The streptomycete strain for the production of anti-parasitic compounds and methods of producing these compounds

 

(57) Abstract:

The invention relates to biotechnology. The resulting strain of Streptomyces avermitilis subspecies niger NRRL 21005 producing avermectins Ala, Alb, A2a, A2b, Bia, Bib, B2a and B2b. The cultivation of this strain in a medium containing sources of carbon, nitrogen and inorganic salts under conditions of aerobic submerged culture is carried out before the production of one or more avermectins. In the method of production of ivermectin after producing strains of avermectins Bia and Bib spend their recovery. The use of a strain of Streptomyces avermitilis subspecies niger NRRL 21005 get compounds with antiparasitic properties. 3 S. p. f-crystals, 2 ill., 3 table.

It is known that the avermectins, which represent a set of related compounds with antiparasitic activity, can be produced by aerobic fermentation of strains of ATSS 31267, and 31271 31272 Streptomyce avermitilis (see, for example, U.S. patent 4 310 519 and 4 042 429). The latter two strains are frozen culture vials and liofilizovannye culture in vitro, respectively, obtained by UV irradiation of ATSS 31267 S. avermitilis.

Strains of S. avermitilis described in the aforementioned U.S. patents, producer the s includes eight different but closely related to the avermectins, described in the literature as A1a, A1b, A2a, A2b, B1a, B1b, B2a and B2b Connections series "a" refer to the natural avermectins, in which the substituent at the 25-position is (S)-sec-butyl, and the series connection "b" refer to the natural avermectins, in which the substituent at the 25-position is isopropyl. The designations "A" and "B" refer to the avermectins, in which the substituent in the 5-position is methoxy or hydroxy, respectively. And finally, the number "1" in the above notation means that the avermectins have a double bond in 22 - and 23-position and the number "2" means that the avermectins have a hydrogen in the 22-position and hydroxy in 23-position.

So, for example, avermectins are compounds of formula I:

< / BR>
where a dotted line indicates a single or double bond;

R1represents H or hydroxy; and R1represents a hydroxy only when the dashed line is a simple link;

R2represents an isopropyl or sec-butyl; and

R3represents methoxy or hydroxy.

It is known that other strains of S. avermitilis produce one or more "natural" avermectins the avermectins where the substituent in the 25-position is either isopropyl or (S)-sec-butyl. For example, strain ADS 31780 S. avermitilis is disclosed in U.S. patent No. 4 378 353; strains ADS 53567 and ATSS 53568 disclosed in the application for Europatent (EP) 276 131; strain ADS 53692 disclosed in EP 276 103; and a description of the NCIB 12121 can be found in EP 214731. However, each of these strains of S. avermitilis is a direct or indirect descendants of the strain ADS 31267 S. avermitilis. Thus, the above strains of S. avermitilis, which have the ability to produce one or more compounds of embryo death, represent the parent strain and its progeny.

In the literature (U.S. Pat. USA N 5 015 662) was described by another strain of S. avermitilis, ATSS 53814. This strain is characterized by its capacity for bioconversion of series connection type milbemycin, and the inability to produce a significant amount of avermectins.

The present invention relates to a new avermectin-producing strain of S. avermitilis, namely subspecies niger NRRL 21005. In addition, the present invention relates to new ways of producing avermectins using subspecies niger NRRL 21005 Streptomyces avermitilis.

The present invention relates to a biologically pure culture of the microorganism Streptomyces avermitilis pottage relates to a method for producing one or more avermectins, namely, that the subspecies niger NRRL 21005 S. avermitilis or avermectin-producing mutant or variant is cultivated in an aqueous culture medium containing assimilable sources of carbon, nitrogen and inorganic salts under aerobic deep conditions until, until there is produced one or more avermectins, and then the specified avermectin or produce avermectins.

In Fig. 1 presents dendogram subspecies niger NRRL 21005 (A58267.2) of Streptomyces avermitilis and other species of Streptomyces avermitilis.

