KR900007642B1 - Novel streptomyces aureus - Google Patents

Abstract

Prodn of tetranactin by mutagen-treated microorganism comprises (a) dispersing spores of streptomyces aureus ATCC 21428 in the TM buffer (pH8.0-8.2), (b) treating the spores with 0.5-2.0 ml/ml of N.T.G. for 15-40 mins, (c) selecting mutants which are much tetranactin producing strains, (d) treating spores of the selected strains with UV light (254 nm), (e) selecting mutants by shaking incubation, (f) culturing the selected mutant in liquid medium at 0.5-1.5 V.V.M of aeration rate and (g) purifying the cultured broth to obtain tetranactin.

Description

Streptomyces aureus HB601 and method for preparing antibiotic tetranactin using the same

The present invention is to produce a macrotetrolide (MARCROTETROLIDE) -based antibiotics using a mutated strain modified from Streptomyces aureus ATCC 21428 to prepare a tetratetin (TETRANACTIN) from the cells It is about how to.

Tetranactin is known to have an excellent pesticidal effect against mites, the main pests of horticultural crops. (US Pat. Nos. 4,127,668, 3,777,023) In particular, this material exhibits strong insecticidal properties even at low concentrations of less than 10 p.pm against apple mites or spotted mites that grow largely in Korea (TAGAO SAGAWA ET AL., J.ECON. ENTO MOL., 65 (2), 372-375, 1972) reported very low toxicity for humans. (KUNIO ANDO ET AL., JAPAN PESTICIDE INFORMATION, NO.36, 36-40) Tetranactin is also widely regarded as a pollution-free, low-toxic pesticide because it is decomposed into safe substances such as carbon dioxide and water under natural environment after use. (HIROSHI SASAKI ET AL., APPLENVIRON.MICROBIOL., 40 (2), 264-268, 1980)

A group of macrotetralide antibiotics isolated from Streptomyces aureus ATCC21428 fermentation broth is known as S-3466 and three types of A, B and C have been described. The general name of the antibiotic S-3466 is tetranactin and the bacteriological properties of Streptomyces aureus ATCC 21428, a tetracactin producing strain, are described in detail in US Pat. No. 3,743,724.

The present invention is a strain obtained by dissolving spores of Streptomyces aureus ATCC21428 in TM BUFFER (TRIS -MALEICBUFFER pH 8.0-8.2) and then treated with NTG 0.5-2.0mg / ml for 15-40 minutes 4-6g of starch, dextrin 3-5g, soyton 1-3g, yeast extract 0.1-0.5g and trace minerals in 100ml of distilled water for 4-6 days shaking cultures to select excellent productivity After further irradiating the spores of this selected strain with ultraviolet (254nm) energy of 95-120J / m ² again, the high-productivity mutants were selected with the medium again. This variant maintained high productivity even after more than 30 generations. The mutant strain was named Streptomyces aureus HB601, and the strain was deposited with the Korean spawn association (deposit date May 13, 1988, accession number KFCC 10606). The use is shown in Table 1, the glucose, maltose, sucrose, lactose, glycerol, inositol, arabinose, xylose, rhamnose, etc. does not show a significant difference from ATCC 21428, but the variant strain of the present invention in the use of starch It is characterized by superiority to the strain. Mutant and probiotics did not show significant differences in peptone agar, YEME agar, trypton agar, NA, PDA, cornmill agar, etc., but not in bennet agar, starch agar, and glycerol asparagine agar. The results are shown in Table 2.

TABLE 1

+: Normal, ++: Good, +++: Very good

[Table 2]

The productivity and purity of tetranactin was dissolved by dissolving sodium hydroxide (NAOH) in SODIUM PICRATE solution, shaking with a dichloromethane (DICHLOROMETHANE) solution containing tetralactin, and recovering the dichloromethane layer. ₂ SO ₄ ) and then absorbance assay at 377 nm (KUNIO ANDO ET AL., KOR.J.APPL.MICROBIOL.BIOENG., 7 (2) 103-115, 1979).

