KR800001610B1 - Process for preparing antibiotic l-13365 - Google Patents

Abstract

Antibiotic L 13365 is produced by fermn. with Actinoplanes sarveparensis. Thus, a preculture of A. sarveparensis was inoculated into 10L of a culture contg. meat exp. 4, peptone 4, yeast ext. 1, NaC1 2.5, soybean meal 10, glucose 50, and CaCO3 5 g/L and incubated at 28-300C with aeration and stirring for 3-4 days. Yield of L13365 FROM 80L broth was 1,14g in 70% purity. Antibiotic L13365 has considerable antibacterial activity, esp. against gram-pos. bacteria.

Description

Method for preparing antibiotic L-13365

1 is the ultraviolet absorption spectrum of antibiotic 13365.

2 is the infrared spectrum of antibiotic 13365.

The present invention relates to a novel antibiotic obtained by fermenting a strain belonging to the Actinopranes family called Actinoplanes Sarveparensis. This novel antibiotic, referred to below as antibiotic L 13365, is a pale yellow crystalline compound with properties such as melting point, infrared and ultraviolet absorption maximum.

As described above, the antibiotic L13365 is prepared by fermenting and culturing a bacterium called Actinopranes sarbeparensis. The bacterium, assigned our conservation number A / 13826, was isolated from the sample soil collected from Sarvepar bilize (India). The bacteria were deposited and poured into CBS [Central Bureau Uoor Schimmekures] Dosterstraat 1-Baarn-Netherlands strain culture, the deposit number is 305.76.

In preparing antibiotic L13365, microorganisms are cultured in an aqueous nutrient medium containing anabolic carbon components, anabolic nitrogen components and inorganic salts under aerobic conditions.

In general, antibiotic-producing strains are pre-incubated in stirred flasks until substantial antimicrobial activity is observed and then inoculated in fermenters containing nutrient fermentation medium.

Incubation is carried out at 25-35 ° C., for a time sufficient to produce substantial antimicrobial activity under aerobic conditions. In order to control the concentration of antibiotics produced during the cultivation, microbiological analysis by means of dilution is performed.

정도로 농축시킨 다음 냉각시키고 방치하여 생성된 침전물을 여과하여 회수한다. 이 침전물은 거의 순수한 항생제 L13365로 이루어져 있다. 모액을 모아서 경유 같은 불활성 비극성 용매 과량에 부여 추가량의 조항생제 L13365를 회수한다. 상기와 같이하여 얻어진 조생성물을 메탄올-아세톤 혼합물에 용해시키고 산성 의수를 첨가하여 침전을 생성시킨다. 두 침전물을 한곳에 모아서 용출계로서 클로로포름-메탄올 혼합물을 사용한 컬럼 크로마토그라피에 의해 더욱 정제시킨다.After removal of the mycelium by filtration, the antibiotics are recovered from the fermented juice filtered by a conventional method such as, for example, extraction with an organic solvent in which the antibiotics are dissolved and not mixed with the aqueous medium. Extraction is performed after adjusting the pH of the filtrate to about 2-4. Suitable organic solvents for extraction are selected from alkanols of 1-6 carbon atoms, (C ₁ -C ₄ ) alkyl esters of lower aliphatic acids or halogenated lower hydrocarbons. The solvent is then separated from the fermentation juice by high speed centrifugation and

Concentrate to a degree, then cool and leave to recover the resulting precipitate by filtration. This precipitate consists of almost pure antibiotic L13365. The mother liquor is collected and an additional amount of the provision L13365 is added to the excess of an inert nonpolar solvent such as diesel. The crude product obtained as above is dissolved in a methanol-acetone mixture and acidic water is added to generate a precipitate. The two precipitates are collected in one place and further purified by column chromatography using a chloroform-methanol mixture as elution system.

Antibiotic L 13365 is finally crystallized in methanol: ethyl acetate (1: 1).

Antibiotic L 13365 exhibits excellent antimicrobial activity in vitro and in vivo. Furthermore, antibiotic L 13365 exhibits significant antimicrobial activity in vitro, particularly against Gram-positive bacteria, as shown in the table below.