In Fig. 2 shows a diagram of the principal components, based on the analysis of fatty acids, and illustrating the relationship between A58267.2 and other types of S. avermitilis.

In one of its variants the present invention relates to a biologically pure culture of the microorganism Streptomyces avermitilis subspecies niger NRRL 21005 (strain NRRL 21005), or avermectin-producing mutant or variant.

For convenience, this new microorganism of the present invention, producing avermectins, was also named culture A58267.2. Culture A58267.2 is an improved strain of culture A58267.21, which is a natural variant of culture A58267. Culture A58267 was isolated from a soil sample collected in Italy.

Taxonomic studies A58267.2 (NRRL 21005) were conducted Frederick P. (Mertz Lilly Research Laboratories). Based on these studies, a new microorganism has been classified as a new subspecies (strain) genus and species of Streptomyces avermitilis, which was given the name "subspecies niger" Streptomyces avermitilis. The classification was carried out on the basis of laboratory classification and analysis of published studies of similar species.

The methods used

Taxonomic studies were carried out with the use of "Methods for characterization of Streptomyces species", Jnt. J. Syst. Bacteriol. 16: 313-340 (1966)], and methods recommended for characterization of Nocardia species [Jordon R. E., D. A. Barnett, J. Handerhan E., and Pano C. H., "Nocardia cveliaca, Nocardia the Nocardin Strain", Jut. J. Syst. Bacteriol, (24): 54-63 (1974)].

To determine the colors that you have used the color chart (ISCC-NBSCentroid Color Charts), the standard sample N 2106 (National Bureau of standards, 1958, U.S. Department of Commerce, Washington, D. C.).

The morphology was investigated using optical microscope and scanning electron microscope (SEM).

The presence of the isomer diaminopimelic acid (DAP), and carbohydrates in hydrolysates of whole cells was established using chromatographic methods Becker and others , "Rapid Differentiation between Nocardia and Streptomyces by Paper Chromatography of whole - cell Hydrolysates", Appl. Environ., 12: 421-423 (1964) and Lechevalier, and others, A University Laboratory Approach, Dietz and Fhayer (eds), Society for Industrial Microbiology, Special Publication N6 Arlington, Virginia, pp. 227 - 233.

Antibiotic resistance was measured by applying sensitive to the antibiotic disks on the surface of the cups with agar seeded with ISP # 2. Resistance was evaluated as ( + ) if it was not observed zone of inhibition, and as ( - ) if the zone of inhibition was observed.

The composition of managenow was determined by the methods described Kroppen - stedt R. M. in: Chemical Methods in Bacterial Systematics", M. Good microbial identification system HP 5898A Miller and others, "Bacterial Identification fy Gas Chromatography of Whole - cell Fatty Acids", Hewlett - Packard Application Note 228-41, (1985).

Methyl esters of fatty acids were obtained from liofilizovannyh whole cells cultured in identical conditions.

NaCl tolerance was determined by adding NaCl to the ISP-the agar N 2 to obtain a desired concentration.

The dendrogram shown in Fig. 1, generated by computer on the basis of akwidaa code distance.

Analysis by the method of principal components, shown in Fig.2, is a two-dimensional analysis was also conducted using computer processing.

Cultural properties.

Culture A58267.2 insufficient grows well in most environments, and does not produce aerial hyphae nor complex environments, nor on any of the nutrient media of a particular composition. The reverse side is painted in a yellow-brown color, except in those cases when the cultivation is carried out on agar Bennett, where is produced the characteristic black pigment. Weak, slightly brownish soluble pigment is produced in ISP medium 2, and reddish-brown soluble pigment is produced in the agar Bennett. These cultural characteristics which are culture A58267.2. Therefore, morphological studies and research of the surface, the dispute could not be conducted. The sporangia, mobile cells, or sclerotia were observed. Culture A58267.2 does not fragment during cultivation on solid or liquid media.

