KR100459289B1 - Method for recovering teicoplanin a2 from fermentation broth - Google Patents
Method for recovering teicoplanin a2 from fermentation broth Download PDFInfo
- Publication number
- KR100459289B1 KR100459289B1 KR10-2002-0030183A KR20020030183A KR100459289B1 KR 100459289 B1 KR100459289 B1 KR 100459289B1 KR 20020030183 A KR20020030183 A KR 20020030183A KR 100459289 B1 KR100459289 B1 KR 100459289B1
- Authority
- KR
- South Korea
- Prior art keywords
- teicoplanin
- filtrate
- filtered
- fermentation broth
- aqueous
- Prior art date
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05B—SPRAYING APPARATUS; ATOMISING APPARATUS; NOZZLES
- B05B11/00—Single-unit hand-held apparatus in which flow of contents is produced by the muscular force of the operator at the moment of use
- B05B11/01—Single-unit hand-held apparatus in which flow of contents is produced by the muscular force of the operator at the moment of use characterised by the means producing the flow
- B05B11/10—Pump arrangements for transferring the contents from the container to a pump chamber by a sucking effect and forcing the contents out through the dispensing nozzle
- B05B11/1042—Components or details
- B05B11/1059—Means for locking a pump or its actuation means in a fixed position
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05B—SPRAYING APPARATUS; ATOMISING APPARATUS; NOZZLES
- B05B11/00—Single-unit hand-held apparatus in which flow of contents is produced by the muscular force of the operator at the moment of use
- B05B11/01—Single-unit hand-held apparatus in which flow of contents is produced by the muscular force of the operator at the moment of use characterised by the means producing the flow
- B05B11/04—Deformable containers producing the flow, e.g. squeeze bottles
- B05B11/042—Deformable containers producing the flow, e.g. squeeze bottles the spray being effected by a gas or vapour flow in the nozzle, spray head, outlet or dip tube
- B05B11/043—Deformable containers producing the flow, e.g. squeeze bottles the spray being effected by a gas or vapour flow in the nozzle, spray head, outlet or dip tube designed for spraying a liquid
Landscapes
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
본 발명은 악티노플라네스 테이코마이세티쿠스 균주를 배양하여 얻어진 발효 브로스를 여과하여 균사체 덩어리와 여액으로 분리하고, 얻어진 균사체 케이크를 15∼30%(v/v)의 아세톤 수용액으로 추출한 후, 이를 여과하여 얻어진 여액을 농축하고, 상기 브로스 용액을 여과하여 얻어진 여액과 합하고, 이를 실란화 실리카겔 컬럼에 걸고, 0.2% 인산나트륨이 함유된 10∼20% 아세토니트릴 수용액으로 세정한 후, 0.2% 인산나트륨이 함유된 27%∼31% 아세토니트릴 수용액으로 용출함을 특징으로 하는 테이코플라닌 A2의 회수방법을 개시한다.In the present invention, the fermentation broth obtained by culturing the Actinoplanes teicomyceticus strain is filtered and separated into a mycelium mass and a filtrate, and the obtained mycelium cake is extracted with 15-30% (v / v) acetone aqueous solution. The filtrate obtained by filtration was concentrated, and the broth solution was combined with the filtrate obtained by filtration, which was hung on a silanized silica gel column, washed with an aqueous 10-20% acetonitrile solution containing 0.2% sodium phosphate, followed by 0.2% phosphoric acid. Disclosed is a method for recovering teicoplanin A 2 , which is eluted with an aqueous 27% to 31% acetonitrile solution containing sodium.
Description
본 발명은 발효액으로부터 테이코플라닌A2의 회수방법에 관한 것이다. 더 상세히는 공지 균주인 악티노플라네스 테이코마이세티쿠스를 배양하여 얻어진 발효 브로스의 용액, 및 이 브로스에서 제거된 균사체 케이크의 추출물을 실란화 실리카겔을 이용하여 경제적이고도 고수율로 테이코플라닌 A2를 회수하는 방법에 관한 것이다.The present invention relates to a method for recovering teicoplanin A 2 from fermentation broth. More specifically, the solution of the fermentation broth obtained by culturing the known strain Actinoplanes teicomyceticus, and the extract of the mycelium cake removed from the broth using tecillinated silica gel are economically and in high yield. A method for recovering A 2 .
