Method of obtaining macrolide compound and streptomyces sp, mortierella sp and micromonosporaceae strains

FIELD: chemistry.

SUBSTANCE: invented group pertains to biotechnologies; in particular, to the method of obtaining macrolide compound 11107D with formula (II) and to Streptomyces sp. FERM BP-8551, Mortierella sp. FERM BP-8547, Mortierella sp. FERM BP-8548 and Micromonosporaceae FERM BP-8550 strains. The initial compound 11107B with formula (I) undergoes biotransformation to the target compound with formula (II) through incubation in the presence of a strain, capable of converting macrolide compound 11107B into 11107D substance and a Mortierella, Streptomyces type or Micromonosporaceae family, or a mycelium cultivated strain sample. The target substance 11107D is separated from the reaction medium.

EFFECT: increased anti-tumour activity and stability of the compound.

8 cl, 9 tbl, 10 ex

 

This invention relates to a method for producing a 12-membered cyclic macrolide compound 11107D with antitumor activity, biological transformation and a new strain used for this receipt.

12-membered cyclic macrolide compound 11107D is a 12-membered cyclic compound having an excellent antitumor activity, which is found together with substance P in the culture product of the strain Streptomyces sp. Mer-11107 (see WO-A02/060890). The 11107D substance meets the substance B having a hydroxyl group in position 16. The productivity of the 11107D substance is worst than the productivity of substances W, and, therefore, is desirable to develop effective way to retrieve it.

The purpose of this invention is the provision of a new method of obtaining a macrolide compound 11107D with the use of macrolide compounds V as source material through a biological way to turn.

The authors of this invention have carried out the testing for the selection of microorganisms capable of converting the hydrogen atom position 16 in the hydroxyl group of the macrolide compounds V screening of a wide range of groups of microorganisms to solve the above problems and as a result have found that the strains belong to easy to the genus Mortierella, classified as belonging to filamentous fungi, a strain belonging to the genus Streptomyces, are classified as belonging to actinomycetes, and strain, belonging to the family Micromonosporaceae, also classified as belonging to actinomycetes have the above-mentioned function of transformation to perform the present invention.

Thus, the present invention relates to the following items(1)-(3).

(1) a method of obtaining a macrolide compound 11107D represented by the formula (II):

http://www.ddbj.nig.ac.jp/http://www.ddbj.nig.ac.jp/http://www.ddbj.nig.ac.jp/

where macrolide compound 11107D derived from macrolide compounds V represented by the formula (I):

by way of biological transformation, which involves the following processes (a) and (b):

(A) incubation process macrolide compounds V represented by the formula (I), in the presence of strain, having the ability to conduct the above mentioned biological transformations and belonging to the genus Mortierella, the genus Streptomyces or the family Micromonosporaceae, or its receipt of cultured mycelium; and

(C) the process of gathering the macrolide compound 11107D represented by the formula (II), from inkubiruemykh solution obtained in stage (A).

(2) the Way of getting the above (1), where the strain belonging the m to the genus Mortierella, is the strain of Mortierella sp. F-1529 (FERM BP-8547) or strain F-1530 (FERM BP-8548).

(3) the Way of getting the above (1), where the strain belonging to the genus Streptomyces, is a strain of Streptomyces sp. AB-1704 (FERM BP-8551), strain a-1544 (FERM BP-8446) or strain a-1545 (FERM BP-8447).

(4) the Method of obtaining the above (1), where the strain belonging to the family Micromonosporaceae, is the strain AB-1896 (FERM BP-8550).

(5) the Strain Streptomyces sp. AB-1704 (FERM BP-8551), having the ability to convert macrolide compounds V represented by the formula (I), macrolide compound 11107D represented by the formula (II).

(6) the Strain Mortierella sp. F-1529 (FERM BP-8547) or strain F-1530 (FERM BP-8548), having the ability to convert macrolide compounds V represented by the formula (I), macrolide compound 11107D represented by the formula (II).

(7) the Strain AB-1896 (FERM BP-8550)with the ability to convert macrolide compounds V represented by the formula (I), macrolide compound 11107D represented by the formula (II).

Detailed description of the invention

In the way that biological transformation of this invention, any microorganism belonging to the genus Mortierella, the genus Strepromyces or the family Micromonosporaceae, can be used regardless of the type species or strain, while he has the ability to turn macrolide compound B represented by the above formula (I), macrolide compound 11107D represented by the above formula (II). However, preferred examples of the microorganism can include a strain of Mortierella sp. F-1529 and strain F-1530 belonging to the genus Mortierella, the strain Streptomyces sp. AB-1704, strain a-1544 and strain a-1545 belonging to the genus Streptomyces, and strain AB-1896, belonging to the family Micromonosporaceae, each of which was isolated from soil.

Strain Mer-11107 was deposited as FERM P-18144 in National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology (1-3, Higadhi 1-chome Tsukuba-shi, Ibaraki-ken 305-8566 Japan) on 19 December 2000 and then converted as depositor FERM BP-7812 in international depositories for the purposes of patenting (International Patent Organism Depositary (IPOD) National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken 305-8566 Japan)) on November 27, 2001.

Strain Mortierella sp. F-1529 was deposited at the international level at the International Patent Organism Depositary National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken 305-8566 Japan) on 12 November 2003 as FERM BP-8547. Strain Mortierella sp. F-1530 was also deposited at the international level at the International Patent Organism Depositary National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken 305-8566 Japan) on 12 November 2003 as FERM BP-8548.

The strain Streptomyces sp. AB-1704 was deposited in International Patent Organism Depositary National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken 305-8566 Japan) as FERM P-18999 5 September 2002 and then transferred to International Deposit FERM BP-8551 12 November 2003, in International Patent OrganismDepositary (IPOD) National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken 305-8566 Japan). Strain a-1544 and strain a-1545 were also deposited in International Patent Organism Depositary (IPOD) National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken 305-8566 Japan) as FERM P-18943 and FERM P18944 23 July 2002 and then transferred to International Deposit FERM BP-8446 and FERM BP-8447 July 30, 2003, respectively, in International Patent Organism Depositary (IPOD) National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken 305-8566 Japan).

Strain AB-1896, belonging to the family Micromonosporaceae, was deposited at the international level at the International Patent Organism Depositary National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken 305-8566 Japan) as FERM-BP 8550 12 November 2003.

The taxonomic properties of the above strains are as follows.

(Taxonomical properties of the strain F-1529)

(1) Morphological characteristics

On each Cup with the agar on the decoction of oat flour (hereinafter referred to as OA depending on circumstances), malt agar (2% of malt extract + 1.5% agar: hereinafter referred to as MEA depending on circumstances) and potato agar with dextrose (hereinafter referred to as PDA, depending on the circumstances of this colony had hopevideo.com shape and color of hyphae was white, showing white shade (1A-1). As for growth, when this strain was cultured at 25°C for one week, these colonies had reached 75 mm, d is ametre on OA-Cup, 75-80 mm in diameter on the MEA Cup and 75-80 mm in diameter on PDA-Cup. The colony had bordered form. Did not observe any staining the back side, no formation of soluble pigment. The color tone has been described in accordance with the "Methuen Handbook of Colour (Kornerup &Wanscher, 1978)".

