pyrroline-5-carboxylate reductase, an important enzyme of the proline biosynthetic pathway, was increased under osmotic conditions by increased 5'-inosinic acid outside the cells. Accumulation of proline (L-proline) inside cells has been reported via Brevibacterium lactofermentum (see Agr, Bio, Chem ., Vol; 53 (9), p. 275-2479, 1989]. In addition to the E. coli (Escherichia coli), Salmonella tie blood bunch Titanium (Salmonella typhimurium), Serratia Mar sense (Serratia marcescens) it is also accumulate proline (L-proline) inside the cell, etc., which reported the regulated by external osmotic pressure [J , Bacteriol ., Vol. 163, p 296, 1985]. Therefore, in order to develop high concentration and high yield of 5'-inosinic acid, it is possible to enhance the osmotic resistance by increasing the ability of proline synthesis (L-proline) and also to increase the synthesis of proline (L-proline) in cells In order to prevent growth and inhibition of biochemical metabolism, it is important to acquire proline analogue resistant strains.

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

First, the microorganism used in the present invention is a variant of Corynebacterium ammoniagenes ATCC6872, the microorganism of the present invention does not require xanthine or guanine while adding adenine, but growth is promoted by adding them. , Urease (Urease) that can magnetize the urea (Urea) is missing, the cell wall synthesis ability is partially missing, has a high sensitivity to the cell wall degrading enzyme Lysozyme (Lysozyme) a large amount of 5'- generated in the cell In addition to the ability to secrete inosinic acid well outside the cell, it is endowed with an introducible resistance trait, which is caused by high concentrations of glucose and various carbon sources added during the culturing process or 5'-inosinic acid in the late stages of culture. Increasing osmotic pressure may inhibit the normal physiological activity of 5'-inosinic acid producing cells. It was developed from the microbial strain (KFCC-10814) (Korean Patent No. 12853), which can prevent the slowing of cell growth and lowering of 5'-inosinic acid, and plays an important role in controlling osmotic pressure (L-proline). We attempted to screen for L-azetidine-2-carboxylic acid resistant strain, an analogue to).

As a result of measuring the degree of resistance of the strain to azetidine carboxylic acid in the minimal medium (Note 1) of the present invention (Azetidine carboxylic acid addition medium, Note 3), the existing CS1019753 (KFCC-10814) was azetidine carboxyl The acid was resistant up to 5 mg / ml and no growth occurred above 10 mg / ml.

Variations of the present invention are chemicals such as X-ray, ultraviolet or N-methyl-N-nitro-N-nitrosoguanine, diethyl sulfate, ethylamine, etc. based on Corynebacterium ammonia genes CS1019753 (KFCC-10814). After treatment with the mutant organic agent, the azetidine carboxylic acid was properly diluted in azetidine carboxylic acid addition medium (Note 3) containing 10 mg / ml, 20 mg / ml and 30 mg / ml, respectively, and then smeared and coated. ) Each colony was cultured in a nutrient medium (Note 1), incubated for 24 hours in a seed medium (Note 4), and then cultured for 3-4 days in a fermentation medium (Note 5). The amount of 5'-inosinic acid accumulated in the fermentation broth was The best CIAC101 (KFCC-11134) was selected.

The media used in the present invention have the following components:

(Note 1) Nutritional medium: peptone 1, gravy 1, sodium chloride 0.25, yeast extract 1, agar 2 pH 7.2.

(Note 2) Minimum medium: Glucose 2, sodium sulfate 0.3, potassium monophosphate 0.1, potassium diphosphate 0.3, magnesium sulfate 0.3, calcium chloride 10mg / l, iron sulfate 10mg / l, zinc sulfate 1mg / l, manganese sulfate 1mg / l, L-cysteine 20 mg / l, calcium pantothenate 10 mg / l, thiamine hydrochloride 5 mg / l, biotin 30 μg / l, adenine 20 mg / l, guanine 20 mg / l, pH 7.3.

(Note 3) Azetidine carboxylic acid addition medium: A medium containing 5, 10, 20, 30 g / l of azetidine carboxylic acid by concentration in Agar-containing medium (Note 2).

(Note 4) Species medium: glucose 5, peptone 0.5, juicy 0.5, yeast extract 1, sodium chloride 0.25, adenine 100 mg / l, guanine 100 mg / l, pH 7.6.

(5) Fermentation medium: 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 Dipping filtrate 20, biotin 30 ㎍ / l, thiamine hydrochloride 5mg / l, adenine 100mg / l, guanine 100mg / l, fructose, glucose and molasses are mixed and added as reducing sugars.

The novel mutant strain CIAC101 isolated from the present invention was deposited with the accession number KFCC-11134 on January 29, 2000 to the Korean spawn association, an international depository organization, and its biochemical properties are shown in Table 1.

Table 1

Characteristics CS1019753 (KFCC-10814) CIAC101 (KFCC-11135) Adenine Demand Demand Guanine (Xanthine) Leak type Leak type Biotin Demand Demand Lysozyme Susceptible Inhibitory Concentration) 8µg / ml 8 μg / ml 3,4-dihydroproline resistance 3500 μg / ml 3500 μg / ml Azetidine carboxylic acid 5mg / ml 30mg / ml

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

That is, microorganisms belonging to the genus Corynebacterium and capable of producing 5'-inosinic acid are cultured under aerobic conditions under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins, and the like.

