KR100264740B1 - A microorganism producing glutamic acid and a producing method of the glutamic acid using the same - Google Patents

Abstract

The present invention relates to Brevibacterium lactofermentum KFCC 10651, which produces glutamic acid, as a parent strain, and is commonly used as a mutagenic agent such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG). Treated according to the method to modify the traits of the parent strain, has a 3,4-dihydroproline resistance and sodium chloride resistance that can continuously produce glutamic acid under high osmotic pressure caused by a certain factor in the fermentation medium, with a low oxygen supply In the present invention relates to a mutant strain having sodium azide resistance that can produce glutamic acid smoothly until the late fermentation.

In addition, the present invention is a method for producing glutamic acid using a microorganism, by culturing the mutant strain of Brevibacterium lactofermentum in a medium containing waste molasses, glucose, starch hydrolyzate, raw sugar, and the like, to obtain glutamic acid from the culture. It is about.

Description

Microorganisms Producing Glutamic Acid and Methods for Preparing Glutamic Acid Using the Same

The present invention relates to a microorganism that produces glutamic acid and a method for producing glutamic acid using the same, and more specifically, 3,4-dihydroproline (3,4) as a variant of Brevibacterium lactofermentum KFCC 10651. It possesses -dehydro-proline resistance and sodium chloride resistance, so it has osmotic resistance and strong resistance to sodium azide, which increases glutamic acid secretion ability even when oxygen supply is insufficient. B. Microorganisms for producing glutamic acid at high concentration and high yield even when the surfactant (cationic, anionic, nonionic surfactant) is added or not added between the beginning and the end of the logarithmic phase during the fermentation process and a method for producing glutamic acid using the same will be.

In a high concentration and high yield fermentation process, microorganisms are inhibited by cell oscillation and activity or continuous production of products due to the osmotic pressure generated by high concentrations of specific media components and products in the culture medium. In glutamic acid fermentation, osmotic pressure is generated by high concentration of sugar in the fermentation medium and glutamic acid produced in the middle and late stages of fermentation, thereby inhibiting the growth and activity of fermented microorganisms or degrading production of glutamic acid. In order to correct the osmotic pressure caused by the substrate or product during the fermentation process, the fermentation microorganism generates excessive amounts of amino acids proline or glutamic acid as an osmoprotectant in the cell.

In addition, the fermentation raw materials in the industrial method for producing glutamic acid using microorganisms have been used, such as glucose, starch hydrolyzate, waste molasses, etc., but due to the high price of glucose and starch hydrolyzate, biotin is present in an excessive amount Lung molasses has been used to produce glutamic acid by adding penicillin antibiotics or surfactants from the beginning to the end of logarithmic growth during fermentation. The addition of penicillin antibiotics in the conventional method, as is well known, since the substance inhibits cell wall synthesis, the ability to form crosslinks of the peptidoglycan chain, a constituent of the cell wall, becomes unstable. This is because glutamic acid produced intracellularly is easily secreted extracellularly through the cell wall.

In general, microorganisms belonging to the genus Brevibacterium or Corynebacterium that produce glutamic acid require biotin for growth, but when biotin is present in fermentation broth, biotin promotes cell membrane synthesis. Since secretion is suppressed and glutamic acid production is reduced, it is necessary to control the amount of biotin in the fermentation broth. However, the use of waste molasses, which is mainly used for glutamic acid fermentation, contains a large amount of biotin and thus the synthesis of cell membranes is well performed. Therefore, development of microorganisms with modified cell membranes has been required to increase the extracellular secretion ability of glutamic acid.

Therefore, the present inventors modified microorganisms to make microorganisms having 3,4-dihydroproline resistance and sodium chloride resistance that can continuously produce glutamic acid even under osmotic pressure caused by specific factors in fermentation medium, and then supply oxygen. The present invention was completed by obtaining an oxygen low constituent mutant having resistance to sodium azide, which is thought to be able to produce glutamic acid even in the latter state even in this small state.

Referring to the method for isolating and obtaining the microorganism of the present invention in detail.