Physiological characteristics

Culture A58267.2 utilized the following carbohydrates when using ISP medium 9 as a basal medium: D - and L-arabinose, cellobiose, D-fructose, D-galactose, D-glucose, glycerol, glycogen, I-Inositol, inulin, D-maltose, D-mannitol, mannose, melibiose, D-raffinose, L-rhamnose, D-ribose, trehalose, xylitol and D-xylose. Culture A58267.2 did not show the ability to recycle some of the carbohydrates used with ISP medium 9 as a basal medium, namely adonica, cellulose, dextrin, dulcita, ethanol, ISO-erythritol, D-lactose, melezitose, a-methyl-D-glucoside, salicin, sorbitol, L-sorbose and sucrose.

Culture A58267.2 is capable of decomposing adenine, malate calcium, casein, starch and urea.

A58267.2 was produced catalase, H2S and liquefied gelatin and had the ability to survive under conditions of temperature 50oC for 8 hours. A58267.2 was found tolerance to NaCl at concentrations up to (in the rata, gipoksantina, testosterone, L-tyrosine, or xanthine. This culture has not produced melanoidin pigments, not restored nitrates, and has not produced a phosphatase, and she did not show resistance to lysozyme at a concentration of 50 mg/ml

Culture A58267.2 found resistance at 2 µg /micrograms/ lincomycin, 30 μg nalidixic acid, 10 units of penicillin G, 300 units of polymyxin b B, and 5 mg of trimethoprim. A58267.2 showed sensitivity to 10 units of bacitracin, 30 µg tsefalotina, 30 µg of Chloromycetin, erythromycin 15 µg, 10 µg oleandomycin, 5 mcg rifampin, 10 mg streptomycin, 30 mcg tetracycline, 10 µg of tobramycin and 30 mcg vancomycin.

Culture A58267.2 were cultured at a temperature in the range from 15 to 37oC. the Optimum temperature is about 30oC.

Analysis of cell wall

Analysis of the cell wall of the parent strain A58267.21 showed that hydrolyzed target cells contained L-diaminopimelic acid (DAP). Sugars present in extracts of whole cells are glucose and ribose. The type of fatty acid is 2C, and major manoyanom is MK-9(H6Measurements revealed the characteristic of the genus Streptomyces type cell wall, and, as is revealed, culture A58267.2 is of type 1 cell wall, the full composition of sugars NC type, and composition of managenow corresponds, mainly, MK-9(H6). These chemotaxonomic properties, as well as cultural and morphological characteristics A58267.2 confirm the assignment of this isolate to the genus Streptomyces [see, Lechevalier and others, "Chemical Composition as a Criterion in the Classification of Aerobic Actinomyceses, Int. J. Syst. Bacteriol. 20; 435-443 (1970)].

Although ATS "Catalogue of Bacterial and Bacteriophages" 18th ed. (1992) revealed 11 strains of Streptomyces avermifilis, however, in the literature of S. Avermitilis, however, in the literature of S. avermitilis ADS 31267 identified as indirect or direct parent 10 of 11 strains. If ADS 31267 (A54508) is not the direct parent strain, often direct parental strain generation is the first generation, ATSS 31272 (A54508.3). Describe the offspring or ATSS 31267, or 31272 in the literature suggests that the offspring has in common with the parent strain characteristics, however, they have some distinctive features (see, for example, the taxonomic description of ATSS 53567 and 53568 in EP 276131). Therefore, as strains for comparison with the strain ADS 21005 (A58267.2) from all direct or indirect parent strains were selected one known strain of S. avermit degree determinative comparison analysis of fatty acids was performed on A58267.2, A54508 and A54508.3. Table 2 shows a comparison of the percentage content of specific fatty acids, found in each strain.

