KR100597309B1 - Method of Preparing [?-HyMeLeu4]Cyclosporin A by Sebekia benihana - Google Patents

Method of Preparing [?-HyMeLeu4]Cyclosporin A by Sebekia benihana Download PDF

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KR100597309B1
KR100597309B1 KR1020040074492A KR20040074492A KR100597309B1 KR 100597309 B1 KR100597309 B1 KR 100597309B1 KR 1020040074492 A KR1020040074492 A KR 1020040074492A KR 20040074492 A KR20040074492 A KR 20040074492A KR 100597309 B1 KR100597309 B1 KR 100597309B1
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유효림
한규범
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Abstract

본 발명은 희귀 방선균의 하나인 세베키아 베니하나 (Sebekia benihana)를 이용하여 사이클로스포린 A로부터 [감마 히드록실메틸루신4]사이클로스포린 A를 얻는 생물전환 제조방법에 관한 것이다. The present invention relates to a bioconversion production method for obtaining [gamma hydroxylmethylleucine 4] cyclosporin A from cyclosporin A using Sebekia benihana, one of the rare actinomycetes.

사이클로스포린 A, [감마 히드록실메틸루신4]사이클로스포린 A, 세베키아 베니하나, 생물전환, 발모제, 탈모방지제 Cyclosporin A, [gamma hydroxylmethyl leucine 4] cyclosporin A, sevecia benihana, bioconversion, hair regrowth, hair loss inhibitor

Description

세베키아 베니하나를 이용한 [감마 히드록실메틸루신4] 사이클로스포린 A의 제조방법 {Method of Preparing [γ-HyMeLeu4]Cyclosporin A by Sebekia benihana} [Method of Preparing [[gamma] -HyMeLeu4] Cyclosporin A by Sebekia benihana} using Sebecia Benihana

도 1은 희귀방선균인 세베키아 베니하나를 이용하여 사이클로스포린 A로부터 [감마 히드록실메틸루신4] 사이클로스포린 A를 얻는 생물전환시 사이클로스포린 A를 용이하게 용해시키기 위하여 사용할 수 있는 각종 첨가물들의 효과를 비교한 그라프이며,FIG. 1 is a graph comparing the effects of various additives that can be used to easily dissolve cyclosporin A during bioconversion to obtain [gamma hydroxylmethylleucine 4] cyclosporin A from cyclosporin A using the rare actinomycetes Severkia bennihana. Is,

도 2는 희귀방선균인 세베키아 베니하나를 이용하여 사이클로스포린 A로부터 [감마 히드록실메틸루신4] 사이클로스포린 A를 얻는 생물전환시 사이클로스포린 A 용해용 첨가물로 사용되는 DMSO (Dimethyl Sulfoxide)의 농도 영향에 대한 그라프이며,2 is a graph showing the effect of concentration of DMSO (Dimethyl Sulfoxide) used as an additive for dissolving cyclosporin A upon obtaining a gamma hydroxylmethylleucine 4 cyclosporin A from cyclosporine A using the rare actinomycetes Severkia bennihana. Is,

도 3은 희귀방선균인 세베키아 베니하나를 이용하여 사이클로스포린 A로부터 [감마 히드록실메틸루신4] 사이클로스포린 A를 얻는 생물전환시 온도 영향을 보여주는 그라프이며, Figure 3 is a graph showing the temperature effect during the bioconversion to obtain [gamma hydroxylmethyl leucine 4] cyclosporin A from cyclosporin A using the rare actinomycetes Severkia Benihana,

도 4는 희귀방선균인 세베키아 베니하나를 이용하여 사이클로스포린 A로부터 [감마 히드록실메틸루신4] 사이클로스포린 A를 얻는 생물전환시 사용되는 균체량이 전환에 미치는 영향에 대한 그라프이며,Figure 4 is a graph of the effect of the amount of cells used in the bioconversion to obtain [gamma hydroxylmethyl leucine 4] cyclosporin A from cyclosporin A using the rare actinomycetes Severkia Benihana,

도 5는 희귀방선균인 세베키아 베니하나를 이용하여 사이클로스포린 A로부터 [감마 히드록실메틸루신4] 사이클로스포린 A를 얻는 생물전환시 사이클로스포린 A의 농도가 전환에 미치는 영향을 나타내는 그라프이며,FIG. 5 is a graph showing the effect of the concentration of cyclosporin A on the conversion during the biological conversion of [gamma hydroxylmethylleucine 4] cyclosporin A from cyclosporin A using the rare actinomycetes Severkia bennihana. FIG.

도 6은 희귀방선균인 세베키아 베니하나를 이용하여 사이클로스포린 A로부터 [감마 히드록실메틸루신4] 사이클로스포린 A를 얻는 생물전환시 pH가 전환에 미치는 영향을 보여주는 그라프이며,FIG. 6 is a graph showing the effect of pH on conversion during bioconversion of [gamma hydroxylmethylleucine 4] cyclosporin A from cyclosporin A using the rare actinomycetes Severkia benihana. FIG.

도 7은 희귀방선균인 세베키아 베니하나를 이용하여 사이클로스포린 A로부터 [감마 히드록실메틸루신4] 사이클로스포린 A를 얻는 생물전환시 균체의 시간대 별 전환능력을 보여주는 그라프이며,FIG. 7 is a graph showing the ability of cells to convert by time when biotransformation of [gamma hydroxylmethylleucine 4] cyclosporin A from cyclosporin A using the rare actinomycetes Svecia benicihana.

도 8은 희귀방선균인 세베키아 베니하나를 이용하여 사이클로스포린 A로부터 [감마 히드록실메틸루신4] 사이클로스포린 A를 얻는 생물전환시 효모추출물과 대두박 성분의 조성 비율에 따른 전환능력을 비교한 그라프이며,8 is a graph comparing the conversion ability according to the composition ratio of the yeast extract and the soybean meal during the biological conversion to obtain [gamma hydroxylmethyl leucine 4] cyclosporin A from cyclosporine A using the rare actinomycetes Severkia bennihana.