테이코플라닌 A2에 관하여는 그의 유효성과 회수방법에 관하여 종래부터 널리 알려져 있으며, 테이코플라닌중, 그의 유효성분으로서는 테이코플라닌 A3-1이 15% 이하, 테이코플라닌 A2군이 85% 이상 함유하는 것이 의약품으로서 유효함이 알려져 있다.Teicoplanin A 2 has been widely known in the past for its effectiveness and recovery method. Among the teicoplanins, teicoplanin A 3-1 is 15% or less and teicoplanin A as its active ingredient. It is known that what contains 85% or more of group 2 is effective as a pharmaceutical.
즉, 미국합중국 특허 제 4,696,817호에서는 브로스로부터 테이코플라닌 A2를 분리 회수하기 위하여 발효 브로스에 20∼50%의 아세톤 또는 n-프로판올을 가하고, 20∼60분간 교반한 후, 발효액/용매를 함유하는 액체를 원심분리 또는 여과하여 균사체를 제거하고, 이를 농축하여 1/2 내지 1/10 양으로 하고, 이 농축액을 냉각시켜 침전물을 생성한 후, 침전물을 여과하여 테이코플라닌 A2를 회수하는 방법이 기재되어 있다. 그러나, 이렇게 얻어진 테이코플라닌 A2는 순도가 낮아 다시 정제하지 않으면 안되고, 또한, 추출시 소모된 혼합물의 회수 및 분리를 위해 다대한 비용이 드는 등의 문제점이 있었다.That is, in US 4,696,817, 20-50% of acetone or n-propanol was added to a fermentation broth to separate and recover teicoplanin A 2 from the broth, followed by stirring for 20 to 60 minutes, and then fermentation broth / solvent was added. The liquid containing is centrifuged or filtered to remove the mycelium, which is concentrated to an amount of 1/2 to 1/10, the concentrate is cooled to form a precipitate, and the precipitate is filtered to give teicoplanin A 2 . The recovery method is described. However, the teicoplanin A 2 thus obtained has low purity and needs to be purified again, and also has a problem such as a large cost for recovery and separation of the mixture consumed during extraction.
미합중국 특허 제4,994,555호에서는 발효 브로스로부터 테이코플라닌을 추출하여 폴리아미드 수지(칼럼 크로마토그래피용 폴리아미드-CC 6, 입자 크기 50-160㎛, 겉보기 밀도 0.20 /㎖, Macherey nagel, 독일)에 흡착시키고, 0.3∼1.0/ℓ의 탄산나트륨 구배를 함유하는 메탄올/물(9/1, V/V)의 혼합물로 용출시킨 다음, 용출액을 분획하여 HPLC로 분석하고, 분획물 중 활성부분을 나타내는 분획을 감압하에서 농축하고, 농축액을 아세톤으로 침전시켜 상등액으로부터 분리 여과하여 회수한후, 건조하여 테이코플라닌 A2를 얻는 것이 개시되어 있다. 그러나, 이 방법은 각종 불순물이 많은 발효 브로스에 흡착 수지를 직접 사용하므로 고순도의 테이코플라닌 A2를 얻는데 어려움이 있다.In US Pat. No. 4,994,555, Teicoplanin was extracted from fermentation broth and adsorbed onto polyamide resin (polyamide-CC 6 for column chromatography, particle size 50-160 μm, apparent density 0.20 / ml, Macherey nagel, Germany). Eluted with a mixture of methanol / water (9/1, V / V) containing a 0.3 to 1.0 / l sodium carbonate gradient, and then the eluate was fractionated and analyzed by HPLC, and the fractions representing the active part of the fractions were decompressed. It is disclosed that the solution is concentrated under reduced pressure, precipitated the concentrate with acetone, collected by filtration from the supernatant, and then dried to obtain teicoplanin A 2 . However, this method has difficulty in obtaining high purity teicoplanin A 2 since the adsorption resin is directly used for fermentation broths containing many impurities.
또한, 미합중국 특허 제 4542018호에서는 테이코플라닌 A2로부터 실란화 실리카겔을 사용하여, 테이코플라닌 A2의 각각의 인자, 즉, 테이코플라닌 A2-1,테이코플라닌 A2-2,테이코플라닌 A2-3,테이코플라닌 A2-4,테이코플라닌 A2-5를 각각 분리하는 방법을 개시하고 있다. 그러나, 이와 같이 각각의 인자를 분리하는 것은 특정 용도에 사용하는 이외는 시판 의약으로서의 경제성은 없다.In U.S. Patent No. 4,542,018 to Tay Plastic nose from non-A 2 using silanized silica gel, teicoplanin A each factor of 2, i.e., teicoplanin A 2-1, teicoplanin A 2 -2 , teicoplanin A 2-3 , teicoplanin A 2-4 and teicoplanin A 2-5 are disclosed. However, the separation of each factor in this way has no economic feasibility as a commercial medicine except for use in specific applications.
