DD201150A1 - METHOD FOR PRODUCING A BIOREGULATOR - Google Patents

Abstract

The invention relates to a process for the preparation of a bioregulator which is capable of stimulating industrially important microorganisms both in their differentiation and in their biosynthetic capacity for anthracycline antibiotics and is therefore of potential use in the production of active substances in the pharmaceutical industry. In particular, the invention relates to a process for the preparation of a hitherto unknown, low molecular weight compound, which can be referred to as 4,5-dihydroxyn-decanoic acid-4-lactone (L-factor) on the basis of the chemical constitution analysis. The microorganism used to make the bioregulator is the leukemomycin-blocked mutant ZIMET 4388668 of the species Streptomyces griseus. The aim of the invention is to obtain a bioregulator whose use in the fermentation industry for optimizing microbial performance, e.g. biosynthetic capacity for antibiotics. This object is achieved by aerobic submerged fermentation of the strain ZIMET 43668 formed in media with suitable C, N sources and mineral salts of the bioregulator, isolated by means of extractive methods of mycelium and culture filtrate and subsequently purified by chromatographic methods.

Description

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Title of the invention

Process for producing a bioregulator

Field of application of the invention

The invention relates to a process for the preparation of a bioregulator, which is able to stimulate industrially important microorganisms both in their differentiation and in their secondary metabolism and therefore in the pharmaceutical and microbiological industry in the production of microbial agents on a fermentative path of potential use. In particular, the invention relates to the production of a bioregulator which induces and stimulates the biosynthesis of anthracycline antibacterials having a cancer chemotherapeutic effect, using a per se known microorganism or its mutants and variants.

Characteristic of the known technical solution

It is known that microorganisms synthesize low-molecular metabolites which are responsible for turning on differentiation processes of a morphological and biochemical nature. If these are species-specific compounds, these differentiation factors are also called autoregulators (Khokhlov, AS 1979, Abstraots Int. Sympos. Antibiotics, Weimar-GDR) * If these factors act beyond the species, terms such as bioregulator or "hormone ¹¹ similar substance use. the chemical nature of bioregulators is very different. In Streptomyces bioregulators are known which enable the formation of aerial mycelium and spores and production of Sekumdärmetaboliten as Antibiotiea

17.NOt 1984 * 971927

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This makes it possible to use bioregulators potentially for optimizing antibiotic production in the microbiological industry. Examples of bioregulators of streptomycetes investigated with varying intensity are the following compounds: cosynthesis factor I from Streptomyces aureofaciens (Miller et al., 1960. J. Amer. Hoem. Soe., J82, 5002) an unspecified inducer of staphylomyein production in S. virginiae (Yanagimoto et al 1971, I.Ferment.Technol.42, 611), the Α-factor in Actinomyces streptomycin! (Kleiner et al., 1976. Bioorg.Khim. (Moscow), 2, 1142-47; Khokhlov et al., 1967. Doc. Akad. Nauk SSSE, V77, 232-35; Tovarova et al., 1970. Isvest Akad.Nauk SSSR (Ser.Biol.) 3_, 427-34; Khokhlov et al., 1973. Z. Allg.Mikrobiol., 1_2, 647-55), the C factor in Streptomyces griseus (Biro et al. , 1980. Eur. J. Biochem., 103 , 359-63), the antibiotic pamamycin in Streptomyces alboniger (McGann and Pogeil, 1979. J. Antibiotics, J32i, 673-78), the sporulation polypeptide in Streptomyces venezuelae (Scribner et al., 1973. Appl. Microbiol.,, 25, 873-79) and the methylenomycin in Streptomyces coelicolor (Kirby et al., 1975. Nature, 254 , 265-67), the latter, however, with an inhibitory effect on aerial mycelia. The occurrence of corresponding differentiation factors has also been reported in fungi (Z, B in the case of penicillium cyclopium, Zendem et al., 1980), Abstract * 1/19, 13 _# Eesngr PharnuGeschäftschaft der DDH, Leipzig, 3 -5 · 11.1980; luckner et ALU, 1977 · Secondary metabolism and cell differentiation · Springer Verlag 1977) · drugs of this type are currently being investigated with regard to their structure and mode of operation or _# in relation to its substitutability for control of fermentation processes and microbial in optimizing Benefits, such as the biosynthesis capacity for antibiotics and other active ingredients »

Object of the invention

The invention serves to prepare a bioregulator which allows both the morphological and the ohmic differentiation of anthracycline-forming streptomyoeten,

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or »can promote. Thus, the range of such bioregulators is extended by a new constitutional ingredient, which is of potential use for optimization tasks of the microbiological industry. "The bioregulator to be produced should, when added to streptomycete cultures, perform neither microfilming and spore formation nor anthracyclines to synthesize and stimulate both differentiation and anthracycline biosynthesis.