도 9은 희귀방선균인 세베키아 베니하나를 이용하여 사이클로스포린 A로부터 [감마 히드록실메틸루신4] 사이클로스포린 A를 얻는 생물전환시 사이클로스포린 A의 첨가 시점에 따른 전환 정도를 비교한 그라프이다.Figure 9 is a graph comparing the degree of conversion according to the time of addition of cyclosporin A during the bioconversion to obtain [gamma hydroxylmethyl leucine 4] cyclosporin A from cyclosporin A using the rare actinomycetes Severkia Benihana.

사이클로스포린 A (Cyclosporin A)는 11개의 아미노산이 환상으로 결합된 올리고 펩타이드로서 장기이식 환자들의 거부반응을 치료하기 위한 강력한 면역억제제이며, 부작용으로 신장독성, 간 독성, 고혈압, 치주조직 비대, 발모효과 등 다양한 생리효과를 가지고 있다. 사이클로스포린 A (Cyclosporin A)는 불완전 균류인 토포클라디움 인플라툼 (Toypocladium inflatum)을 호기성 상태에서 액체배양 할 경우에 생성되는 24가지의 구조적으로 유사한 대사물질들 (사이클로스포린 A에서 Z까지) 가운데 가장 많이 생성되는 물질이다. 사이클로스포린 A (Cyclosporin A)와 그 유사물 들은 사이클로스포린 신데테이즈 (Cyclosporin Synthetase)라는 다기능 단일 효소에 의해서 11개의 아미노산이 순서대로 활성화되고, N-메틸레이션(N-methylation)되고, 펩티드결합을 형성하는 Non-ribosomal 기작으로 생합성된다. Cyclosporin A is an oligopeptide with a ring of 11 amino acids, which is a powerful immunosuppressant to treat rejection of organ transplant patients.Its side effects include renal toxicity, hepatotoxicity, hypertension, periodontal tissue enlargement, hair growth effects, etc. It has various physiological effects. Cyclosporin A is the most abundant of the 24 structurally similar metabolites (cyclosporin A to Z) that are produced when liquid cultures of incomplete fungi, Topocladium inflatum, are aerobically cultured. It is a substance. Cyclosporin A and its analogs are 11 amino acids activated in sequence, N-methylated, and form peptide bonds by a multifunctional single enzyme called Cyclosporin Synthetase. Biosynthesis by non-ribosomal mechanism.

사이클로스포린 A의 여러 부작용 중에서 모 과대성장 (발모) 부작용을 이용한 발모제로 개발 가능성이 그동안 여러 연구에 의해 수행되어 왔었다. 그 중에는 동물 모발실험 (Arch, Dermatol. Res., 1996; 288; 408-410), 인간원형 탈모증 (J. Am. Acad. Dermatol., 1990; 22; 242-250), 인간 남성형 탈모증 (J. Am. Acad. Dermatol., 1990; 22; 251-253 및 Skin Pharmacol., 1994; 7; 101-104) 및 화학요 법에 의한 탈모 동물에서도 탈모억제 효과 (Clin. Lab. Invest., 1995; 190; 192-196 및 Am. J. Pathol., 1997; 150; 1433-1441) 등 매우 광범위하게 연구되었고, 마우스 등판 실험 결과로 비교 한다면 시판되고 있는 미녹시딜 보다 약 100 배 정도 우수한 효과를 보여주고 있다. Among the various side effects of cyclosporin A, the possibility of developing it as a hair regrowth agent using the side effect of hair growth (hair growth) has been conducted by several studies. Among them are animal hair experiments (Arch, Dermatol. Res., 1996; 288; 408-410), alopecia areata (J. Am. Acad. Dermatol., 1990; 22; 242-250), human alopecia areata (J. A. Acad. Dermatol., 1990; 22; 251-253 and Skin Pharmacol., 1994; 7; 101-104) and hair loss inhibitory effects in hair loss animals by chemotherapy (Clin. Lab. Invest., 1995; 190 192-196 and Am. J. Pathol., 1997; 150; 1433-1441), and compared with the results of mouse backing experiments, showing about 100 times better effectiveness than commercial minoxidil.

사이클로스포린 A (Cyclosporin A)의 인체 내 대사과정을 모사하기 위하여 다양한 세균과 진균류를 이용하여 사이클로스포린 A (Cyclosporin A)를 생물전환시켰을 때, 희귀 방선균인 세베키아 베니하나 (Sebekia benihana)에 의해서 [감마 히드록실메틸루신4]사이클로스포린 A ([γ-hyMe Leu4]-cyclosporin A)로 35%, [감마히드록실루신4]사이클로스포린 A ([γ-HyLeu4]-cyclosporin A)로 4.5%, [감마메틸루신4,감마히드록실메틸루신6]사이클로스포린 A ([γHyMeLeu4, γHyMeLeu6]-cyclosporin A)로 8.6%가 전환되는 것이 확인되었다 (J. Antibiotics, 1996; 49; 781-787). [Gamma hydride] was used by the rare actinomycetes Sebekia benihana when biotransforming Cyclosporin A using various bacteria and fungi to simulate the metabolic processes of Cyclosporin A in humans. 35% with loxylmethylleucine 4] cyclosporin A ([γ-hyMe Leu4] -cyclosporin A), 35% with [gammahydroxylcin 4] cyclosporin A ([γ-HyLeu4] -cyclosporin A), [gammamethylleucine 4 It was confirmed that 8.6% conversion to gammahydroxymethylleucine 6] cyclosporin A ([γHyMeLeu4, γHyMeLeu6] -cyclosporin A) (J. Antibiotics, 1996; 49; 781-787).

또한, 대한민국 특허 10-0316604 및 미국특허 6521595에 따르면 생물전환된 사이클로스포린 A (Cyclosporin A)의 유도체중의 하나인 [감마 히드록실메틸루신4]사이클로스포린 A ([γ-hyMe Leu4]-cyclosporin A)는 사이클로스포린 A (Cyclosporin A)가 가지고 있는 면역억제의 독성은 감소되고, 발모효과는 그대로 유지된다고 보고가 되어 있다. In addition, according to Korean Patent 10-0316604 and US Patent 6521595 [gamma hydroxylmethyl leucine 4] cyclosporin A ([[gamma] -hyMe Leu4] -cyclosporin A), one of the derivatives of the bioconverted cyclosporin A, Cyclosporin A has been reported to reduce the toxicity of immunosuppression and maintain the hair growth effect.