에서 조결정 90g을 얻은 것으로 보고되고 있다.(미합중국 특허 제3,743,724호) 상기의 방법은 배양 기간이 2-6일로 짧고, 균주가 이용하는 탄소원의 농도가 낮으므로 생성되는 테트라낙틴의 양이 적다는 단점이 있다.Currently, the production of tetranactin by microorganism is 2% by weight of glucose, 1% by weight of soluble starch, 1.5% by weight of soybean meal, 0.5% by weight of sodium chloride, 0.025% by weight of potassium diphosphate, 1.5% by weight of magnesium sulfate heptahydrate, 0.4% by weight of calcium carbonate. %, Incubate for 3 days at 27 ℃ in a medium containing 0.02% by weight of antifoam, inoculated 1% of the culture medium in the same culture medium of the same composition as the culture medium, and then aeration 0.75VVM, stirring Culture medium 20 by incubating at 27 ℃ for 3 days at a speed of 250rpm

(US Pat. No. 3,743,724) The above method has a short incubation period of 2-6 days and a low concentration of tetranactin due to the low concentration of carbon source used by the strain. There is this.

를 셀라이트(CELITE)로 여과하여 얻은 균사체에 아세튼 15

를 가하여 상온에서 12시간 추출하고, 아세톤추출물을 감압하에 농축시킨후 에틸아세테이트(ETHYLACETATE) 3

로 2회 반복 추출하여 살충력이 있는 조 추출물을 얻었다.In the present invention, the production method of tetranactin was improved by optimizing the medium and culture conditions using a mutant strain having a high concentration of 4% by weight or more, preferably 4-12% by weight of starch. Medium contains 4-12% by weight of starch, glucose, mannitol, glycerol, dextrin, maltose, maltose, etc., and 0.2-2% by weight of peptone, soyton, corn steep liquor, tryptone, soybean meal, and yeast extract as carbon sources. %, And a small amount of inorganic salts were used, and the aeration rate of 0.8-1.5VVM, the agitation rate of 400-900rpm, the initial pH of 5.8-7.8, preferably 25-30 ° C at the initial pH of 6.5-7.5. Incubated for 5-8 days. In some cases, a culture method of maintaining a constant pH of about 7.0-7.6 after 2-4 days of culture was used. The seed culture was incubated for 2-4 days using a medium containing the same carbon source, nitrogen source, and inorganic salts, and inoculated to 2-5% of the culture medium. On the other hand, the well-known technology for the separation and purification of tetranactin produced and accumulated in fermentation broth is fermentation broth 60

On the mycelium obtained by filtration with CELITE 15

Extracted at room temperature for 12 hours, and the acetone extract was concentrated under reduced pressure, followed by ethyl acetate (ETHYLACETATE) 3

Repeated extraction twice with crude insecticide was obtained.

로 먼저 용출한후, 다시 노르말-헥산과 아세톤 1:1비의 혼합용액 1

로 용출하고 마지막으로 에칠아세테이트 1

로 용출하여, 유효성분을 함유하고 있는 에칠아세테이트 용출액을 감압하에서 농축하여 18g의 유효성분을 분리하고 -10℃에서 노르말-헥산으로 결정화하여 조 테트라낙틴 결정을 얻은 것으로 보고하고 있다.(KUNIO ANDO ET AL., J.ANTIBIOT.64(6), 347-352, 1971) 미합중국 특허 제3,777,023호에 보고된 정제방법은 10

발효 배양액을 원심분리한후 2

의 아세톤을 가하여 2회 추출한후 감압하에 농축하여 아세톤을 제거하였다.The crude extract was purified using silica gel column chromatography (SILICA GELCOLUMN CHROMA TOGRAPHY) to isolate the active ingredient. Elution solvent in column chromatography is normal-hexane (n-HEXANE) 1

First eluted with a mixture of normal-hexane and acetone 1: 1 ratio 1

Elute and finally Ethyl Acetate 1

It was reported that the crude ethyl acetate was obtained by eluting with ethyl acetate and concentrating the eluate containing the active ingredient under reduced pressure to separate 18 g of the active ingredient and crystallizing with normal-hexane at -10 ° C. (KUNIO ANDO ET AL., J.ANTIBIOT. 64 (6), 347-352, 1971) The purification method reported in US Pat. No. 3,777,023 is 10.