The result of observation under an optical microscope vegetative hyphae were hyaline, had a smooth surface, had no walls, and had a width of 4-5 microns. Swollen a structure similar to that of spores with thick wall having a spherical shape and approximate size of 26.5-33 μm, was observed in the hyphae. In culture medium for exposure to this test, any structure that seemed to have the sexual organ, were not formed even under cultivation during the period not exceeding 3 weeks.

(2) Analysis of gene 18S rRNA

Mycelium of the strain F-1529, cultivated on the Cup with agar, were subjected to DNA extraction using a set of Fast Prep FP120 (manufactured by Q-Bio gene) and a set of Fast DNA (manufactured by Q-Bio gene). PCR was performed using pellet puRetaq Ready-To-Go (manufactured by Amersham Biosciences) and PCR primers NS1 and NS8 shown in tables 1 and 2. PCR products were purified using the kit QIAquick PCR Purification Kit (manufactured QIAGEN) and then treated with a set ABI Prism BigDye Terminator Kit (manufactured Applied Biosystems). As Sequeira primers used NS1, NS2,NS3, NS4, NS5, NS6, NS7 and NS8 shown in tables 1 and 2. The reaction products were purified using a set of Dye EX 2.0 Spin Kit (manufactured QIAGEN) and subjected to sequencing analysis using the analyzer ABI PRISM 3100 Genetic Analyzer (manufactured Applied Biosystems). Then sequenced fragments were joined together using Auto Assembler (manufactured Applied Biosystems) to obtain the full nucleotide sequence.

Table 1< / br>
The primers used to determine the nucleotide sequence of the gene 18S rRNA (forward direction)
The sequence numberNameSequence
1NS15'-gtagtcatatgcttgtct-3'
2NS35'-gcaagtctggtgccagcagcc-3'
3NS55'-aacttaaaggaattgacggaag-3'
4NS75'-gaggcaataacaggtctgtgatg-3'

Table 2< / br>
The primers used to determine the nucleotide sequence of the gene 18S rRNA (reverse direction)
The sequence numberNameThe sequence
5NS25'-cgttcagaccacggtcgtcgg-3'
6NS45'-ttgaatttccttaactgccttc-3'
7NS65'-ctccgttattgtccagacactac-3'
8NS85'-aggcatccacttggacgcct-3'

Thus obtained gene RNA 18S this strain has the nucleotide sequence described in Sequence No. 9 (SEQ ID NO:9).

The DNA sequence is known strain was obtained from a DNA Database of Japan (http://www.ddbj.nig.ac.jp./) to test the homology of genes 18S rRNA. As a result, this gene 18S rRNA was 99% (803 grounds against the course of transcription) homology with the gene 18s rRNA Mortierella hyalina (GenBank access number AY157493), 98% (full sequence) homology with the gene 18S rRNA Mortierella chlamydospora (GenBank access number AF157143) and 98% (full sequence) homology with the gene 18S rRNA Mortierella multidivaricata (GenBank access number AF157144).

From the above mycological properties of the authors of this invention have determined that this strain belongs to the genus Mortierella.

Taxonomical properties of the strain F-1530

(1) Morphological characteristics

On each Cup with the agar on the decoction of oat flour, malt agar and potato agar with dextrose this colony had cotton (fluffy) the form and color of hyphae was white, showing white Otten is to (1A-1). As for growth, when this strain was cultured at 25°C for one week, colonies of this strain was reached 80-85 mm in diameter on OA-Cup, 85 mm in diameter on the MEA Cup and 85 mm in diameter on PDA-Cup. The colony had bordered form. Did not observe any staining the back side, no formation of soluble pigment. The color tone has been described in accordance with the "Methuen Handbook of Colour (Kornerup &Wanscher, 1978)".

The result of observation under an optical microscope vegetative hyphae were hyaline, had a smooth surface, had no walls, and had a width of 2.5 to 5 μm. Swollen a structure similar to that of spores with thick wall having a spherical shape and a size of approximately 10 μm were observed in the hyphae. In culture medium for exposure to this test any structure that seemed to have the sexual organ, were not formed even under cultivation during the period not exceeding 3 weeks.

(2) Analysis of gene 18S rRNA

Gene 18S rRNA F-1530 analyzed in the same manner as in the case of strain F1529.

Thus obtained gene 18S rRNA of strain F-1530 had the nucleotide sequence described in Sequence No. 10 (SEQ ID NO:10).

The DNA sequence is known strain was obtained from a DNA Database of Japan (http://www.ddbj.nig.ac.jp./) to test the homology of genes 18S rRNA. As a result, this gene 18S rRNA was 100% (one progress against transcription) homology with the gene 18s rRNA Mortierella hyalina (GenBank the access number AY157493), 98% (full sequence) homology with the gene 18S rRNA Mortierella chlamydospora (GenBank access number AF157143) and 98% (full sequence) homology with the gene 18S rRNA Mortierella multidivaricata (GenBank access number AF157144).

From the above microbial characteristics, the authors of this invention have determined that this strain belongs to the genus Mortierella.

(Taxonomical properties of the strain AB-1704)

(1) Morphological characteristics

In this strain of vegetative hyphae stretched aerial hyphae type located right vertical rows of plastic hyphae. At the end of these Mature aerial hyphae were formed chains of spores. The size of these spores was approximately 0.6-0.8 x 1.0 - 1.1 µm, the surface of the spores was smooth and special bodies, such as the sporangium, sclerotium and flagella were observed.

(2) Cultural characteristics on various media

Cultural characteristics of this strain after incubation at 28°within two weeks on various media are shown in table 3. The color tone is described using names and color codes are shown in parentheses Tresner''s Color wheels.

Table 3
WednesdayGrowthAerial hyphaeThe color of the vegetative hyphaeactuarily pigment
Yeast extract - malt agar (ISP-2)GoodThick Ivory (2db)Clearly yellowish brown (4gc)No
Agar broth oat flour (ISP-3)GoodRich Ivory (2db)Color light yellow melon (EA)No
Inorganic salts-starch agar (ISP-4)GoodThick Putty - ivory (1 1/2 EU-2db)The color of the bark of the cork oak (4ie)No
Glycerol-asparagine agar (ISP-5)GoodThick Color of parchment (1 1/2 db)Clearly yellowish brown (4gc)No
Peptone-yeast extract-iron agar (ISP-6)GoodAbundant White (a)Color light yellow melon (EA)No
Tyrosinaemia agar (ISP-7)GoodThick Ivory (2db)Clearly yellowish brown (4gc)No

(3) the Use of different carbon sources

Various carbon sources were added to the agar Pridham-Gottlieb and cultivated at 28°within 2 weeks. The growth of this the strain shown in table 4.

Table 4
D-glucose+Inositol-
L-arabinose±L-rhamnose+
D-xylose+D-mannitol+
D-fructose+Raffinose-
Sucrose±

(+: positive, ±: weakly positive; -: negative)

(4) Different physiological properties

Different physiological properties of this strain are as follows.