그러나 세베키아 베니하나 등 미생물에 의한 생물전환시 그 수율이 미미하므로 이러한 결과를 산업적으로 이용하기에는 적당하지 않다는 문제점이 있었다. Kuhnt의 논문 (J. Antibiotics. 1996: 49(8): 781-787)에서 기술된 방법 역시 세베 키아 베니하나에 의한 사이클로스포린 A로부터 [감마 히드록실메틸루신4]사이클로스포린 A ([γ-hyMe Leu4]-cyclosporin A)로의 전환율은 35%에 불과하고 생성된 [감마 히드록실메틸루신4]사이클로스포린 A ([γ-hyMe Leu4]-cyclosporin A)의 농도가 낮아 이 역시 산업적으로 이용하기에는 생산성이 낮다는 단점이 있다 (포도당 (Glucose) 0.7%, 효모추출물 (Yeast Extract) 0.45%, 맥아추출물 (Malt Extract) 0.5%, 수용성 풀 (Soluble Starch) 1%, 탄산칼슘 (CaCO3) 0.005% 배지에서 사이클로스포린 A를 100 mg/L 첨가하고, 96 시간 동안 배양함).However, when the bioconversion by microorganisms such as Severkia Benihana, the yield is insignificant, there was a problem that these results are not suitable for industrial use. The method described in Kuhnt's paper (J. Antibiotics. 1996: 49 (8): 781-787) was also derived from [gamma hydroxylmethylleucine 4] cyclosporine A ([[gamma] -hyMe Leu4]) by cyclosporin A by Sevekia Benihana. conversion to -cyclosporin A) is only 35% and the concentration of [gamma-hydroxymethylleucine 4] cyclosporin A ([γ-hyMe Leu4] -cyclosporin A) is low, which is also low for industrial use. (Glucose 0.7%, Yeast Extract 0.45%, Malt Extract 0.5%, Soluble Starch 1%, Calcium Carbonate (CaCO3) 0.005% 100% cyclosporine A in medium) mg / L added and incubated for 96 hours).

세베키아 베니하나 (Sebekia benihana)를 이용하여 사이클로스포린 A로부터 [감마 히드록실메틸루신4]사이클로스포린 A ([γ-hyMe Leu4]-cyclosporin A)을 생물전환하여 얻는 방법에 대한 연구는 현재까지 극히 미미한 실정인 바, 발모제로서의 [감마 히드록실메틸루신4]사이클로스포린 A의 산업화에 걸림돌이 되는 현재의 낮은 생산성을 향상시키기 위하여 본 발명자 들은 세베키아 베니하나를 이용하여 사이클로스포린 A (Cyclosporin A)로부터 [감마 히드록실메틸루신4]사이클로스포린 A ([γ-hyMe Leu4]-cyclosporin A)로의 생물전환시 배지성분조절 및 배양조건을 최적화하고 사이클로스포린 A를 용해시키기 위한 첨가물 선정 및 사이클로스포린 A의 적정 투여 시점을 설정함으로써 세베키아 베니하나의 생물전환 능력을 크게 증가시키는 것을 본 발명의 주 목적으로 하였다.The research on the bioconversion of [gamma hydroxylmethyl leucine 4] cyclosporin A ([γ-hyMe Leu4] -cyclosporin A) from cyclosporine A using Sebekia benihana has been extremely minimal. In order to improve the current low productivity which is an obstacle to the industrialization of [gamma hydroxylmethylleucine 4] cyclosporin A as a hair regrowth agent, the present inventors have used [Cyclosporin A] from [Cyclosporin A] using sebecia benihana. Sebecia by optimizing medium composition and cultivation conditions, selecting additives to dissolve cyclosporin A, and setting an appropriate time point for cyclosporin A upon bioconversion to [γ-hyMe Leu4] -cyclosporin A) The main purpose of the present invention was to significantly increase Benihana's bioconversion capacity.

[실시예]EXAMPLE

실시예 1: 세베키아 베니하나 (Sebekia benihana)의 기본배양 및 사이클로스포린 A와 [감마 히드록실메틸루신4]사이클로스포린 A의 분석 Example 1 Basic Culture of Sebekia benihana and Analysis of Cyclosporin A and [Gamma Hydroxylmethylleucine 4] Cyclosporin A

세베키아 베니하나가 사이클로스포린 A (Cyclosporin A)로부터 [감마 히드록실메틸루신4]사이클로스포린 A로의 생물전환시 최적의 조건을 찾아내기 위하여 다음과 같은 실험을 수행하였다.The following experiments were performed to find the optimal conditions for the bioconversion of Severkia Benihana from Cyclosporin A to [gamma hydroxylmethylleucine 4] cyclosporin A.

생물전환에 사용된 세베키아 베니하나의 균주는 한국생명공학연구원 생물자원센터에 보관된 균주 KCTC 9173를 사용하였으며, 이의 배양배지는 포도당 (Glucose) 0.7%, 수용성 풀 (Soluble Starch) 1%, 효모추출물 (Yeast Extract) 0.45%, 맥아추출물 (Malt Extract) 0.5%, 탄산칼슘 (CaCO3) 0.005%가 포함된 배지를 사용하였고, 배양 온도는 30℃, 배양 시간은 6일로 하였다 (J. Antibiotics, 1996; 49; 781-787). The Sevekia Benihana strain used for bioconversion was used strain KCTC 9173 stored at the Korea Institute of Bioscience and Biotechnology, and its culture medium was glucose (Glucose) 0.7%, Soluble Starch (1%), yeast. A medium containing 0.45% of Yeast Extract, 0.5% of Malt Extract, and 0.005% of calcium carbonate (CaCO3) was used, and the incubation temperature was 30 ° C. and the incubation time was 6 days (J. Antibiotics, 1996 49; 781-787).

