KR940004000B1 - Production of mildiomycin - Google Patents

Abstract

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Description

Preparation of Mildiomycin

The present invention relates to a method for fermentative preparation of Mildiomycin.

Mildiomycin is an antibiotic named “antibiotic B-98891” found by culturing Mildiomycin-producing strains belonging to the genus Striptobertylium as a compound of formula (I).

Attention has been focused on its activity, in particular as a medicament or acaricide, which prevents and treats a broad spectrum of plant diseases. Therefore, it was required to increase the yield of Mildiomycin, and incubated Mildiomycin-producing strains in a medium containing at least 3 mM (mmol / l) N-methyl compound or in a medium containing 5-hydroxymethylcytosine. Fermentative methods have been attempted characterized by the following [US, in particular, 4007267; US Patent 4334022; US Patent 4296107; British Patent 1507193; "The Journal of Antibiotics" Vol. 31, No. 511-518, 519-524 (1978); "Journal of Pesticide Science" Vol. 4, No. 3, 349-353 (1979); "Tetrahedron" Vol. 37, 1317-1327 (1981).

However, in the known fermentative method of producing Mildiomycin, the ability of the Mildiomycin-producing strain used to produce Mildiomycin is limited, thereby improving the yield of Mildiomycin by improving the substances contained in the medium. There is a limit to this. Thus, this method is not satisfactory for producing large amounts of Mildiomycin on an industrial scale.

As a result of steady research to improve the Mildiomycin-producing ability of the Mildiomycin-producing strains, the inventors have found that Mildiomycin-producing strains have the following structural formula:

(II )의 세린히드록사메이트 또는 구조식 :

Serine hydroxamate or structural formula of (II):

( III )의 카나바닌에 대하여 내성을 갖게 하면 밀디오마이신-생산 능력이 의외로 개선되며, 공지의 배지를 이용하여 배양하더라도 밀디오마이신 생산의 효율성이 높아지는 것을 발견하였다. 이러한 발견에 기초를 두고 본 발명을 완성하였다.

It was found that the resistance to cannabanin of (III) surprisingly improved the Mildiomycin-producing ability and increased the efficiency of Mildiomycin production even when cultured using a known medium. Based on this finding, the present invention has been completed.

In more detail, this invention relates to following (1)-(4).

(1) from a culture broth produced after culturing Mildiomycin-producing strains belonging to Streptoverticilium and resistant to serine hydroxyxamate or cannabanine in the medium to assimilate and accumulate Mildiomycin A method for producing Mildiomycin, characterized by recovering Mildiomycin.

(2) The method for producing Mildiomycin according to (1), wherein the medium contains at least 3 mM N-methyl compound.

(3) The method for producing Mildiomycin according to (1) or (2), wherein the medium contains 5-hydroxymethylcytosine.

(4) streptovertylsilium limofaciens resistant to serine hydroxyxamate or cannabanin and having the ability to produce mydiomycin.

In the method for producing Mildiomycin of the present invention, Mildiomycin-producing strains (hereinafter referred to as "strains of the present invention") belonging to Streptoverticilium and resistant to serine hydroxyxamate or cannabanin are used. . The strain of the present invention has the following properties, for example.

[1] morphological characteristics:

이다. 포자는 통상적으로 3∼16의 사슬로서 형성되며, 각 포자의 표면은 매끄럽거나 또는 무사마귀가 있다. 공지의 배지에서는 포자낭, 편모, 보속 균체 등이 전혀 관찰되지 않는다.On various known media, the strain of the present invention forms a parasitic mycelium representing a rotifer having a secondary reproductive body. In rare cases, rings and hooks are found on synthetic agar media (eg, glucose, asparagine agar). Spores are ovoid to cylindrical in shape

to be. Spores are typically formed as chains of 3 to 16, with the surface of each spore smooth or wart free. In known media, no spore sac, flagella, binding cells and the like are observed at all.

[2] culture characteristics:

The culture characteristics of the strains of the present invention are shown in Table 1. Unless otherwise indicated, the explanation is the result of 21 days of incubation at 28 ° C. The color names shown in () in the table are based on the Color Harmony Manual, 4th ed., Container Corporation of America.

TABLE 1

[3] physiological properties

(1) growth temperature range; Grows at 15-38 ° C, better growth and formation of parasitic mycelium is observed at 28-36 ° C.

(2) gelatin liquefied (incubated at 24 ° C. for 28 days); Not liquefied

(3) hydrolysis of starch; positivity

(4) reduction of nitrates; Negative bacterium.nitrate. Juicy (ISP-NO.8) and Xzapec Solution

(5) coagulation of scheme milk; Positive, fabtonization; positivity

(6) production of temptation pigments; Positive (peptone, yeast, iron agar medium), negative (tyrosine agar medium)

(7) Use of carbon sources (Freedam, High Live Agar Badges):

Iii) a well-used carbon source; Inositol, D-galactose, D-glucose, maltose, D-mannose, starch, glycerin, sodium acetate, sodium succinate, sodium citrate.

Ii) the carbon source from which the equivalent is used; D-fructose, trehalose.

Iii) an unused carbon source; Erythritol, adonitol, D-sorbitol, D-mannitol, dulcitol, D-xylose, L-arabinose, L-sorbose, rhamnose, melvios, sucrose, lactose, raffinose, salicycin, esculin , Inulin.

(8) pigmentation on proteinaceous medium; weakness

(9) cracks on bulon-agar medium; radish

이고 카나바닌에 대해서는 최소한

이다.(10) resistance to serine hydroxyxamate or cannabanin; Minimum growth inhibition concentrations are at least for serine hydroxyxamate

And at least for cannabanine

to be.

[4] other characteristics; According to the enzymatic method described in Journal of Fermentation Technology, Vol. 63, No. 1, 17-21 (1985), the inactivation to produce 5-hydroxymethyl cytosine is at least 0.18 nmol / min / mg.protein. .

