KR960014621B1 - Streptomyces lavendofoliae dk-506, and processing method of aclacinomycin-a - Google Patents
Streptomyces lavendofoliae dk-506, and processing method of aclacinomycin-a Download PDFInfo
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Abstract
Description
제1도는 HPLC를 이용한 아클라시노마이신의 분리 및 확인.1 shows the isolation and identification of aclacinomycin using HPLC.
본 발명은 스트렙토마이세스(Streptomyces)속에 속하는 신규 미생물 및 이를 이용하여 항암제인 아클라시노마이신(aclacinomycin) A를 제조하고 이를 분리정제하는 방법에 관한 것이다. 아클라시노마이신은 안쓰라싸이클린(anthracycline) 계열의 항생물질로 항생작용 외에 항암작용을 갖는 매우 유용한 물질이기 때문에 이에 대한 연구개발이 매우 시급한 실정이다. 아클라시노마이신은 그 구조에 따라 크게 A와 B로 나누어지며 실제 미생물의 배양시에는 이외에 비슷한 구조를 갖는 여러물질들이 혼재되어 생성된다. 이들중 아클라시노마이신 B는 A에 비하여 부작용이 심하고 항 종양 활성이 낮기 때문에 실제 임상에 적용되기가 어려우며 기타 물질들은 그 생산성이 매우 낮기 때문에 연구자체가 미진한 상태이다. 아클라시노마이신은 세개의 데옥시피라노즈(deoxypyranose) 잔기로 구성되어 있으며 DNA 염기 사이의 인터킬레이션(intekoalation)으로 인해 핵산의 합성을 저해함으로서 사이토톡식(cytotoxic)한 작용을 나타낸다. 또한 같은 안쓰싸이클린 계열의 다우노루비신(daunorubicin),독소루비신(doxorubicin)그리고 칼미노마이신(carminomycin)등과는 달리 아클라시노마이신은 RNA의 합성을 선택적으로 저해하는 효과를 나타낸다. 아클라시노마이신은 넓은 항암작용을 나타내는 물질로서 암이나 백혈병등의 치료에도 효과가 있으며 다우노루비신이나 독소루비신에 비하여 부작용이 현저하게 적기 때문에 매우 각광을 받는 새로운 항암물질이다. 종래에 알려져 있는 미생물 발효에 의한 아클라시노마이신의 제조방법으로는 스트렙토마이세스 갈릴레우스(Streptomyces galilaeus)를 배양하는 방법(U.S.Pat.NO.3,988,315)이 알려져 있다.The present invention relates to a novel microorganism belonging to the genus Streptomyces, and a method for preparing and separating and purifying aclacinomycin A, an anticancer agent, using the same. Aklacinomycin is an anthracycline family of antibiotics, and since it is a very useful substance having anti-cancer activity in addition to antibiotics, the research and development of this is very urgent. Aclacinomycin is largely divided into A and B according to its structure. In addition, in the cultivation of microorganisms, various substances having a similar structure are mixed. Among these, aclosinomycin B has a severe side effect and low antitumor activity compared to A, so it is difficult to be applied to the actual clinical practice, and other substances have very low productivity. Aclacinomycin is composed of three deoxypyranose residues and exhibits cytotoxic activity by inhibiting the synthesis of nucleic acids due to intertelation between DNA bases. In addition, unlike the same cyclin-type daunorubicin, doxorubicin, and carminomycin, aclacinomycin has an effect of selectively inhibiting the synthesis of RNA. Aclaccinomycin is a new anticancer substance that has a wide anticancer activity and is effective in treating cancer and leukemia, and has much less side effects than daunorubicin or doxorubicin. As a known method for producing aclacinomycin by microbial fermentation, a method of culturing Streptomyces galilaeus (U.S. Pat. NO. 3,988, 315) is known.
이에 따르면 스트렙토마이세스 갈릴레우스는 동일한 배양조건에서 A와 B를 비롯하여 약 18개 정도의 구조 유사물질을 생산하는 것으로 밝혀졌으며 이들의 생산성은 매우 낮아 주 산물인 아클라시노마이신 A의 경우 46mg/1, B의 경우 23mg/1로 나타났다. 따라서 이 균을 이용하여 경제성을 고려한 생산에는 무리가 따르며 생산성이 더욱 증가된 새로운 생산 균주가 필요하다.It has been shown that Streptomyces galileus produces about 18 structural analogues, including A and B, under the same culture conditions, and their productivity is very low and 46 mg / A for the main product, Aklacinomycin A. 1, B was 23mg / 1. Therefore, economical production using this bacterium requires new production strains with increased productivity and increased productivity.