6일 동안 배양된 균을 원심분리하여 균만을 수득한 뒤 생물전환 시험을 수행하였으며, 생물전환 시 사용된 배지는 포도당 (Glucose) 0.7%, 대두박 (Soybean Powder) 1%, 수용성 풀 (Soluble Starch) 1%, 탄산칼슘 (CaCO3) 0.005%가 포함된 배지이며, 전환 온도는 30℃, 시간은 24시간이었다.  The microorganisms cultured for 6 days were centrifuged to obtain only the microorganisms, followed by a bioconversion test. The medium used for bioconversion was glucose (Glucose) 0.7%, soybean powder (1%), water-soluble pool (Soluble Starch) It was a medium containing 1%, calcium carbonate (CaCO3) 0.005%, the conversion temperature was 30 ℃, time was 24 hours.

생물전환 후 배양액에 동량의 메탄올을 첨가하여 사이클로스포린 A (Cyclosporin A)와 [감마 히드록실메틸루신4] 사이클로스포린 A ([γ-hyMe Leu4]-cyclosporin A)를 추출한 뒤 원심분리하여 균체를 제거하고 상등액은 액체크로마토그래피 (HPLC)로 분석하였다. 이때 사용한 액체크로마토그래피의 조건은 C-18 컬럼을 사용하였으며, 용매조건은 물 (Water)과 아세토나이트릴 (Acetonitrile)의 비율이 3:7, 컬럼 의 온도는 55℃의 조건으로 분석하였다. After bioconversion, the same amount of methanol was added to the culture medium to extract cyclosporin A and [gamma hydroxylmethyl leucine 4] cyclosporin A ([γ-hyMe Leu4] -cyclosporin A), followed by centrifugation to remove the cells and the supernatant. Silver was analyzed by liquid chromatography (HPLC). The liquid chromatography was used for the C-18 column, the solvent condition was the ratio of water (water) and acetonitrile 3: 3, the column temperature was analyzed at 55 ℃.

실시예 2: 각종 첨가물에 따른 사이클로스포린 A로부터 [감마 히드록실메틸루신4]사이클로스포린 A로의 생물전환 비교실험Example 2: Comparison of bioconversion from cyclosporin A to [gamma hydroxylmethylleucine 4] cyclosporin A according to various additives

멸균된 생물전환용 배지 (대두박 1%, 포도당 0.7%, 수용성 풀 1%, 탄산칼슘 0.005%)를 각각의 삼각플라스크에 분배 후, 실시예 1에서 얻은 세베키아 베니하나 균을 30% 첨가하고, DMSO (Dimethyl Sulfoxide), 소포제, 유화제 또는 에탄올과 같은 다양한 첨가물에 녹인 사이클로스포린 A (Cyclosporin A)를 최종농도 500 mg/L가 되도록 첨가한 후 온도 30℃에서 24시간 동안 생물전환하였다. 본 비교실험에서 대조군은 사이클로스포린 A 가루를 용해용 첨가물 없이 직접 첨가한 것이며, 도 1에서 보이는 것과 같이 사이클로스포린 A를 DMSO에 녹여 최종농도 0.5%로 첨가한 뒤 생물전환하는 것이 가장 좋음을 확인하였다.After dispensing the sterilized bioconversion medium (1% soybean meal, 0.7% glucose, 1% water-soluble pool, 0.005% calcium carbonate) into each Erlenmeyer flask, 30% of the Severkibenihana bacteria obtained in Example 1 were added, Cyclosporin A dissolved in various additives such as DMSO (Dimethyl Sulfoxide), an antifoaming agent, an emulsifier or ethanol was added to a final concentration of 500 mg / L and then bioconverted at a temperature of 30 ° C. for 24 hours. In this comparative experiment, the control group was directly added cyclosporin A powder without dissolving additives, and as shown in FIG. 1, cyclosporin A was dissolved in DMSO and added to a final concentration of 0.5%.

실시예 3: 첨가물 DMSO (Dimethyl Sulfoxide)농도에 따른 사이클로스포린 A로부터 [감마 히드록실메틸루신4]사이클로스포린 A로의 생물전환 비교Example 3: Comparison of Bioconversion from Cyclosporin A to [Gamma Hydroxymethylleucine 4] Cyclosporin A with Additive DMSO (Dimethyl Sulfoxide) Concentration

실시예 2에서 DMSO를 사이클로스포린 A 용해용 첨가물로 사용하는 것이 전환에 좋은 영향을 미치는 것이 확인되었으므로 본 실험에서는 세베키아 베니하나의 생물전환시 DMSO의 첨가 농도가 전환에 미치는 영향을 확인하였다. 멸균된 생물전환용 배지 (대두박 1%, 포도당 0.7%, 수용성 풀 1%, 탄산칼슘 0.005%)를 각각의 삼각플라스크에 첨가한 뒤, 여기에 실시예 1에서 얻은 세베키아 베니하나 균체 30% (균체 침전량 백분율 %)를 첨가한 다음, DMSO에 녹아있는 사이클로스포린 A를 최종 DMSO 농도가 0.1, 0.25, 0.5, 1, 2, 3, 4% 가 되도록 생물전환용 배지에 첨가하고 온도 30℃에서 24시간 동안 생물전환 한 뒤 이를 액체 크로마토그래피로 분석하였다. 도 2에서 나타나는 것과 같이 DMSO를 최종농도 0.5%가 되도록 첨가하는 것이 가장 좋은 결과를 주었다. In Example 2, it was confirmed that the use of DMSO as an additive for dissolving cyclosporin A had a good effect on the conversion, and thus, in this experiment, the effect of the concentration of DMSO on the conversion was confirmed in the bioconversion of Severkia venihana. Sterilized bioconversion medium (1% soybean meal, 0.7% glucose, 1% water-soluble grass, 0.005% calcium carbonate) was added to each Erlenmeyer flask, followed by 30% of the Severkibenihana bacteria obtained in Example 1 ( % Of cell settling), and then cyclosporin A dissolved in DMSO was added to the bioconversion medium so that the final DMSO concentration was 0.1, 0.25, 0.5, 1, 2, 3, 4% and at a temperature of 30 ° C. for 24 hours. After bioconversion, it was analyzed by liquid chromatography. As shown in Figure 2, the addition of DMSO to the final concentration of 0.5% gave the best results.