따라서, 테이코플라닌 A2를 경제적이고도 순도가 높으며, 수율이 높은 방법은 아직까지 개발되지 않고 있는 실정이어서, 이러한 문제점을 개선한 방법이 요망되어 왔다.Therefore, since the economic and purity of teicoplanin A 2 and the high yield are not developed yet, the method which improved this problem has been desired.
본 발명자는 상기 문제점을 해결하기 위하여 예의 연구한 결과, 공지의 방법으로 악티노플라네스 테이코마이세티쿠스 균주를 배양하여 얻어진 발효 브로스의 용액을 여과하여 균사체 덩어리를 분리하고, 얻어진 균사체 케이크를 아세톤 수용액으로 추출하고, 이를 여과하여 얻어진 여액을 농축한 후, 전술한 브로스 용액의 여액과 합하고, 이를 실란화 실리카겔 컬럼에 걸고, 일정량의 용출액으로 용출하여버리고, 그런 다음 동일 용출액으로 용출함으로서 테이코플라닌 A2를 경제적이고도 고수율로 얻을 수 있음을 발견하고, 본 발명을 완성하게 이르렀다.As a result of intensive studies to solve the above problems, the present inventors filtered a solution of fermentation broth obtained by culturing Actinoplanes teicomyceticus strain by a known method to separate the mycelium masses, and acetone obtained from the mycelium cake. Extraction with aqueous solution, the filtrate obtained by filtration is concentrated, combined with the filtrate of the broth solution described above, it is suspended on a silanized silica gel column, eluted with a certain amount of eluent, and then eluted with the same eluent. It was found that nin A 2 can be obtained economically and with high yield, and the present invention has been completed.
즉, 본 발명은 테이코플라닌 A2의 회수방법을 제공하는 것이다.That is, the present invention provides a method for recovering teicoplanin A 2 .
도 1은 실시예 5에서 얻은 테이코플라닌 A2를 HPLC로 분석한 결과를 나타내는 도이다.1 is a diagram showing the results of HPLC analysis of teicoplanin A 2 obtained in Example 5. FIG.
도 2는 시판되는 테이코플라닌 A2(G 사)의 HPLC로 분석한 결과를 나타내는 도이다.Fig. 2 is a diagram showing the results of HPLC analysis of commercial teicoplanin A 2 (G company).
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 공지의 방법으로 악티노플라네스 테이코마이세티쿠스 균주를 배양하여 얻어진 발효 브로스의 용액으로부터 테이코플라닌 A2의 회수하는 것이다. 균주를 배양하여 얻어진 발효 브로스는 균사체와 용액으로 이루어져 있다. 이를 여과하여 균사체 덩어리와 여액으로 분리하고, 얻어진 균사체 케이크를 15∼30%(v/v)의 아세톤 수용액으로 추출하면 테이코플라닌 A2가 용해되어 나오고, 이를 여과하여 얻어진 여액을 농축한 후, 전술한 브로스 용액을 여과하여 얻어진 여액과 합한다. 이를 실란화 실리카겔 컬럼에 걸고, 0.2% 인산나트륨이 함유된 10∼20% 아세토니트릴 수용액으로 용출하면 불순물이 제거된다. 이 때, 20% 이상의 아세토니트릴 수용액으로 용출하면, 유효성분인 테이코플라닌 A2도 용출되어 함께 빠져나가므로 바람직하지 않고, 또한 10% 이하의 아세토니트릴 수용액으로 용출하면, 불순물이 용출되지 않거나, 용출시간이 대폭 늘어나 바람직하지 않다. 그런 다음, 0.2% 인산나트륨이 함유된 27%∼31% 아세토니트릴 수용액으로 용출한다. 이때, 27%이하의 아세토니트릴 수용액을 사용하면, 목적하는 테이코플라닌 A2가 추출되지 않고, 31%를 초과하는 아세토니트릴 수용액을 사용하는 경우에는 테이코플라닌 A1과 같은 다른 물질이 함께 추출되어 나오므로 사용할 수 없다.The present invention recovers teicoplanin A 2 from a solution of fermentation broth obtained by culturing Actinoplanes teicomyceticus strains by a known method. Fermentation broth obtained by culturing the strain consists of a mycelium and a solution. The filtrate was separated into a mycelial mass and a filtrate, and the obtained mycelium cake was extracted with 15-30% (v / v) acetone aqueous solution to dissolve teicoplanin A 2 , and the filtrate was concentrated by filtration. The broth solution mentioned above is combined with the filtrate obtained by filtration. This is suspended over a silanized silica gel column and eluted with an aqueous 10-20% acetonitrile solution containing 0.2% sodium phosphate to remove impurities. At this time, eluting with 20% or more of acetonitrile aqueous solution is not preferable because elutoniplanin A 2 , which is an active ingredient, is also eluted and escaped together, and eluting with 10% or less of acetonitrile aqueous solution does not elute impurities. As a result, the elution time is greatly increased, which is undesirable. Then elute with an aqueous 27% to 31% acetonitrile solution containing 0.2% sodium phosphate. At this time, if an aqueous solution of acetonitrile of 27% or less is used, the desired teicoplanin A 2 is not extracted, and if an aqueous solution of acetonitrile of more than 31% is used, other substances such as teicoplanin A 1 It can not be used because it is extracted together.