-182(세린히드록사메이트-내성), 스트렙토베르티실륨 리모파시엔스

(세린히드록사메이트-및 카나바닌-내성) 및 스트렙토베르티실륨 리모파시엔스

(카나바닌-내성)이다. 상기 스트렙토베르티실륨 리모파시엔스

-182는 일본국 재단법인 발효 연구소(IFO)에 1985년 6월 28일부터 IFO-14448의 수탁 번호로 기탁되어 있으며 일본국 통상 산업성 공업 기술원 미생물 공업 기술 연구소(FRI)에 1985년 7월 8일부터 수탁 번호 FERM P-8334로 기탁되어 있고 부다페스트 협약하의 기탁으로 전환되어 FERM BP-1127의 기탁 번호로 기탁되어 있다. 상기 균주는 또한 1986년 8월 6일자로 한국 종균 협회(KFCC)에 KFCC-10242의 수탁 번호로 기탁되어 있다. 균주 스트렙토베르티실륨 리모파시엔스 CV^R-48은 IFO에 1985년 6월 28일부터 IFO-14449의 수탁 번호로 기탁되어 있으며, FRI에 1985년 7월 8일 부터 FERM P-8333의 수탁번호로 기탁되어있고 또한 부다페스트 협약하의 기탁으로 전환되어 FERM BP-1126의 기탁 번호로 기탁되어 있다. KFCC에는 1986년 8월 6일에 KFCC-10243의 수탁번호로 기탁 되었다. 균주 스트렙토베르티실륨 리모파시엔스 CV^R-16은 IFO에 1986년 7월 15일부터 IFO-14527의 수탁 번호를 기탁되어 있으며, FRI에서 1986년 7월 25일부터 FERM P-8874의 수탁 번호로 기탁되어 있고, 또한 부다페스트 협약하의 기탁으로 전환되어 FERM BP-1134의 기탁 번호로 기탁되어 있다. KFCC에는 1986년 8월 6일에 KFCC-10244의 수탁 번호로 기탁되었다.Strains of the present invention having the above-described characteristics are streptoverticillium limofaciens and the like, for example streptovertylsilium limofaciens

-182 (serine hydroxyxamate-resistant), streptoverticilium limofaciens

(Serine hydroxamate- and cannabanin-resistant) and streptoverticilium limofaciens

(Canavanin-resistant). The streptovertyllium limofaciens

-182 has been deposited with the IFO-14448 accession number from the International Fermentation Research Institute (IFO) on June 28, 1985, and on July 8, 1985, with the Institute of Microbiological Technology (FRI), Japan Institute of Trade and Industry. Deposited as accession number FERM P-8334 and converted to deposit under the Budapest Convention and deposited with accession number FERM BP-1127. The strain was also deposited with the accession number of KFCC-10242 to the Korean spawn association (KFCC) on August 6, 1986. The strain Streptoverticillium limofaciens CV ^R -48 has been deposited with the IFO under accession number IFO-14449 from 28 June 1985 to FRI under accession number FERM P-8333 from 8 July 1985. Deposited and converted to a deposit under the Budapest Convention and deposited with the accession number of FERM BP-1126. The KFCC was deposited with the accession number of KFCC-10243 on August 6, 1986. Strain Streptovertyllium limofaciens CV ^R- 16 has been deposited with the IFO an accession number of IFO-14527 from July 15, 1986, and has been assigned to the FERM P-8874 accession number from July 25, 1986 by FRI. Deposited and converted to a deposit under the Budapest Convention and deposited with a deposit number of FERM BP-1134. The KFCC was deposited with accession number KFCC-10244 on 6 August 1986.

In the culturing of the method for producing mydiomycin of the present invention, a medium containing a carbon source, a digestible nitrogen source and an inorganic salt, which can be assimilated by a microorganism, can be used. If necessary, small amounts of effective micronutrients, growth promoters, precursors and other substances can be added to the batch. Generally assimilable carbon sources include, for example, starch, dextrin, glucose, maltose, sucrose, molasses, corn syrup, oat jelly, as well as organic acids or fats such as acetic acid, succinic acid and polyhydric acids such as oils or glycerin. Alcohols are included and these may be used alone or in combination. Digestive nitrogen sources may include organic nitrates such as yeast extract, casein, meat juice, cottonseed meat, corn steep liquor, and flesh, as well as inorganic nitrates such as various ammonium salts, nitrates and urea, and these alone or in combination Can be used. The medium may also further contain iron, magnesium, manganese, cobalt, copper sodium, potassium, calcium, zinc salts as inorganic salts, if necessary. In addition, when the microorganisms used are required, the medium may appropriately contain special nutrients such as amino acids, vitamins and nucleic acid bases. The concentration of these additives is not particularly limited within the range in which the object of the present invention can be achieved, and may be selected within the range generally used in known fermentation processes.

를 갖는 사차 암모늄염이 바람직하다. 덧붙여, 배지내에서

기로 전환될 수 있는 화합물, 예를 들어 N, N-메틸렌비스아크릴아미드도 N-메틸 화합물로서 이용할 수 있다. N-메틸 화합물은 일반적으로 약50∼1000 바람직하게는 90∼130의 분자량을 갖는다. N-메틸 화합물은 수용성 또는 물에 불용성일 수 있지만, 수용성 N-메틸 화합물을 유리하게 사용할 수 있다. 이러한 N-메틸 화합물의 예는 N-메틸 산아미드, N-메틸아미노 화합물, N-메틸아민, N-메틸암모늄 화합물, N,N-메틸렌비스아크릴아미드 등이다. 예를 들어, N,N-디메틸아세트아미드, N-메틸-아크릴아미드 등의 N-메틸 산 아미드; N-메틸우레아, 1,1,3,3-테트라메틸우레아, 2-디메틸아미노에탄올 등의 N-메틸아미노 화합물; 트리메틸아민, 디메틸아민 등의 N-메틸아민; 레시틴, 콜린, 베타인, 테트라메틸암모늄 등의 N-메틸 암모늄 화합물 등을 유리하게 사용할 수 있다. N-메틸암모늄 화합물, 특히 콜린, 베타인 또는 테트라메틸암모늄을 사용하였을 때 만족할 만한 결과를 얻을 수 있다. 이들 N-메틸 화합물은 단독으로 또는 2종 이상의 혼합물로써 사용할 수 있다.In order to increase the yield of Mildiomycin, it is desirable to add N-methyl compounds to the medium. Such N-methyl compounds include compounds having one or more nitrogen atoms substituted by 1 to 4 methyl groups in their molecules. Among them, one having a nitrogen atom substituted by the methyl group (s) in the molecule is preferable, in particular a trimethyl ammonio group in which the nitrogen atom is substituted by a 3methyl group, that is,