The microorganism CJ971010 (KFCC 11039) of the present invention has a parent strain of Brevibacterium lactofermentum KFCC10651 and causes mutations such as UV irradiation and N-methyl-N'-nitro-N-nitrosoguanidine (NTG). After treatment according to the zero conventional method, a variety of strains capable of growing in the medium (3) added with 3,4-dihydroproline by concentration were obtained for several weeks, and again, agar containing up to 2.2 mol of sodium chloride was obtained. By spreading on the medium (Note 4), a complex strain CJ696 mutant strain that can be grown in 3,4-dihydroproline medium and also in sodium chloride medium can be obtained, and this strain is induced as the parent strain of the CJ696 strain. Zero-treated and coated on sodium azide addition medium (Note 5) to obtain a strain that is resistant to sodium azide, which was named as CJ971010 and deposited in the Korean spawn association.

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

(2) Minimum medium: Glucose 10g / l, Potassium phosphate 1g / l, Dipotassium phosphate 0.5g / l, Ammonium sulfate 0.5g / l, Urea 1g / l, Iron sulfate 20mg / l, Magnesium sulfate 0.5 g / l, manganese sulfate 20 mg / l, thiamine hydrochloride 200 g / l, biotin 200 g / l, molasses 5 g / l, pH7.0.

(Note 3) Medium containing 3,4-dihydroproline: (Note 2) A medium in which 600 g / ml to 1200 g / ml of 3,4-dihydroproline was added to a minimum medium.

(Note 4) Sodium chloride added medium: (Note 2) Medium containing 1.7 mol to 2.2 mol of sodium chloride in the minimum medium.

(Note 5) Sodium azide addition medium: (Note 2) Medium containing 1.0 g / m to 14 g / m of sodium azide added to the minimum medium.

The biochemical properties of the novel mutant strain CJ971010 (KFCC 11039) of the present invention are as described in Table 1, Table 2 and Table 3. According to these contents, the microorganism of the present invention directly accumulates glutamic acid during fermentation culture, If a high concentration of carbon source is present in the culture medium or a large amount of glutamic acid is produced in the culture medium, the effect of osmotic pressure can be excluded to prevent the normal physiological activity inhibition by the osmotic pressure generated by the high concentration of solutes outside the cells. It is considered to be a special mutant with high resistance to 3,4-dihydroproline, high resistance to sodium chloride, and high resistance to sodium azide.

Table 1

Comparison of resistance by 3,4-dihydroproline concentration

Strain 3,4-dihydroproline (g / ml) 0 200 400 600 800 1000 KFCC10651 +++ +++ + + － － CJ971010 +++ +++ +++ + + ±

Incubation at 30 ° C. for 72 hours, +; Growth; Not growing.

TABLE 2

Comparison of resistance by sodium chloride concentration

Strain Sodium chloride (mol) 0 1.0 1.5 1.7 1.9 2.0 KFCC10651 +++ +++ +++ + + + CJ971010 +++ +++ +++ +++ +++ +++

96 hours of incubation at 30 ° C, Inc .; Growth; Not growing.

TABLE 3

Comparison of resistance by sodium azide concentration

Strain Sodium azide (g / ml) 0 One 2 4 8 10 KFCC10651 +++ +++ +++ +++ ± － CJ971010 +++ +++ +++ +++ +++ +

Incubation at 30 ° C. for 72 hours, +; Growth; Not growing.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1

Use strain:

Microorganisms CJ971010 (KFCC), KFCC10651 of the present invention.

Species Badge:

Lung molasses 0.5%, glucose 1%, peptone 1%, gravy 0.5%, yeast extract 1%, sodium chloride 0.25%, pH7.2.

Fermentation Medium:

Waste molasses 1%, glucose 2%, potassium potassium phosphate 0.1%, potassium diphosphate 0.05%, ammonium sulfate 0.05%, urea 0.9%, iron sulfate 0.002%, magnesium sulfate 0.05%, manganese sulfate 0.002%, thiamine hydrochloride 200 g / l, biotin 10 g / l, pH7.2.

Fermentation Method:

3 ml of the seed medium was dispensed into a test tube with a diameter of 18 mm, inoculated using strain after pressurization and sterilization according to a conventional method, and shake cultured at 30 ° C. for 18 hours to use as a seed culture solution. 40 ml of the fermentation broth was dispensed into a 500 ml shaking flask for shaking, and sterilized by autoclaving at 120 ° C. for 10 minutes to incubate 1 ml of the seed culture solution and incubated for 48 hours. The rotation speed was adjusted to 180 times per minute at a temperature of 30 ° C. and pH 7.2. After incubation, the amount of glutamic acid in the medium was 15.72 g / l for KFCC10651 and 19.82 g / l for CJ971010.

