KR0134131B1 - Cephalosporium which produces cephalosporin and process for cephalosporin - Google Patents
Cephalosporium which produces cephalosporin and process for cephalosporinInfo
- Publication number
- KR0134131B1 KR0134131B1 KR1019940033568A KR19940033568A KR0134131B1 KR 0134131 B1 KR0134131 B1 KR 0134131B1 KR 1019940033568 A KR1019940033568 A KR 1019940033568A KR 19940033568 A KR19940033568 A KR 19940033568A KR 0134131 B1 KR0134131 B1 KR 0134131B1
- Authority
- KR
- South Korea
- Prior art keywords
- cephalosporin
- kfcc
- cephalosporium
- microorganism
- potassium cyanide
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
- C12P35/06—Cephalosporin C; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/745—Cephalosporium ; Acremonium
- C12R2001/75—Cephalosporium acremonium ; Acremonium strictum
Abstract
Description
본 발명은 세파로스포린 C를 생산하는 미생물및 이를 이용한 세파로스포린 C의 제조방법에 관한 것으로, 좀 더 구체적으로는 세파로스포리움 아크레모니움 KFCC 10782의 변이주로서 미생물의 미토콘드리아에서 일어나는 전자전달계의 호흡억제제인 시아나이드 (CN)에 대해서 내성을 가지며 탄소원및 에너지원으로써 혼합오일을 이용하여 고농도의 세파로스포린 C를 생산하는 미생물과 이러한 미생물을 이성화 액당, 혼합오일, 콘스팁리커, 땅콩분등이 함유된 배지에서 발효 배양하여 세파로스포린 C를 고농도로 생산하는 세파로스포린 C의 제조방법에 관한것이다.The present invention relates to a microorganism producing cephalosporin C and a method for producing cephalosporin C using the same. More specifically, the present invention relates to an electron transport system that occurs in the mitochondria of microorganisms as a mutant of the Separosporium acremonium KFCC 10782. Resistant to cyanide (CN), a respiratory inhibitor, and microorganisms that produce high concentrations of cephalosporin C using mixed oils as carbon and energy sources, and these microorganisms, such as isomerized sugars, mixed oils, cornspicker, peanut powder, etc. It relates to a method for producing cephalosporin C fermentation culture in the medium containing this to produce a high concentration of cephalosporin C.
종래 미생물을 이용하여 세파로스포린 C를 생산하는 방법의 경우, 미생물의 산소 요구성이 매우 높아 발효가 진행됨에 따라 용존 산소량을 30% 수준으로 유지시켜 주어야 한다. 이를 위해 발효조의 RPM과 내압을 상승시켜야 하고 이를 충족시키지 못할 경우 세파로스포린 C의 생산성이 저하되는 단점이 있다.In the case of the conventional method for producing C Separosporin C using a microorganism, the oxygen demand of the microorganism is very high, so as the fermentation proceeds to maintain the dissolved oxygen amount to 30% level. To this end, it is necessary to increase the RPM and internal pressure of the fermenter, and if it fails to meet this, there is a disadvantage in that the productivity of Separosporin C is reduced.
이에 본 발명자등은 상기 지적된 문제점을 해결하고자, 미행물의 미토콘드리아에서 일어나는 전자전달계의 호흡억제제인 시아니이드 (CN)에 대해서 내성을 가지는 변이주들 중에서 산소요구성이 낮고 세파로스포린 C를 고농도로 생살할 수 있는 미생물을 획득하여 본 발명을 완성하였다.In order to solve the above-mentioned problems, the inventors of the present invention have a low oxygen urine composition and have a high concentration of cephalosporin C among the mutants having resistance to cyanide (CN), which is a respiratory inhibitor of an electron transport system occurring in mitochondria of the microorganisms. The present invention was completed by obtaining a viable microorganism.
본 발명은 호흡억제제인 포타슘시아나이드의 농도가 10μg/ml 이상인 조건에서 생육가능하고, 산소요구성이 낮은 세파로스포린 C를 생산하는 미생물인 세파로스포리움 아크레모니윰 KFCC-10845을 이성화액당, 혼합오일, 땅콩분, 콘스팁리커등이 함유된 배지중에서 배양하여 세파로스포린 C를 고농도로생산함을 특징으로 한다.The present invention is a microorganism capable of growing under the condition that the concentration of potassium cyanide, a respiratory inhibitor, is 10 μg / ml or more, and produces cephalosporin C with low oxygen urine composition. It is characterized by the production of cephalosporin C at a high concentration by culturing in a medium containing mixed oil, peanut powder, corn steep liquor and the like.