[table]

Moreover, as mentioned above, antibiotic L 13365 also shows excellent bioantibacterial activity against experimental infectious bacteria in mice. More particularly, the ED 50 value of antibiotic L 13365 for the experimental infection caused by Staphylococcus aureus, Strepcoccus haemolyticus and Diplococcus pneumoniae is as follows.

The new antibiotic is effective against microorganisms that are resistant to commonly used antibiotics.

In Table I, as a representative example, the minimum inhibitory concentrations (MIC) of antibiotic L 13365 against Staphylococcus aureus resistant to some antibiotics are reported.

TABLE I

As a result of oral or parenteral administration to mice, the toxicity of antibiotic L 13365 is very low, as LD 50 value is 1000 mg / kg or more.

[Gross Experiments on Colonies of Actino Pranes Sarveparensis A / 13826]

The bacterium develops well in a variety of nutritional parents. Colonies in oatmeal agar are 3-4 mm in diameter and exhibit irregular contours and rough surfaces. Mycelium in air was largely absent.

[Microscope experiment]

Spore sacs are generated in some KEPCO media. The spore sacs are regular globules 13-20μ in diameter. Very active residents are spherical but in some cases 1.5-2μ in diameter. Because of this feature, strain A / 13826 is included in Actinoplanes and actinoplanes sarveparensis C.B.S. It is named 305.76. Various standard badges presented by Sheeting and Gotrib (Intern.J.Syst.Bact., 16, 313-340,1966) and other badges presented by Waxman (The Actinomyces, Volume II, The Williams & End) Culture characteristics of Actinoprene sarveparensis CBS305.76 cultured on Wilkins Company, 1961) are shown in Table II. Culture characteristics were observed 6-14 days after incubation at 30 ° C. The use of carbon sources tested by the method of Freedam and Gotrib (Intern. J. Syst. Bact, 56, 107, 1948) is described in Table III.

Table IV describes the physiological properties of the strains.

Actinoplanes Sarveparensis C.B.S. 305.76

TABLE II

Chromaticity measurements were performed by the method of Maretz and Paul (Haerz, A and M Reg. Paul 1950 Chromaticity Dictionary, 2nd Edition M. Grow-Hillbook Company, Inc., New York).

For the number of culture media refer to those given by Shirring and Coat Rib. (Intern, J. Syst. Bact., 16, 313-340, 1966)

TABLE III

TABLE IV

[Production and Separation of Antibiotic]

In order to produce antibiotics, the strain Actinoplanes sarveparensis C.B.S. Pre-culture 305.76 aerobically in the medium until main antibacterial activity appears at pH about 6-about 10. For example, a stirred flask culture consists of the following composition per gram / 1.

The flasks are stirred at about 28-30 ° C. for about 24 hours and the preculture (1 L) is inoculated into fermentation beds containing 10 L of the following culture medium, respectively.

Each batch of culture is incubated at 28-30 ° C. under aerobic stirring. A time interval is used for the microbiological analysis of antibacterial activity by agar dilution using Staphylococcus aureus as a test group. The highest antimicrobial activity occurs after fermentation for 72-96 hours.

[Isolation and Purification of Antibiotic L13365]

The culture (80 liters) is filtered using 1% Kracel (W / V) as the filter aid. The mycelium is removed and the filtered solution is acidified to pH 2.5 with 10% Hcl and extracted twice with about 50% of the volume of ethyl acetate.

The organic phase is separated from the aqueous layer by high speed centrifugation, dried over Na ₂ SO, concentrated in vacuo at 40-50 ° C. until about 1/300 of the original volume and finally cooled to about 0-10 ° C.

The crude precipitate collected in the filter is washed with a small amount of ethyl acetate and dried in vacuo at 45 ° C. 1.140 g of antibiotic L13365 is obtained with a purity of 70%, which is measured by spectrophotometry.

The mother liquor from the filtration is poured into 20 times more diesel oil to obtain 2.550 g of antibiotic with a purity of 20-25% (measured by spectrometry). This material is suspended in a small amount of methanol: acetone (9: 1) mixture and all insolubles are filtered off and diluted with water. 10% HCl was added to the solution to a pH of about 2.5, and the precipitate collected by centrifugation was further dissolved in a minimum amount of butanol at about 45-50 ° C. The solution was concentrated in vacuo to about 1/5 of the original volume and then cooled to about 4 ° C. The precipitate obtained by collecting and vacuum drying is antibiotic L13365 (0.450 g) having a purity of 80% (measured by spectrophotometry). The product thus obtained is then subjected to conventional purification procedures.