The dendrogram generated using the computer, and based on the profile of fatty acids, shown in Fig. 1. The dendrogram illustrates the related ratio A58267.2 with other strains of Streptomyces avermitilis as shown by aquidaba code distance. Culture, showing the relationship below 10,00, was interpreted as synonymous, i.e. belonging to the same species. Relationship A58267.2 with respect to S. avermitilis is above 10,00.

The principal component analysis relates to the field of multivariate statistical analysis that deals with the internal correlations of the set of variables. In this analysis, the greatest amount of variance within the original data or the data of the test results expressed in terms of principal components [Alderson G., "The Application and Relevance of Nonhierarchic Methods in Bacterial Taxonomy", b "Computer-assisted Bacterial Systematics", Goodfellow, M., and others (ed.), Academic Press, New York (1985)]. Thus, there may be constructed a chart illustrating the variation and variability. Then, by estimating this variance can be estimated is the R on the composition of fatty acids, was built a two-dimensional chart of main components, illustrating the relationship between culture A58267 and its parent strain A58267.21 other types of S. avermitilis. The data obtained is shown in Fig.2.

As in the dendrogram, and the diagram of the principal components formed two different cluster. A58267.2 and A58267.21 form one cluster, and A54580 and A54580.3 form another cluster. These two clusters are similar enough, which indicates that the strains in these clusters are related, but they also have sufficient distinction, which suggests that neither the cluster nor the individual strains in these clusters are not identical.

Differences between A58267.2 and A54840.3 systematized in table 3.

As previously established, differences between strains of S. vermitelis A58267.2 and A54508/A54508.3 are insufficient to qualify A58267.2 as a separate type. However, these differences are sufficient to distinguish A58267.2 in a separate subspecies (strain) that is different from known strains of Streptomyces vermitilis. Thus, A58267.2 was classified as a subspecies of S. avermitilis, and in particular as a subspecies niger NRRL 21005 Streptomyces avermitilis. The name "niger" this strain was due to its colors is the own production of one or more avermectins, which is that the subspecies niger NRRL 21005 S. avermitilis, or avermectin-producing mutant or variant is cultivated in an aqueous culture medium containing assimilable sources of carbon, nitrogen, and inorganic salts in the deep aerobic conditions until, until there is produced one or more avermectins, then these produce avermectins. Received the avermectins can be selected using different procedures for isolation and purification are well known to the specialists.

As in the case of other organisms, the characteristics of avermectin-producing culture of the present invention, subspecies niger NRRL 21005 Streptomyces avermitilis, can be modified. Mutants of this strain can be obtained by known methods, for example, by processing the strain of various physical and chemical mutagens, such as ultraviolet radiation, x-ray radiation, gamma radiation, and chemical compounds such as n-methyl-N'-nitro-N-nitrosoguanidine. Natural options specified strain can also be obtained by standard methods, for example by screening cultures of the parent strain. In the scope of the present invention includes such natural variants and and is.

For the cultivation of a culture of S. avermitilis of the present invention may be used any of the available suitable environments. For example, the preferred carbohydrate sources in large-scale fermentation are glucose, mannose, and especially, potato dextrin. However, it can also be used and other sources of carbohydrates such as ribose, xylose, fructose, galactose, mannitol, etc.

Preferred nitrogen sources is soy flour, although it can also be used an enzyme or acid-hydrolyzed casein, yeast, liver meal, meat gelatin, fish meal, etc.

In the culture medium can be introduced nutrient inorganic salts, such as commonly used soluble salts such as the chloride, carbonate, sulfate, nitrate zinc, sodium, magnesium, calcium, ammonium, etc.

The culture medium should also contain the main trace elements necessary for the growth and development of the organism. Such trace elements commonly found in other components as impurities in amounts sufficient to meet the needs of micro-organism growth. If necessary environment for large-scale fermentatively molecular weight of about 2000.

Examples of preferred concentrations of the components of culture medium below in example 1.