실시예 4: 세베키아 베니하나 (Sebekia benihana)를 이용하여 사이클로스포린 A로부터 [감마 히드록실메틸루신4]사이클로스포린 A로의 생물전환시 최적 온도 조건Example 4 Optimal Temperature Conditions for Bioconversion from Cyclosporin A to [Gamma Hydroxylmethylleucine 4] Cyclosporin A Using Sebekia benihana

실시예 1에서 얻은 세베키아 베니하나 (Sbekia benihana) 균체 30% (균체 침전량 백분율 %)에 멸균된 생물전환용 배지 (대두박 1%, 포도당 0.7%, 수용성 풀 1%, 탄산칼슘 0.005%)를 첨가한 뒤, DMSO (Dimethyl Sulfoxide)에 녹인 사이클로스포린 A (Cyclosporin A)를 최종농도 500 mg/L 되도록 첨가하고, 온도 25℃, 30℃, 37℃에서 24시간 동안 생물전환하였다. 그 결과 25℃에서 25 mg/L/day, 30℃에서 130 mg/L/day, 37℃에서 50 mg/L/day로 전환되었으며 30℃에서 가장 전환이 좋은 것을 확인하였다 (도 3). To 30% of the Sbekia benihana cells obtained in Example 1 (% of cell precipitation), sterilized bioconversion medium (1% soybean meal, 0.7% glucose, 1% aqueous pool, 0.005% calcium carbonate) was added. Then, cyclosporin A (Cyclosporin A) dissolved in DMSO (Dimethyl Sulfoxide) was added to a final concentration of 500 mg / L and bioconverted for 24 hours at a temperature of 25 ℃, 30 ℃, 37 ℃. As a result, it was converted to 25 mg / L / day at 25 ℃, 130 mg / L / day at 30 ℃, 50 mg / L / day at 37 ℃ was confirmed that the best conversion at 30 ℃ (Fig. 3).

실시예 5: 세베키아 베니하나 (Sbekia benihana)를 이용하여 사이클로스포린 A (Cyclosporin A)를 생물전환시 사용되는 최적 균체 농도Example 5 Optimal Cell Concentration Used for Bioconversion of Cyclosporin A Using Sbekia benihana

멸균된 생물전환용 배지 (대두박 1%, 포도당 0.7%, 수용성 풀 1%, 탄산칼슘 0.005%)를 각각의 삼각플라스크에 첨가한 뒤, 여기에 실시예 1에서 얻은 세베키아 베니하나 균을 각각 5, 10, 30, 50, 70% (균체 침전량 백분율 %) 첨가 후, DMSO (Dimethyl Sulfoxide)에 녹인 사이클로스포린 A (Cyclosporin A)를 최종농도 500 mg/L가 되도록 첨가하고, 온도 30℃에서 24시간 동안 생물전환하였다. 도 4에서 보이는 것과 같이 30%의 균체를 첨가하여 전환하는 조건에서 가장 많은 농도의 [감마 히드록실메틸루신4] 사이클로스포린 A ([γ-hyMe Leu4]-cyclosporin A)를 얻을 수 있었으며, 70% 균체를 접종한 조건에서는 상대적으로 가장 낮은 농도의 [감마 히드록실메틸루신4] 사이클로스포린 A ([γ-hyMe Leu4]-cyclosporin A)를 얻을 수 있었다. 따라서 30% (균체 침전량 백분율 %) 정도의 균을 접종하여 사이클로스포린 A로부터 [감마 히드록실메틸루신4]사이클로스포린 A를 생물전환으로 얻는 것이 가장 좋은 조건임을 알 수 있었다.Sterilized bioconversion medium (1% soybean meal, 0.7% glucose, 1% water soluble grass, 0.005% calcium carbonate) was added to each Erlenmeyer flask, and each of the Svechia Benihana bacteria obtained in Example 1 was added 5 After addition of 10, 30, 50, 70% (% of cell precipitation), cyclosporin A dissolved in DMSO (Dimethyl Sulfoxide) was added to a final concentration of 500 mg / L, and at a temperature of 30 ° C. for 24 hours. Bioconversion. As shown in FIG. 4, the highest concentration of [gamma hydroxylmethyl leucine 4] cyclosporin A ([γ-hyMe Leu4] -cyclosporin A) was obtained under conversion conditions by adding 30% of the cells, and 70% of the cells. In the inoculated conditions, the lowest concentration of [gamma hydroxylmethyl leucine 4] cyclosporin A ([γ-hyMe Leu4] -cyclosporin A) was obtained. Therefore, it was found that the best condition was to obtain [gamma hydroxylmethyl leucine 4] cyclosporin A from the cyclosporin A by bioconversion by inoculating a bacterium of about 30% (percentage of cell settling percentage%).

실시예 6: 사이클로스포린 A (Cyclosporin A)의 첨가 농도에 따른 [감마 히드록실메틸루신4]사이클로스포린 A로의 생물전환 비교Example 6: Comparison of bioconversion to [gamma hydroxylmethylleucine 4] cyclosporin A according to the concentration of cyclosporin A

