KR830001258B1 - Method for producing L-glutamine by microorganisms - Google Patents

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Description

Method for producing L-glutamine by microorganisms

The present invention obtains glutamic acid-producing strains of the genus Corynebacterium isolated from soil when producing L-glutamine by fermentation method, and transforms this strain into parent strains, thereby requiring L-loycin for growth and promoting growth by alanine. The present invention relates to a method for producing L-glutamine using a new strain, wherein the L-glutamine producing strain is cultured aerobicly in a medium containing saccharides as a main ingredient to accumulate a large amount of L-glutamine and recovers and recovers it.

L-glutamine is a kind of amino acid, which is widely used in medicine for nutrition, digestive ulcer, alcoholism, and brain function.

The conventional method for producing L-glutamine by fermentation is a method of culturing glutamic acid producing strains in Japanese Patent Publication No. 39-7391 in a medium containing an excess of nitrogen source, and Japanese Patent Publication No. 51-16588 for antibiotics. In addition, a method of adding a surfactant into a culture medium or a method of separating and culturing sulfa resistant strains in Japanese Patent Publication No. 49-81587 is known. However, in a nitrogen-excess medium, the normal growth of the strain is poor, so that a large amount of metal salt In addition to removing the growth inhibition of bacteria caused by excess nitrogen, the culture of sulfa resistant strains and the cultivation of antibiotics or surfactants, or the growth conditions and strains of the strains Depending on the growth conditions, the treatment time, concentration, etc. of the drug is different, there were various industrially difficult problems .

In order to solve these problems, the present invention has conducted fermentation for the accumulation of L-glutamine in nutrient medium containing sugar as a main ingredient by using nutrient-constituting mutants in Corynebacterium. In addition, the invention is a new and industrially useful method that does not require the addition of sulfa or antibiotics in culture.

Specifically, the present invention requires the growth of L- with the production of L-glutamine by the microorganisms and strains of the genus Corynebacterium MWM-791012 (KFCC-10032) having the characteristics that the growth is promoted by alanine ) Is a method invention characterized by accumulating a large amount of L- glutamine in the culture medium by aerobic culturing in a nutrient medium containing sugar as a main ingredient.

The new strain of the present invention mutated the glutamic acid producing strain of Corynebacterium NO 462 isolated from the soil to the parent strain to isolate various nutritional requirements, antibiotic resistant strains, L- glutamine analog resistant strains, etc. -While studying the fermentative production method of glutamine, it is known that the new strain MWM-791012 (KFCC-10032), which requires L-leucine for growth and promotes growth by alanine, significantly increases L-glutamine production accumulation. Will be completed.

Separation method of the new strain of the strain used in the present invention is a strain strain of the genus Corynebacterium producing glutamic acid isolated from the soil as a parent strain in the normal mutation treatment, that is, UV irradiation or NTG (N-methyl-N'-methyl- Chemical strains such as N-nitrosoguanidine) and DES (diethsulfide) were treated with penicillin enrichment method or replica method, and then cultured in isolated liquid medium and analyzed for accumulation of L-glutamine. To isolate the new strain of the present invention.

, 황산망간 10mg/

, 치아민염산염 1mg/

, 육즙 0.5%, L-로이신 50mg/

, 알라닌 50mg/

, pH7.2 탄산칼슘 5%(별도멸균첨가)이다.The composition of the separation medium was 10% glucose, 3% ammonium chloride, 1% monobasic phosphate, 1% dibasic phosphate, and 500 mg magnesium sulfate /

Manganese Sulfate 10mg /

Chiamine Hydrochloride 1mg /

, Juicy 0.5%, L-leucine 50mg /

Alanine 50mg /

, pH7.2 calcium carbonate 5% (additional sterilization).

In the culture method in the separation medium, 10 ml of the medium was inoculated into 50 ml triangle flasks, and the inoculated mutant strains were inoculated, followed by shaking culture for 30 hours at 30 ° C.

The results of examining the bacteriological properties of the present invention strain isolated and screened in this manner are as described in detail below.

The experimental method was performed according to the method described in a manual of microbiblogical method (H.J. Conn and M.W. Jennison, Society of American bacteriologist, 1957).