본 발명자들은 여러가지 스트렙토마이세스에 속하는 균주를 대상으로 발효법에 의한 아클라시노마이신 생산균주를 조사한 결과 스트렙토마이세스 라벤도폴리아(Streptomyces lavendofoliae, ATCC 15872)가 소량의 아클라시노마이신 A,B 및 Y를 생산함을 알고 생산성 향상을 위해 돌연변이 방법으로 처리하였다. 그 결과 새로운 미생물인 스트렙토마이세스 라벤도폴리아 DK-506(한국과학기술원 유전공학연구소 유전자은행에 기탁)이 모균주보다 월등히 많은 양의 아클라시노마이신 A를 균체내와 배지중에 축적함을 확인하였다.The inventors of the present invention examined the strains of aclosinomycin produced by the fermentation method of strains belonging to various Streptomyces, Streptomyces lavendofoliae (ATCC 15872) is a small amount of alaccinomycin A, B and Y Knowing to produce the was processed by the mutation method to improve productivity. As a result, the new microorganism Streptomyces Ravendofolia DK-506 (deposited to the Genetic Research Institute of Korea Institute of Science and Technology Genetics) accumulates a much larger amount of aclacinomycin A in the cell and medium than the parent strain. .
본 발명에 사용한 미생물은 아클라시노마이신 생성균인 야생주 스트렙토마이세스 라벤도폴리아(ATCC 15872)를 친주로 하고 자외선 조사나 NTG(N-메칠-N'-니트로-N-니트로소구아니딘)등에 의한 통상의 변이처리 방법을 사용하여 친주보다 많은 양의 아클라시노마이신 A를 생산하는 신 균주 DK-506(KCTC 8537P)을 분리하였다.The microorganisms used in the present invention are wild-type Streptomyces lavendofolia (ATCC 15872), which is an acecinomycin-producing bacterium, and is irradiated with ultraviolet rays or NTG (N-methyl-N'-nitro-N-nitrosoguanidine). Conventional mutagenesis methods were used to isolate the new strain DK-506 (KCTC 8537P), which produces a greater amount of aclacinomycin A than the parent strain.
본 발명에서 변이주의 분리방법의 예를들면 완전고체배지에서 30℃에서 6-7일간 배양된 균체에 멸균된 증류수와 유리섬유(glass wool) 여과기를 이용하여 포자만을 모은 후 0.05M(Tris-Malate) 완충용액(pH 6.5)으로 3회 세척하여 동일 완충용액으로 포자의 농도가 106-108/ml 되도록 한 다음 NTG를 500㎍/ml 되도록 가하여 30℃에서 20분간 처리하였다.In the present invention, for example, the separation method of the mutant strain 0.05M (Tris-Malate) after collecting only the spores using sterilized distilled water and a glass wool filter on the cells incubated for 6-7 days at 30 ℃ in a complete solid medium After washing three times with buffer (pH 6.5), the concentration of spores was changed to 10 6 -10 8 / ml with the same buffer, and then NTG was added to 500㎍ / ml and treated at 30 ° C for 20 minutes.
NTG 처리가 완료되면 원심분리나 여과에 의하여 포자만을 회수하고 멸균생리식염수로 3회 세척한 다음 완전배지에 도말하고 30에서 7-10℃일간 배양하여 변이주를 분리하였다.After NTG treatment was completed, only spores were recovered by centrifugation or filtration, washed three times with sterile saline solution, plated in complete medium, and cultured at 30 to 7-10 ° C. to isolate mutants.
본 발명에서 사용한 완전배지의 조성은 다음과 같다.The composition of the complete medium used in the present invention is as follows.
(주1) 완전배지 : 포도당 1.0%, 카제인 가수분해물 0.2%, 육즙 0.1%, 효모 추출물 0.1%, 한천 1.5%, pH 7.0-7.2(1) Complete medium: glucose 1.0%, casein hydrolyzate 0.2%, gravy 0.1%, yeast extract 0.1%, agar 1.5%, pH 7.0-7.2
본 발명의 균주(KCTC 8537P)와 친주(ATCC 15872) 그리고 또다른 생산균주인 스트렙토마이세스 갈릴레우스(ATCC 31133)의 형태적 특성과 생리적 특성을 비교한 결과 다음과 같다.As a result of comparing the morphological characteristics and physiological characteristics of the strain (KCTC 8537P) and the parent strain (ATCC 15872) and another production strain Streptomyces galileus (ATCC 31133) of the present invention.
