KR880001945B1 - Process for preparing l-glutamic acid by microorganism - Google Patents

Abstract

Prepn. of L-glutamic acid by culture of a mutant, Brevibacterium sp. strain CS 850815 (KFCC 10183) having a resistance against azaserine and sulfamylamide comprises (a) inoculating 1 liter of preculture of a mutant KFCC 10183 into a 3 liter far-fermentor contg. 13 liter medium consisting of 100 sugar beet molasses, 1 MgSO4.7H2O, 0.4 FeSO4.7H2O, 0.01 MnSO4.7H2O, 0.01 thiamine.HCl, 30 CSL, 0.02 alanine, 0.02g glycine and 300 μg biotin/dl and culturing at pH 7.8, 1 vvm, and 350 rpm; and (b) adding Tween 60 up to 0.5g/1 after 6hr of incubation and culturing for further 30hr to give 65% yield based on sugar.

Description

Method for producing L-glutamic acid by microorganisms

The present invention belongs to the genus Brevibacterium and is resistant to purine nucleotide biosynthesis inhibitors azaserine and sulfanamide, a mutant strain that has the ability to produce L- glutamic acid is promoted growth by adding one or two of alanine or glycine The present invention relates to a method for producing L-glutamic acid by microorganisms characterized in that aerobic culture of CS 850815 (KFCC 10183) is performed to collect L-glutamic acid accumulated in the medium.

That is, the inventors of the present invention, while studying to obtain more productive L-glutamic acid-producing bacteria, belong to the genus Brevibacterium and have resistance to azaserine and sulfanylamide, which are purine nucleotide biosynthesis inhibitors. Successfully isolated and grown strains having high L-glutamine productivity among the mutant strains in which growth is promoted by the addition of one or two of alanine or glycine have completed the present invention.

Variant CS 850815 (KVCC 10183) of the present invention, after treatment with the parent strain Brevibacterium flaboom NO.2247 with ultraviolet irradiation or N-methyl-N'-nitrosoguanidine, the amount of azaserine that the parent can not grow And mutant CS 850330 which was mutated to be resistant to azaserine and sulfanylamide by culturing in a solid medium containing sulfanyl amide and then fractionated to be resistant to azaserine and sulfanyl amide. However, in order to solve the shortcomings of CS 850330, which is a primary mutant strain whose growth time is poor due to sluggish growth, it is repeated by the above-described method again by adding one or two kinds of alanine or glycine in the mutant strains. Variation CS 850815 (KFCC 10183) of the present invention was obtained in which growth was promoted and L-glutamic acid was produced in a high yield in the medium, and the culture time was also normal.

Tolerances of the modified strains CS 850815 (KFCC 10183) and CS 850330 of the present invention against azaserine and sulfanylamide are shown in Table 1 and Table 2, and the growth promoting effect by alanine or glycine is shown in Table 3.

TABLE 1

Relative Growth for Azaserine

* Provincial: Brevibacterium plaboom NO.2247

TABLE 2

Relative Growth for Shalpanilamide

* Friend: Brevybacteriumflameum NO.2247

TABLE 3

Growth Influence of Mutant of the Invention and Growth Promotion Effect by Alanine or Glycine

Tolerance of each strain shown in Table 1 and Table 2 was measured as follows.

Glucose 1.0g / dl, Urea 0.15g / dl, Yuan 0.15g / dl, KH ₂ PO ₄ 0.1g / dl, K ₂ HPO ₄ 0.1g / dl, MgSO ₄ 7H ₂ O 0.01g / dl, CaCl ₂ 2H ₂ O 0.1 mg / dl, thiamine hydrochloride 10 μg / dl, biotin 3 μd / dl, and azaserine in the amounts shown in Table 1 or sulfanilamide in the amounts shown in Table 2, adjusted to pH 7.0 to 115 ° C. Sterilized cells in sterile water for 24 hours in a medium (peptone 1g / dl, homo extract 1g / dl, Nacl 0.5g / dl, pH 7.0) in a medium (20ml / 500ml flask) sterilized in Aseptically inoculated and flask shake culture for 24 hours.

In addition, the growth and production promoting effect shown in Table 3 was the same composition (except azaserine and sulfanilamide) of the same medium as the medium of which the resistance of the present invention strain CS850815 (KFCC 10183) to azaserine and sulfanylamide was measured. And alanine or glycine in the amounts shown in Table 3, respectively, and obtained in the same manner as the method of obtaining the data in Table 1 and Table 2 and expressed in the relative growth in each table.

As a method of producing and accumulating L-glutamic acid using the mutant strain of the present invention, a method employed in conventional culture of L-glutamic acid producing bacteria was used.

That is, as a carbon source, carbohydrates such as glucose, sucrose and sugarcane, molasses, beet molasses, and starch saccharification solution containing them are used as nitrogen sources such as ammonium salt, ammonia water, ammonia gas, and urea.