For this purpose it is dissolved in a minimum amount of chloroform: methanol (85:15) mixture, chromatographed on silica-ceit column (1: 1 v / v) and then preactivated at 100 ° C. and washed with the chloroform / methanol mixture. .

Elution and thin layer chromatography control of the parts were carried out in the same mixture. The collected portions are concentrated in vacuo to a small amount according to the thin layer chromatography table. After addition of diethyl ether, a precipitate was formed, consisting of antibiotic L13365 with a purity of 95% (as determined by spectrometry).

The precipitate is dissolved in a methanol: ethyl acetate (1: 1) mixture, heated to 45 ° C. and all insolubles are filtered off. Cooling at 4 ° C. and left at the same temperature for one night yields pure antibiotic L13365 as a light yellow needle.

[Physical and Chemical Properties of Antibiotic L13365]

Antibiotic L13365 is a light yellow crystalline powder with acidic properties. Analysis of the acid hydrolyzate of antibiotic L13365 in 6N hydrochloric acid in a sealed tube at 110 ° C. revealed four amino acids, three of which were identified as aspartic acid, threonine and glycine by an automated amino acid analyzer. . Moreover, antibiotic L13365 has the following characteristics.

1) Melting Point: 210 ℃

2) Elemental analysis: C = 51.35 H = 5.05 N = 10.50 S = 11.05 C (separately) = 22.05

3) UV and visible absorption spectrum

Ultraviolet absorption spectra were measured with a UNIICAM SP800 ultraviolet spectrometer using solutions of L13365 dissolved in methyl cellosolve (MCS) -buffers at various pHs.

The front side of the spectrum is shown in FIG.

4) Fluorescence Spectrum:

The antibiotic has a strong fluorophore and the solution of the product dissolved in 0.1 N sodium hydroxide at 240 nm shows maximum emission spectra at 355 and 490 nm.

Fluorescence spectra were measured with a Perkin Elmer model MPF-44 fluorescence spectrometer.

5) infrared spectrum

A characteristic band of absorption in Newsol was observed at the following wave number (cm-1).

3400 (curved), 3350 (sharp), 3200 (curved), 3100 (sharp), 2930, and 2870 (new sol)

2800-2400, 2380 (atmospheric carbon dioxide) 1750 (art), 1670 (art), 1620 (art), 1540 (art), 1480 (art), 1460 and 1380 (new sol), 1420 (art), 1340 (art) , 1320 (Sharp), 1280 (Sharp), 1240 (Sharp), 1195 (Sharp), 1170 (Sharp), 1145 (Sharp), 1120 (Sharp), 1075 (Broad), 1015 (Sharp), 990 (Sharp) , 935 (art), 920 (broad), 900 (art), 865 (art), 640 (art), 770 (art), 770 (art), 770 (art), 755 (art), 740 (art) , 720 (new sol).

Infrared spectra were measured with a Perkin-Elmer model 157 spectrometer.

The front side of the spectrum is shown in FIG.

6) Non-radiance

+125° (농도는 메탄올:클로로포름 1:1중에서 0.8%)

+ 125 ° (concentration 0.8% in methanol: chloroform 1: 1)

7) Solubility

The compound is dissolved in dimethylsulfoxide, dimethylformamide and chloroform / methanol mixture, slightly soluble in chloroform, methanol, methanol / ethyl acetate mixture sodium bicarbonate solution and glacial acetic acid, and water and other common organic solvents are insoluble.

8) characteristic reactions

9) pH

The ionization function pka 7.8 was determined by potentiometric titration of 0.1 N sodium hydroxide of antibiotic L13365 in a methyl cellosolve: water 4: 1 solution. The equivalent measured together is 1400.

10) Perform chromatography

Claims (1)

Incubate ActinoPranes Sarveparensis (Bacterial Accession No .: CBS NO.305.76) in a medium containing anabolic carbon sources, anabolic nitrogen sources and inorganic salts until a substantial amount of antibiotic L13365 is produced under deep aerobic conditions. Method for preparing antibiotic L13365, characterized in that the antibiotic is separated from.

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