Quaternary ammonium salts having are preferred. In addition, in badge

Compounds that can be converted into groups, such as N, N-methylenebisacrylamide, can also be used as N-methyl compounds. N-methyl compounds generally have a molecular weight of about 50-1000 and preferably 90-130. N-methyl compounds may be water soluble or insoluble in water, but water soluble N-methyl compounds may be advantageously used. Examples of such N-methyl compounds are N-methyl acidamides, N-methylamino compounds, N-methylamines, N-methylammonium compounds, N, N-methylenebisacrylamide and the like. N-methyl acid amides such as N, N-dimethylacetamide and N-methyl-acrylamide; N-methylamino compounds, such as N-methylurea, 1,1,3,3- tetramethylurea and 2-dimethylaminoethanol; N-methylamine, such as trimethylamine and dimethylamine; N-methyl ammonium compounds, such as lecithin, choline, betaine, tetramethylammonium, etc. can be used advantageously. Satisfactory results can be obtained when using N-methylammonium compounds, in particular choline, betaine or tetramethylammonium. These N-methyl compounds can be used individually or as a mixture of 2 or more types.

The present invention also encompasses a method characterized by supplying betaine-containing beet molasses, lecithin-containing flesh or eggs containing choline and lecithin mode to the medium to provide at least 3 mM N-methyl compound within the scope of the present invention. The concentration of N-methyl compound in the medium can be appropriately selected within the range that does not inhibit the growth of microorganisms and should be adjusted to 3mM or more. The concentration is preferably 4 mM to 200 mM, more preferably 7 to 50 mM, and the effect obtained by adding an N-methyl compound is not so remarkable as compared with the effect obtained at 200 mM or less at a concentration exceeding 200 mM. The amount of natural material containing the N-methyl compound may be selected within a range that allows the N-methyl compound to be provided at the above-mentioned concentrations, but such concentrations may be higher than those normally used to achieve this concentration. As necessary, the growth of microorganisms is inhibited by components other than the N-methyl compound of natural substances, thereby inhibiting the production of midiomycin. In this case, it is preferable to use N-methyl compounds in parallel with natural substances. The timing of adding the N-methyl compound to the medium is preferable before the microorganism is added to the medium in view of easy manipulation and effect, but may be added at an appropriate time during the culture.

In addition, the method of the present invention may be added with 5-hydroxymethylcytosine or a compound capable of producing this compound in a medium in order to improve the harvest of Mildiomycin. The concentration of 5-hydroxymethylcytosine or a compound capable of producing it in the medium is preferably 0.01 to 1.0% (W / V) and more preferably 0.03 to 0.5% (W) in view of 5-hydroxymethylcytosine. / V). The timing of adding these to the medium is most preferable just before the culture in view of the effect, but may be added at an appropriate time during the culture.

While surface culture is possible, aerobic deep culture is generally the most useful. In performing deep aerobic culture, the pH of the medium is preferably about acidic to about alkali, and the culture temperature is preferably maintained at 15 to 40 ° C, particularly at 24 to 34 ° C. Of course, the culture conditions can be adjusted and selected depending on the particular strains of microorganisms used, external conditions and other factors to obtain satisfactory results. In any case, it is generally possible to obtain culture broths containing a very large amount of Mildiomycin 4-14 days after the start of the culture. In order to recover Mildiomycin from this broth, a separation method that is commonly used to recover metabolites of microorganisms from culture can be used. For example, because Mildiomycin is a water soluble base and in most cases is formed in the liquid portion of the culture broth, cells may be first removed by filtration or centrifugation. The liquid portion thus obtained is brought into contact with a suitable adsorbent, for example activated carbon, adsorbent resin, cation-exchange resin, activated alumina or silica gel or molecular sieve so that the effective fraction is adsorbed thereto. The effective fraction is desorbed using an aqueous solution of a water-soluble organic solvent such as acetone, methanol, ethanol, propanol, butanol, as well as an aqueous solution of an acid, an alkali, a buffer solution and an inorganic or organic salt as a solvent. This separation method can be suitably combined and carried out. By concentrating and crushing the effective fraction, it is possible to recover midimycin in the free compound or salt state.

The serine hydroxyxamate- or cannabanin-resistant Mildiomycin-producing strain belonging to the genus Streptorbertylium, which is used in the method for producing Mildiomycin of the present invention, may be, for example, the Mildiomycin-producing strain belonging to the Genus Streptoverticilium Can be obtained by inducing mutations into serine hydroxamate- or cannabanin-resistant strains. Mildiomycin-producing strains belonging to the genus Streptorbertysilium to be mutated are frequently used, for example, Streptovertylsilium limofaciens E5-24-5 (hereinafter referred to as "parent strain"). This parent strain has been deposited with the IFO under accession number IFO-14125 since June 12, 1981, and with FRI accession number under FERM P-6052 since June 24, 1981, and under the Budapest Convention. Converted and deposited under the accession number of FERM BP-1125. In addition, KAIST was deposited with the accession number of KFCC-10241 on August 6, 1986. Spores or mycelium of Mildiomycin-producing strains belonging to Streptoverticillium to irradiate serine hydroxyxamate- or cannabanin-resistant strains are irradiated with ultraviolet rays or the strains

( Ⅳ )의 N-메틸 N'-니트로-N-니트로소구아니딘과 접촉시키거나, 상기 균주를 구조식

(Ⅴ)의 D-4-아미노-3-이속사졸리돈(관용명 "시클로세린")을 함유하는 배지 위에서 성장시키거나 또는 이들 방법을 적절히 배합하여 실시할 수 있다.

Contacting with N-methyl N'-nitro-N-nitrosoguanidine (IV) or the strain

It can be performed on the medium containing D-4-amino-3-isoxazolidone (common name "cycloserine") of (V), or these methods are combined suitably.

)조사함으로써 변이를 일으킨다.For example, when the spores or mycelium of the Mildiomycin-producing strain belonging to the genus Streptorverticilium are to be irradiated with ultraviolet rays to cause mutations, 10 to 100 watts, preferably 30 to 60 watts UV lamps are used. Spores or mycelium of the parent strain can be irradiated with ultraviolet light. The wavelength of an ultraviolet-ray is 130-350 nm normally, Preferably it is 190-290 nm. The irradiation time is usually 30 seconds to 5 minutes, and preferably 60 to 90 seconds. Spores or mycelium of the parent strain are suspended in, for example, sterile water and then irradiated with ultraviolet light. For example, spores of a parent strain suspended in sterile water are placed 30 cm below a 30 watt UV lamp and are stirred for 90 seconds (wavelength; 2537).