상기에서 균사체 케이크로부터 소량의 용매를 사용하여 추출한 조테이코플라닌 농축액이나 여과된 발효브로스 여액을 양이온교환수지(Duolite C-20, Purolite C-100H)나 음이온교환수지(Purolite MN150, Diaion WA10) 또는 흡착수지(HP2MG, SP850) 등으로 전처리를 한 후, 실란화 실리카겔(YMC ODS-A 12nm, S-150㎛) 칼럼을 통해 회수할 수 있다.Joteicoplanin concentrate or filtered fermentation broth filtrate extracted using a small amount of solvent from the mycelium cake is cation exchange resin (Duolite C-20, Purolite C-100H) or anion exchange resin (Purolite MN150, Diaion WA10) Alternatively, after pretreatment with an adsorptive resin (HP2MG, SP850) or the like, it may be recovered through a silanized silica gel (YMC ODS-A 12nm, S-150㎛) column.
이렇게 얻어진 용출액을 당해 분야의 통상의 방법, 예를 들면, 활성탄 처리, 탈염, 동결건조 등의 조작을 수행함으로써 고순도의 테이코플라닌 A2를 얻을 수 있다.The eluate thus obtained can be subjected to conventional methods in the art, for example, activated carbon treatment, desalting, lyophilization and the like to obtain high purity teicoplanin A 2 .
(실시예)(Example)
이하, 실시예를 들어 본 발명을 구체적으로 설명한다. 그러나, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, an Example is given and this invention is demonstrated concretely. However, the scope of the present invention is not limited to these Examples.
실시예 1Example 1
발효브로스 10ℓ를 여과하여 균사체 케이크로 남는 균사체 덩어리를 분리한다. 분리된 균사체 케이크를 20% 아세톤 수용액에 가하여, 추출하고, 감압하 농축한 다음, 상기의 발효브로스를 여과한 여액과 합한 후, 실란화 실리카겔(YMC-gel ODS-A 120nm, S-150㎛, YMC co., ltd. 일본) 1ℓ로 채운 유리 칼럼을 걸었다. 정제수 3ℓ, 및 0.2% 인산나트륨이 함유된 20% 아세토니트릴 수용액 3ℓ의 순서로 세척하여 불순물을 제거한 후, 0.2% 인산나트륨이 함유된 31% 아세토니트릴 수용액으로 수지를 용출하였다. 용출 초기 1.3베드 부피는 버리고, 나머지는 회수하여 총 3베드부피의 용출액을 회수하였다. 용출액을 활성탄으로 처리한 후, 여과하였다. 농축, 탈염한 후, 동결 건조하여 순도 90%의 테이코플라닌 A2를 70%의 수율로 얻었다.10 L of fermented broth is filtered to separate the mycelium mass remaining as mycelium cake. The isolated mycelium cake was added to a 20% acetone aqueous solution, extracted, concentrated under reduced pressure, and the fermentation broth was combined with the filtrate, and then silanated silica gel (YMC-gel ODS-A 120 nm, S-150 μm, YMC co., Ltd. Japan) hanged a glass column filled with 1 liter. After washing with 3 L of purified water and 3 L of 20% acetonitrile aqueous solution containing 0.2% sodium phosphate to remove impurities, the resin was eluted with 31% acetonitrile aqueous solution containing 0.2% sodium phosphate. The initial 1.3 bed volume of elution was discarded and the rest was recovered to recover a total of 3 bed volumes of eluate. The eluate was treated with activated carbon and then filtered. Concentrated and desalted, lyophilized to obtain Teicoplanin A 2 with a purity of 90% in a yield of 70%.