Explanation of the essence of the invention

The invention has for its object to describe a technologically simple method, with the aid of the bioregulator, also known as leukemomycin biosynthesis-inducing factor (L-factor), prepared from a microorganism by cultivating it on an easily accessible medium using cheaper starting materials with Can be detected by means of a specific method and isolated and purified by chromatographic methods.

According to the invention the object is achieved in that under microbial and sterile culture conditions in a liquid nutrient medium with appropriate carbon, nitrogen sources and mineral salts of the microorganism or its mutants and variants are bred »The microorganism, which is used in this method ist'naoh mutagenesis a population of the leukemic myrman, Streptomyces griseus JA 5142 (Fleck and Strauss, 1975. Z. Allgmicrobiol., 15, 455-503). The producer of the L-factor, which after mutation has lost both the differentiation and the anthracycline biosynthetic capacity, is under the registration number ZIMET 43 668 in the depository of the Central Institute for Microbiology and Experimental Therapy of the Academy of Sciences of DDl, 6900 Jena, Beutenbergstr. 11, deposited ·. '

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The cultivation of the strain ZIMET 43 668 and its variants and mutants is carried out under aerobic conditions. Lyophilically dried spore material or glucose / gelatin (5%) lyophilized Bäycelfragmente are inoculated on suitable Agarnährböden and subsequently in liquid, previously sterilized nutrient media and the resulting Myoel is in sioh known manner at a temperature between 25 and 37 ⁰ C. , preferably 28 ⁰ G, over a period of 2 to 6 days, preferably 4 days, cultured at an acidity at the beginning of the fermentation process between pH 6.2 and 7.0 and at the end of the process between pH 7.5 and pH 8.3 is located. The nutrient medium consists of carbon and nitrogen sources as well as inorganic salts. As sources of carbon and nitrogen it is possible to use the starting materials customary in the antibiotics industry. The fermentation of the L-factor-forming agent ZIMET 43 668 can be carried out in steep-breasted bottles and round-bottomed flasks of different contents, in glass fritters and in V2A-Tank £. The isolation of the L-factor is carried out by extracting it from mycelium and culture filtrate, preferably from the latter by organic solvents, preferably butyl acetate or chloroform. By concentration of the butyl acetate extracts of culture filtrates, a crude product was obtained, which was purified by column chromatography on organophilic dextrins, and the resulting active fractions were bound to dry cellulose powder, then eluted with water. The aqueous eluates are extracted with ether, the resulting products are again säulenchromatografieoh on buffered silica gel at pH 7.0 or thin-layer chromatography on neutral kieselge! enriched, then, the products obtained by preparative gas chromatography of all accompanying. components freed *

The L-factor is a slightly yellowish oil at room temperature, which is determined by the mass spectrometric investigation (Hooh resolving mass spectrometer JEOL JMS-D-100, direct inlet system, 55 ⁰ C, 75 eV electron flow)

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has a molecular weight of 186 and the empirical formula C ₁₀ H ₁₈ O ₃ , (FIG. 2) M ⁺ : m / z 186.1253. (186.1256, for G ₁₀ H ₁₈ O ₃ ). The characteristic maxima in the ΙΕ spectrum (GOGl ₃ ) are: 715, 721, 740, 895, 905, 990, 1030, 1155, 1187, 1420, 1774, 3610 cm ^-1 .

The maximum absorption in the UV range is 279 nm (EtOH), based on the building block analysis and according to ^ C u. 'H nuclear magnetic resonance spectrometry obtained findings (Fig, 2, Tab. 1 and 2), the L-factor represents a previously unknown 4,5-dihydroxy-n-decanoic acid 4-lactone with the constitutional formula given in Figure 1. Zur more detailed characterization of the L-factor in Figure 2, the mass spectrometry obtained Fragmentierungssehema and in Tables 1 and 2 the

13 1

Assignments of the ^ C or H nuclear magnetic resonance signals are listed ·

The L-factor 4,5-dihydroxy-n-decanoic acid 4-laeton shows the following Rf values on silica gel foil η:

0.10 for solvent system benzene: ether = 3: 1 (v / v) x 0.20 for solvent system benzene: ether = 1: 1 (v / v). 0.80 for solvent system benzene: acetone = 5 * 3 (v / v) · Use of silica gel plates lead to the following fif values of the L-factor:

0.50 for solvent system benzene: acetone = 8: 2 (v / v). 0.17 for eluent system CHCl ₃ : .Ether = 6: 4 '(v / v). On cellulose films, the L-factor in the eluent system H ₂ O gives an Ef value of 0.5, on washed, neutral chromatography paper in the same eluent Sf 0.65.