After centrifugation of the fermentation broth 2

Acetone was added and extracted twice, followed by concentration under reduced pressure to remove acetone.

의 노르말-헥산으로 2회 추출하고 감압농축하여 황적색의 물질을 얻은후 100g의 실리카겔을 충진한 컬럼에서 노르말-헥산, 노르말-헥산과 에칠아세테이트 등의 혼합용매를 이용하여 분리한 후, 감압하에서 농축하고 저온에서 일주일 경과후 2g의 조결정을 얻었다. 상기 조결정 700mg을 다시 클로로포름과 에칠아세테이트 혼합용매에 녹인후 다시 컬럼 크로마토그라피를 이용하여 얻은 유효 용출액을 농축하여 소량의 노르말-헥산에 늑여 -10℃에서 결정화하여 마름모꼴의 테트라낙틴 결정을 얻은 것으로 보고하고 있다. 미합중국특허 제3,743,724호에 의한 정제방법에서는 발효배양액 20

를 황산으로 pH를 3으로 조절하고 80℃에서 30분 동안 가열후 2%의 규조토를 가하여 여과함으로써 여과의 용이성을 제고시켰고, 여과하고 얻은 균사체를 10

의 메탄올로 추출후 농축하고 5

의 노르말-헥산으로 재추출하여 S-3466복합물 조결정 90g을 얻었다.The concentrate 1

Extracted twice with normal-hexane, concentrated under reduced pressure to obtain a yellowish red substance, and then separated from a column filled with 100 g of silica gel using a mixed solvent such as normal-hexane, normal-hexane and ethyl acetate, and concentrated under reduced pressure. After 1 week at low temperature to obtain a crude crystal of 2g. 700 mg of the crude crystals were again dissolved in a mixed solvent of chloroform and ethyl acetate, and the effective eluate obtained by column chromatography was concentrated again and crystallized in a small amount of normal-hexane at -10 ° C. to give a lozenge tetralactin crystal. Doing. Fermentation broth 20 in the purification method according to US Pat. No. 3,743,724

The pH of the mixture was adjusted to 3 with sulfuric acid, heated at 80 ° C. for 30 minutes, and then filtered by adding 2% of diatomaceous earth to enhance the ease of filtration.

Extracted with methanol, and concentrated.

90 g of S-3466 composite crude crystal was obtained by reextracting with normal-hexane.

It is reported that 150 g of S-3466 A, 720 mg of S-3466 B, and 912 mg of S-3466 C were obtained by dissolving 2 g of S-3466 crude crystals in a small amount of chloroform and using column chromatography packed with 400 g of silica gel.

As such, known purification methods have a lot of problems in industrial use because purification costs are high and purification yield is very low because many purification steps are required for extracting and separating active ingredients from fermentation broth.

The present inventors used the mutant strain (KFCC 10606) obtained by artificially mutating from Streptomyces aureus ATCC 21428 carbon sources such as starch, glucose, dextrin, nitrogen sources such as corn steep liquor, soy flour, peptone, soyton, and various After aerobic shaking culture in a nutrient medium containing inorganic salts, the production of tetralactin was accumulated more than two times after 6-8 days, and the culture was centrifuged to recover the precipitate, and from the precipitate to acetone. The active ingredient was extracted and separated with ethyl acetate, chloroform and the like to obtain a tetranactin crude extract.

The crude extract was crystallized with acetone to develop a method for purifying tetracactin of high purity in a simple and short time, thereby increasing productivity and improving purification efficiency. The present invention will be described in detail by the following examples.

Example 1

Use strain: Streptomyces aureus mutant strain (KFCC 10606)

Seed culture medium

This culture medium

100 ml of the seed culture medium was placed in a 500 ml flask and autoclaved at 121 ° C. for 20 minutes, and then inoculated with a platinum tooth inoculated with a platinum strain (KFCC 10606) incubated for 3-5 days in an agar slope medium (east-malt extract agar medium). Species cultures were prepared by time incubation.