As the carbon source, carbohydrates such as glucose, fructose, sterilized pretreated molasses (i.e., 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, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used, and vitamins and nutrient-constituting bases may be added as necessary. The culture is carried out under aerobic conditions, for example by shaking culture or aeration stirred culture, preferably at a temperature of 20-40 ° C. The pH of the medium is preferably maintained near the neutral during incubation. The culture was performed for 3-4 days and the 5'-inosinic acid accumulated by the direct fermentation method was analyzed according to the conventional method.

<Example>

Example 1 Isolation of Azetidine Carboxylic Acid Resistant CIAC101

Variants of the present invention were suspended in phosphate buffer (pH 7.0) or citrate buffer (pH 5.5) at 10 ⁷ -10 ⁸ cells / ml, with Corynebacterium ammonia genes CS1019753 (KFCC-10814) as parent strains. Methyl-N-nitro-N-nitrosoguanine was added to a final concentration of 10-50 μg / ml and treated for 20-40 minutes at room temperature or 32 ° C to induce mutations, followed by 0.85 saline over two times. After washing, the azetidine carboxylic acid was appropriately diluted in azetidine carboxylic acid addition medium (Note 3) containing 10 mg / ml, 20 mg / ml, and 30 mg / ml, respectively, and smeared to obtain a colony. Each colony was cultured in a nutrient medium (Note 1), incubated for 24 hours in a seed medium (Note 4), and then cultured for 3-4 days in a fermentation medium (Note 5), and the amount of 5'-inosinic acid accumulated in the fermentation broth was increased. The best strains were selected with CIAC101 (KFCC-11134), Table 2 shows the azetidine carboxylic acid resistance concentrations of the existing strains and strains of the present invention.

Table 2.

Characteristics CS1019753 (KFCC-10814) CIAC101 (KFCC-11134) Azetidine carboxylic acid resistance concentration 5 mg / ml 30 mg / ml

Example 2: Erlenmeyer flask fermentation titer experiment

Strains used: CIAC101

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 broth: 1.5 potassium phosphate 1.5, 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 needle feed solution 20 , Biotin 30 ㎍ / l, thiamine hydrochloric acid 5mg / l, adenine 100mg / l, guanine 100mg / l, fructose, glucose and molasses are mixed and added to the reducing sugar 6 used.

Fermentation method: 3 ml of the seed medium was dispensed into a test tube with a diameter of 18 mm, inoculated using strain after autoclaving according to a conventional method, and shaken at 30 ° C. for 24 hours to use as a seed culture solution. 40 ml of the fermentation broth were dispensed into a 500 ml shake Erlenmeyer flask and autoclaved at 120 ° C. for 10 minutes to inoculate 3 ml of the seed culture solution and incubated for 3-4 days. The rotation speed was adjusted to 200 times per minute at a temperature of 30 ° C. and pH 7.0. The 5'-inosinic acid accumulation in the medium was 18.1 g / l.

Example 3: Fermentation Titer Test in 5-L Fermenter

Use strain: CIAC101

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 30 ㎍ / ml, thiamine hydrochloride 5mg / l, adenine 200mg / l, guanine 100mg / l, fructose, glucose and molasses are mixed and added to 18 as total reducing sugar.

Fermentation method: 40ml of the seed medium was dispensed into a 500ml shaking Erlenmeyer flask and inoculated using strain after autoclaving according to the conventional method, and shaken at 30 ° C. for 24 hours to use as a seed culture solution. 1600ml of fermentation broth was placed in a 5L-test fermenter and autoclaved at 120 ° C for 15 minutes to inoculate 200ml of the seed culture solution and incubated for 3-4 days. Rotation speed was adjusted to 900 times per minute, temperature 28-34 ℃, pH 7.0. In addition, when the reducing sugar became 2 in the fermentation broth, primary, secondary and tertiary additional sugars were added such that 8 were mixed as fructose, glucose and molasses as final reducing sugars. The 5'-inosinic acid accumulation in the medium was 67.3 g / l.

The microorganism according to the present invention is a proline analogue of the acetidine carboxylic acid (L-azetidine-2-carboxylic acid) resistant strain CIAC101 (KFCC-) from the known strain Corynebacterium ammonia genes (KFCC-10814) developed by our company 11134), it is possible to produce 5'-inosine acid more economically by producing high concentration and high yield of 5'-inosine acid by direct fermentation method.

Claims (4)

Belonging to the genus Corynebacterium, capable of growing under the concentration of L-azetidine-2-carboxylic acid of 30 mg / ml, resistant to osmotic pressure, and in fermentation broth when cultured by direct fermentation 5 A microorganism of Brevibacterium ammonegenes CIAC101 (KFCC-11134) characterized by being able to accumulate inosinic acid at high concentration and high yield. The microorganism according to claim 1, which requires adenine and biotin for growth and is a guanine or xanthine leaky mutant. The microorganism according to claim 1, wherein the minimum growth inhibitory concentration by lysozyme, which is a cell wall degrading enzyme, is 8 µg / ml. The microorganism of claim 1, wherein the concentration of 3,4-dihydroproline is resistant to growth at 3500 μg / ml.