The detection of the action of the L-factor is carried out by means of a special method which allows to detect both the stimulation of aerial mycelium and sporulation as well as leukemomycin biosynthesis. Details of the microbiological analysis are described in Eritt et al (1981).

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EXAMPLES

1. Mycelium fragments of the strain ZIMBT 43 668, which had been cultivated on the streptomyoete standard oatmeal agar for 7 days, are used as inoculum for pre-breeding cultures in the following Submers medium: 0.6% Bacto-peptone; 0.3% yeast extract; 0.05% CaCO 3 f! Tap water ad 100%; pH 7.2; Sterilization. 120 ⁰ C, 30 min The Vorzucht cultures are in 100 ml Steilbrustflasohen a 20 ml fill and 48 h at 28 ⁰ C on a shaking incubated (180 U / min).. The submerged mycelium thus obtained is used by incremental scaling to inoculate 400 1 fermentors with submersible medium of the following composition: 3% glucose; 1% soy flour; 0.3% NaCl; 0.3% CaCO ₃ , pH 6.5 after sterilization (115 ⁰ C, 30 min.) · During the fermentation at 25 ⁰ C, which is carried out at a feed rate of 280 U / min and an air supply of 400 l / min Foaming may be controlled by adding small amounts of sunflower oil or other silicone-based antifoaming agents. After 96 hours of perforation, the highest content of Lr factor is determined ·

2 "300 l of a culture solution of strain ZIMET 43 668 obtained according to example 1 are adjusted to a pH value between pH 5 to 7, separated into mycelium and culture filtrate with the aid of a separator and worked up in accordance with the scheme shown in FIG. The extraction of the Eulturfiltrates with 60 1 butyl acetate to 50 1 extract, which after neutralization with NaHCO «and concentration in vacuo 200 g of crude product of the L-factor ·

3 # The extraction of the L-factor from emers cultures can be done from the myoel 12 © hr of old cultures if the medium has the following composition:

Oatmeal 2%

Agar-agar 1.5%

pH 7.8

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Incubation takes place at 25 to 29 ⁰ C * The oatmeal agar medium prior to inoculation mii a eemipermeablen cellulose film a result, the strain ZIMET can be 43,668 NaOH 120 h of incubation with the cellulose film without Agarbestandteile lift off the semi-solid medium and extract covered · 10 g of the mycelium (wet weight) obtained in this way are extracted with 500 ml of chloroform for 24 hours at room temperature, then filtered off and the mycelial residue is discarded. NaOH Concentration of the chloroform extract in vacuo gives 110 mg of L-lactone-containing crude product.

4. In each case 50 g of crude product obtained according to Example 2 and 3 from culture filtrate and Emersmyoel are dissolved in 200 ml of methanol and filtered off from insoluble fractions. The resulting filtrate is applied to a 150 × 8 cm column filled with organophilic dextrins (bed height 80 cm), the active fractions are collected after methanol elution, combined and concentrated in vacuo. The residue obtained from 1 g is taken up in 100 ml of diethyl ether and treated with 20 g of dry cellulose powder for column chromatography. Bach the removal of the ether in vacuo, the residue on a column of 100 χ 1.5 cm, filled with dry cellulose powder to a bed height of 10 cm, abandoned. The mixture is then eluted with 2 1 of water, before the eluates obtained with 2 1 diethyl ether extracted in 3 portions, the combined ether extracts over Wa ₂ SO. The residue of 0.5 g is then applied to a column (100 χ 1.5 cm), which with buffered silica gel (pH 7.0, KH ₂ PO ^) in benzene / ether (1: 1 , v / v) is filled. After elution with the same system, according to Eritt et al. (1981) separated the active fractions and combined (yield: 75 mg). Further purification of the L-factor is carried out by preparative thin-layer chromatography on neutral silica gel bases, using benzene / ether (1: 1, v / v, twice run) · Active fractions can be due to yellow fluorescence in the UV light as well as due to characteristic biological

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red violet staining by 1% vanillin in conc. H ₂ SO ^ are detected · After elution of the active zone with ether about 30 mg of a # 90 pure preparation of the L-factor are obtained.