의 발효조에 본배양 배지 10

를 넣고 121℃에서 20분간 가압멸균후 종배양액 200ml을 접종하여 통기량 1V.V.M., 교반속도 500rpm의 조건으로 28℃에서 8일간 배양하였다. 멸균후 초기 pH는 6.4였다. 친균주인 ATCC 21428도 동일한 배지 및 배양 조건으로 배양하였다. 배양일수에 따른 pH, 건조균체량, 잔존전분량 및 테트라낙틴 생산량은 표 3과 같다.This culture has a total capacity of 13.7

Culture medium in fermenter

After incubation at 121 ° C. for 20 minutes, inoculated with 200 ml of the seed culture solution was incubated at 28 ° C. for 8 days under conditions of aeration 1V.VM and agitation speed of 500 rpm. The initial pH after sterilization was 6.4. The parent strain ATCC 21428 was also cultured in the same medium and culture conditions. The pH, dry cell mass, remaining starch amount and tetranactin production amount according to the culture days are shown in Table 3.

TABLE 3

The amount of dry bacteria was measured by washing a predetermined amount of the culture solution three times with the same volume of saline solution and drying it at 80 ° C for 48 hours.

Example 2

Use strain: Streptomyces aureus mutant strain (KFCC 10606)

Seed culture medium under pressure sterilization of a mutated platinum larvae cultured on agar slope medium (glucose 15g / l, corn starch 30g / l, corn steep liquor l5g / l, yeast extract 1g / l, calcium carbonate 5g / l, magnesium sulfate) 0.5g / l) was incubated 72 hours after inoculation to prepare a culture medium.

After the sterilization of the culture medium described in Table 4, the seed culture solution was inoculated at a volume ratio of 3%, and cultured at 28 ° C. for 8 days under conditions of aeration rate of 0.8 V.V.M. and agitation speed of 700 rpm. Media composition 1, 2, 3 Each culture was recovered and the results of measuring the productivity of tetranactin are shown in Table 5.

TABLE 4

TABLE 5

Example 3

Use strain: Streptomyces aureus mutant strain (KFCC 10606)

씩 넣어 가압 멸균후 종배양액 400ml을 접종하여 통기량 1.2V.V.M., 교반속도 550rpm의 조건으로 28℃에서 8일간 배양하되, 1대의 발효조는 수산화칼륨을 사용하여 초기 pH릍 7.0으로 맞추고 배양 3일후부터는 수산화칼륨과 초산을 사용하여 배양이 끝날때까지 pH를 7.4로 일정하게 유지시켰다. 다른 1대의 발효조는 배양초기부터 pH를 조절하지 않았다. 상기 두 발효조에서 회수한 배양액의 배양일수에 따른 테트라낙틴의 생산성은 표 6과 같다.The composition of the seed culture medium and the main culture medium is the same as in Example 1. Each of the two fermenters was used with 20 culture medium.

After each sterilization, the solution was inoculated with 400ml of the seed culture solution and incubated at 28 ° C for 8 days under the condition of aeration 1.2VVM and agitation speed of 550rpm. One fermenter was adjusted to initial pH 릍 7.0 using potassium hydroxide and 3 days after incubation. Potassium and acetic acid were used to keep the pH constant at 7.4 until the end of the incubation. The other fermentor did not adjust pH from the beginning of the culture. The productivity of tetranactin according to the culture days of the culture liquids recovered from the two fermentors is shown in Table 6.

TABLE 6

Example 4

중 10

를 취하여 고속 원심분리기로 원심분리하여(6,000rpm,15분) 상등액은 버리고 침전물을 회수하였다. 침전물에 아세톤 2

를 가하고 상온에서 12시간 방치한후 감압여과하여 여액을 진공회전농축기로 농축하여 유효성분을 추출하였다.Cultures (Production Rate: 9,200 μg / ml) Recovered from Medium Composition 1 of Example 2 30

Of 10

The mixture was centrifuged with a high speed centrifuge (6,000 rpm, 15 minutes), and the supernatant was discarded to recover the precipitate. Acetone 2 in sediment

After adding for 12 hours at room temperature and filtered under reduced pressure, the filtrate was concentrated with a vacuum rotary concentrator to extract the active ingredient.