실시예 2Example 2
발효브로스 10ℓ를 여과하여 균사체 케이크로 남는 균사체 덩어리를 분리한다. 분리된 균사체 케이크를 20% 아세톤 수용액에 가하여, 추출하고, 감압하 농축한 다음, 양이온 교환수지(Duolite C-20, Purolite C-100H)에 통탑을 하면서, 실란화 실리카겔(YMC-gel ODS-A 12nm, S-150㎛, YMC co., ltd. 일본) 1ℓ로 채운 유리 칼럼을 통과시켰다. 정제수 3ℓ, 및 0.2% 인산나트륨이 함유된 20%아세토니트릴 수용액 3ℓ의 순서로 세척하여 불순물을 제거한 후, 0.2% 인산나트륨이 함유된 31% 아세토니트릴 수용액으로 수지를 용출하였다. 용출초기 1.3베드 부피는 버리고, 나머지는 회수하여 총 3베드부피의 용출액을 회수하였다. 용출액을 1/10으로 농축, 탈염한 후 동결 건조하여 순도 92%의 테이코플라닌 A2를 63%의 수율로 얻었다.10 L of fermented broth is filtered to separate the mycelium mass remaining as mycelium cake. The separated mycelium cake was added to a 20% acetone aqueous solution, extracted, concentrated under reduced pressure, and then subjected to a sieve column with a cation exchange resin (Duolite C-20, Purolite C-100H), and then silanized silica gel (YMC-gel ODS-A). 12 nm, S-150 μm, YMC co., Ltd. Japan) was passed through a glass column filled with 1 L. After washing with 3 L of purified water and 3 L of 20% acetonitrile aqueous solution containing 0.2% sodium phosphate to remove impurities, the resin was eluted with 31% acetonitrile aqueous solution containing 0.2% sodium phosphate. The initial 1.3 bed volume of the elution was discarded and the rest was recovered to recover a total of 3 bed volumes of the eluate. The eluate was concentrated to 1/10, desalted and lyophilized to obtain teicoplanin A 2 with a purity of 92% in a yield of 63%.
실시예 3Example 3
발효브로스 10ℓ를 여과하여 균사체 케이크로 남는 균사체 덩어리를 분리한다. 분리된 균사체 케이크를 20% 아세톤 수용액에 가하여, 추출하고, 감압하 농축한 다음, 음이온 교환수지(Purolite MN150, Diaion WA10)에 통탑을 하면서, 실리카겔(YMC-gel ODS-A 12nm, S-150㎛, YMC co., ltd. 일본) 1ℓ로 채운 유리 칼럼을 통과시켰다. 정제수 3ℓ, 및 0.2% 인산나트륨이 함유된 20%아세토니트릴 수용액 3ℓ의 순서로 세척한 후, 0.2% 인산나트륨이 함유된 31% 아세토니트릴 수용액으로 수지를 용출하였다. 용출초기 1.3베드 부피는 버리고, 나머지는 회수하여 총 3베드부피의 용출액을 회수하였다. 용출액을 1/10으로 농축, 탈염한 후 동결 건조하여 순도 91∼92%의 테이코플라닌 A2을 55∼63%의 수율로 얻었다.10 L of fermented broth is filtered to separate the mycelium mass remaining as mycelium cake. The separated mycelium cake was added to a 20% acetone aqueous solution, extracted, concentrated under reduced pressure, and then subjected to a column through an anion exchange resin (Purolite MN150, Diaion WA10), followed by silica gel (YMC-gel ODS-A 12 nm, S-150 μm). , YMC co., Ltd. Japan) was passed through a glass column filled with 1 liter. After washing with 3 L of purified water and 3 L of 20% acetonitrile aqueous solution containing 0.2% sodium phosphate, the resin was eluted with 31% acetonitrile aqueous solution containing 0.2% sodium phosphate. The initial 1.3 bed volume of the elution was discarded and the rest was recovered to recover a total of 3 bed volumes of the eluate. The eluate was concentrated to 1/10, desalted and lyophilized to obtain teicoplanin A 2 with a purity of 91 to 92% in a yield of 55 to 63%.