For producing significant quantities of avermectins are preferred depth of the aerobic cultivation is carried out in fermenters. A small amount of culture A58267.2 can be obtained by cultivation in shake flask. Because the lag is the period when the production of embryo death is usually associated with inoculation large fermenters spore-forming microorganism, it is preferable to use an actively growing seed. Such vegetative planting material prepared by inoculation of a small volume of culture medium spore-forming or micelle fragments of the microorganism, and get in the fresh, actively growing culture of the microorganism. Then vegetative planting material is transferred to a larger vessel and begin stage production of avermectins. Environment for vegetative inoculum can be the same as in the implementation of more large-scale fermentati, but you can use any other suitable environment.

In the method of the present invention avermectins produced with IPoA temperature producing embryo death is from about 29oC to about 31oC.

As usually practiced in the submerged aerobic cultivation, sterile air fed into the vessel from below, and the medium is stirred using a standard turbine stirrers, maximum absorption of oxygen during the fermentation in used conditions, thus, not greater than about 0.16 mm/l/min In a fully partitioned 115-liter fermenter, the rate of aeration and mixing should be sufficient to maintain the level of dissolved oxygen, at least 45% saturation of oxygen in the internal pressure in the vessel of 5.0 ATM.

The production of avermectins can be controlled during the fermentation by testing samples of the nutrient medium by comparing with the existing standard using a variety of methods, such as HPLC.

After production in submerged aerobic fermentation, the avermectins can be isolated from the fermentation medium by known methods commonly used for this purpose. Usually the avermectins produced in the fermentation culture A58267.2 or its avermectin-producing mutant or variant, present in both the filtered broth, and feature the culture broth can be dried and mixed with food for these animals.

More preferably, if the avermectins are extracted from the whole culture broth, and the allocation of individual compounds and embryo death is carried out by solvent extraction and chromatographic fractionation using different solvent system and chromatographic techniques. The technique of separation and allocation of avermectins are described in U.S. patent No. 4 429 042: and Miller, T. W., and others, Antimicrob. Agents Chemother. 15(3); 368-371 (1979).

Preferred methods of separation and allocation described in the examples below.

After the acquisition and allocation of avermectins their use, in particular, for the treatment of animals suffering from parasitic infections, as well as deworming drugs, insecticides and acaricides. The use of these compounds as antihelminthic means well known in veterinary medicine and is widely described in the literature (see, for example, U.S. Pat. USA NN 4 042 429 and 4 310 519).

In addition, can be obtained (not optional) ivermectin, which is a well-known derivative of the combination of the reconstructed embryo death B1a and B1b ways described Charbula, and others, in U.S. patent No. 4 199 569, which is introduced into the present description by reference.

For Bo be treated as a restriction of the scope of the present invention in all respects.

In the following examples: spectral data13C-nuclear magnetic resonance for avermectin compounds A1a, A1b, A2a, A2b, B2a, B1b, B2a and B2b, in a solution of deuterated chloroform were obtained using a spectrometer (General Electric Model QE 300 (Femont, CA). Solution volume of each sample is approximately 0.7 ml Chemical shifts for each connection embryo death regarding deuterochloroform are given in ppm (77 ml). Data FABMS (mass spectroscopy by fast atom bombardment) were obtained using a spectrometer ZABII-SE (VG Analytical, Manchester, England).

Example 1

Fermentation A58267.2

A. Catalogna bulb

The culture of niger strain NRRL 21005 Streptomyces avermitilis, stored in liquid nitrogen, and used for inoculation of 0.5 ml of a vegetative medium of the first stage having the following composition:

The growing environment

Ingredient: Amount (g/l)

Tripticos - 30,0

Yeast extract - 3,0

MgSO47H2O - 2,0

Glucose - 5,0

Maltose - 4,0

Deionized water, Hon. Col. - up to 50 ml

The unadjusted pH 7.0; after sterilization, the pH was not adjusted; pH after sterilization - 6,9.