멸균된 생물전환용 배지 (대두박 1%, 포도당 0.7%, 수용성 풀 1%, 탄산칼슘 0.005%)를 각각의 삼각플라스크에 첨가한 뒤, 여기에 실시예 1에서 얻은 세베키아 베니하나 균을 30% (균체 침전량 백분율 %) 넣고, 사이클로스포린 A를 DMSO (Dimethyl Sulfoxide)에 용해 후 최종 농도로 100 mg/L, 200 mg/L, 500 mg/L, 1 g/L, 2 g/L가 되도록 첨가 후, 온도 30℃에서 24시간 동안 생물전환 하였다. 전환 결과는 도 5에 나타내었으며, [감마 히드록실메틸루신4] 사이클로스포린 A ([γ- hyMeLeu4]-cyclosporin A)로 가장 많이 전환된 것은 사이크로스포린 A를 2 g/L로 첨가하였을 때 얻은 약 300 mg/L/day 농도이었으나, 첨가한 사이클로스포린 A의 농도에 비하여 전환된 [감마 히드록실메틸루신4] 사이클로스포린 A ([γ-hyMeLeu4]-cyclosporin A)의 양 즉, 전환율은 가장 낮았으며, 사이클로스포린 A를 100 mg/L로 적게 첨가하였을 때전환율은 60%로 높았으나 얻어진 [감마 히드록실메틸루신4] 사이클로스포린 A ([γ-hyMeLeu4]-cyclosporin A)의 양이 약 60 mg/L/day로 낮으므로 생물전환시 비효율적이었다. 따라서 전환율과 얻어지는 [감마 히드록실메틸루신4] 사이클로스포린 A의 양 등을 종합적으로 고려해보면 사이클로스포린 A 500 mg/L 첨가 농도가 가장 효율적인 임이 확인되었다.  Sterilized bioconversion medium (1% soybean meal, 0.7% glucose, 1% water soluble grass, 0.005% calcium carbonate) was added to each Erlenmeyer flask, and 30% of the bacterium of Svekia benai obtained in Example 1 was added thereto. (Cellulose precipitate percentage%), cyclosporin A was dissolved in DMSO (Dimethyl Sulfoxide) and added to the final concentration to 100 mg / L, 200 mg / L, 500 mg / L, 1 g / L, 2 g / L Biotransformation was carried out at 30 ° C. for 24 hours. The conversion results are shown in Figure 5, the most converted to [gamma hydroxylmethyl leucine 4] cyclosporin A ([γ-hyMeLeu4] -cyclosporin A) was obtained when the addition of cyclosporin A at 2 g / L The concentration of [gamma hydroxylmethylleucine 4] cyclosporin A ([[gamma] -hyMeLeu4] -cyclosporin A) converted to the concentration of cyclosporin A added at 300 mg / L / day was the lowest, and the cyclosporin When A was added as low as 100 mg / L, the conversion was high as 60%, but the amount of [gamma hydroxylmethyl leucine 4] cyclosporin A ([γ-hyMeLeu4] -cyclosporin A) obtained was about 60 mg / L / day. Low and inefficient for bioconversion. Therefore, considering the conversion rate and the amount of [gamma hydroxylmethyl leucine 4] cyclosporin A obtained, it was confirmed that the concentration of 500 mg / L of cyclosporine A was the most efficient.

실시예 7: 세베키아 베니하나 (Sebekia benihana)를 이용하여 사이클로스포린 A로부터 [감마 히드록실메틸루신4]사이클로스포린 A로의 생물전환 시 최적 pH 조건 설정Example 7 Setting Optimal pH Conditions for Bioconversion from Cyclosporin A to [Gamma Hydroxymethylleucine 4] Cyclosporin A Using Sebekia benihana

pH가 각각 6, 7, 8로 조절된 생물전환용 배지 (대두박 1%, 포도당0.7%, 수용성 풀 1%, 탄산칼슘 0.005%)를 발효조에 넣고 멸균한 뒤, 세베키아 베니하나 균을 5% 접종하고, 온도 30℃로 배양하였다. 배양 36시간 되는 시점에서 DMSO (Dimethyl Sulfoxide)에 녹인 사이클로스포린 A를 최종농도 500 mg/L가 되도록 모든 발효조에 첨가하고, 60시간을 더 배양하였다. 배양 도중 변하는 pH는 2N NaOH와 2N HCl을 이용하여 조절하였다. 비교 결과는 도 6에 나타내었다.The bioconversion medium (soybean meal 1%, glucose 0.7%, water soluble grass 1%, calcium carbonate 0.005%) adjusted to pH 6, 7, 8, respectively, was put into a fermenter and sterilized. Inoculation was incubated at a temperature of 30 ° C. At 36 hours of incubation, cyclosporin A dissolved in DMSO (Dimethyl Sulfoxide) was added to all fermenters to a final concentration of 500 mg / L, and further cultured for 60 hours. The pH change during the culture was adjusted using 2N NaOH and 2N HCl. Comparative results are shown in FIG. 6.

pH 7과 pH 8의 양상이 비슷하게 나타났고, 배양 48시간 때에는 pH 8의 조건이 pH 7 의 조건에서 보다 많은 양의 [감마 히드록실메틸루신4] 사이클로스포린 A를 생산하였으나, 배양 60시간에는 pH 8에서의 전환량이 pH 7의 조건에서의 전환양보다 줄어드는 것이 확인되었다. pH 7의 조건은 전환량이 일정하게 유지가 되고 배양 60시간 이후까지도 지속적으로 전환량이 상승하고 있는 양상을 보이기 때문에 발효조 배양에서의 pH조건은 중성인 7이 가장 최적조건임을 확인하였다.The pattern of pH 7 and pH 8 was similar, and at 48 hours of culture, the conditions of pH 8 produced more [gamma hydroxylmethylleucine 4] cyclosporin A at pH 7, but at 60 hours of culture, pH 8 It was confirmed that the amount of conversion at was lower than the amount of conversion at the condition of pH 7. The pH 7 condition was consistently maintained and the conversion was continuously increased even after 60 hours of incubation. Therefore, pH 7 in the fermenter culture was confirmed to be the most optimal condition.

실시예 8: 세베키아 베니하나 (Sebekia benihana)를 이용하여 사이클로스포린 A로부터 [감마 히드록실메틸루신4]사이클로스포린 A로의 생물전환시 배지 조성에 따른 생물전환 능력 차이 비교Example 8 Comparison of Bioconversion Ability in Different Media Compositions During Bioconversion from Cyclosporin A to [Gamma Hydroxymethylleucine 4] Cyclosporin A Using Sebekia benihana