실시예 4Example 4
발효브로스 10ℓ를 여과하여 균사체 케이크로 남는 균사체 덩어리를 분리한다. 분리된 균사체 케이크를 20% 아세톤 수용액에 가하여, 추출하고, 감압하 농축한 다음, 흡착수지(Diaion 825, 850)에 통과시키고 30% 메탄올 수용액으로 세척한 후, 80∼90% 메탄올 수용액으로 수지를 용출시켰다. 용출액을 감압하 농축하여 실란화 실리카겔(YMC-gel ODS-A 12nm, S-150㎛, YMC co., ltd. 일본) 1ℓ로 채운 유리 칼럼을 통과시켰다. 정제수 3ℓ, 및 0.2% 인산나트륨이 함유된 20%아세토니트릴 수용액 3ℓ로 세척한 후, 0.2% 인산나트륨이 함유된 31% 아세토니트릴 수용액으로수지를 용출하였다. 용출초기 1.3베드 부피는 버리고 나머지는 회수하여 총 3베드부피의 용출액을 회수하였다. 용출액을 1/10으로 농축하고 활성탄을 처리한 후 여과하였다. 농축, 탈염하여 동결 건조하여 순도 92%의 테이코플라닌 A2를 26%의 수율로 얻었다.10 L of fermented broth is filtered to separate the mycelium mass remaining as mycelium cake. The isolated mycelium cake was added to a 20% acetone aqueous solution, extracted, concentrated under reduced pressure, passed through an adsorbent resin (Diaion 825, 850), washed with 30% aqueous methanol solution, and then the resin was washed with 80-90% aqueous methanol solution. Eluted. The eluate was concentrated under reduced pressure and passed through a glass column filled with 1 liter of silanized silica gel (YMC-gel ODS-A 12 nm, S-150 μm, YMC co., Ltd. Japan). After washing with 3 L of purified water and 3 L of 20% acetonitrile solution containing 0.2% sodium phosphate, the resin was eluted with 31% acetonitrile solution containing 0.2% sodium phosphate. The initial 1.3 bed volume was discarded and the rest was recovered to recover a total of 3 bed volumes of eluate. The eluate was concentrated to 1/10 and treated with activated charcoal and filtered. Concentrated, desalted and lyophilized to give teicoplanin A 2 with a purity of 92% in a yield of 26%.
실시예 5Example 5
발효브로스 10ℓ를 여과하여 균사체 케이크로 남는 균사체 덩어리를 분리한다. 분리된 균사체 케이크를 20% 아세톤 수용액에 가하여, 추출하고, 감압하 농축한 다음, 흡착수지(Diaion HP2MG)에 통과시키고 30% 메탄올 수용액으로 세척한 후, 55∼65% 메탄올 수용액으로 수지를 용출시켰다. 용출액을 감압하 농축하여 실란화 실리카겔(YMC-gel ODS-A 12nm, S-150㎛, YMC co., ltd. 일본) 1ℓ로 채운 유리 칼럼을 통과시켰다. 정제수 3ℓ, 및 0.2% 인산나트륨이 함유된 20%아세토니트릴 수용액 3ℓ로 세척한 후, 0.2% 인산나트륨이 함유된 31% 아세토니트릴 수용액으로 수지를 용출하였다. 용출초기 1.3베드 부피는 버리고 나머지는 회수하여 총 3베드부피의 용출액을 회수하였다. 용출액을 1/10으로 농축하고 활성탄을 처리한 후, 여과하였다. 농축, 탈염하여 동결 건조하여 순도 97%의 테이코플라닌 A2를 43%의 수율로 얻었다.10 L of fermented broth is filtered to separate the mycelium mass remaining as mycelium cake. The separated mycelium cake was added to a 20% acetone aqueous solution, extracted, concentrated under reduced pressure, passed through an adsorption resin (Diaion HP2MG), washed with 30% methanol aqueous solution, and the resin was eluted with 55-65% methanol aqueous solution. . The eluate was concentrated under reduced pressure and passed through a glass column filled with 1 liter of silanized silica gel (YMC-gel ODS-A 12 nm, S-150 μm, YMC co., Ltd. Japan). After washing with 3 L of purified water and 3 L of 20% acetonitrile solution containing 0.2% sodium phosphate, the resin was eluted with 31% acetonitrile solution containing 0.2% sodium phosphate. The initial 1.3 bed volume was discarded and the rest was recovered to recover a total of 3 bed volumes of eluate. The eluate was concentrated to 1/10, treated with activated carbon, and filtered. Concentrated, desalted and lyophilized to obtain teicoplanin A 2 with a purity of 97% in a yield of 43%.