Inoculated vegetative medium is incubated in width is 2 inches (5.08 cm) at 250 rpm/min The flask was protected by two bio-protective filters, and the angle of the shaker table was 0oC.

B. Bioreactor for fermentation A58267.2

To obtain a large amount of inoculum 10 ml of incubated medium of the first stage, prepared as described above in Part A, was used for inoculation of 400 ml of a vegetative medium for the second phase having the same composition as the medium for the first stage. This Wednesday the second stage were incubated in a wide-mouthed 2-liter Erlenmeyer flask at 30oC for 48 hours on a shaker rotating in a circular orbit in 2 inches (5.08 cm) at 250 rpm/min the flask was protected by two bio-protective filters, and the angle of inclination of the vibration table was 0oC.

This growing environment of the second stage (400 ml) was used for inoculation of 107 l of sterile nutrient production medium having the following composition:

Nutrient production environment

Ingredient: Amount (g /l)

Soy flour - 5,0

Potato dextrin - 80,0

Oat flour for baby food - 5,0

Raw molasses - 5,0

CaCO3- 2,0

Glycerin - 1.0 ml

Mineral uterine mixture of čapek - 2.0 ml

Deionized water, Hon. to the donkey sterilization pH 7.0. Added antifoam SAG 471 at 0.2 g/l (Union Carbide Sistersille, Zap.Virginia).

Inoculated productive environment left in 115-liter fermenter with a mixer at 10-11 days at a temperature of 30oC for fermentation. Dissolved oxygen was maintained at a level of about 45% saturation of air, because the stirring was carried out at low speed (from about 137 to about 255 rpm).

Example 2

The total allocation of avermectins

Whole fermentation broth from 115 l, obtained in accordance with the description in example 1 was brought to a pH of 6.5 using 5 N. HCl. Then to the solution was added an equal volume of MeOH and the resulting solution was filtered through a ceramic filter Mebralox (U. S. Filter /SCT, Bazet, France). Thereafter, the filtrate was passed through a column containing 10 liters of HP-20SS (Dialon) (Mitsubishi Chemical Industries Ltd, Tokyo, Japan). Then the column was suirable linear gradient of 50-100% MeOH in water. The collected fractions were analyzed by HPLC on a column [4.6 mm (int.dia.) x 30 cm] containing Dynamax C18(Rainin Company, Woburn, MA). The column was suirable using isocrates gradient MeOH: CH3CN:(0,2% HOAc, pH, increased to 5.0 using NaOH) = 44:44:12, a flow rate of 1.5 ml/min based On the HPLC analysis, the fraction of merged pools was extracted with CHCl3and the extracts evaporated, resulting in a received 18,25 g of oily product (Pool A) and 6.48 in g of oily product (Pool B).

Example 3

Processing A Pool

Pool A (18,25 g) from example 2 was dissolved in 45 ml of MeOH and was chromatographically on klenke (5 cm (EXT. dia.) x 1.1 m) containing Sephadex LH-20 (Pharmacia, Piscataway, New Jersey). The column was suirable MeOH and fractions were analyzed by HPLC. Fractions containing avermectins were combined into two pools and evaporated, resulting in the received Pool C (10,96 g) and Pool I (1.45 g).

Example 4.

Processing Pool C

Material from the Pool C (2 g of the material obtained in example 3) was dissolved in 4.5 ml of MeOH and was chromatographically in column (41,4 mm (int.dia.) x 25 cm), containing Dynamax - 60A C18(Rainin, Woburn, Massachusetts). The column was suirable using a linear gradient of CH3CN:MeOH:H2O= 40:40:20 - 46: 46: 8 for 100 minutes, with a flow rate of 15 ml/min On the basis of HPLC analysis was obtained 4 pool: Pool E (489 mg), Pool F (624 mg), Poole G (656 mg), and Pool H (177 mg).