본 발명의 미생물은 세파로스포리움 아크레모니움 KFCC 10782를 친주로 하여 자외선 조사, N-메틸-N'-니트로소구아니딘 (NTG)등의 변이유발제로 통상적인 방법에 따라 처이한 후 포타슘시아나이드 (KCN)가 1-50μg/ml의 농도로 함유되어 있는 액체 또는 한천배지 (포도당 10g/L, 황산마그네숨 0.5g/L, 인산제1칼륨 0.5g/L, 인산제2칼륨 1g/L, pH 7.0)에서 7-10일간 배양하였다. 이때 고농도의 포타슘시아나이드에 대해 내성을 가지는 균주는 생육이 가능하므로, 이에 포타슘시아나이드가 최소저해농도 (Minimun Inhibitory Concentration, MIC)로 함유된 배지에서 자라는 균주를 분리하였다. 그런 다음 포타슘시아나이드 내성균주중 산소 요구성이 낮고 세파로스포린 C 생산성이 향상된 변이주를 분리하여 이를 1994년 월 일자로 한국종균협회에 기탁하였다 (균주명 : 세파로스포리움 아크레모니움, 기탁번호 ; KFCC-10845).The microorganism of the present invention is potassium cyanide after being treated according to a conventional method with a mutagenesis agent such as UV irradiation, N-methyl-N'-nitrosoguanidine (NTG), with Separosporium acremonium KFCC 10782 as a parent. Liquid or agar medium containing (KCN) at a concentration of 1-50 μg / ml (glucose 10g / L, magnesium sulfate 0.5g / L, potassium potassium phosphate 0.5g / L, potassium diphosphate 1g / L, pH 7.0) for 7-10 days. At this time, since a strain resistant to high concentration of potassium cyanide can be grown, a strain growing in a medium containing potassium cyanide at a minimum inhibitory concentration (MIC) was isolated. Then, mutant strains with low oxygen demand and improved cephalosporin C productivity among potassium cyanide-resistant strains were isolated and deposited with the Korean spawn association as of January 1994 (Corporate name: Sepharoseporium acremonium, Deposit No .; KFCC-10845).
상기 균주의 균학적 성질은 시아나이드 내성, 산소 요구성및 세파로스포린 C 생산성을 제외하고는 친주의 성질과 동일하다. 본 발명에 따른 미생물 KFCC-10845의 특성은 하기표에 기재된 바와 같다.The bacteriological properties of the strains are identical to those of the parent strains except for cyanide resistance, oxygen demand and cephalosporin C productivity. The characteristics of the microorganism KFCC-10845 according to the present invention are as described in the following table.
1. 포타슘시아나이드에 대한 내성1. Resistance to potassium cyanide
KFCC 10782와 KFCC-10845을 포타슘시아나이드의 함량을 변화시킨 한천배지중에서 배양하였으며, 생육여부에 따라 포타슘시아나이드에 대한 내성을 하기 표 1에서 나타낸 바와 같이 비교하였다.KFCC 10782 and KFCC-10845 were cultured in agar medium with varying content of potassium cyanide, and resistance to potassium cyanide was compared as shown in Table 1 according to growth.
+ ; 생육, - ; 생육치 못함+; Growth,-; Lack of growth
2. 산소 소비량의 측정2. Measurement of oxygen consumption
KFCC 10782와 KFCC-10845의 균체당 산소 소비량을 비교하기 위하여 플라스크에서 배양한 동량의 균체로 Warburg 장치를 이용하여 산소 소비량을 측정하여 그 상대값을 표 2에 나타내었다.In order to compare the oxygen consumption per cell of KFCC 10782 and KFCC-10845, oxygen consumption was measured using the Warburg apparatus with the same amount of cells cultured in a flask, and the relative values are shown in Table 2.
3. 산소 소모속도및 이산화탄소 배출속도 비교3. Comparison of oxygen consumption rate and carbon dioxide emission rate
KFCC 10782와 KFCC-10845의 산소 요구성을 비교하기 위하여, 산소 소모속도 (Oxygen Uptake Rate)와 이산화탄소 배출속도 (Carbon dioxide Evolution Rate)을 30L 발효조에서 측정하여 표 2에 나타내었다.To compare the oxygen requirements of KFCC 10782 and KFCC-10845, the oxygen uptake rate and carbon dioxide evolution rate were measured in a 30L fermenter and are shown in Table 2.
4. 내압 및 RPM비교4. Internal pressure and RPM comparison
KFCC 10782와 KFCC-10845를 30-L 발효조에서 배양하였으며, 내압및 RPM을 측정하여 산소 요구성을 표 4에서 나타낸 바와 같이 간접적으로 비교하였다.KFCC 10782 and KFCC-10845 were incubated in a 30-L fermenter, and the oxygen demand was measured and the oxygen demand was indirectly compared as shown in Table 4.
이하, 본 발명을 하기 실시예에서 좀 더 구체적으로 설명한다.Hereinafter, the present invention will be described in more detail in the following Examples.
