KR920005976B1 - Method for preparation of l-glutamire - Google Patents

Abstract

A L-glutamine is prepd. by fermenting corynetacterium MWM 891129, KFCC 10695 (I) with L-cysteine requirement at 30 deg.C under aerobic condition in the medium contg. 14 % glucose, 3.5 % ammonium chloride, 2 % ammonium sulfate, 4 % corn steep liquor, 0.1 % KH2PO4, 0.1 % K2HPO4, 0.002 % magnesium sulfate, 0.005 % ferrous sulfate, 0.002 % manganese sulfate, 0.5 % urea, 100 mg/l L- cysteine, 5 % calcium carbonate, etc., and separating from the culture broth. The (I) is prepd. by the mutation of corynebacterium sp. No. 462 with UV irradiation, NTG or DES. The yield of L-glutamine is high.

Description

Method for producing L-glutamine by microorganisms

In the present invention, when producing L-glutamine by fermentation method, strains of Corynebacterium genus isolated from soil were obtained by UV and NTG treatment (N-methyl-N'-methyl-, N-nitroso guanidine). As a result of investigating amino acid nutritional requirements of strains that cannot be grown in the smallest medium, the present invention relates to a method for producing L-glutamine by aerobic cultivation method using sugar as a main ingredient of mutant strains capable of growing by addition of L-cystine.

L-glutamine is a kind of amino acid, which is used as a nutrient, a medicine for treating digestive ulcers, an alcohol poisoning drug, a brain function improving agent, and the like, and recently, it has been spotlighted as a raw material for moisturizing cosmetics.

As a conventional method for producing L-glutamine by microorganisms, Japanese Patent Publication No. 39-7391 discloses a method of culturing L-glutamic acid producing strains in a medium containing excess cuticle source, and Japanese Patent Publication No. 49-31547. The method for separating and culturing sulfa resistant strains is to add antibiotics to the culture medium in Japanese Patent Publication No. 53-12490, and to add L-glutamine by adding antibiotics and surfactant to Japanese Patent Publication No. 51-15688. Methods for accumulating and producing L-glutamine are known from Korean Patent Publication No. 83-1260, using L-leucine and L-alanine demanding strains.

Separation method of the new strain used in the present invention has the nutritional requirements of L-cystine in the mutant strains obtained by carrying out the usual mutation treatment with strain NO462 of Corynebacterium producing L-glutamic acid isolated from soil When strain MWM-891129 (KFCC-10695) was added to a certain concentration of L-cystine in a fermentation broth, a strain was selected to generate and accumulate a large amount of L-glutamine.

In other words, strain NO 462 of Corynebacterium is used as a parent strain, and NTG (N-methyl-N'-methyl-N-nitrosoguanidine) or DES (diethyl sulfate), which is a conventional mutation treatment, that is, UV irradiation or chemically modified organic agent ), And then cultured in a medium containing L-cystine above a certain concentration, and then analyzing it. A strain having a requirement for L-cystine accumulates a larger amount of L-glutamine in the culture medium than the parent strain Corynebacterium NO 462. It was shown that this strain was isolated.

The composition of the first medium for isolation of the new strain was 3% glucose, 1% ammonium chloride, 0.1% potassium monophosphate, 0.2% potassium diphosphate, iron sulfate 2mg / l, thiamine hydrochloride 100μg / l, biotin 50 Equal to μg / l.

This chemical property of the present invention use modified strain MWM-891129 (KFCC-10695) obtained using this medium composition was found to have the properties shown in the following Tables 1 and 2.

TABLE 1

Growth Comparison between Parents (Corinebacterium NO 462) and Mutants (M`VM-891129).

++; Growth good, +; Normal growth,-: Poor growth

Note 1) Complete medium: 1% Fabton, 0.5% Yeast Extract, 0.5% Juicy, 0.25% Sodium Chloride, Note 2) Add 100 mg / L of L-cystine.

As shown in Table 1, MWM-891129 (KFCC-10695), which requires L-cystine, was isolated by obtaining amino acids necessary for growth from the amino acid pool among the strains that were unable to grow in the minimum medium.

As a result of comparative analysis of the growth and L- glutamine production by the L-cystine addition concentration of the new strain of the present invention screened and separated in this manner is as shown in Table 2.