＋＋＋; 매우 우수, ＋＋; 우수, ＋; 보통, ±; 미약, ―;모자람+++; Very good, + +; Excellent, +; Usually, ±; Weak;
본 발명에서 배양액 및 균체내의 아클라시노마이신의 분석 방법은 메타놀과 클로로포름을 20:1로 섞어 산물의 추출을 시도한 후 HPLC를 사용하였으며 이때의 고정상으로는 실리카(sillica)컬럼을 사용하였고, 유동상은 클로로포름(cholroform) : 벤젠(benzen) : 에틸아세테이트(ethylacetate) : 메타놀(methanol) : 개미산(formic acid) : 물(water) : 트리에칠아민(triethyl amine)을 30 : 30 : 14 : 9 :3 .5 : 0.02(v/v)의 비율로 혼합사용하였으며 1.9ml/min의 유속으로 흘려보내면서 432nm에서의 흡광도를 측정하였다. 별도로 아클라시노마이신 A 및 B 표준품을 사용하여 얻은 흡광 지체시간과 시료흡광의 지체시간을 비교하여 균체내의 아클라시노마이신 A와 B의 농도를 산출하였다.In the present invention, the method of analyzing aclacinomycin in the culture medium and the cells was mixed with methanol and chloroform 20: 1 and tried to extract the product, and then HPLC was used as the stationary phase. (cholroform): Benzene: Ethyl acetate: Methanol: Formic acid: Water: Triethylamine 30: 30: 14: 9: 3. The mixture was used at a ratio of 5: 0.02 (v / v), and the absorbance at 432 nm was measured while flowing at a flow rate of 1.9 ml / min. Separately, the absorption delay time of the sample absorption and the absorption delay time obtained using the aclosinomycin A and B standards were compared to calculate the concentrations of the aclacinomycin A and B in the cells.
본 발명의 미생물을 이용하여 아클라시노마이신 A를 제조하는 방법은 통상의 발효에 사용하는 탄소원, 질소원, 무기물 기타 영양물질을 함유하는 배지에 배양하여 배양액 및 균체내의 축적된 아클라시노마이신 A를 회수 하였다.The method for producing aclacinomycin A using the microorganism of the present invention is to culture the accumulated aclacinomycin A in the culture medium and cells by culturing in a medium containing a carbon source, nitrogen source, minerals and other nutrients used in conventional fermentation Recovered.
본 발명에서 사용하는 주요 탄소원으로는 전분, 대두밀등을 사용하며 질소원으로는 대두밀 외에 황산암모늄등을 사용한다. 무기물로는 탄산칼슘, 황산 마그네슘 황산아연, 황산철, 황화수산화나트륨등을 사용한다. 배양은 통상의 호기적 조건하에서 배양온도 28℃-30℃, pH 7.2에서 4-5일간 배양하며 배양중의 pH 조절은 유기 또는 무기 알칼리성 물질, 암모니아수, 탄산 칼슘등을 사용한다. 배양완료액에서 아클라시노마이신 A를 회수하는 방법은 아세톤과 클로로포름등의 유기용매를 이용한 추출과 실리신산을 이용한 칼럼크로마토그라피등을 이용한 통상의 저분자량의 분리방법을 사용한다.As the main carbon source used in the present invention, starch, soybean mill and the like are used, and nitrogen source is ammonium sulfate or the like in addition to soybean mill. Calcium carbonate, magnesium sulfate zinc sulfate, iron sulfate, sodium sulfide, etc. are used as the inorganic substance. The culture is incubated for 4-5 days at a culture temperature of 28 ° C.-30 ° C. and pH 7.2 under normal aerobic conditions. The pH of the culture is controlled using organic or inorganic alkaline materials, ammonia water, calcium carbonate and the like. The method for recovering acrycinomycin A from the culture completed solution is conventional extraction using acetone and chloroform, using a low molecular weight separation method using column chromatography using silicic acid, and the like.
이하 실시예를 들어 본 발명을 구체적으로 설명한다.The present invention will be described in detail with reference to the following Examples.