실시예1에 기술된 조건에서 6일 동안 배양된 세베키아 베니하나 (Sebekia benihana) 균을 멸균된 대두박 1% 배지 (대두박 1%, 포도당 0.7%, 수용성 풀 1%, 탄산칼슘 0.005%), 효모추출물 1% 배지 (효모추출물 1%, 수용성 풀 1%, 포도당 0.7%, 탄산칼슘 0.005%), 그리고 효모추출물 0.5% 대두박 0.5% 복합배지 (효모추출물 0.5%, 대두박 0.5%, 포도당 0.7%, 수용성 풀 1%, 탄산칼슘 0.005%)에 각각 5% 접종하고 배양시간 24시간 후부터 12시간 간격으로 샘플을 채취하였다. 채취된 샘플을 원심분리하여 얻어진 균체를 사이클로스포린 A가 500mg/L 첨가된 생물전환용 배지가 들어있는 삼각플라스크에 30% 접종하고 30℃에서 24시간 생물전환시킨 다음 이를 동량의 메탄올로 추출하여 액체 크로마토그래피로 분석하였다. 생물전환시 온도는 30℃이었고 배지 조성에 따른 전환능력 비교 결과는 도 7에 나타내었다. 효모추출물 0.5% 대두박 0.5% 복합배지에서 사이클로스포린 A로부터 [감마 히드록실메 틸루신4]사이클로스포린 A로의 전환능력이 가장 좋았다. Sebekia benihana bacteria cultured for 6 days under the conditions described in Example 1 were treated with sterile soybean meal 1% medium (1% soybean meal, 0.7% glucose, 1% water soluble grass, 0.005% calcium carbonate), yeast Extract 1% medium (1% yeast extract, 1% water soluble grass, 0.7% glucose, 0.005% calcium carbonate), and yeast extract 0.5% soybean meal 0.5% complex medium (yeast extract 0.5%, soybean meal 0.5%, glucose 0.7%, water soluble) Pool 1%, calcium carbonate was inoculated 5% each 5% and samples were taken at intervals of 12 hours after 24 hours of incubation time. Centrifuged samples were inoculated 30% in a Erlenmeyer flask containing cyclosporine A with 500mg / L of bioconversion medium, bioconverted at 30 ° C for 24 hours, and extracted with the same amount of methanol to give liquid chromatography. Analysis by graphy. The temperature at the time of bioconversion was 30 ° C and the results of comparison of the conversion capability according to the medium composition are shown in FIG. In yeast extract 0.5% soybean meal 0.5% complex medium, the conversion from cyclosporin A to [gamma hydroxyl methylusin 4] cyclosporin A was the best.

실시예 9: 효모추출물과 대두박 복합배지의 다양한 첨가 농도에 따른 세베키아 베니하나의 생물전환능력 비교Example 9 Comparison of Bioconversion Capacity of Severkia Benihana According to Various Concentrations of Yeast Extract and Soybean Meal Complex Medium

실시예 8에서 효모추출물 0.5% 대두박 0.5% 복합배지의 생물전환능력이 가장 좋음을 확인하였으므로, 본 실시예에서는 효모추추물과 대두박의 첨가 농도에 따른 세베키아 베니하나의 생물전환능력을 확인하고자 다음과 같은 실험을 수행하였다.   In Example 8, it was confirmed that the bioconversion capacity of the yeast extract 0.5% soybean meal 0.5% complex medium was the best, in this example, to determine the bioconversion capacity of Severkia Benihana according to the concentration of yeast extract and soybean meal The same experiment was performed.

세베키아 베니하나를 실시예 1에 기술된 배지에서 6일 동안 배양 후 발효조에 5%를 접종하였다. 발효조 배양 배지는 포도당 0.7%, 수용성 풀 1%, 탄산칼슘 0.005%가 기본적으로 첨가된 대두박 1% 배지, 대두박 0.5% 배지, 효모추출물 0.5% 배지, 효모추출물 1% 배지, 대두박 0.5% 효모추출물 0.2% 복합배지, 대두박 0.5% 효모추출물 0.5% 복합배지, 대두박 0.5% 효모추출물 1% 복합배지, 대두박 1% 효모추출물 0.5% 복합배지, 대두박 1% 효모추출물 1% 복합배지, 그리고 대두박 1% 효모추출물 2% 복합배지이었다. 배양시작 24시간부터 60시간까지 12시간 간격으로 샘플을 채취하고 원심분리하여 얻어진 균체를 사이클로스포린 A가 500mg/L 첨가된 생물전환용 배지가 들어있는 삼각플라스크에 30% 접종하고 30℃에서 24시간 생물전환시킨 다음 이를 동량의 메탄올로 추출하여 액체 크로마토그래피로 분석하였다. 그 결과를 도 8에 나타내었는데 이를 살펴보면 대두박이나 효모추출물 단독 첨가보다 복합 첨가 시 세베키아 베니하나의 사이클로스포린 A로부터 [감마 히드록실메틸루신4]사이클로스포린 A로의 전환능력이 증대되었음을 알 수 있는데 대두박, 효모추출물이 각각 0.5% 및 1%가 첨가된 복합배지에서 사이클로스포린 A로부터 [감마 히드록실메틸루신4]사이클로스포린 A로의 전환능력이 가장 좋았다.   Severkia Benihana was incubated in the medium described in Example 1 for 6 days and then inoculated with 5% in the fermentor. Fermenter culture medium consists of soybean meal 1% medium, soybean meal 0.5% medium, yeast extract 0.5% medium, yeast extract 1% medium, soybean meal 0.5% yeast extract 0.2 % Complex medium, soybean meal 0.5% Yeast extract 0.5% Complex medium, soybean meal 0.5% Yeast extract 1% Complex medium, Soybean meal 1% Yeast extract 0.5% Complex medium, Soybean meal 1% Yeast extract 1% Complex medium, and Soybean meal 1% Yeast extract 2% composite medium. Samples were collected at 12-hour intervals from 24 to 60 hours at the start of the culture, and the cells obtained by centrifugation were inoculated by 30% in an Erlenmeyer flask containing 500 mg / L of cyclosporine A and then incubated at 30 ° C for 24 hours. After conversion it was extracted with the same amount of methanol and analyzed by liquid chromatography. The results are shown in FIG. 8, which shows that the conversion ability of the sebecia benihana cyclosporin A to [gamma hydroxylmethylleucine 4] cyclosporine A was increased when the compound was added more than the soybean meal or the yeast extract alone. The conversion from cyclosporin A to [gamma hydroxylmethylleucine 4] cyclosporin A was the best in the mixed medium containing 0.5% and 1% of the extract, respectively.