By investigating causes variation.

, 바람직하게는

의 농도로 용해시켜 사용할 수 있다. 덧붙여, 스트렙토베르티실륨 속에 속하는 밀디오마이신-생산균주를 시클로세린(V)-함유 배지상에서 자라게하여 변이를 일으키는 방법에서는, 밀디오마이신-생산균주를 시클로세린(V)-함유 배지상에서

, 바람직하게는

의 비율로 배양하는 것이 바람직하다. 다른 배양 조건은 세린히드록사메이트 또는 카나바닌에 대해 내성을 갖는 밀디오마이신-생산균주의 배양과 관련하여 상술한 사항들로 부터 적절하게 선택할 수 있다.In a method of mutating a midiomycin-producing strain belonging to the streptoverticilium, by contacting with N-methyl N'-nitro-N-nitrosoguanidine (IV), spores or single cells of the strain are combined with compound (IV). It is preferable to make contact. This contact can be carried out by any method, for example by mixing normally. The temperature at which the contact is carried out is 0 to 50 ° C, preferably 10 to 40 ° C, and the required time is usually 10 seconds to 3 hours, preferably 10 minutes to 2 hours. Spores or single cells of Mildiomycin-producing strains were e.g. w / v sucrose 1-6% (w / v) sodium glutamate, 0.001-0.1% (w / v) Tween 80 (Kao) Atlas Co., Ltd.) and sterile water, and dispersed in a suitable dispersion medium previously steam sterilized at 110 ~ 130 ℃ for 10 to 30 minutes. Compound (IV) was added to a buffer solution, for example triaminomethanehydrochloric acid buffer (pH 7.5).

, Preferably

It can be dissolved and used at a concentration of. In addition, in the method of causing mutations by growing Mildiomycin-producing strains belonging to the Streptorverticilium genus on a cycloserine (V) -containing medium, the Mildiomycin-producing strains are produced on a cycloserine (V) -containing medium.

, Preferably

It is preferable to incubate at a ratio of. Other culture conditions may be appropriately selected from those described above in connection with the cultivation of Mildiomycin-producing strains resistant to serine hydroxyxamate or cannabanin.

, 바람직하게는

) 또는 카나바닌 (

, 바람직하게는

)을 함유하는 배지에 펴거나 또는 이식한 후, 배지에서 자라는 균주를 분리한다. 또는 변이 처리하여 수득한 현탁액, 혼합용액 또는 생장율, 5-히드록시메틸시토신 생합성의 억제제인 아미노프테린(

, 바람직하게는

)을 함유하는 배지에 펴바른 후 균주가 자라도록 하고, 한천을 덩어리로 절단하여 각 덩어리의 항생 역가를 공지의 방법으로 분석함으로써(시험 생명체, 로도토룰라루브라) 강한 항생 역가를 나타내는 것을 선별한다. 이렇게 함으로써 세린히드록상메이트에 대한 내성을 갖는 뛰어난 밀디오마이신-생산균주의 선별을 확인한다. 상술한 세린히드록사메이트 또는 카나바닌에 대해 내성을 갖는 밀디오마이신-생산균주의 제조방법을 반복함으로써 세린히드록사메이트 또는 카나바닌에 대한 내성이 증강된 밀디오마이신-생산균주를 수득할 수 있다. 세린히드록상메이트에 대해 내성을 갖는 밀디오마이신-생산균주 또는 카나바닌에 대해 내성을 갖는 밀디오마이신-생산균주 또는 카나바닌에 대해 내성을 갖는 밀디오마이신-생산균주를 상술한 방법대로 변이 시킴으로써 세린히드록사메이트와 카나바닌 모두에 대해 내성을 갖는 밀디오마이신 생산균주를 얻을 수 있다. 이들 균주도 또한 스트렙토베르티실륨 속에 속하며 세힌히드록사메이트 또는 카나바닌에 대해 내성을 갖는 본 발명의 균주의 범주안에 포함된다.From the variants obtained by the above-described mutation induction method, Mildiomycin-producing strains resistant to serine hydroxyxamate or cannabanin are selected. Selection is made after selection, which is commonly used in the field of actinomycetes. For example, suspensions, mixtures or growths of strains obtained by mutating the serine hydroxyxamate in amounts that may interfere with the growth of the parent strain (

, Preferably

) Or canavanin (

, Preferably

After spreading or transplanting into a medium containing), the strain growing in the medium is isolated. Or aminopterin, an inhibitor of suspension, mixed solution or growth rate, 5-hydroxymethylcytosine biosynthesis,

, Preferably

After spreading on medium containing), strains are grown, and the agar is cut into agglomerates, and the antibiotic titer of each agglomerate is analyzed by a known method (test organism, Rhotorlorulalubra) to select a strong antibiotic titer. . This confirms the selection of an excellent Mildiomycin-producing strain that is resistant to serine hydroxate. Mildiomycin-producing strains having enhanced resistance to serine hydroxyxamate or cannabanine can be obtained by repeating the above-described method for producing a Mildiomycin-producing strain resistant to serine hydroxyxamate or cannabanine. . By mutating Mildiomycin-producing strains resistant to serine hydroxymate or Mildiomycin-producing strains resistant to cannabanin or Mildiomycin-producing strains resistant to cannavanin by the above-described method Mildiomycin producing strains resistant to both serine hydroxyxamate and cannabanin can be obtained. These strains also fall within the scope of the strains of the invention belonging to the streptoverticilium and resistant to either cerhinoxamate or cannabanin.

Mildiomycin obtained by the above-described method of the present invention can be safely used as an active ingredient of a medicament for the prevention and treatment of powdery mildew or as an active ingredient of a fungicide or acaricide for plants (US Patent 4007267, British Patent 1507193). number).

Mildiomycin-producing strains of the present invention belonging to the streptovertisilium and resistant to serine hydroxyxamate or cannabanine, as well as excellent resistance to serine hydroxyxamate or cannabanine, as well as to 5-hydroxymethylcytosine It shows high activity in biosynthesis.