(a) the temperature Range of growth (yeast extract-malt agar, incubation for 2 weeks) 5°-33°

(b) the Range of optimum temperature of growth (yeast extract-malt agar, incubation for 2 weeks) 15°-33°

(C) Liquefaction of gelatin (glucosamina medium with gelatin) positive

(d) the Coagulation of milk (medium with skim milk) positive

(e) Peptonize milk environment (with skim milk) positive

(f) Hydrolysis of starch (inorganic salts-starch-containing agar) positive

(g) Education melanoides pigment (peptone-dragieva the extract-iron agar) negative

(h) Production of hydrogen sulfide (peptone-yeast extract-iron agar) negative

(i) reduction of nitrate (broth containing 0.1% potassium nitrate) positive

(j) Resistance to sodium chloride (yeast extract-malt agar, incubation for 2 weeks) - grows in the salt content of 7% or less

(5) Chemotaxonomy

LL-diaminopimelic acid detected from the cell wall of this strain.

Of the above microbial characteristics, the authors of this invention have determined that this strain belongs to the genus Streptomyces.

(Taxonomical properties of the strain A-1544)

(1) Morphological characteristics

Aerial hyphae spiral type stretched from vegetative hyphae in this strain. Chains of spores, consisting of approximately 10-20 cylindrical spores, formed on the end of the Mature aerial hyphae. The size of these spores was approximately 1.0×1,2-1,4 μm, the surface of the spores was barbed, and specific organs, such as the sporangium, sclerotium and flagella were observed.

(2) Cultural characteristics on various media

Cultural characteristics of this strain after incubation at 28°within two weeks on various media are shown in table 5. The color tone is described using names and color codes are shown in parentheses Tresner''s Color wheels./p>

Table 5
WednesdayGrowthAerial hyphaeThe color of the vegetative hyphaeSoluble pigment
Yeast extract - malt agar (ISP-2)GoodThick Silver-grey (32fe)Color light yellow melon (EA)No
Agar broth oat flour (ISP-3)GoodAbundant Light-gray-silver-grey (d-3fe)Color light yellow melon (EA)No
Inorganic salts-starch agar (ISP-4)GoodRich Silver gray (3fe)Color light yellow melon (EA)No
Glycerol-asparagine agar (ISP-5)GoodAbundant Color of ashes-(5fe)Color light yellow melon (EA)No
Peptone-yeast extract-iron agar (ISP-6)GoodNoColor light yellow melon (EA)Pale blackish brown
Tyrosinaemia agar (ISP-7)GoodAbundant Hidden-gray (2fe)Color light yellow is Oh melons (EA) No

(3) the Use of different carbon sources

Various carbon sources were added to the agar Pridham-Gottlieb and cultivated at 28°within 2 weeks. Growth of this strain are shown in table 6.

Table 6
D-glucose+Inositol-
L-arabinose+L-rhamnose+
D-xylose+D-mannitol+
D-fructose+raffinose-
Sucrose-

(+: positive, -: negative)

(4) Different physiological properties

Different physiological properties of this strain are as follows.

(a) the temperature Range of growth (yeast extract-malt agar, incubation for 2 weeks) 15°-41°

(b) the Range of optimum temperature of growth (yeast extract-malt agar, incubation for 2 weeks) 20°-37°

(C) Liquefaction of gelatin (glucosamina medium with gelatin) positive

(d) the Coagulation of milk (medium with skim milk) positive

(e) Peptonize the Oia milk environment (with skim milk) positive

(f) Hydrolysis of starch (inorganic salts-starch-containing agar) positive

(g) Education melanoides pigment (peptone-yeast extract-iron agar) positive (tyrosine agar) negative

(h) Production of hydrogen sulfide (peptone-yeast extract-iron agar) positive

(i) reduction of nitrate (broth containing 0.1% potassium nitrate) negative

(j) Resistance to sodium chloride (yeast extract-malt agar, incubation for 2 weeks) - grows in the salt content of 7% or less

(5) Chemotaxonomy

LL-diaminopimelic acid detected from the cell wall of this strain.

Of the above microbial characteristics, the authors of this invention have determined that this strain belongs to the genus Streptomyces.

(Taxonomical properties of the strain A-1545)

(1) Morphological characteristics

In this strain of vegetative hyphae stretched aerial hyphae type located right vertical rows of plastic hyphae. At the end of these Mature aerial hyphae were formed chains of spores from approximately 50 spores. The size of these spores was approximately 0.8×and 1.0 μm, the surface of the spores was smooth, and specific organs, such as the sporangium, sclerotium and flagella were observed.

(2) Cultural characteristics different from the meals

Cultural characteristics of this strain after incubation at 28°within two weeks on various media are shown in table 7. The color tone is described using names and color codes are shown in parentheses Tresner''s Color wheels.

Table 7
WednesdayGrowthAerial hyphaeThe color of the vegetative hyphaeSoluble pigment
Yeast extract - malt agar (ISP-2)GoodThick Grayish-yellowish-pink (5cb)Color light yellow melon - clearly yellowish brown (EA-4gc)No
Agar broth oat flour (ISP-3)ModerateThin Grayish-yellowish-pink (5cb)Pearl pink (3ca)No
Inorganic salts-starch agar (ISP-4)GoodThin Grayish-yellowish-pink (5cb)Color light ivory (sa)No
Glycerol-asparagine agar (ISP-5)GoodAbundant Grayish-yellowish-pink (5cb)Pearl pink (sa)No
Peptone draw gavoi-extract-iron agar (ISP-6) ModerateNoColor light yellow melon (EA)No
Tyrosinaemia agar (ISP-7)GoodAbundant Grayish-yellowish-pink (5cb)Color light yellow melon (EA)No

(3) the Use of different carbon sources

Various carbon sources were added to the agar Pridham-Gottlieb and cultivated at 28°within 2 weeks. Growth of this strain are shown in table 8.

Table 8
D-glucose+Inositol±
L-arabinose+L-rhamnose+
D-xylose+D-mannitol+
D-fructose+Raffinose+
Sucrose-

(+: positive, ±: weakly positive; -: negative)

(4) Different physiological properties

Different physiological properties of this strain are as follows.

(a) the temperature Range of growth (yeast extract-malt agar, incubation for 2 weeks) 10°S° With

(b) the Range of optimum temperature of growth (yeast extract-malt agar, incubation for 2 weeks) 20°-33°

(C) Liquefaction of gelatin (glucosamina medium with gelatin) negative

(d) the Coagulation of milk (medium with skim milk) positive

(e) Peptonize milk environment (with skim milk) positive

(f) Hydrolysis of starch (inorganic salts-starch-containing agar) positive

(g) Education melanoides pigment (peptone-yeast extract-iron agar) negative (tyrosinaemia agar) negative

(h) Production of hydrogen sulfide (peptone-yeast extract-iron agar) positive

(i) reduction of nitrate (broth containing 0.1% potassium nitrate) negative

(j) Resistance to sodium chloride (yeast extract-malt agar, incubation for 2 weeks) - grows in the salt content of 7% or less

(5) Chemotaxonomy

LL-diaminopimelic acid detected from the cell wall of this strain.

Of the above microbial characteristics, the authors of this invention have determined that this strain belongs to the genus Streptomyces.

(Taxonomical properties of the strain AB-1896)

(1) Morphological characteristics

Strain AB-1896 showed a good or moderate growth on the cult of the social environments used to identify this strain, at 28°C for 7-14 days. Hyphae (air) were not observed during cultivation and on each of the vegetative hyphae observed one dispute. Specific organs, such as the sporangium, sclerotium and flagella were observed.