5. The 90% pure preparation of the L-factor is obtained according to Example 4 and subsequently completely purified by preparative gas chromatography. The pre-purified preparation of the 1-factor is freed from concomitant components when placed on a glass column (150 χ 0.6 cm) filled with 2 $ 07 101 on Chromosorb W (AW, DMCS, 80-100 mesh) in isothermal operation (120 ⁰ C), is chromatographed in argon gas stream. The substance appears after a retention time of approx. 15 min. Among the accompanying components of the L-factor, with a share of approx. $ 5 of the total amount used, there is a biologically equally active C-5 isomer (diastereomer) of the L -vFaktors, which has a shorter Eetentionszeit (13 min.) Under the above conditions. (Tab. 1 and 2)

6. The purification of the L-pactor is carried out as in Example 3 with the difference that instead of the column chromatography with silica gels, the thin layer chromatography is carried out with neutral silica gel polishes · each 100 mg obtained by cellulose chromatography crude product according to Example 4 to a 20 χ 20 cm film and developed in the eluent system benzene / ether (1: 1, v / v) by running twice in the same solvent.

Claims (6)

- ⁹ - 232491 2 Erfindungsansprüehe 1, A method for producing a bioregulator, characterized in that the strain ZIMET 43 668 of the species Streptomyces griseus under aerobic conditions in liquid nutrient media containing a carbon, nitrogen source and mineral salts, at a temperature of 25 to 37 ⁰ C, during a From 2 to 6 days, and the resulting bioregulator is extracted from the ffiyoel and / or culture filtrate at an acidity of pH 4 to 7, by means of customary organic solvents, subsequently concentrated by conventional methods and purified by means of oxy-chromatographic methods. Method according to item 1, characterized in that; the bioregulator from strain ZIMET 43 668 is further purified by gel chromatography on organophilic dextrins in organic solvents as eluent and enriched by chromatography with cellulose and neutral silica gels. 3. The method of item 1> and 2, characterized in that high-purity bioregulator is obtained by preparative gas chromatography on impregnated carriers. 2491 Chemical work-up of the bioregulator 4,5-dihydroxy-n-decanoic acid 4-lactone (L-factor) 3QQ 1 Culture solution of strain ΖΙΜΕΐ 43 668 (pH 5.0) Butyl acetate extract of KL (50 l ) residue after concentration in vacuo (200 g) column chromatography on organophilic dextran gels Active fractions (40 g) ¹ 'inactive fractions adsorption on dry cellulose powder elution with H ₂ O.
ether extraction Residue of the ether extract ( 2 g ) column chromatography (silica gel, KH ^ PO.-buffered _Λ \ P 7.0) Active fractions (300 was ' Preparative thin-layer chromatography (Silica gel films, benzene: ether - 1: 1, 90% pure Biore | g; ulator (110 mg) (Yield: 33 # relative to those in culture) filtrate). Preparative gas chromatography OY 101 on öhromosorb W) Gas chromatographically uniform bioregulator Selection of active fractions by Eritt et al * (1981) microbiological method (Z. Alig. Mikrobiol.). 2324-91 2 Table 1: Assignments of the ¹ ^ C nuclear magnetic resonance signals of 4,5-dihydroxy-n-decanoic acid 4-lactone (! -Faktor) and the diastereomeric Nebenkomponen te (Fourier transform NMH spectrometer Varian 100/15, 26 , 16 MHz, TMS as internal standard, off-resonant multiplicities: Sr-singlet; Dt doublet; T: triplet; Qt quartet ι ι Table 2: Assignment of the H-NMR signals of the bioregulator 4,5-dihydroxy-n-decanoic acid 4-lactone (L-Jaktor) and a small concomitant G-5-diastereomeric compound (values in parentheses). All data refer to TMS as internal standard, CDCl ₃ as solvent, instrument: Bruker WP 200 / SY Fourier-transform spectrometer, 200.13 MHz. Proton chemical shift (Ppm) Coupling constants (Hz) C-2H, C-2 H _B C-3 H _n 2.ι (2:58) 2.52 (2.52) 2.27 (2.25) FROM BC BD CD CM DX MX 6.5 (6.5). For this h pages drawings