Example 5

Selection and embryo death AIa

Pool B from example 2 was dissolved in 30 ml of MeOH and was chromatographically as described in example 3. The column was suirable MeOH and fractions were analyzed by HPLC. The fractions containing was referable on a column (2.5 cm (EXT.dia.) x 30 cm), containing Chomegabond MC18 (ES Industries, Berlin, New Jersey) using a gradient of CH3CN: MeOH:H2O= 40:40:20 - 42:42:16 within 90 minutes at a flow rate of 5 ml/min In the described procedure was obtained 63 mg avermectin Ala; FAB-Ms m/z = 909 [Aver.A1a+ Na]+; NMR (75 MHz, CDCl3) : 11,96; 12,88; 15,03; 16,30; 17,62; 18,32; 19,82; 20,18; 27,44; 30:50; 34,18; 34,43; 35,13; 36,52; 39,66; 40,41; 45,61; 56,32; 56,41; 57,69; 67,18; 68,07; 68,15; 68,32; 74,83; 76,01; 76,89; 77,41; 78,15; 80,43; 80,50; 81,92; 118,25; 118,31; 119,58; 124,79; 135,10; 135,98; 136,08; 137,58; 139,85; 173,81; 94,88; 98,44; 68,33; 127,74.

Example 6

The selection of avermectins A2a and BIb

Material from the Pool F (from example 4) was dissolved in 2.5 MeOH and chromatographically on the column as described in example 4. The column was suirable isocratically using MeOH:H2O= 85:15, flow rate 15 ml/min, resulting in a net avermectin A2a (202 mg); FAB-MS: m/z = 927 [Aver.A2a+ Na]+; 13C-NMR (75 MHz, CDCl3) : 173,4; 139,83; 137,48; 135,79; 135,60; 124,80; 119,65; 118,43; 117,47; 99,56; 98,41; 94,78; 81,67; 80,50; 80,36; 79,27; 78,15; 77,50; 76,80; 75,90; 70,67; 68,79; 68,25; 68,12; 68,06; 67,55; 67,18; 57,60; 56,38; 45,54; 41,06; 40,70; 39,62; 36,27; 35,61; 35,04; 34,41; 34,26; 34,07; 27,18; 20,20; 19,86; 18,31; 17,65; 15,06; 13,72; 12,37; 11,75; and avermectin B1b (79 mg): FAB-MSm/z = 881 [Aver.BIb+ Na]+;13C-NMR (75 MHz, CDCl3) : 173,56; 139,54; 137,95; 137,80; 135,97; 135,07; 127,72; 124,70: 120,31; 118,28; 118,00; 98,43; 95,63; 94,88; 81,83; 80,38; 80,32; 79,32; 79,22; 78,22; 77,26; 75R>
The selection of avermectins B1a A1b and

Material from the Pool of G (from Example 4) was dissolved in 3 ml of CH3CN and chromatographically on a column (2.5 cm (EXT.dia.) x 30 cm) containing Chromegabond MC18 (ES Industries, Berlin, new Jersey) for 100 minutes using a linear gradient of CH3CN : H2O = 73 : 27 to 80 : 20 at a flow rate of 5 ml/min in three separate series, resulting in a net avermectin A1b (145 mg); FAB-MS m/z = 875 [Aver.A1b+ Na]+;13C-NMR (75 MHz, CDCl3) : 173,39; 139,61; 137,35; 135,50; 134,87; 127,58; 119,45; 118,29; 118,07; 98,22; 94,41; 94,69; 81,68; 80,29; 80,22; 79,10; 77,99; 77,28; 77,00; 76,65; 75,73; 68,08; 68,05; 67,91; 66,98; 57,43; 57,21; 56,18; 45,39; 40,24; 39,45; 36,36; 34,30; 34,05; 30,65; 28,09; 20,82; 20,03; 19,66; 18,15; 17,48; 16,31; 14,87; 14,64; 124,63; and avermectin B1a (245 mg); FAB-M M/z = 895 [Aver.B1a+ Na]+; 13C-NMR (75 MHz, CDCl3) : 173,40; 139,53; 137,84; 137,70; 136,08; 135,08; 127,74; 124,69; 120,23; 118,23; 117,94; 98,42; 95,69; 94,87; 94,87; 81,69; 80,23; 80,15; 79,13; 78,05; 75,69; 74,61; 68,12; 68,03; 67,93; 67,46; 67,01; 56,26; 56,17; 45,43; 40,28; 39,51; 36,33; 34,93; 34,23; 34,05; 30,32; 27,25; 19,99; 19,66; 18,24; 17,50; 16,15; 14,85; 12,74; 11,81.