(2) Cultural characteristics on various media

Cultural characteristics of this strain after incubation at 28°within two weeks on various media are shown in table 9. The color tone is described using names and color codes are shown in parentheses Tresner''s Color wheels.

Table 9
WednesdayGrowthThe color of the vegetative hyphaeSoluble pigment
Yeast extract-malt extract agar (ISP-2)Good Berver (3li)Tomato (3ec)No
Agar broth oat flour (ISP-3)Moderate Color of the bark of the cork oak (4ie)Color light yellow melon (EA)No
Inorganic salts-starch agar (ISP-4)Good Berver (3li)Color light yellow melon (EA)No
Glycerol-asparagine agar (ISP-5)Moderate the Light-olive-dull-brown (1li) Pearl pink (sa)No
Peptone-yeast extract-iron-containing agar (ISP-6)Good Berver (3li)Color light yellow melon (EA)No
Tyrosinaemia agar (ISP-7)Moderate Light - olive-gray (1 1/2ge)Pearl pink (sa)No

(3) the Use of different carbon sources

Various carbon sources were added to the agar Pridham-Gottlieb and cultivated at 28°within 2 weeks. Growth of this strain are shown in table 10.

Table 10
D-glucose+Inositol-
L-arabinose+L-rhamnose-
D-xylose+D-mannitol-
D-fructose+Raffinose+
Sucrose+

(+: positive, -: negative)

(4) Different physiological properties

Different physiological properties of this strain are as follows.

(a) the temperature Range of growth (yeast ek is a path-malt agar, incubation for 2 weeks) 20°-41°

(b) the Range of optimum temperature of growth (yeast extract-malt agar, incubation for 2 weeks) 25°-37°

(C) Liquefaction of gelatin (glucosamina medium with gelatin) negative

(d) the Coagulation of milk (medium with skim milk) negative

(e) Peptonize milk environment (with skim milk) positive

(f) Hydrolysis of starch (inorganic salts-starch-containing agar) positive

(g) Education melanoides pigment (peptone-yeast extract-iron agar) negative (tyrosinaemia agar) negative

(h) Production of hydrogen sulfide (peptone-yeast extract-iron agar) negative

(i) reduction of nitrate (broth containing 0.1% potassium nitrate) positive

(j) Resistance to sodium chloride (yeast extract-malt agar, incubation for 2 weeks) - grows in the salt content of 4%

(5) Chemotaxonomy

Diaminopimelic acid of mesotype detected from the cell wall of strain AB-1896. As the main structural sugars whole mycelium were detected xylose and mannose. Type acyl in the peptidoglycan of the cell wall was the type glycolyl. As the main menkenovich components were detected MK-9 (H4), MK-9 (H 6), MK-10 (H4) and MK-10 (H6).

(6) Analysis of the gene 16S rRNA

The culture broth of strain AB-1896 collected and then subjected to DNA extraction using a set of Fast DNA (manufactured by Q-Bio gene). PCR was performed under the reaction conditions 96°C/20 seconds, 50°C/20 seconds, and 72°/one minute 30 cycles in General. As primers used 9F (5'-GTGTTTGATCCTGGCTCAG-3') (sequence No. 11) and 536R (5'-GTATTACCGCGGCTGCTG-3') (sequence No. 12). The PCR product was purified using a kit MinElute PCR Purification Kit (manufactured QIAGEN) to obtain samples for sequencing.

Sequencing was performed using the analyzer ABI PRISM 310 Genetic Analyzer (manufactured Applied Biosystems) and set BigDye Terminator Kit in accordance with their standard protocols. As primers used 9F and 536R.

Thus obtained nucleotide sequence of about 500 nucleotides on the 5'-terminal side of the gene 16S rRNA of this strain is described in sequence No. 13.

DNA sequences of known strains were obtained from the DNA Database of Japan (http://www.ddbj.nig.ac.jp./) for testing homology 400-500 bases on the 5'-terminal side of the gene 16S rRNA. As a result, this gene 16S rRNA had 98% homology with the gene 16s rRNA Micromonospora sp. DSM44396 (GenBank access number AJ560637), 98% homology with the genome of Micromonospora purpureochromogenes (GenBank access number H) and 95% homology with the genome of M. ChalceaIFO12135 (GenBank the access number D85489), which is a type strain of the genus Micromonospora and 95% homology with the gene Verrucosispora gifhornensis (GenBank access number Y15523), which is a type strain of the genus Verrucosispora.

Although the strain AB-1896 had almost the same characteristics with characteristics of the genus Micromonospora, it did not correspond exactly to the genus Micromonospora in the sense that arabinose was not detectable, and mannose were detected as the main structural sugar just mycelium. Strain AB-1896 also did not correspond exactly to the genus Verrucosispora in the sense that disputes in specific Verrucosispora gifhornensis were observed on the medium used for the identification of this strain. The authors of this invention have determined that the strain AB-1896 is actinomycetes belonging to Micromonosporaceae, taking into account the above taxonomical properties.

In accordance with this invention, the first process (A) macrolide compound W, which is the starting material (substrate), incubated in the presence of the above strains or products obtained with the use of their cultivated mycelium, and optionally in the presence of oxygen. This processing may be carried out by addition of the substrate in the culture broth with the cultivation of the above strains in aerobic conditions or, depending on circumstances, the addition of this substrate fact the český in a suspension solution of cultured mycelium of the above strains or product obtained by homogenization of the cells, noise gas containing oxygen, for example air.

This substrate can be added to the culture broth before cultivation after passage of a fixed time after the start of cultivation. The above mycelium can be obtained by the inoculation of any of the above strains in a medium containing nutrients and cultivation under aerobic conditions. The cultivation of this strain to obtain drugs or cultivation of mycelium of this strain, which is carried out in situations when the substrate is added, can be carried out in accordance with the fundamental conventional method of cultivation of microorganisms. However, this is usually the cultivation is conducted preferably under aerobic conditions using, for example, culture in shake flasks and tank culture in accordance with the culture in liquid medium.

As a medium used for culturing may be used in any environment, provided that it contains nutrients that microorganisms belonging to the genus Mortierella, the genus Streptomyces or the family Micromonosporaceae, can use, and can be used in a variety of synthetic environment synthetic environment and the natural environment. As for the composition of the medium, can be used in owani different carbon sources, such as glucose, galactose, sucrose, maltose, fructose, glycerin, dextrin, starch, molasses and soybean oil, either independently or in combination.

As for the nitrogen source, there can be used a sole source of nitrogen or a combination of organic nitrogen sources, such as pharmaceutical environment, peptone, meat extract, soybean meal, fish meal, flour from gluten, casein, yeast, amino acid, yeast extract, NZ-casein and urea, and inorganic nitrogen sources such as sodium nitrate and ammonium sulfate. In addition, can be added and used, for example, salts such as sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, sodium phosphate, potassium phosphate, copper sulphate, ferrous sulphate, manganese chloride or cobalt chloride; salts of heavy metals; vitamins, such as vitamin b or Biotin; and agents include, such as cyclodextrins, if necessary. Further, when an appreciable foaming during cultivation in the environment can be added appropriately protivovspenivayushchie agents as needed. Adding protivovspenivayushchie agent should be added in a concentration of not having a harmful effect on the receipt of a target substance.