Microscopic appearance

19) The new strain of the present invention was inoculated into a minimum medium based on Gray and Tatum, but did not grow and was grown by adding leucine to the minimum medium.

Therefore, it was found that the new strain of the present invention requires leucine to grow, and when leucine and alanine are added together, growth is promoted more than when leucine is added alone, and thus, the new strain of the present invention requires leucine to grow. It is certain that growth is promoted by.

In addition, when the parent strain of the present invention strain was cultured in an aerobic method containing saccharide medium containing glutamine, the amount of glutamine accumulated only as a trace, but the accumulation of glutamic acid was remarkable. However, when the new strain of the present invention was cultured in the same medium, the accumulation of glutamic acid was hardly observed, but the production of glutamine was significantly increased.

20) Production of organic acid: Trace amounts of α-ketoglutal acid and lactic acid are produced in glucose medium

21) party to sucrose

As a result of classifying and identifying the new strain of the present invention having the above bacteriological properties using Burgy's Manual of Determinative Bacteriology, edition 7,8, it was found that it belongs to the genus Corynebacterium MWM. Named -791012 and compared with the physiological properties of the parent strain before the mutation treatment, as shown in the following Table 1 showed a different physiological properties of the strain.

TABLE 1

[Bacterial Properties of Chinju (Corinebacterium NO 462) and Mutant Juju (MWM-791012)]

, 치아민 1mg/

, 미량원소혼합액 1ml (미량원소혼합액 조성 : 소딩움테트라보레이트 90mg, 암모니움멜리보데이트 40mg, 염화철 1g, 황산아연 800mg등을 혼합하여 증류수 1

에 용해한것)Note 1) Minimum medium: Gray and Tatum medium: 5% glucose, 0.5% ammonium chloride, 0.1% ammonium nitrate, 0.2% sodium sulfate, 0.01% magnesium sulfate, 0.1% primary phosphate, 0.3% secondary phosphate Biotin 10r /

, Chimine 1mg /

, 1 ml of trace element mixture (mixture of trace elements: Sodiumum tetraborate 90mg, ammonium melibodate 40mg, iron chloride 1g, zinc sulfate 800mg, etc., distilled water 1

Dissolved in)

Note 2) +++: Good growth +: Normal growth ±: Growth failure-: Inability to grow

*: Analyzed by incubating for 72 hours in fermentation broth (see Example 1).

As described in Table 1, the mutant strain MWM-791012 of the present invention is a L- leucine-required strain and is characterized by promoting growth by alanine and accumulating a large amount of glutamine.

The new mutant of the present invention is not yet theoretically clear on the cause of the significant accumulation of L- glutamine in the culture medium, but alanine, L that accumulates in the specific enzyme activity of L- glutamine biosynthesis or accumulates as a by-product of L-glutamine fermentation Synthetic enzymes such as leucine may be damaged and affect the activity of L-glutamine synthesis enzymes.

As fermentation medium used in the present invention, industrially inexpensive saccharide raw materials, such as glucose, fructose, waste molasses, and starch hydrolyzate, can be used, and as inorganic mineral sources, ammonia gas, ammonia water, ammonium chloride, yuan, urea, ammonium phosphate, etc. As a natural nutrient, corn steep liquor (CSL), gravy, yeast extract, peptone, soybean meal can be used.Inorganic salts are magnesium sulfate, zinc chloride, primary phosphate, secondary phosphate, manganese sulfate, etc. It is possible to use a mixed medium.

The culture method is incubated for 2-3 days while maintaining the temperature of 25-35 ℃, pH5.5-7.5, the accumulated L- glutamine is adsorbed to the ion exchange resin by the conventional method, the eluent is obtained and treated with ethanol. Purification to obtain crude crystals.

The following experimental method is described in detail in the Examples.