As a result of the present embodiment, it was confirmed that the microorganism of the present invention can produce high concentration of glutamic acid even without adding penicillin antibiotic or surfactant even if the concentration of biotin in the fermentation broth is excessive.

Example 2

Use strain:

Same as Example 1.

Primary species:

Waste molasses 0.5%, glucose 1%, potassium potassium phosphate 0.1%, potassium diphosphate 0.05%, ammonium sulfate 0.05%, urea 0.1%, iron sulfate 0.002%, magnesium sulfate 0.05%, manganese sulfate 0.002%, thiamine hydrochloride 200 g / l, biotin 10 g / l, pH7.0.

Secondary species:

Waste molasses (converted to invert sugar) 2.0%, glucose 1%, cornstriki 3%, magnesium sulfate 0.05%, urea 0.2%, 0.1% potassium phosphate, 0.05% potassium diphosphate, 0.002% iron sulfate, sulfuric acid Manganese 0.002%, ammonium sulfate 0.3%, thiamine hydrochloride 200 g / l, biotin 50 g / l, antifoam slightly, pH7.0.

Fermentation Medium:

Waste molasses (converted to invert sugar) 8.0%, (NH ₄ ) ₂ H ₂ PO ₄ 0.15%, iron sulfate 0.002%, magnesium sulfate 0.05%, manganese sulfate 0.002%, constipiki 3%, ammonium sulfate 0.3%, thiamine Hydrochloride 200 g / l, antifoam slightly, pH7.0.

Primary seed culture:

40 ml of the primary seed medium was dispensed into a 500 ml shaking Erlenmeyer flask, pressurized and sterilized at 120 ° C. for 10 minutes, inoculated with the strain after cooling, and incubated at 180 ° C. per minute and shaken at 30 ° C. for 20 hours.

Secondary Cultivation:

After dispensing 1.2 liters of the secondary seed medium into the experimental fermenter with a capacity of 2.6 liters, autoclaving at 121 ° C for 10 minutes, and cooling, inoculating 5 ml of the culture medium of the primary seed culture solution and supplying 0.5 liters of air per minute. Shake culture was performed at 900 rpm for 30 hours at 30 ℃.

Fermentation Method:

1.8 liters of the fermentation broth were dispensed into a 5 liter test fermentation tank, sterilized by autoclaving at 121 ° C. for 10 minutes, cooled, and then inoculated with about 150 ml of the secondary seed culture solution, while supplying 2.5 liters of air per minute. Shaking culture was carried out at 900 rpm, 37 ℃.

Incubated under the above conditions, 0.3unit / ml of penicillin was added between the beginning and the end of the logarithmic growth period, and the culture was continued. The pH of the fermentation broth was continuously adjusted to pH 7.7 with 28% ammonia water. When the concentration of the residual sugar in the culture was 1.5%, sterile pulmonary molasses or a mixture of waste molasses and raw sugar in a constant ratio was frequently supplied. Further culture was continued until the sum of the sugars added to the initial fermentation medium with molasses or mixed sugar became 20% of the amount of fermentation broth. After completion of the culture, the concentration of glutamic acid was 142.7 g / l for KFCC10651 and 151.7 g / l for CJ971010.

Example 3

Strains used, primary and secondary seed cultures, fermentation broths, primary and secondary seed cultures were carried out in the same manner as in Example 2.

Fermentation Method:

When the OD was about 0.3 (OD ₅₆₂ 100), the procedure was the same as in Example 2 except that the non-ionic surfactant Tween 40 was added to 70 g / ml compared to the fermentation broth. After completion of the culture, the concentration of glutamic acid was 141.5 g / l for KFCC10651 and 151.2 g / l for CJ971010.

As described above, the novel mutant CJ971010 of the present invention possesses 3,4-dihydroproline resistance and sodium chloride resistance, which can continuously produce glutamic acid even under high osmotic pressure, and is resistant to sodium azide, providing oxygen Even in this low state, glutamic acid can be continuously produced in effective yields until the end of fermentation.

Claims (2)

KFCC 11039, a glutamic acid producing microorganism CJ971010, which is a variant of Brevibacterium lactofermentum and has 3,4-dihydroproline resistance, sodium chloride resistance and sodium azide resistance. Method for producing glutamic acid using microorganisms comprising culturing KFCC 11039, a variant strain of Brevibacterium lactofermentum, in culture medium containing waste molasses, glucose, starch hydrolysate and raw sugar .