The cultivation conditions can be appropriately selected within that specific microbe is the first strain grows well and can produce the above-mentioned substance. For example, a pH of approximately 5-9 and preferably close to neutral pH. The temperature of fermentation usually support at 20-40°and preferably 24-30°C. the Period of fermentation is approximately 1-8 days and usually about 2-5 days. The above-mentioned fermentation conditions can appropriately be changed depending on the type and properties of the microorganism used, environmental conditions, etc. and there is no need to talk about that can be selected optimal conditions.

The drug culture of the mycelium also receive suspendirovanie mycelium is separated by centrifugation or filtration or homogenized in a suitable solution, after completion of the cultivation. Examples of the solution used for the suspension of the mycelium, include the above-mentioned medium or buffer solutions such as Tris-acetic acid, Tris-hydrochloric acid, sodium succinate, sodium citrate, sodium phosphate and potassium phosphate, either independently or in combinations. pH buffer solution of 5.0-9.0 and preferably 6,0-7,5.

Substance W as a substrate can be added to the culture broth or the suspension solution of the mycelium or in the form of powder as such or in the form of a solution dissolved in a water-soluble solvent, for example ethanol, methanol, acetone or is metilsulfate. The number of added substances W is preferably 50-5000 mg per 1 l of the culture broth in the case of the culture broth. After addition of the substrate carried out such procedures as the shaking flasks or cultivation in the tank culture at 20-40°for about 1-5 days to conduct the reaction under aerobic conditions, whereby the substance W as the substrate becomes the 11107D substance.

Then, in process (C) target 11107D substance extracted from inkubiruemykh solution obtained in the process (A). Selects appropriate methods from a wide variety of known purification methods which are usually used for the excretion of metabolites of microorganisms, and use them to highlight the 11107D substance from the reaction mixture in the process (A). For example, extraction with an organic solvent, such as methanol, ethanol, butanol, acetone, ethyl acetate, butyl acetate, chloroform or toluene; various types of ion-exchange chromatography; gel filtration chromatography using Sephadex LH-20; treatment of the adsorption and desorption absorption chromatography using a hydrophobic adsorbent resin such as Diaion HP-20, active carbon or silica gel, or thin layer chromatography; or high performance liquid chromatography using a column with reversed phase, etc. may be the used independently or in combination, or used again, whereby can be isolated and purified 11107D substance.

Examples

The invention will be explained in more detail using examples that are not intended to limit the scope of the present invention. In the following examples, all designations % are masses. percent (wt.%), if there are no other indications.

Referential example 1. Obtaining substances V as source material

One loop of culture on the sloped agar (medium ISP-2) strain Streptomyces sp. Mer-11107 (FERM BP-7812) was inoculable in an Erlenmeyer flask of 500 ml containing 50 ml of seed medium (2.0% glucose, 1.0% of soybean meal (ESUSAN-MEAT manufactured Ajinomoto Co. Ltd.), 0.5% of yeast extract (manufactured Oriental Yeast Co., Ltd.), 0.25% sodium chloride, 0.32 per cent calcium carbonate, pH 6.8 before sterilization), and cultivated at 28°within two days from the receipt of the first seed culture broth. 0.1 ml of this culture broth was inoculable in an Erlenmeyer flask of 500 ml containing 100 ml of the same seed medium and cultured at 28°C for one day to obtain a second seed culture broth. This second seed culture broth (800 ml)thus obtained was inoculable in the tank 200 liters, containing 100 l production nutrient medium (5% soluble starch, 0.8% of the pharmaceutical environment, 0.8% of the flour is rakoviny, 0.5% of yeast extract and 0.1% calcium carbonate, pH 6.8 before sterilization) and were cultured for five days under the following conditions, to obtain the culture broth.

The temperature of cultivation: 28aboutWith

Mixing: 90 rpm

Internal pressure: 20 kPa

Part of the culture broth (10 l), thus obtained, was extracted with 10 l of 1-butanol and then received butanolic layer was evaporated to dryness to obtain 100 g of the crude active fraction. This crude active fraction was applied on a Sephadex LH-20 (1500 ml; manufactured Pharmacia, Co. Ltd.) and suirable a mixture of tetrahydrofuran-methanol (1:1) as solvent. Elyuirovaniya fraction from 540 to 660 ml was concentrated to dryness to obtain a residue (660 mg). The obtained residue was dissolved in a mixture of ethyl acetate and methanol (9:1, vol/about.) and subjected to column chromatography on silica gel (WAKO GEL C-200, 50 g). The column was suirable mixture (2 l), consisting of n-hexane and ethyl acetate (1:9, vol/vol.), faction, erwerbende from 468 to 1260 ml was collected and evaporated to obtain 25 mg of the crude active fraction.

The crude active fraction was subjected to preparative high performance liquid chromatography (HPLC) under the following conditions preparative HPLC and fractions, erwerbende at retention time of 34 minutes, was collected. After removal of the acetone is trila appropriate fractions were absoluely using HPLC under the following conditions preparative HPLC (C) obtaining V (retention Time: 37 minutes 6 mg).

Conditions preparative HPLC As:

Column: YMC-PACK ODS-AM SH-343-5AM ϕ20 mm × 250 mm (manufactured YMC Co.)

Temperature: room temperature

The flow rate: 10 ml/min

Detection: 240 nm

Eluent: acetonitrile/0.15% aqueous potassium dihydrophosphate (pH 3.5) (2:8-8:2, vol/about., 0-50 min, linear gradient).

Conditions preparative HPLC In:

Column: YMC-PACK ODS-AM SH-343-5AM ϕ20 mm × 250 mm (manufactured YMC Co.)

Temperature: room temperature

The flow rate: 10 ml/min

Detection: 240 nm

Eluent: methanol/water (2:8-10:0, from./about., 0-40 min, linear gradient).

Example 1. Selection of strain AB-1704

One loop of culture on the sloped agar (0.5% of soluble starch, 0.5% of glucose, 0.1% fish meat extract (manufactured WAKO Pure Chemical Industries, Ltd.), 0.1% of yeast extract (manufactured Oriental Yeast Co., Ltd.), 0,2% NZ-casein (manufactured Humko Sheffield Chemical Co.), 0.2% sodium chloride, 0.1% calcium carbonate, and 1.6% agar (manufactured by WAKO Pure Chemical Industries, Ltd.)) strain, isolated from soil, was inoculable in the test tube 65 ml, containing 7 ml of seed medium (a 2.0% soluble starch, 1.0% glucose, 0.5% polypath (manufactured by Nihon Pharmaceutical Co., Ltd.), 0.5% of yeast extract (manufactured Oriental Yeast Co., Ltd.) and 0.1% calcium carbonate) and cultivated at 28°C for three days on a rotary shaker to obtain a seed culture broth.

The ZAT is 0.5 ml of this seed culture of peptone was inoculable in the test tube 65 ml, containing 7 ml of a production culture medium (2.0% of soluble starch, 1.0% glucose, 0.5% polypath (manufactured by Nihon Pharmaceutical Co., Ltd.), 0.5% of yeast extract (manufactured Oriental Yeast Co., Ltd.) and 0.1% calcium carbonate) and cultivated at 28°C for three days on a rotary shaker. Then prepare a solution of 25 mg/ml substrate substance W in ethanol and 0.2 ml of this solution was added to the culture. After the addition was shaken at 28°C for 48 hours for the reaction conversion. After the reaction, the reaction mixture was analyzed by using HPLC in the following conditions (a) analytical HPLC with obtaining strain AB-1704 (FERM BP-8551), which formed the 11107D substance in this reaction mixture.