실험예Experimental Example
상기 실시예 5에서 얻어진 테이코플라닌 A2와 시판되는 테이코플라닌 A2(제조원: G사)를 하기 조건에서 HPLC로 분석하여 순도를 측정한 바, 실시예 5의 테이코플라닌 A2의 순도는 97.7%이었고, 시판 테이코플라닌 A2는 82.9%이었다. 이 실험으로부터 본 발명의 방법에 의하면, 종래의 방법에 비해 고순도로 테이코플라닌 A2를 회수할 수 있음을 알 수 있다.Teicoplanin A 2 obtained in Example 5 and commercially available teicoplanin A 2 (manufacturer: G Company) were analyzed by HPLC under the following conditions, and the purity thereof was measured. The purity of 2 was 97.7% and the commercial teicoplanin A 2 was 82.9%. According to the method of the present invention from the experiments, it can be seen that compared with the conventional method to recover the teicoplanin A 2 in high purity.
HPLC 분석조건HPLC analysis conditions
실험조건 HPLC (JASCO 1500 series)Experimental HPLC (JASCO 1500 series)
검출기 자외부 흡광광도계(측정파장: 254㎚)Detector Ultraviolet Absorbance Spectrometer (Wavelength: 254 nm)
컬럼 옥타데실릴화한 8∼10㎛의 실리카 겔을 충전한 내경 4㎜, 길이Inner diameter 4mm, length filled with the column octadecylylated silica gel of 8-10 micrometers, length
250㎜의 스테인레스관 (제조원: Merck)250mm stainless steel pipe (Merk)
이동상Mobile phase
이동상 A: 인산2수소나트륨 1.95g에 아세토니트릴/물(1/9) 혼액을Mobile Phase A: Acetonitrile / water (1/9) mixture was mixed with 1.95 g of sodium dihydrogen phosphate.
넣어 용해한 다음, 수산화나트륨 시액으로 pH를 6.0으로Dissolve, and then adjust the pH to 6.0 with sodium hydroxide solution.
조정하고, 500㎖로 하였다.It adjusted to 500 ml.
이동상 B: 인산2수소나트륨 1.95g에 아세토니트릴/물(7/3) 혼액을Mobile phase B: Acetonitrile / water (7/3) mixture was mixed with 1.95 g of sodium dihydrogen phosphate.
넣어 용해한 다음, 수산화나트륨 시액으로 pH를 6.0으로Dissolve, and then adjust the pH to 6.0 with sodium hydroxide solution.
조정하고, 500㎖로 하였다.It adjusted to 500 ml.
유속 2㎖/min.Flow rate 2 ml / min.
이상에서 설명한 바와 같이, 본 발명 방법에 의하면, 종래의 방법에 비해 발효액으로부터 테이코플라닌 A2를 용이하고도 고순도로 회수할 수 있다.As described above, according to the method of the present invention, teicoplanin A 2 can be recovered easily and with high purity from the fermentation broth as compared with the conventional method.
Claims (2)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2002-0030183A KR100459289B1 (en) | 2002-05-30 | 2002-05-30 | Method for recovering teicoplanin a2 from fermentation broth |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2002-0030183A KR100459289B1 (en) | 2002-05-30 | 2002-05-30 | Method for recovering teicoplanin a2 from fermentation broth |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20030092504A KR20030092504A (en) | 2003-12-06 |
| KR100459289B1 true KR100459289B1 (en) | 2004-12-03 |
Family
ID=32385087
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR10-2002-0030183A KR100459289B1 (en) | 2002-05-30 | 2002-05-30 | Method for recovering teicoplanin a2 from fermentation broth |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR100459289B1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100476818B1 (en) * | 2002-07-19 | 2005-03-17 | 종근당바이오 주식회사 | Purification method for teicoplanin A2 |
| KR100474653B1 (en) | 2004-04-16 | 2005-03-14 | 동국제약 주식회사 | Preparation Method of Teicoplanin with High Productivity and Purity |
| EP2592089A1 (en) | 2011-11-11 | 2013-05-15 | LEK Pharmaceuticals d.d. | Process of purification of teicoplanin |
| EP2592090A1 (en) | 2011-11-11 | 2013-05-15 | LEK Pharmaceuticals d.d. | Process of purification of teicoplanin |
| CN107267581B (en) * | 2017-08-04 | 2020-11-20 | 福建省微生物研究所 | Fermentation medium for producing teicoplanin |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4696817A (en) * | 1983-10-11 | 1987-09-29 | The Dow Chemical Company | Extraction of teichomycin A2 from whole culture fermentation broth |
| KR910004634A (en) * | 1989-08-07 | 1991-03-29 | 안시환 | Purification Recovery of Sepharoserin C Using Polyphase Resin Column |
| US5085990A (en) * | 1987-09-07 | 1992-02-04 | Gruppo Lepetit S.P.A. | Teicoplanin-like derivatives |