Experimental Example I

2% sucrose, 0.5% soluble starch, 0.12% ammonium nitrate, 0.25% potassium dihydrogen phosphate, 0.005% magnesium sulfate 0.0025% casamino acid, 0.0025% homoextract and 2.0% powdered agar (% Is a w / v.) Serine hydroxyxamate and cannabanin at various concentrations are added to the agar medium (pH 7.0). The medium thus prepared was steam sterilized at 120 ° C. for 15 minutes and then divided into petri dishes by 20 ml. After cooling, the spores of the following test strains are spread over each medium. After incubation of the medium for 10 days at 28 ° C, the minimum concentration of MIC (minimum inhibitory concentration) for inhibiting growth is determined as shown in Table 2.

Test strain:

(1) streptoverticilium limofaciens E5-24-5

(2) streptoverticilium limofaciens

(3) streptoverticilium limofaciens

(4) streptoverticilium limofaciens

TABLE 2

Experimental Example 2

-182,

-48 및

의 포자를 접종하고, 28℃에서 20시간 동안 200rpm으로 회전 진탕 배양함으로써 종(種) 배양액을 수득한다. 그 후에 11%의 글루코스, 3.0%의 소르비톨, 3.0%의 카제인, 0.5%의 옥수수 침지액, 2.0%의 프로폴로, 0.5%의 황산암모늄, 0.5%의 질산암모늄, 0.1%의 콜린 클로라이드, 0.005%의 황산망간, 0.005%의 황산제일철 및 0.003%의 황산구리(%는 w/v이다)로 이루어진 배지를 제조하고, 20%(w/v)의 가성소다 수용액을 가하여 pH를 7.0으로 조절한다. 배지에 탄살칼슘을 1%(w/v)의 농도로 가한 후 충분히 혼합한다. 이렇게 하여 제조한 배지를 25m씩 200ml 에톨렌마이어 플라스크(우레탄 마개)에 넣고, 120℃에서 20분간 증기 멸균한다. 각 배지 (25ml)에 상기 종 배양액을 2ml씩 접종한 후, 회전 진탕기에서 200rpm으로, 28℃에서 3∼5일간 배양한다. 문헌[Journal of Fermentation Technology, Vol.63,17(1985에 기술된 방법에 따라 무세포 추출 용애글 제졸하고, 용소반을 실시한 후 5-히드록메틸시토신세엘을 결정함으로써 표 3에 나타낸 5-히도록 시메틸 시토신-생산활성를 얻는다.2.5% glucose, 2.0% corn steep liquor, 1.0% cottonseed flour [trademark; Proflo, Trader Oil Mill Co.,], a medium consisting of 0.3% ammonium sulfate, 0.05% magnesium sulfate and 0.3% calcium carbonate (% is w / v) was prepared and 20% ( The pH is adjusted to 7.0 by dropwise addition of an aqueous solution of caustic soda. 25 ml of the medium thus prepared was divided into Erlenmeyer flasks (urethane stoppers), and steam sterilized for 15 minutes at 120 ° C. Streptoverticilium limofaciens E5-24-5,

-182,

-48 and

Spores and seed cultures were obtained by rotary shaking culture at 200 rpm for 20 hours at 28 ° C. Then 11% glucose, 3.0% sorbitol, 3.0% casein, 0.5% corn steep liquor, 2.0% propolo, 0.5% ammonium sulfate, 0.5% ammonium nitrate, 0.1% choline chloride, 0.005% A medium consisting of manganese sulfate, 0.005% ferrous sulfate and 0.003% copper sulfate (% is w / v) was prepared, and the pH was adjusted to 7.0 by adding 20% (w / v) aqueous sodium hydroxide solution. Calcium carbohydrate is added to the medium at a concentration of 1% (w / v) and then mixed sufficiently. The medium thus prepared is placed in a 200 ml ethrenmeyer flask (urethane stopper) for 25 m each, and steam sterilized at 120 ° C. for 20 minutes. After inoculating 2 ml of each of the seed cultures in each medium (25 ml), the incubator was incubated at 200 rpm in a rotary shaker for 3 to 5 days at 28 ° C. [Journal of Fermentation Technology, Vol. 63, 17 (5-Hydrogen shown in Table 3 by crystallization of cell-free extractive agglomerates according to the method described in 1985, solvolysis and then determining 5-hydroxymethylcytosineceel). To obtain the methyl methyl cytosine-producing activity.

TABLE 3

* Average value of 3, 4 and 5 days cultured

Example 1

(1) Collect the spores and parasitic mycelium by scraping the oblique culture of Streptoverticilium limofaciens E5-24-5 (FERM P-6052, FERM BP-1125, IPO-1412, KFCC-10241) It is placed in 30 ml of a dispersion medium consisting of% sucrose, 2% sodium glutamate and 0.01% Tween 80 (steam sterilization at 120 ° C. for 15 minutes). Grind the mixture sufficiently using a Potter equalizer. The product is filtered using Toyo Filter Paper Co., Ltd. (manufactured by Toyo Filter Paper Co., Ltd.) to obtain a suspension of the filtrate (25 ml) of spores and parasitic mycelia fragments.

농도의 시클로세린을 함유하며, 2.0%의 슈크로스, 0.5%의 가용성 전분, 0.2%의 질산 암모늄, 0.1%의 인산이수소칼륨, 0.05%의 염화칼륨, 0.05%의 황산 마그네슘, 0.0015%의 황산 철 및 2.0%의 분말 한천(%는 w/w이다)로 이루어진 한천 판 배지에 상기에서 수득한 혼합액을 펴바른 후 28℃에서 14일간 정체 배양한다. 생성된 집락중에서, 형태학적으로 정상이며, 기생 균사체를 갖는 100집락을 분리한다. 멸균 유리봉을 이용하여 이들 집락을 각각 분쇄한다. 이들 분쇄된 물질에 약 10ml의 멸균수를 가하고, 도요 필터 페이퍼 101호를 이용하여 여과함으로써 영액을 얻는다. 이 여액을 시클로세린을 함유하지 않는 한천판 배지에 펴바른다. 배지를 28℃에서 10일간 정체 배양한다. 생성된 집락을 각각 사면 한천 배지(조성물은 상술한 한천 판 배지의 것과 동일하다)에 이식한다. 이들 배지를 28℃에서 10일간 정체 배양 한다. 사면 배지의 표면을 긁어서 생성된 기생 균사체의 포자를 수집하고, 이를 (1)에 설명한 방법대로 현탁, 파쇄 및 여과지에 의한 여과를 실시하여 현탁액 100 시료를 제조한다.(2) The same amount of solution of N-methyl N'-nitro-N-nitrosoguanidine dissolved in trisaminomethane hydrochloride buffer solution (0.1M, pH 7.5) was added to the suspension obtained in (1) at a concentration of 2 mg / ml. Add. The mixture is shaken at 28 ° C. for 90 minutes.