Conditions analytical HPLC (a)

Column: CAPCELL PAK C18 SG120 ϕ4.6 mm × 250 mm (manufactured SHISEIDO CO.)

Temperature: 40°

The flow speed: 1 ml/min

Detection: 240 nm

Eluent: acetonitrile/0.15% aqueous potassium dihydrophosphate (pH 3.5) (3:7-5:5, vol/about., 0-18 min, linear gradient), acetonitrile/0.15% of potassium dihydrophosphate (pH 3.5) (5:5-85:15, vol/about., 18-22 minutes, linear °)

Retention time: the 11107D substance of 9.9 min, substance W of 19.4 minutes

Example 2. Selection of strain A-1544 and strain A-1545

One loop of culture on the sloped agar (yeast-malt agar) strain isolated from soil, was inoculable in flask Arlem the hyères 250 ml, containing 20 ml of seed medium (2.4% of soluble starch, 0.1% glucose, 0.5% of soybean meal (ESUSAN-MEAT manufactured Ajinomoto Co. Ltd.), 0.3% of meat extract (manufactured Difco), 0.5% of yeast extract (manufactured Difco), 0.5% tripton-peptone (manufactured Difco) and 0.4% calcium carbonate), and cultivated at 28°C for three days in a rotary shaker to obtain a seed culture broth.

Then 0.6 ml of this seed culture broth was inoculable in an Erlenmeyer flask of 500 ml containing 60 ml of the production culture medium (2.0% of soluble starch, 2.0% glucose, 2.0% of soybean meal (ESUSAN-MEAT manufactured Ajinomoto Co. Ltd.), 0.5% of yeast extract (manufactured Oriental Yeast Co., Ltd.), 0.25% sodium chloride, 0.32 per cent calcium carbonate, of 0.0005% sulphate of copper, of 0.0005% chloride of manganese, of 0.0005% zinc sulfate, pH 7.4 before sterilization) and cultivated at 28°C for four days in a rotary shaker. 2 ml of the obtained culture was poured in test tubes 15 ml were Then prepared solution of 20 mg/ml substrate substance W in dimethyl sulfoxide and added to 0.05 ml of this solution. After adding the test tubes were shaken at 28°C for 23 hours for transformation. After the reaction, the reaction mixture was analyzed by using HPLC in the following conditions (b) analytical HPLC with obtaining strain A-1544 (FERM BP-8446) and strain A-1545 (FERM-BP-8447)that education is obyvali the 11107D substance in this reaction mixture.

Conditions analytical HPLC (b)

Column: CAPCELL PAK C18 SG120 ϕ4.6 mm × 250 mm (manufactured SHISEIDO CO.)

Temperature: 40°

The flow speed: 1 ml/min

Detection: 240 nm

Eluent: acetonitrile/water (50:50, vol/vol.). Isocratic

Retention time: the substance W 7,2 min, the 11107D substance of 3.6 minutes

Example 3. The transformation strain AB-1704 scale Kolb

One loop of culture on the sloped agar (0.5% of soluble starch, 0.5% of glucose, 0.1% fish meat extract (manufactured WAKO Pure Chemical Industries, Ltd.), 0.1% of yeast extract (manufactured Oriental Yeast Co., Ltd.), 0,2% NZ-casein (manufactured Humko Sheffield Chemical Co.), 0.2% sodium chloride, 0.1% calcium carbonate, and 1.6% agar (manufactured by WAKO Pure Chemical Industries, Ltd.)) strain Streptomyces sp. AB-1704, isolated from soil, was inoculable in the Erlenmeyer flask containing 100 ml of seed medium (a 2.0% soluble starch, 1.0% glucose, 0.5% polypath (manufactured by Nihon Pharmaceutical Co., Ltd.), 0.5% of yeast extract (manufactured Oriental Yeast Co., Ltd.) and 0.1% calcium carbonate) and cultivated at 28°C for three days on a rotary shaker to obtain a seed culture broth. Then 2 ml of this seed culture broth was inoculable in each of the 150 Erlenmeyer flasks having a capacity of 500 ml containing 100 ml of production nutrient medium (a 2.0% soluble starch, 1.0% glucose, 0.5% polypath (manufactured by Nihon Pharmacetical Co., Ltd.), 0.5% of yeast extract (manufactured Oriental Yeast Co., Ltd.) and 0.1% calcium carbonate), and cultivated at 28°C for two days on a rotary shaker.

Preparing a solution of 20 mg/ml substrate substance W in ethanol and of 0.44 ml of this solution was added to the obtained culture (100 ml/500 ml-Erlenmeyer flask, 150 flasks). After adding the flasks were shaken at 28°C for 9 hours for the reaction conversion. After completion of the reaction these cultures were collected and divided into the culture supernatant and mycelia by centrifugation at 2700 rpm for 10 minutes. The mycelium was extracted with 5 l of methanol and filtered to obtain a solution of the methanol extract. This methanol extract was evaporated to remove methanol, combined with the culture supernatant and was extracted with 10 l of ethyl acetate. Received an ethyl acetate solution was evaporated to obtain 2090 mg of the crude active fraction. The crude active fraction was dissolved in 4 ml of a mixture of tetrahydrofuran-methanol (1:1, vol/about.) and 6 ml of 50% aqueous solution of acetonitrile was subjected to chromatography on an ODS column (manufactured by YMC Co., ODS-AM 120-S50 ϕ3.6 cm × 43 cm) and suirable 40% aqueous solution of acetonitrile. Elyuirovaniya fraction from 336 ml to 408 ml was concentrated to dryness under reduced pressure to obtain 560 mg of the residue. Next, the residue was dissolved in 10 ml of 50% vodno the solution of methanol, was subjected to chromatography on an ODS column (manufactured by YMC Co., ODS-AM 120-S50 ϕ3.6 cm × 40 cm) and suirable 50% aqueous solution of methanol. Elyuirovaniya fraction from 1344 ml to 1824 ml was concentrated to dryness under reduced pressure to obtain 252 mg of 11107D substance.

Example 4. The transformation strain And-1545-scale Kolb

One loop of culture on the sloped agar (yeast-malt agar) strain A-1545 (FERM BP-8447) was inoculable in the Erlenmeyer flask 250 ml, containing 25 ml of seed medium (2.0% of soluble starch, 2.0% glucose, 2.0% of soybean meal (ESUSAN-MEAT manufactured Ajinomoto Co. Ltd.), 0.5% of yeast extract (manufactured Difco), 0.25% sodium chloride and 0.32% calcium carbonate, pH 7.4 before sterilization), and cultivated at 28°C for two days on a rotary shaker to obtain a seed culture broth. 0.75 ml of this broth was poured in a test tube for serum (manufactured Sumimoto Bakelite Co., Ltd.) and added an equal amount of 40% aqueous solution of glycerol. After mixing, they were frozen at -70°obtaining frozen inoculum. Frozen seed stock was thawed, 0.25 ml of it was inoculable in the Erlenmeyer flask 250 ml, containing 25 ml of seed medium (2.0% of soluble starch, 2.0% glucose, 2.0% of soybean meal (ESUSAN-MEAT manufactured Ajinomoto Co. Ltd.), 0.5% of yeast extract (manufactured Oriental Yeast Co., Ltd.), 0.25% chloride hydroxide is I and 0.32% of calcium carbonate, pH 7.4 before sterilization) and cultivated at 28°C for two days on a rotary shaker to obtain a seed culture broth. Then this seed culture broth (0.5 ml) was inoculable in an Erlenmeyer flask of 500 ml containing 100 ml of production nutrient medium (2.0% of soluble starch, 2.0% glucose, 2.0% of soybean meal (ESUSAN-MEAT manufactured Ajinomoto Co. Ltd.), 0.5% of yeast extract (manufactured Oriental Yeast Co., Ltd.), 0.25% sodium chloride and 0.32% calcium carbonate, pH 7.4 before sterilization), and cultivated at 28°C for three days on a rotary shaker.