실시예 1Example 1
아클라시노마이신 생산을 위해 먼저 사면배지(pH 7.2)에 본 발명의 균주 DK-506을 접종하여 28℃에서 5일간 배양한 다음 이를 종 배양배지에 접종하였다. 종 배양배지의 제조는 pH를 7.8로 조절한 후 500ml 용량의 삼각 플라스크에 50ml를 분주하여 121℃에서 15분간 살균하여 준비하였으며 균을 접종하여 회전식 진탕 배양기에서 240rpm으로 2일간 배양하였다. 산물의 생산을 위한 발효배지는 pH 7.5로 조절한 다음 종 배양액을 10% 되도록 접종하여 통기량 1.0vvm, 교반속도 500rpm, 배양온도 28℃의 조건하에서 5일간 배양하였다.For the production of aclacinomycin, the strain DK-506 of the present invention was first inoculated into a slope medium (pH 7.2) and incubated at 28 ° C. for 5 days, and then inoculated into a seed culture medium. The seed culture medium was prepared by sterilizing 50 ml in a 500 ml Erlenmeyer flask and sterilizing for 15 minutes at 121 ° C. after the pH was adjusted to 7.8 and inoculated with the bacteria and incubated at 240 rpm in a rotary shake incubator for 2 days. Fermentation medium for the production of the product was adjusted to pH 7.5 and inoculated to 10% seed culture was incubated for 5 days under conditions of aeration rate 1.0vvm, stirring speed 500rpm, culture temperature 28 ℃.
본 발명에서 사용한 배지의 조성은 각각 다음과 같다.The composition of the medium used in the present invention is as follows.
(주2) 사면배지 : 전분 2%, 인산수소칼륨 0.05%, 황산마그네슘 0.05%, 질산 칼륨 0.1%, 식염 0.05%, 황산제이철 0.001%, 한천 1.5-2%(pH 7.2)(Note 2) Slope medium: starch 2%, potassium hydrogen phosphate 0.05%, magnesium sulfate 0.05%, potassium nitrate 0.1%, salt 0.05%, ferric sulfate 0.001%, agar 1.5-2% (pH 7.2)
(주3) 종 배양배지 : 대두분 1.1%, 전분 2.6%, 황산암모늄 0.1%, 탄산칼슘 0.8%, 식염 0.3%, 해바라기 기름 3%(pH 7.8)(3) Species culture medium: soy flour 1.1%, starch 2.6%, ammonium sulfate 0.1%, calcium carbonate 0.8%, salt 0.3%, sunflower oil 3% (pH 7.8)
(주4) 발효배지 : 전분 9.0%, 대두분 1.9%, 황산암모늄 0.15%, 탄산칼슘 0.8%, 황산마그네슘 0.1%, 황산제1철 0.002%, 황화수소나트륨 0.3%, 황산아연 0.001%(pH 7.5)(4) Fermentation medium: starch 9.0%, soy flour 1.9%, ammonium sulfate 0.15%, calcium carbonate 0.8%, magnesium sulfate 0.1%, ferrous sulfate 0.002%, sodium hydrogen sulfide 0.3%, zinc sulfate 0.001% (pH 7.5 )
상기와 동일한 방법으로 친주를 배양하여 각 배양액으로부터 아클라시노마이신 A와 B의 생성량을 조사한 결과 표 2와 같다.The parent cell was cultured in the same manner as described above, and the amount of alaccinomycin A and B produced from each culture solution was examined.
상기 표 1에서와 같이 본 발명균주에서의 아클라시노마이신 A의 생산능이 친주에 비하여 두배 이상 증가하였으며 B의 생산능은 오히려 감소한 것으로 보아 본 발명의 균주는 정확하게 알 수는 없지만 이차 대사산물인 아클라시노마이신 생합성 경로의 일부가 변환된 균임을 추측할 수 있다.As shown in Table 1 above, the production capacity of aclacinomycin A in the strain of the present invention was more than doubled compared to the parent strain, and the production capacity of B was rather reduced, so that the strain of the present invention is not exactly known, but it is a secondary metabolite. It can be inferred that part of the clasinomycin biosynthetic pathway is a transformed bacterium.
실시예 2Example 2
본 발명의 균주 스트렙토마이세스 라벤도폴리아 DK-506을 이용한 아클라시노마이신 생산시 발효조건중 통기량을 0.5vvm으로 낮추어 5일간 배양했을때의 아클라시노마이신 A 와 B의 생성량은 다음 표3과 같다.The production amount of aclacinomycin A and B when the aspirinomycin production using the strain Streptomyces Ravendopolya DK-506 of the present invention was lowered to 0.5vvm in a fermentation condition and then cultured for 5 days. Same as
아클라시마이신의 발효생산시 발효배지내에 산소의 공급량이 줄어들면 전체적으로 균체의 성장속도가 느려지게 되며 따라서 아클라시노마이신의 생성량도 감소하게 된다.When the amount of oxygen in the fermentation broth is reduced during fermentation production of aklashimamycin, the growth rate of the cells is slowed as a whole, and thus the production amount of aclacinomycin is also reduced.