TABLE 2

Growth and glutamine accumulation (mg / ml) of strains by L-cystine addition.

Note (1) Growth: Measure the absorbance at dilution 610nm culture medium cultured for 72 hours in a separate medium.

Note (2) Gln accumulation amount: L-glutamine accumulation amount (mg / ml) incubated for 72 hours in a fermentation medium (Example 1).

As shown in Table 2, the optimum concentration of cystine of the mutant strain MWM-891129 was found to be 50-100 mg / l.

The mechanism of metabolism that L-cystine promotes L-glutamine biosynthesis in strain MWM-891129 (KFCC-10695) used in the present invention is not exactly known, but as shown in Figure 1, the metabolism process of L-cystine biosynthesis As a result, the accumulation of L-glutamine is expected to improve somewhat.

That is, it is assumed that the metabolism of L-serine and acetyl CoA, which is a kind of amino acid, does not flow toward L-cystine, thereby promoting the accumulation of L-glutamine.

Culture conditions for L-glutamine fermentation include glucose, raw sugar, and starch hydrolyzate as carbon sources, and nitrogen sources include ammonia gas, ammonia water, ammonium chloride, yuan, urea, ammonium phosphate, and other natural nutrients. It can be used if the medium is mixed with an inorganic metal salt.

The culture method is incubated for 2-3 days aerobicly while maintaining the pH at 5.5-7.5 within the temperature of 30 ℃.

L-glutamine accumulated in the culture complete solution is adsorbed and separated by an ion exchange resin according to a conventional method to obtain an eluate, and purified and treated with ethanol to obtain L-glutamine crude crystals.

It will be described in detail in the following Examples.

Example 1

Strains used: Corynebacterium MWM-891129 (KFCC-10695).

Species medium composition: glucose 5%, ammonium sulfate 0.5%, gravy 0.25%, first carry phosphate 0.05%, second carry phosphate 0.05%, urea 0.3%, magnesium sulfate 0.01%, biotin 20㎍ / ℓ, thiamine hydrochloride 1mg / l, pH7.0.

Fermentation medium composition: 14% glucose, 3.5% ammonium chloride, 2% yuan, 4% corn slicker (CSL), 0.1% monobasic phosphate, 0.1% dibasic phosphate, 0.002% magnesium sulfate, 0.005% iron sulfate, Manganese sulfate 0.002%, zinc sulfate 0.002%, thiamine hydrochloride, 3 mg //, urea 0.5%, L-cystine 100 mg //, calcium carbonate 5% (additionally sterilized), pH7.2.

Cultivation method: Dispensing the seed medium having the composition of the medium in a sterile 500ml flask and 50ml each to autoclave at 120 ℃, 15 minutes and then inoculated the new strain MWM-891129 of the present invention and incubated shaking for 30 hours at 30 ℃ This is used as the seed culture medium.

20 ml of the fermentation broth was dispensed into 500 ml shake flasks, inoculated with 2 ml of the spawn culture medium prepared by autoclaving at 120 ° C. for 15 minutes, and then incubated at 30 ° C. for 72 hours.

After completion of the fermentation, the amount of L-glutamine accumulation in the culture was 45.5 mg / ml.

Example 2

In the fermentation medium of Example 1, except for calcium carbonate, all the medium was inserted into a 30 L small fermentation tank 16 / directly sterilized and inoculated with 900 ml of the previously prepared seed culture solution and incubated at 30 ° C., 350 rpm, and 1 vvm for 72 hours. The pH was adjusted between 6.8 and 6.0. After fermentation, L-glutamine accumulation was 54.7 mg / ml.

Example 3

Instead of glucose in the same medium as in Example 2, starch hydrolysates were added in the same manner as in Example 1 using a fermentation broth added with 14% glucose.

At the end of fermentation, L-glutamine accumulation in the culture was 51.2 mg / ml.

Claims (1)

In the production of L-glutamine by microorganisms, Corynebacterium MWM-891129 (KFCC-10695), an L-cystine-required strain that requires L-cystine for growth, is aerobicly cultured in a nutrient medium containing sugar as a main ingredient. Method for producing L- glutamine by a microorganism, characterized in that.