Contain concentrations of cycloserine, 2.0% sucrose, 0.5% soluble starch, 0.2% ammonium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% potassium chloride, 0.05% magnesium sulfate, 0.0015% iron sulfate And agar plate media consisting of 2.0% of powder agar (% is w / w), and then the culture solution obtained above is subjected to stagnation at 28 ° C. for 14 days. Of the resulting colonies, 100 colonies that are morphologically normal and with parasitic mycelium are isolated. Each of these colonies is crushed using sterile glass rods. About 10 ml of sterile water is added to these pulverized materials, and a filtration liquid is obtained by filtering using Toyo Filter Paper 101. The filtrate is spread on agar plate medium containing no cycloserine. The medium is incubated for 10 days at 28 ° C. The resulting colonies are transplanted into sloped agar medium (the composition is the same as that of the agar plate medium described above). These media are incubated at 28 ° C. for 10 days. Spores of the parasitic mycelium generated by scraping the surface of the slope medium are collected and subjected to suspension, crushing and filtration by filter paper as described in (1) to prepare a suspension 100 sample.

) 100시료를 각각

의 아미노프테린을 함유하는 대응하는 수의 한천 판 배지 표면전체에 펴바른 후 28℃에서 10일간 정체 배양한다.(3) the suspension prepared in (2) above (the number of living microorganisms; about

100 samples each

After spread over the whole surface of the agar plate medium of the corresponding number containing the aminopterin, the cells were subjected to stagnation at 28 ° C for 10 days.

A cork perforator is used to cut the cylindrical fragments (diameter; 6 mm, height; about 4 mm) from the agar plate medium and transfer them to the medium for analysis of antimicrobial titers (test organisms; Rhododola lubra, Bulon agar). Medium, pH 7.0) The treated medium is left at 30 ° C. overnight. Five samples in suspension show relatively large (diameter; 14 mm or more) holes (growth inhibition range).

생명체를 함유하도록 한다. 각각

의 세린히드록사메이트를 함유하는 대응하는 수의 한천 판 배지에 희석한 현탁액

씩을 펴바른 후 28℃에서 14일간 정체 배양한다. 100배지 중에서 7배지에서 생장이 관찰되는데, 이중에는 상기 (3)에서 커다란 운륜(억제구역)을 나타낸 다섯개의 시료 모두가 포함된다. 다섯개 중에서 상기 (3)에서 가장 커다란 운륜(억제구역)을 나타낸 한 변이체만을 스트렙토베르티실륨 리모파시엔스

로서 분리한다.(4) 100 samples of the suspension obtained in (2) were diluted with sterile water,

Contains life. each

Suspension diluted in agar plate medium of the corresponding number containing serine hydroxyxamate

After spreading each well, incubate for 14 days at 28 ℃. Growth was observed in 7 out of 100 medium, including all five samples showing a large cloud (inhibition zone) in (3). Of the five, only one variant exhibiting the largest wheel drive (inhibition zone) in (3) above was streptovertyllium limofaciens.

Separate as.

Example 2

을 이용하여 실시예 (1) 및 (2)와 비슷한 방법으로 그의 현탁액 시료 150개를 제조한다. 실시예 1의 (3) 및 (4)와 비슷한 방법으로 이들 시료로부터 스트렙토베르티실륨 리모파시엔스

를 분리한다(단, 공정 (4) 에서는

농도의 세린히드록사메이트를 함유하는 한천 판 배지를 이용한다).Obtained in Example 1 instead of streptoverticilium limofaciens E5-24-5

To prepare 150 suspension samples thereof in a similar manner to Examples (1) and (2). Streptoverticilium limofaciens from these samples in a similar manner to (3) and (4) of Example 1

(However, in step (4)

Agar plate medium containing the concentration of serine hydroxyxamate is used).

Example 3

를 이용하여 실시예 1 의 (1) 및 (2)와 비슷한 방법으로 그의 현탁액 시료 200개를 제조한다. 실시예 1의 (3) 및 (4)와 비슷한 방법으로 이들 시료로부터 스트렙토베르티실륨 리모파시엔스

(FERM P-8334, FERM BP-1127, IPO-14448, KFCC-10242)를 분리한다(단, 공정 (4)에서는

농도의 세린히드록사메이트를 함유하는 한천 판 배지를 이용한다).Obtained in Example 2 instead of streptoverticiryllium limofaciens E5-24-5

200 samples of the suspension thereof were prepared in a similar manner to (1) and (2) of Example 1 by using. Streptoverticilium limofaciens from these samples in a similar manner to (3) and (4) of Example 1

(FERM P-8334, FERM BP-1127, IPO-14448, KFCC-10242) are separated (however, in step (4)

Agar plate medium containing the concentration of serine hydroxyxamate is used).

Example 4

를 이용하여 실시예 1의 (1) 및 (2)와 비슷한 방법으로 그의 현탁액 시료 300개를 제조한다. 실시예 1의 (3) 및 (4)와 비슷한 방법으로 이들 시료로부터 스트렙토베르티실륨 리모파시엔스

를 분리한다(단, 공정 (4)에서는

농도의 세린히드록사메이트를 함유하는 한천 판 배지를 이용한다).Obtained in Example 3 instead of streptoverticilium limofaciens E5-24-5

300 suspension samples thereof were prepared in a similar manner to (1) and (2) of Example 1 by using. Streptoverticilium limofaciens from these samples in a similar manner to (3) and (4) of Example 1

(However, in step (4)

Agar plate medium containing the concentration of serine hydroxyxamate is used).

Example 5

을 이용하여 실시예 1의 (1) 및 (2)와 비슷한 방법으로 그의 현탁액 시료 100개를 제조한다.