를 각각 10

씩 나누어 미합중국 특허 제3,743,724호 및 제3,777,023호에 의한 방법으로 정제를 실시하였다. 결정화가 끝난후, 상기의 세가지 정제방법으로 회수한 결정들을 건조기에서 60℃로 건조시킨후 흡광도 측정법으로 그 순도를 결정하고 결정의 양을 전자식 천평으로 칭량하여 정제수율을 계산하였으며 그 결과는 표 7과 같다.The concentrate was reextracted with ethyl acetate, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure. 100 ml of normal-hexane was added to the concentrate, and the components dissolved in normal-hexane were discarded. 50 ml of acetone at 45 ° C. was added to the remaining precipitate, and crystallized at −5 ° C. to obtain white crystals after 2 days. Remaining culture 20

10 each

The purification was carried out by the method according to US Pat. Nos. 3,743,724 and 3,777,023. After crystallization, the crystals recovered by the above three purification methods were dried at 60 ° C. in a drier, the purity was determined by absorbance measurement, and the amount of crystals was weighed by electronic balance to calculate the purification yield. Same as

TABLE 7

Example 5

중 10

를 취하여 원심분리하고 침전물을 회수하였다. 침전물에 500ml의 아세톤을 가하여 상온에서 12시간 추출한후 감압여과하고 여액을 진공 회전 농축기로 농축하여 아세톤을 제거하였다.Culture solution (production amount: 9,510 μg / ml) recovered from the case of adjusting the pH of Example 3 20

Of 10

Was taken and centrifuged to recover the precipitate. 500 ml of acetone was added to the precipitate, followed by extraction at room temperature for 12 hours, followed by filtration under reduced pressure. The filtrate was concentrated with a vacuum rotary concentrator to remove acetone.

200 ml of ethanol was added to the concentrated solution, and the mixture was transferred to an aliquot. Then, 500 ml of chloroform was added, shaken vigorously for 5 minutes, and left for 10 minutes to separate the chloroform layer. The chloroform layer was dehydrated with anhydrous sodium sulfate and concentrated with a vacuum concentrator.

는 공지의 방법에 준하여 정제를 행하였다. 결정화가 끝난후 상기 두가지 방법으로 정제한 결정들을 회수하여 실시예 4와 동일한 방법으로 순도, 결정량 및 정제수율을 측정하였으며 그 결과는 표 8과 같다.The concentrate was dissolved in 50 ml of acetone at 45 ° C. and crystallized at −5 ° C. Remaining culture 10

Purification was carried out according to a known method. After crystallization was completed, the crystals purified by the above two methods were recovered, and the purity, crystallite amount, and purification yield were measured in the same manner as in Example 4, and the results are shown in Table 8.

TABLE 8

Notice: KUNIO ANDO ET AL., J.ANTIBIOT., 64 (6), 347-352, 1971

Claims (4)

The spores of Streptomyces aureus ATCC 21428 were dissolved in TM BUFFER (pH 8.0-8.2), treated with NT G 0.5-2.0 mg / ml for 15-40 minutes, shaken and cultured in medium to select strains. Streptomyces aureus HB601 (Accession No.: Streptomyces aureus HB601), characterized in that the starch utilization is 4-12% by weight in the vegetation. KFCC 10606) Macrotetrolide antibiotics were accumulated by incubating Streptomyces Aureus HB601 with aeration volume of 0.8-1.5VVM in a medium composed of 6-12% by weight nitrogen source, 0.1-2% by weight nitrogen source and trace mineral salts. Method for producing an antibiotic tetranactin, characterized in that impurities are removed by normal-hexane in the purification process. The method of claim 2, wherein the purification process after the accumulation of the macrotetralide antibiotics is separated by tetrachlorotin with chloroform, and determined by acetone to improve the yield. The method according to claim 2 or 3, wherein the initial pH of the culture medium is maintained at 6.5-7.5 when the Streptomyces aureus HB601 is cultured, and the pH is maintained at 7.2-7.6 after 3 days of culture. Manufacturing method.