[실시예]EXAMPLE
본 발명에서 사용한 배지성분은 하기표 5-7과 같다.Medium components used in the present invention are shown in Table 5-7.
친주인 KFCC 10782와 본 발명 균주인 KFCC-10845을 하기 발효 방법에 따라 배양하여 세파로스포린 C 생성량을 측정하였다.The parent strain KFCC 10782 and the strain KFCC-10845 of the present invention were cultured according to the following fermentation method, and the amount of cephalosporin C was measured.
상기 표 5의 1차 종배지를 50ml씩 500ml 용량의 진탕용 삼각플라스크에 넣고, 살균시킨 후 균주를 식균하여 30℃에서 220 rpm으로 90-96시간 1차 종배양하였다.The primary seed medium in Table 5 was placed in a 500 ml shake Erlenmeyer flask for 50 ml each, and after sterilization, the strains were phagocytized and primary seed culture was carried out at 30 ° C. at 220 rpm for 90-96 hours.
상기 표 6의 2차 종배지를 7-L 용량의 시험용 발효조에 각 5-L씩 분주하고 120℃에서 30분간 살균, 냉각시킨후 1차 종배양에서 얻은 배양완료액을 15 ml씩 접종한 다음, 매분당 0.5-1.0L의 공기를 공급하면서 30℃에서 600-900rpm으로 80-90시간 종배양하였다.After dispensing 5-L each of the secondary seed medium in Table 6 into a 7-L test fermenter, sterilizing and cooling at 120 ° C. for 30 minutes, and inoculating 15 ml of the culture completion solution obtained in the primary seed culture. , 80-90 hours incubation at 600-900rpm at 30 ℃ while supplying 0.5-1.0L of air per minute.
상기 표 7의 발효배지를 30L용량의 시험용 발효조에 19.4L씩 분주하고 120℃에서 30분간 살균, 냉각시킨후 2차 종배양액을 1.6L씩 접종한 다음, 매분당 0.5-1.0L의 공기를 공급하면서 300-600rpm, 25-30℃에서 140시간 발효 배양하였다. 배양중 잔존 혼합 오일 농도가 4-5%가 되면 살균된 혼합오일을 수시로 공급하였다. 배양중 pH는 암모니아수를 사용하여 5.0-6.0으로 조절하였다. 발효 완료후 배지중의 세파로스포린 C의 농도는 다음과 같다.The fermentation broth of Table 7 was dispensed 19.4L into a 30L test fermenter, sterilized and cooled at 120 ° C for 30 minutes, and then inoculated with 1.6L of secondary seed culture solution, and then supplied with 0.5-1.0L of air per minute. Fermentation was carried out for 140 hours at 300-600rpm, 25-30 ℃. When the residual mixed oil concentration in the culture was 4-5%, sterilized mixed oil was frequently supplied. The pH of the culture was adjusted to 5.0-6.0 using ammonia water. After the fermentation was completed, the concentration of cephalosporin C in the medium was as follows.
이와 같이 본 발명 미생물 KFCC-10845은 탄소원및 에너지원으로서 혼합 오일을 이용하여 세파로스포린 C를 고농도로 생산할 수 있었다.As described above, the microorganism KFCC-10845 of the present invention was able to produce cephalosporin C at a high concentration using a mixed oil as a carbon source and an energy source.
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| KR1019940033568A KR0134131B1 (en) | 1994-12-06 | 1994-12-06 | Cephalosporium which produces cephalosporin and process for cephalosporin |
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| KR1019940033568A KR0134131B1 (en) | 1994-12-06 | 1994-12-06 | Cephalosporium which produces cephalosporin and process for cephalosporin |
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| KR960023039A KR960023039A (en) | 1996-07-18 |
| KR0134131B1 true KR0134131B1 (en) | 1998-04-14 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100446108B1 (en) * | 1997-10-24 | 2004-10-26 | 씨제이 주식회사 | Cephalosporin c-producing microorganism having improved lipase activity and method for producing cephalosporin c using the same |
| KR100446110B1 (en) * | 1997-10-24 | 2004-10-28 | 씨제이 주식회사 | Cephalosporin c-producing microorganism having tolerance against high concentration of glycerol |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111850078A (en) * | 2020-07-21 | 2020-10-30 | 伊犁川宁生物技术有限公司 | Fermentation method of cephalosporin C |
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1994
- 1994-12-06 KR KR1019940033568A patent/KR0134131B1/en not_active IP Right Cessation
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100446108B1 (en) * | 1997-10-24 | 2004-10-26 | 씨제이 주식회사 | Cephalosporin c-producing microorganism having improved lipase activity and method for producing cephalosporin c using the same |
| KR100446110B1 (en) * | 1997-10-24 | 2004-10-28 | 씨제이 주식회사 | Cephalosporin c-producing microorganism having tolerance against high concentration of glycerol |
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| KR960023039A (en) | 1996-07-18 |
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