실시예 10: 사이클로스포린 A (Cyclosporin A)의 최적 첨가시기 Example 10 Optimal Addition of Cyclosporin A

실시예 9에서 대두박 0.5% 효모추출물 1% 복합배지가 세베키아 베니하나의 생물전환 능력을 가장 좋게 함을 확인하였으므로, 동 배지 조건 하에서 배양시기별 사이클로스포린 A의 첨가 시점이 사이클로스포린 A로부터 [감마 히드록실메틸루신4]사이클로스포린 A로의 생물전환에 미치는 영향을 확인하여 최적 생물전환 방법을 확정하고자 하였다. 먼저 실시예 1의 방법에 따라 6일 동안 배양된 세베키아 베니하나를 대두박 0.5%, 효모추출물 1%, 포도당 0.7%, 수용성 풀 1%, 탄산칼슘 0.005%가 함유된 생물전환 용 배지에 접종하고 배양시작 후 24 시간 (배양초반), 36 시간 (배양중반), 47 시간 (배양후반) 시점에서 각각 500 mg/L의 사이클로스포린 A를 첨가하고 12시간 간격으로 시료를 채취하여 사이클로스포린 A와 [감마 히드록실메틸루신4] 사이클로스포린 A의 양를 액체크로마토그래피로 분석하였다. 결과 도 9에서 보는 것과 같이 사이클로스포린 A를 배양초반 보다는 배양중반 이후에 첨가하는 것이 바람직한 것을 확인하였다. Since the soybean meal 0.5% yeast extract 1% complex medium in Example 9 showed the best biotransformation capacity of Sevekia Benihana, the addition time of cyclosporin A by cultivation time under the same medium conditions from cyclosporin A [gamma hydroxyl To determine the optimal bioconversion method by checking the effect on the bioconversion to methyllucin4] cyclosporin A. First, inoculated in Severkia Benihana cultured for 6 days according to the method of Example 1 in a bioconversion medium containing 0.5% soybean meal, 1% yeast extract, 0.7% glucose, 1% water-soluble grass, 0.005% calcium carbonate At 24 hours (early culture), 36 hours (mid culture), and 47 hours (low culture), 500 mg / L of cyclosporin A was added and samples were taken at 12-hour intervals. Oxylmethylleucine 4] The amount of cyclosporin A was analyzed by liquid chromatography. Results As shown in FIG. 9, it was confirmed that cyclosporin A was preferably added after the mid-culture rather than the early stage of the culture.

희귀 방선균인 세베키아 베니하나 (Sbekia benihana)를 이용하여 인체 면역억제력과 발모효과를 가지고 있는 사이클로스포린 A (Cyclosporin A)에서 발모효과는 그대로 유지하면서 면역억제력은 감소된 [감마 히드록실메틸루신4]사이클로스포 린 A ([γ-hyMe Leu4]-cyclosporin A)를 제조함에 있어, 본 발명의 핵심 사항인 사이클로스포린 A 용해용 첨가제 DMSO (Dimethyl Sulfoxide)를 사용하고 대두박 및 효모추출물을 적절히 배합한 배지를 사용하며 사이클로스포린 A의 투여시점을 배양중반이후로 설정함으로써 세베키아 베니하나 (Sebekia benihana)의 사이클로스포린 A로부터 [감마 히드록실메틸루신4]사이클로스포린 A로의 생물전환 능력을 크게 향상시킬 수 있으며 결과적으로 [감마 히드록실메틸루신4]사이클로스포린 A의 산업화가 가능하게 하는 효과를 제공한다. Using the rare actinomycetes Sbekia benihana, cyclosporin A, which has human immunosuppressive and hair growth effects, maintains the hair growth effect while reducing the immunosuppressive effect [gamma hydroxylmethyl leucine 4] In the preparation of [γ-hyMe Leu4] -cyclosporin A, cyclosporine A dissolving additive DMSO (Dimethyl Sulfoxide) is used, and a medium containing soybean meal and yeast extract is used. By setting the point of administration of cyclosporin A after mid-culture, it is possible to greatly improve the bioconversion ability of Sebekia benihana from cyclosporin A to [gamma hydroxylmethylleucine 4] cyclosporin A and consequently [gamma hydroxyl] It provides the effect of allowing the industrialization of methylleucine 4] cyclosporin A.

Claims (7)

삭제delete 삭제delete 희귀 방선균 세베키아 베니하나 (Sebekia benihana)를 이용하여 사이클로스포린 A를 [감마 히드록실메틸루신4] 사이클로스포린 A로 생물전환시 사이클로스포린 A를 용해시키기 위해 첨가물 DMSO (Dimethyl Sulfoxide)를 사용하는 방법.A method of using the additive DMSO (Dimethyl Sulfoxide) to dissolve cyclosporin A upon bioconversion of cyclosporin A to [gamma hydroxylmethylleucine 4 ] cyclosporin A using the rare actinomycetes Sebekia benihana. 제3항에 있어서, 상기 첨가물 DMSO의 사용농도가 0.1 내지 4%인 것을 특징으로 하는 방법.The method of claim 3, wherein the concentration of the additive DMSO is 0.1 to 4%. 제3항에 있어서, 상기 첨가물 DMSO의 사용농도가 0.5%인 것을 특징으로 하는 방법.4. The method according to claim 3, wherein the concentration of the additive DMSO is 0.5%. 제3항에 있어서, 생물전환용 배지의 대두박 농도가 0.5% 내지 5% 그리고 효모추출물 농도가 0.5% 내지 10%인 것을 특징으로 하는 방법.4. The method according to claim 3, wherein the soybean meal concentration of the bioconversion medium is 0.5% to 5% and the yeast extract concentration is 0.5% to 10%. 제3항에 있어서, 사이클로스포린 A의 첨가시기가 배양 시작 후 35시간 내지 48시간 인 것을 특징으로 하는 방법.The method of claim 3, wherein the cyclosporin A addition time is 35 hours to 48 hours after the start of the culture.
KR1020040074492A 2004-09-17 2004-09-17 Method of Preparing [?-HyMeLeu4]Cyclosporin A by Sebekia benihana KR100597309B1 (en)

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