농도의 카나바닌을 함유하는 한천 판 배지에 각각의 현탁액을 펴 바른다. 생성된 100배지를 28℃ 에서 14일간 정체 배양한다. 오직 한 시료에서만 나타나는 집탁을 2.0%의 슈크로스, 0.5%의 가용성 전분, 0.12%의 질산 암모늄, 0.25%의 인산이수소칼륨, 0.005%의 황산 마그네슘, 0.0025%의 카스아미노산, 0.0025%의 효모엑기스 및 2.0%의 한천(%는 w/v이다)으로 이루어진 한천 사면 배지(pH 7.0)에 이식하고, 28℃에서 10일간 정체 배양함으로써 세리히드록사메이트와 카나바닌에 대해 내성을 갖는 변이체

(FERM P-8333, FERM BP-1126, IPO-1449, KFCC-10243)을 수득한다.Obtained in Example 4 instead of streptoberrythisilium limofaciens E5-24-5

100 suspension samples thereof were prepared in a similar manner as in Example 1 (1) and (2).

Each suspension is spread on agar plate medium containing a concentration of cannabanine. The resulting 100 medium is incubated for 14 days at 28 ℃. The haze that appears in only one sample was 2.0% sucrose, 0.5% soluble starch, 0.12% ammonium nitrate, 0.25% potassium dihydrogen phosphate, 0.005% magnesium sulfate, 0.0025% casamino acid, 0.0025% yeast extract. And a variant resistant to serichydramate and cannabanin by transplanting into agar slope medium (pH 7.0) consisting of agar (% is w / v) of 2.0% and stagnant culture at 28 ° C for 10 days.

(FERM P-8333, FERM BP-1126, IPO-1449, KFCC-10243) are obtained.

Example 6

-182 및

-48의 포자를 접종한다; 회전 진탕기내에서 200pm으로 28℃에서 20분간 배양하여 각각의 종 배양액을 수득한다. 그리고 11%(w/v)의 글루코스, 3.0%의 소르비톨, 3.0%의 카제인, 0.5%의 옥수수 침지액, 2.0%의 프로플로, 0.5%의 황산암모늄, 0.5%의 질산 암모늄, 0.1%의 콜린 클로라이드, 0.005%의 황산 망간, 0.005%의 황산철 및 0.003%의 황산 구리(%는 w/v이다)로 이루어진 배지를 제조하고 20%(w/v)의 가성 소다 수용액을 적가하여 pH 7.0으로 조절한다. 이 배지에 탄산 칼슘을 1%(w/v)의 농도로 가하고, 혼합물을 충분히 혼합하고, 25ml씩을 200ml 에톨렌마이어플라스크(우레탄 마개)에 넣은 후 120℃에서 20분간 증기 멸균함으로써 발효 배지를 수득한다. 이렇게 하여 제조한 각각의 배지에 상술한 종 배양액 2ml씩을 접종한다. 회전 진탕기를 이용하여 200rpm 으로 28℃에서 10일간 발효시킨다. 배양 후, 문헌[Journal of Fermentation Technology 62, 537 (1984)]에 설명된 방법에 따라 고수율 액체 크로마토그래피하여 밀디오마이신을 정량한다. 상술한 방법으로 밀디오마이신의 생산 시험을 실시하고 결과를 표 4에 나타낸다.2.5% glucose, 2% corn steep liquor, 1.0% cottonseed flour [trademark; Proflo, Trader Oil Mill Co. Product), 0.3% ammonium sulfate, 0.05% magnesium sulfate, and 0.3% calcium carbonate (% is w / v), dropwise adding 20% (w / v) aqueous solution of caustic soda to adjust the pH of the medium. 7.0 is set. 25 ml of this medium is placed in a 200 ml Etlenmeyer flask (urethane stopper) and steam sterilized at 120 ° C. for 15 minutes. Streptovertylthium limofaciens E5-24-5,

-182 and

Inoculate spores of -48; Each species culture was obtained by incubating for 20 minutes at 28 ° C. at 200 pm in a rotary shaker. And 11% (w / v) glucose, 3.0% sorbitol, 3.0% casein, 0.5% corn steep liquor, 2.0% proplo, 0.5% ammonium sulfate, 0.5% ammonium nitrate, 0.1% choline A medium consisting of chloride, 0.005% manganese sulfate, 0.005% iron sulfate and 0.003% copper sulfate (% is w / v) was prepared and 20% (w / v) aqueous caustic soda was added dropwise to pH 7.0. Adjust Calcium carbonate was added to this medium at a concentration of 1% (w / v), the mixture was sufficiently mixed, 25 ml of each was placed in a 200 ml Ethylene Meier flask (urethane stopper), and steam sterilized at 120 ° C. for 20 minutes to obtain a fermentation medium. do. Each medium thus prepared is inoculated with 2 ml of the above-described seed culture solution. Fermentation is carried out at 28 rpm for 10 days at 200 rpm using a rotary shaker. After incubation, mildiomycin is quantified by high yield liquid chromatography according to the method described in Journal of Fermentation Technology 62, 537 (1984). The production test of Mildiomycin is conducted by the method mentioned above, and the result is shown in Table 4.

TABLE 4

Example 7

을 발효시킨다. 발효 브로쓰를 수집하여 1l를 원심분리 함으로써 상층액을 분리하고, 이를

의 이온-교환 수지 앰버라이트 IRC-50(

-형; Rohm, & Hass Co.)으로 채운 컬럼에 통과시킨다. 컬럼을 물로 세탁하고,

의 0.5%(w/v) 수성 암모니아로 용출시킨다(실시예 6과 같은 방법으로 정량한 후) 활성 분획을 250ml의 크로마토그래피용 활성탄(Takeda Chemical Industries, Ltd. 제조)으로 채운 컬럼에 통과시켜 흡착되도록 한다. 1.25l의 아세톤-물(3:7) 혼합물을 이용하여 용출시킨다. 용출시킨 활성 분획을 수집하여 농축시킨다. 농출물에 500ml의 메탄올을 가하여 조 밀디오마이신을 침전시킨다. 분말인 조 밀디오마이신을 여과하여 수집하고, 6.5g의 분말을 물에 용해시키고, 이를 125ml의 앰버라이트 CG-50(H⁺-형; Rohm & Hass Co. 제조)을 채운 컬럼에 통과시킨다. 컬럼을 50ml의 물로 세척하고, 1.25l의 0.5%(w/v) 수성 암모니아로 용출시킨다. 유효 분획을 수집하고 250ml의 활성탄으로 채운 컬럼에 통과시켜 흡착시킨다. 1.25l의 아세톤-0.1N 포름산(2:8)을 이용하여 분별 용출시키고, 유효 분획을 농축시킨다. 농축물에 메탄올을 가하여 밀디오마이신을 침전시킨다. 생선된 침전물을 여과하여 수집한 후 감압하에 건조시켜 5.5g의 밀디오마이신 포르메이트를 수득한다. 포르메이트의 물리화학적 특성중에, 융점, 선광도(c=1.0