According to other needs, inorganic ions such as phosphate and magnesium salts were added to the medium, and micronutrients such as thiamine and biotin were appropriately used as necessary.

In addition, alanine or glycine was appropriately added to promote growth, and biotin-inhibiting agents such as antibiotics or nonionic surfactants were added to the medium according to conventional methods.

The culture was carried out under aerobic conditions, the incubation temperature was 30-40 ℃, pH during the culture was adjusted to 6-9, ammonia water or urea was used for pH adjustment.

L-glutamic acid was collected from the fermentation broth using ion exchange resin and other known methods.

Example 1

Glucose 10g / dl, KH ₂ PO ₄ 0.1g / dl, MgSO ₄ 7H ₂ O 0.04g / dl, FeSO ₄ 7H ₂ O 1mg / dl, MnSO ₄ 7H ₂ O 1mg / dl Thiamine hydrochloride 10μg Prepare a medium having a composition of / dl, biotin 30 μg / dl, CSL (as TN) 3 g / dl, alanine 10 mg / l (pH 7.0), and dispense 30 ml each into a 500 ml shake flask and heat at 115 ° C. for 10 minutes. Sterilized.

Mutant strain CS 850815 (KFCC 10183) and its parent strain of the present invention were inoculated in this medium, and shake cultured at 31.5 ° C. The pH of the culture solution during the cultivation was maintained at 6-9 by appropriately adding 40 g / dl of urea water.

6 hours after the start of the culture, penicillin was added to a concentration of 0.5-10 unit / l and fermentation was terminated after 30 hours.

The yield per unit of L-glutamic acid accumulated in the culture was obtained, and the results are shown in Table 4.

TABLE 4

Yield of L-Glutamic Acid

Example 2

Sugar cane molasses as reducing sugar 10 g / dl, KH ₂ PO ₄ 0.1 g / dl, MgSO ₄ · 7H ₂ O 0.04 g / dl, FeSO ₄ · 7H ₂ O 1 mg / dl, MnSO ₄ · 7H ₂ O 1 mg / dl, thiamine hydrochloride 10 μg / dl glycine 5 mg / l (pH 7.0) was prepared, the 30 ml each was dispensed into a 500 ml shake flask and heat sterilized for 10 minutes at 115 ℃.

The mutant strains and parent strains of the present invention were inoculated into the culture medium, and shaken at 31.5 ° C.

In addition, 40 g / dl of urea water was appropriately added to maintain the pH of the culture medium during culture.

After 6 hours from the start of the culture, polyoxyethylene sorbitan monopalmitic acid was added to a concentration of 0.01 g / dl, and fermentation was terminated after 30 hours of culture.

A large yield of L-glutamic acid accumulated in the culture was obtained, and the results are shown in Table 5.

TABLE 5

L-glutamic acid yield

Example 3

In the fermentation broth of Example 1, a medium prepared by adding beet molasses as a reducing sugar instead of 10 g / dl as a reducing sugar and 2 mg / l each of alanine and glycine as a substitute for 10 mg / l of alanine was added to 30 l jar fermenter. Into a pre-co-sterilized 2000 ml shake flask, 3 g / dl, KH ₂ PO ₄ 0.1 g / dl, MgSO ₄ 0.01 g / dl, FeSO ₄ · 7H ₂ O1 mg / dl, thiamine hydrochloride 10 μg / dl biotin 3 μg / Let the seed medium having the composition / dl and inoculated 500ml each, inoculated the mutant strain and the parent strain of the present invention at 115 ℃ and inoculated 1L seed culture medium incubated for 16 hours at 30 ℃ to maintain a pH 7.8 with ammonia water and stirring the aeration ( IVVM, 350 rpm).

After 6 hours from the start of the culture, polyoxyethylene sorbitan monopalmitic acid was added to a concentration of 0.05 g / dl, and the fermentation was terminated after 30 hours of culture.

A large yield of L-glutamic acid accumulated in the culture was obtained, and the results are shown in Table 6.

TABLE 6

L-glutamic acid yield

Example 4

In the fermentation broth of Example 1, the medium prepared by adding starch saccharified solution as reducing sugar instead of glucose, 10 μg / dl as biotinol 0.3 μg / dl, and glycine 5 mg / l instead of alanine was cultured in the same manner as in Example 3. After the end of fermentation, the yield of L-glutamic acid accumulated in the culture was obtained, and the results are shown in Table 7.

TABLE 7

L-glutamic acid yield

Claims (1)

Mutant CS850815 belonging to the genus Brevibacterium, resistant to purine nucleotide biosynthesis inhibitors azaserine and sulfanylamide and having the ability to produce L-glutamic acid, which is promoted by the addition of one or two of alanine or glycine KFCC 10183) culturing aerobically in a saccharide medium to collect L- glutamic acid produced in the medium to collect L- glutamic acid.