Example 8

The selection of avermectins A2b, B2a, and B2b

Material from Pool E (from example 4) was dissolved in 1.9 ml of CH3CN and chromatographically on the column described in example 7. The column was suirable for 100 minutes, a linear gradient of CH3CN : H2O = 65:35 to 75:25, at a speed of flux is/SUB> + Na]+;13C-NMR (75 MHz, CDCL3) : 173,60; 139,83; 137,47; 135,92; 135,54; 124,75; 119,52; 118,21; 117,44; 99,53; 98,37; 94,69; 81,53; 80,43; 80,30; 79,21; 78,10; 77,34; 76,81; 75,92; 72,24; 69,75; 68,24; 68,10; 68,05; 67,46; 67,14; 57,65; 56,38; 56,31; 45,52; 40,99; 40,63; 39,60; 36,37; 35,95; 34,47; 34,14; 27,86; 20,68; 20,16; 19,80; 18,29; 17,59; 15,05; 13,85; 13,55; avermectin B2a (125 mg); FAB-MS m/z = 913 [Aver.B2a+ N]+;13C-NMR (75 MHz, CDCl3) : 173,63; 139,68; 138,01; 135,62; 135; 124,72; 120,34; 117,92; 117,54; 99,63; 98,47; 94,77; 81,55; 80,35; 79,31; 79,08; 78,17; 76,07; 72,35; 69,86; 68,43; 68,32; 68,10; 67,68; 67,51; 67,25; 56,3; 56,3; 45,67; 41,11; 40,72; 39,75; 36,55; 36,07; 34,57; 34,24; 34,17; 27,96; 20,80; 20,21; 19,97; 18,41; 17,68; 15,17; 13,95; 13,67; and avermectin B2b (145 mg); FAB-MSm/z = 899 [Aver.B2b+ Na]+;13C-NMR (75 MHz, CDCl3) : 173,09; 139,44; 137,65; 137,57; 135,44; 124,50; 120,90; 117,76; 117,34; 99,41; 98,26; 94,63; 81,47; 80,19; 79,20; 79,10; 78,04; 75,72; 70,57; 69,66; 68,10; 68,03; 67,97; 67,45; 67,36; 67,05; 56,28; 56,24; 45,45; 40,57; 39,52; 36,17; 35,46; 34,90; 34,26; 34,09; 33,91; 27,04; 20,00; 19,69; 18,16; 17,51; 14,91; 13,56; 12,33; 11,59;

1. The strain of Streptomyces avermitilis subspecies NRRL 21005 - producer of avermectins.

2. Method of producing one or more avermectins A1a, A1b, A2a, A2b, B1a, B1b, B2a and B2b, including the cultivation of the producer strain in a culture medium containing sources of carbon, nitrogen and inorganic salts, in the submerged culture conditions for the production of specified embryo death or avermectins, characterized in that as the strain producedthis is the producer strain is cultivated in an aqueous culture medium containing assimilable sources of carbon, nitrogen and inorganic salts in submerged aerobic cultivation until then, until you have produced connection B1a and B1b, and the connection B1a and B1b restore, characterized in that as the producer strain used subspecies niger NRRL 21005 microorganism Streptomyces avermitilis.

 

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