및 c=1.0, 0.1N-HCL에서의

), 271nm 및 280nm에서의 자외선 흡광도는 문헌["The Journal of Antibiotics", 31권 6호 523(1978)]에 나타나 있는 것과 완전히 동일하다.Streptoverticillium limofaciens under the same conditions as in Example 6

Ferment. Collect the fermentation broth and centrifuge 1 l to separate the supernatant,

Ion-exchange resin of Amberlite IRC-50 (

-brother; Pass through a column filled with Rohm, & Hass Co.). Wash the column with water,

Was eluted with 0.5% (w / v) aqueous ammonia (after quantification in the same manner as in Example 6) and the active fraction was passed through a column filled with 250 ml of activated carbon for chromatography (manufactured by Takeda Chemical Industries, Ltd.) for adsorption. Be sure to Eluate with 1.25 l of acetone-water (3: 7) mixture. The eluted active fractions are collected and concentrated. 500 ml of methanol is added to the concentrate to precipitate crude Mildiomycin. The crude crude dimycin is collected by filtration and 6.5 g of powder are dissolved in water and passed through a column filled with 125 ml of Amberlite CG-50 (H ⁺ -form; manufactured by Rohm & Hass Co.). The column is washed with 50 ml of water and eluted with 1.25 l of 0.5% (w / v) aqueous ammonia. The effective fractions are collected and adsorbed by passing through a column filled with 250 ml of activated carbon. Fractional elute with 1.25 l of acetone-0.1N formic acid (2: 8) and concentrate the effective fraction. Methanol is added to the concentrate to precipitate Mildiomycin. Fish precipitates are collected by filtration and dried under reduced pressure to yield 5.5 g of Mildiomycin formate. Among the physicochemical properties of formate, melting point, optical intensity (c = 1.0)

And at c = 1.0, 0.1N-HCL

), Ultraviolet absorbance at 271 nm and 280 nm is exactly the same as that shown in "The Journal of Antibiotics", Vol. 31, No. 523 (1978).

Example 8

의 L-카나바닌을 함유하는 한천 판 배지에 상기에서 얻은 혼합 용액을 펴 바르고, 28℃에서 14일간 정체 배양시킨다. 생성된 집락을 사면 한천 배지(성분은 상술한 한천 배지의 것과 동일하다)에 이식한다. 이들 배지를 28℃에서 10일간 정체 배양함으로써 카나바닌에 대해 내성을 갖는 14변이체를 수득한다.N-methyl dissolved in trisaminomethane-hydrochloric acid (0.1 N, pH 7.5) in a suspension obtained according to a method similar to that of Example 1 (1) using streptoverticilium limofaciens E5-24-5 An equivalent amount of N'-nitro-N-nitrosoguanidine solution is added to bring the concentration to 2 mg / ml. The mixture is shaken at 28 ° C. for 90 minutes.

The mixed solution obtained above is spread on agar plate medium containing L-canabannin, and cultured at 28 ° C. for 14 days. The resulting colonies are transplanted into agar medium (components are the same as those of the agar medium described above). The cultivation of these media at 28 ° C. for 10 days yields 14 variants resistant to cannabanin.

(FERM P-8874, FERM BP-1134, IPO-14527, KFCC-10244)으로 명명한다. 이러한 방법으로 부모 균주 및 변이체

에 의해 생산된 밀디오마이신의 양을 표 5에 나타내었다.Under similar conditions as in Example 6, variants that are resistant to cannavanin and the parent strain (E5-24-5) are fermented and their Mildiomycin productivity is compared. Among them, one variant with the highest productivity was isolated and streptovertylsilium limofaciens

(FERM P-8874, FERM BP-1134, IPO-14527, KFCC-10244). In this way parent strains and variants

The amount of Mildiomycin produced by is shown in Table 5.

TABLE 5

이 부모 균주보다 훨씬 뛰어난 밀디오마이신-생산능력을 갖는 것을 확실히 할 수 있다.Variants from the Table

It can be ensured that Mildiomycin-producing ability is much better than this parent strain.

Example 9

을 발효시킨다. 발효 브로쓰(1l)을 수집하고, 실시예 7과 비슷한 방법에 따라 브로쓰로부터 밀디오마이신을 분리 및 정제한다. 3.1g의 밀디오마이신 포르메이트가 얻어진다. 포르메이트의 물리화학적 성질 중에서, 융점, 선광도 및 271nm 및 280nm에서의 자외선 흡광도는 문헌(실시예 7에 개체)에 보고된 것과 완전히 동일하다.Under similar conditions as in Example 6, streptoverticiryllium limofaciens

Ferment. Fermentation broth (1 l ) is collected and Mildiomycin is isolated and purified from the broth according to a method similar to Example 7. 3.1 g of Mildiomycin formate are obtained. Among the physicochemical properties of the formate, the melting point, the optical intensity and the ultraviolet absorbance at 271 nm and 280 nm are exactly the same as reported in the literature (individual in Example 7).

Accordingly, Mildiomycin production of the present invention is characterized by cultivating Mildiomycin-producing strains which are resistant to serine hydroxyxamate or cannabanin and belonging to Streptoverticillium, to produce Mildiomycin very efficiently. The method is an industrially advantageous fermentative method for mass production of Mildiomycin.

Claims (3)

Mildiomycin-producing strains belonging to Streptoverticilium and resistant to serine hydroxyxamate or cannabanin were cultured in the medium to allow the strain to assimilate and accumulate Mildiomycin, and then from the culture broth produced. A method for producing Mildiomycin, characterized by recovering diomycin. 2. The method of claim 1, wherein the medium contains at least 3 mM N-methyl compound. 3. The method for producing midiomycin according to claim 1 or 2, wherein the medium contains 5-hydroxymethylcytosine.