Each of the obtained culture broth (100 ml/500 ml-Erlenmeyer flask, 10 flasks) were subjected to centrifugation at 3000 rpm for 10 minutes to collect cells of the microorganism and these cells are suspended in 100 ml of 50 mm phosphate buffer solution (pH 6.0). Then prepare a solution of 100 mg/ml substrate substance W in dimethyl sulfoxide and added to 1 ml of this solution. After the addition was shaken at 28°within 24 hours for the reaction conversion. After completion of the reaction, this reaction solution was collected and divided into the culture supernatant and mycelia by centrifugation at 5000 rpm for 20 minutes. The supernatant was extracted with 1 l of ethyl acetate. The mycelium was extracted with 500 ml of methanol and then filtered to obtain methane is a high extract. This methanol extract was evaporated to remove methanol and extracted with 1 l of ethyl acetate. Each of an ethyl acetate layers were washed with water, dried and dehydrational over anhydrous sodium sulfate and the combined layers were evaporated to obtain 937 mg of the crude fraction. The crude fraction was subjected to column chromatography on silica gel (Russ. name gel 60, 50 g) and suirable 1200 ml of a mixture of ethyl acetate-n-hexane (90:10;./about.) to obtain 234 mg fractions containing the 11107D substance. The obtained active fraction was subjected to preparative high performance liquid chromatography (HPLC) under the following conditions (C) preparative HPLC and the resulting eluate was analyzed using HPLC under the following conditions analytical HPLC (with). The solvent was removed from the fractions containing the thus obtained 11107D substance, receiving 80 mg of 11107D substance.

Conditions preparative HPLC (C)

Column: CAPCELL PAK C18 UG120 ϕ30 mm × 250 mm (manufactured SHISEIDO CO.)

The flow rate: 20 ml/min

Detection: 240 nm

Eluent: acetonitrile/water (30:70, about./vol.). Isocratic

Conditions analytical HPLC (C)

Column: CAPCELL PAK C18 SG120 ϕ4.6 mm × 250 mm (manufactured SHISEIDO CO.)

Temperature: 40°

The flow speed: 1 ml/min

Detection: 240 nm

Eluent: acetonitrile/water (35:65, about./vol.). Isocratic

Retention time: the substance 1110D 7,8 minutes

Example 5. The transformation strain A-1544-scale Kolb

Each of the cultures of strain A-1544 (FERM BP-8446) (100 ml/500 ml-Erlenmeyer flask, 10 flasks)obtained by the method similar to the method described in example 4 was subjected to centrifugation at 3000 rpm for 10 minutes to collect cells of the microorganism and these cells are suspended in 100 ml of 50 mm phosphate buffer solution (pH 6.0). Then prepare a solution of 100 mg/ml substrate W in dimethyl sulfoxide and added to 1 ml of this solution. After the addition was shaken at 28°within 24 hours for the reaction conversion. After completion of the reaction, this reaction solution was collected and divided into the culture supernatant and mycelia by centrifugation at 5000 rpm for 20 minutes. The supernatant was extracted with 1 l of ethyl acetate. The mycelium was extracted with 500 ml of acetone and then filtered to obtain the acetone extract. This acetone extract is evaporated to remove the acetone and then the residue was extracted with 1 l of ethyl acetate. Each of an ethyl acetate layers were washed with water, dried and dehydrational over anhydrous sodium sulfate and the combined layers were evaporated to obtain 945 mg of the crude fraction. The crude fraction was subjected to column chromatography on silica gel (Russ. name gel 60, 50 g) and suirable 100 ml of a mixture of ethyl acetate-n-hexane (50:50;./vol.), 200 ml of a mixture of etelaat is-n-hexane (75:25; about./about.) and the mixture (600 ml) ethyl acetate-n-hexane (90:10;./about.) obtaining 463 mg fractions containing the 11107D substance. The obtained active fraction was subjected to preparative high performance liquid chromatography (HPLC) under the conditions of preparative HPLC (C)described in example 4, and the analytical conditions HPLC (C)described in example 4. The solvent was removed from the fractions containing the thus obtained 11107D substance, obtaining 304 mg 11107D substance.

Example 6. The selection of the strain F-1529 and strain F-1530

One loop of culture on the sloped agar (potato agar with dextrose) strain isolated from soil, was inoculable in the Erlenmeyer flask 250 ml, containing 20 ml of seed medium (2.0% of potato starch, 1.0% glucose, 2.0% of soybean meal (ESUSAN-MEAT manufactured Ajinomoto Co. Ltd.), 0.1% potassium dihydrophosphate and 0.05% heptahydrate magnesium sulfate), and were cultured at 25°C for three days on a rotary shaker to obtain a seed culture broth. Next, 0.6 ml of this seed broth was inoculable in the Erlenmeyer flask of 500 ml containing 60 ml of the production culture medium (2.0% of potato starch, 1.0% glucose, 2.0% of soybean meal (ESUSAN-MEAT manufactured Ajinomoto Co. Ltd.), 0.1% potassium dihydrophosphate and 0.05% heptahydrate magnesium sulfate) and was cultured at 28°C for four days on a rotary shaker. 2 ml of the obtained Kul the Ural broth was poured in test tubes 15 ml Each test tube was subjected to centrifugation at 3000 rpm for 5 minutes to collect the cells of the microorganism and then suspended in 2 ml of 50 mm phosphate buffer solution (pH 7.0). Then prepare a solution of 20 mg/ml substrate substance W in dimethyl sulfoxide and added to 0.05 ml of this solution. After the addition was shaken at 28°C for 23 hours for the reaction of hydroxylation. After the reaction, the reaction mixture was analyzed by HPLC under analytical conditions (C), described in example 4, to obtain the strain F-1529 (FERM BP-8547) and strain F-1530 (FERM BP-8548), both of which had a peak of 11107D substance in HPLC.