Example 1

Use strain: Corynebacterium MWM-791012 (KFCC-10032)

,pH7.0Species medium: glucose 5%, peptone 1%, yeast extract 1%, sodium chloride 0.25%, urea 0.3%, biotin 30r /

, pH7.0

, 황산철 10mg/

, 황산망간 10mg/

, 황산아연 10mg/

, 치아민염산염 1mg/

, 요소 0.5%, pH7.0, L-로이신 400mg/

, 알라닌 30mg/

, 탄산칼슘 5%(별도멸균첨가) pH7.2Fermentation medium: glucose 10%, ammonium chloride 3.5%, yuan 2%, corn steep liquor (CSL) 0.5%, gravy 0.7%, potassium monophosphate 0.2%, potassium diphosphate 0.2%, magnesium sulfate 300mg /

Iron sulfate 10mg /

Manganese Sulfate 10mg /

Zinc sulfate 10mg /

Chiamine Hydrochloride 1mg /

Urea 0.5%, pH7.0, L-Leucine 400 mg /

Alanine 30mg /

, Calcium carbonate 5% (additional sterilization) pH7.2

Cultivation method: 50 ml of the seed medium was dispensed into 500 ml shake flasks and autoclaved according to the conventional method, followed by inoculation of the strain MWM-791012 of the present invention, followed by shaking culture at 30 ° C. for 24 hours to make a seed culture. 20 ml of the fermentation broth was inserted into a 500 ml shake flask and autoclaved at 120 ° C. for 10 minutes, followed by 2 ml inoculation of the prepared seed culture solution and incubated at 30 ° C. for 72 hours.

After fermentation, L-glutamine accumulation in the culture was 49..6 mg / ml.

Example 2

를 사입하여 멸균한 5

소형 발효조에 미리 준비한 종균배양액을 200ml접종하고 600rpm, 1.5vvm의 조건으로 30℃에서 48시간 배양한다. pH조절은 암모니아수로 6.0-7.0으로 조절한다.Medium 2 without calcium carbonate in the fermentation broth of Example 1

Injected and sterilized 5

Inoculate 200 ml of the seed culture medium prepared in advance in a small fermenter and incubate at 30 ° C. for 48 hours under conditions of 600 rpm and 1.5 vvm. pH control is adjusted to 6.0-7.0 with ammonia water.

At the end of the culture, L-glutamine accumulation was 56.3 mg / ml.

을 균체를 제거한후 상법에 따라 이온교환수지에 흡착 분리하여 용리액을 얻고 여기에 에탄올을 처리 L-글루타민조결정 48.4g을 얻었다.Fermentation complete amount 1

After removing the cells, the eluate was obtained by adsorptive separation into an ion exchange resin according to a conventional method, and 48.4 g of L-glutamine crude crystals were obtained by treating ethanol.

Example 3

를 5

소형 발효조에 사입하여 120℃에서 15분간 멸균한다.Fermentation medium 2 added in terms of glucose to 10% glucose instead of glucose in the fermentation medium of Example 2

5

Insert into a small fermenter and sterilize for 15 minutes at 120 ℃.

Inoculate 200ml of the previously prepared spawn culture solution and incubate at 30 ° C. for 48 hours under conditions of 600rpm and 1.5vvm. Other culture methods are carried out in the same manner as in Example 2.

At the end of fermentation, L-glutamine accumulation in the culture was 52.7 mg / ml.

Example 4

를 5

소형발효조에 사입하여 120℃에서 15분간 멸균한다.Fermentation medium 2 added to convert the starch hydrolyzate to 10% glucose instead of glucose in the fermentation medium of Example 2

5

Insert into a small fermentation tank and sterilize at 120 ℃ for 15 minutes.

Inoculate 200ml of the prepared seed culture medium in advance and incubate at 30 ° C. for 48 hours under conditions of 600rpm and 1.5vvm. Other culture methods are carried out in the same manner as in Example 2.

At the end of fermentation, L-glutamine accumulation in the culture was 53.4 mg / ml.

Claims (1)

Nutrition medium containing sugar as a main ingredient of the strain MWM-791012 (KFCC-10032) in the corynebacterium, which requires L-leucine for growth and promotes growth by alanine in producing L-glutamine by microorganisms. A method for producing L-glutamine by fermentation, characterized in that the aerobic culture to accumulate a large amount of L- glutamine in the culture medium.