| US5486465A (en) * | 1990-10-01 | 1996-01-23 | Gruppo Lepetit S.P.A. | Teicoplanin recovery process |
| KR20030034949A (en) * | 2001-10-29 | 2003-05-09 | 씨제이 주식회사 | Process for the purification of teicoplanin |
-
2002
- 2002-05-30 KR KR10-2002-0030183A patent/KR100459289B1/en active IP Right Grant
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4696817A (en) * | 1983-10-11 | 1987-09-29 | The Dow Chemical Company | Extraction of teichomycin A2 from whole culture fermentation broth |
| US5085990A (en) * | 1987-09-07 | 1992-02-04 | Gruppo Lepetit S.P.A. | Teicoplanin-like derivatives |
| KR910004634A (en) * | 1989-08-07 | 1991-03-29 | 안시환 | Purification Recovery of Sepharoserin C Using Polyphase Resin Column |
| US5486465A (en) * | 1990-10-01 | 1996-01-23 | Gruppo Lepetit S.P.A. | Teicoplanin recovery process |
| KR20030034949A (en) * | 2001-10-29 | 2003-05-09 | 씨제이 주식회사 | Process for the purification of teicoplanin |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20030092504A (en) | 2003-12-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| DK173231B1 (en) | Method for Extraction of Glycopeptidic Antibiotics | |
| JP2006014720A (en) | Method for refining vancomycin hydrochloride | |
| KR100476818B1 (en) | Purification method for teicoplanin A2 | |
| KR100459289B1 (en) | Method for recovering teicoplanin a2 from fermentation broth | |
| JP2756279B2 (en) | Hirudin isolation and purification method | |
| CN108250270A (en) | A kind of method of the enrichment extraction Daptomycin from zymotic fluid | |
| JP2021524464A (en) | Method for preparing recombinant human insulin or its analog precursor | |
| JP3992497B2 (en) | High purity acarbose manufacturing method | |
| CN1336434A (en) | Prepn. and affinity chromatographic purification process of kallidinogen enzyme | |
| CN117244533A (en) | Adsorbent for recycling and pretreatment of acetonitrile waste liquid and preparation method and application thereof | |
| CN105238841B (en) | Cephalosporin adsorbs the recycling of DCPC and method for transformation in waste liquid | |
| CN109666051B (en) | Purification method of kasugamycin | |
| CN109929013A (en) | A kind of purification and refining methd of Pneumocandin B0 | |
| US5180670A (en) | Method for purification of mitomycin C | |
| CN112300229A (en) | Method for purifying acarbose from acarbose fermentation liquor | |
| CN113416223B (en) | Method for separating and purifying salidroside and product thereof | |
| CN106589075B (en) | Purification method of teicoplanin | |
| CN102617727B (en) | Thymalfasin compound and novel preparation method thereof | |
| JP2971128B2 (en) | Purification method of mitomycin C | |
| CN109705174A (en) | The extracting method of tobramycin | |
| CN109988211B (en) | Purification method of glycochenodeoxycholic acid sodium salt | |
| KR800001596B1 (en) | Recovery method of cephalosporin c using multiion exchange column | |
| KR950009201B1 (en) | Purification of vancomycin by using ion exchange resin and alumina resin | |
| CN108164382B (en) | Method for comprehensively extracting flavone compounds from hops | |
| CN112812142A (en) | Method for refining monosialotetrahexosyl ganglioside sodium |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant | ||
| FPAY | Annual fee payment |
Payment date: 20121122 Year of fee payment: 9 |
|
| FPAY | Annual fee payment |
Payment date: 20131119 Year of fee payment: 10 |
|
| FPAY | Annual fee payment |
Payment date: 20141121 Year of fee payment: 11 |
|
| FPAY | Annual fee payment |
Payment date: 20151120 Year of fee payment: 12 |
|
| FPAY | Annual fee payment |
Payment date: 20161114 Year of fee payment: 13 |
|
| FPAY | Annual fee payment |
Payment date: 20171113 Year of fee payment: 14 |
|
| FPAY | Annual fee payment |
Payment date: 20181108 Year of fee payment: 15 |
|
| FPAY | Annual fee payment |
Payment date: 20191114 Year of fee payment: 16 |