실시예 3Example 3
본 발명 균주 스트렙토마이세스 라벤도폴리아 DK-506을 이용한 아클라시노마이신 생산시 발효조건중 변화시키거나 초기 발효배지의 pH를 변화시키면 30℃, pH 7.0-7.5로 배양했을때에 비하여 아클라시노마이신의 생성량이 감소된다.In the production of aclacinomycin using the strain Streptomyces lavendopolya DK-506 of the present invention, the change in the fermentation conditions or the pH of the initial fermentation broth was increased to 30 ° C. and pH 7.0-7.5 compared to that of incubation The amount of mycin produced is reduced.
실시예 4Example 4
실시예 1에서와 같이 배양하여 얻은 배양액 3l를 20%의 아세톤이 포함된 부틸 아세테이트와 혼합하여 2 : 1의 부피비로 혼합하여 잘 섞은 후 용매층으로의 추출을 시도하였다. 이 추출액 1,200ml을 진공 감압 농축기로 농축한 후 500ml의 클로로포름으로 재추출 및 농축하였다. 농축된 추출물에 포화 하이드로 카본인 n-핵산 100ml를 가하여 아클라시노마이신 A를 침전시키고 이 침전물을 모아 실리신산으로 준비된 흡착 크로마토그라피를 실시하였다. 이때 사용한 컬럼은 길이 40cm, 내경 3cm의 크기를 사용하였으며 산물의 용출을 위하여 사용한 유기용매는 클로로포름과 매칠알콜을 20 : 1의 부피 조성비로 준비하여 사용하였다. 흡착 크로마토그라피를 실시한 후 활성분획을 모아 농축시키고 2차로 같은 앰벌라이트 XAD -2컬럼 크로마토그라피로 실시하여 각종 색소들을 제거하였다. 이때 산물의 용출을 위하여 사용한 유기용매는 앞의 것과 동일한 것으로 사용하였다. 아클라시노마이신 A가 확인된 활성분획 200ml를 모아 농축한 후 동결건조 방법을 이용하여 최종산물을 획득하였다. 그 결과 약 220mg의 아클라시노마이신 A를 얻었으며 다양한 유기용매 시스템의 HPLC(고속 액체크로마토그라피)를 실시하여 표준품과 비교 조사하여 그 순도를 확인하였다. (제1도)3 l of the culture solution obtained by culturing as in Example 1 was mixed with butyl acetate containing 20% acetone, mixed at a volume ratio of 2: 1, and mixed well, and then extracted into a solvent layer. 1,200 ml of the extract was concentrated with a vacuum depressurizer, and then extracted and concentrated again with 500 ml of chloroform. 100 ml of saturated hydrocarbon n-nucleic acid was added to the concentrated extract to precipitate aclacinomycin A. The precipitates were collected and subjected to adsorption chromatography prepared with silicic acid. The column used was 40 cm in length and 3 cm in diameter. The organic solvent used for elution of the product was prepared by using chloroform and methyl alcohol in a volume ratio of 20: 1. After performing the adsorption chromatography, the active fractions were collected and concentrated, and the same Amberlite XAD-2 column chromatography was performed secondarily to remove various pigments. At this time, the organic solvent used for elution of the product was used as the same as the previous one. 200 ml of the active fractions identified with acrycinomycin A were collected and concentrated, and the final product was obtained by lyophilization. As a result, about 220 mg of aclacinomycin A was obtained, and HPLC (high-speed liquid chromatography) of various organic solvent systems was carried out to compare with a standard to confirm the purity. (Figure 1)
실시예 5Example 5
실시예 4와 같은 컬럼 크로마토그라피를 이용한 아클라시노마이신 분리 정제시 용매의 조성비를 달리하여 사용했을 경우 최종적으로 수득할 수 있는 아클라시노마이신 A의 양이 감소됨을 알 수 있다(표 5).It can be seen that the amount of acrycinomycin A that can be finally obtained is reduced when the composition ratio of the solvent is used in the separation and purification of aclacinomycin using column chromatography as in Example 4 (Table 5).
Claims (4)
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