Example 7. The transformation strain F-1529-scale Kolb

One loop of culture on the sloped agar (potato agar with dextrose) strain Mortierella sp. F-1529 (FERM BP-8547) was inoculable in the Erlenmeyer flask 250 ml, containing 25 ml of seed medium (2.0% of potato starch, 1.0% glucose, 2.0% of soybean meal (ESUSAN-MEAT manufactured Ajinomoto Co. Ltd.), 0.1% potassium dihydrophosphate and 0.05% heptahydrate magnesium sulfate), and were cultured at 25°C for two days on a rotary shaker to obtain a seed culture broth. Next, 0.6 ml of this seed broth was inoculable in the Erlenmeyer flask of 500 ml containing 60 ml of the production culture medium (2.0% of potato starch, 1.0% glucose, 2.0% of soybean meal (ESUSAN-MEAT manufactured Ainomoto Co. Ltd.), 0.1% potassium dihydrophosphate and 0.05% heptahydrate magnesium sulfate) and were cultured at 25°C for three days on a rotary shaker.

Each of the obtained culture broth (60 ml/500 ml-Erlenmeyer flask, 18 flasks) were subjected to centrifugation at 3000 rpm for 5 minutes to collect the cells of the microorganism and then suspended in 60 ml of 50 mm phosphate buffer solution (pH 7.0). Then prepare a solution of 100 mg/ml substrate W in dimethyl sulfoxide and added to 0.6 ml of this solution. After the addition was shaken at 25°C for 22 hours to conduct reaction conversion. After completion of the reaction, the culture broth was separated into supernatant and mycelia by centrifugation at 5000 rpm for 20 minutes. The supernatant was extracted with 1 l of ethyl acetate. The mycelium was extracted with 500 ml of acetone and then filtered to obtain a solution of acetone extract. This solution acetone extract was evaporated to remove acetone and then was extracted with 1 l of ethyl acetate. Each of an ethyl acetate layers respectively washed with water, dehydrational and dried over anhydrous sodium sulfate and then combined together and evaporated to obtain 1,21 g of the crude fraction comprising the 11107D substance. The crude fraction comprising the 11107D substance was subjected to column chromatography on silica gel (Russ. name gel 60, 50 g) and e who were yirawala 1200 ml of a mixture of ethyl acetate-n-hexane (90:10; about./about.) obtaining 369 mg fractions containing the 11107D substance.

The obtained fraction was subjected to preparative high performance liquid chromatography (HPLC) under the conditions of preparative HPLC (C)described in example 4, to obtain elyuirovaniya fractions containing the 11107D substance. Then the solvent was removed to obtain 180 mg of the 11107D substance.

Example 8. The transformation strain F-1530 scale Kolb

One loop of culture on the sloped agar (potato agar with dextrose) strain Mortierella sp. F-1530 (FERM BP-8548) was inoculable in the Erlenmeyer flask 250 ml, containing 25 ml of seed medium (2.0% of potato starch, 1.0% glucose, 2.0% of soybean meal (ESUSAN-MEAT manufactured Ajinomoto Co. Ltd.), 0.1% potassium dihydrophosphate and 0.05% heptahydrate magnesium sulfate), and were cultured at 25°C for two days on a rotary shaker to obtain a seed culture broth. Next, 0.6 ml of this seed broth was inoculable in the Erlenmeyer flask of 500 ml containing 60 ml of the production culture medium (2.0% of potato starch, 1.0% glucose, 2.0% of soybean meal (ESUSAN-MEAT manufactured Ajinomoto Co. Ltd.), 0.1% potassium dihydrophosphate and 0.05% heptahydrate magnesium sulfate), and were cultured at 25°C for three days on a rotary shaker.

Each of the obtained culture broth (60 ml/500 ml-Erlenmeyer flask, 18 flasks) were subjected to centrifugation at 3000 rpm in those who tell 5 minutes to collect the cells of the microorganism and then suspended in 60 ml of 50 mm phosphate buffer solution (pH 7.0). Then prepare a solution of 100 mg/ml substrate W in dimethyl sulfoxide and added to 0.6 ml of this solution. After the addition was shaken at 25°C for 22 hours to conduct reaction conversion. After completion of the reaction, the culture broth was separated into supernatant and mycelia by centrifugation at 5000 rpm for 20 minutes. The supernatant was extracted with 1 l of ethyl acetate. The mycelium was extracted with 500 ml of acetone and then filtered to obtain a solution of acetone extract. This solution acetone extract was evaporated to remove acetone and then was extracted with 1 l of ethyl acetate. An ethyl acetate layers respectively washed with water, dehydrational and dried over anhydrous sodium sulfate and then combined together and evaporated to obtain 0,89 g of the crude fraction comprising the 11107D substance. The crude fraction comprising the 11107D substance was subjected to column chromatography on silica gel (Russ. name gel 60, 50 g) and suirable 1200 ml of a mixture of ethyl acetate-n-hexane (90:10;./about.) and then 500 ml of ethyl acetate to obtain 163 mg of the fraction containing the 11107D substance. The obtained fraction was subjected to preparative high performance liquid chromatography (HPLC) under the conditions of preparative HPLC (C)described in example 4, to obtain elyuirovaniya fractions containing the 11107D substance. The solvent is then removed from the receipt of the m 30 mg 11107D substance.

Example 9. Selection of strain AB-1896

One loop of culture on the sloped agar (0.5% of soluble starch, 0.5% of glucose, 0.1% fish meat extract (manufactured WAKO Pure Chemical Industries, Ltd.), 0.1% of yeast extract (manufactured Oriental Yeast Co., Ltd.), 0,2% NZ-casein (manufactured Humko Sheffield Chemical Co.), 0.2% sodium chloride, 0.1% calcium carbonate, and 1.6% agar (manufactured by WAKO Pure Chemical Industries, Ltd.)) strain, isolated from soil, was inoculable in the test tube 65 ml, containing 5 ml of seed medium (a 2.0% soluble starch, 1.0% glucose, 0.5% polypath (manufactured by Nihon Pharmaceutical Co., Ltd.), 0.5% of yeast extract (manufactured Oriental Yeast Co., Ltd.) and 0.1% calcium carbonate) and cultivated at 28°within ten days on a rotary shaker to obtain a seed culture broth. Then 0.1 ml of this seed culture broth was inoculable in the test tube 65 ml, containing 5 ml of a production culture medium (2.0% of soluble starch, 1.0% glucose, 0.5% polypath (manufactured by Nihon Pharmaceutical Co., Ltd.), 0.5% of yeast extract (manufactured Oriental Yeast Co., Ltd.) and 0.1% calcium carbonate) and cultivated at 28°C for three days on a rotary shaker. Then prepare a solution of 40 mg/ml substrate substance W in ethanol and 0.05 ml of this solution was added to the culture. After the addition was shaken at 28°within 24 hours for the reaction of the hydroxy is investing. After this reaction, the culture broth was subjected to HPLC analysis under the following analytical conditions (d) to obtain the strain AB-1896, which was formed by the 11107D substance.

Conditions analytical HPLC (d)

Column: UNISON UK-C18, ϕ4.6 mm × 50 mm (manufactured Imtakt)

Temperature: 30°

The flow rate: 2 ml/min

Detection: 240 nm

Eluent: water/acetonitrile/formic acid(1000:10:1 - 10:1000:1 about./about./about., 0-4 minutes, linear gradient)

Retention time: the 11107D substance of 2.5 minutes

Example 10. The transformation strain AB-1896-scale test-tubes

One loop of culture on the sloped agar (0.5% of soluble starch, 0.5% of glucose, 0.1% fish meat extract (manufactured WAKO Pure Chemical Industries, Ltd.), 0.1% of yeast extract (manufactured Oriental Yeast Co., Ltd.), 0,2% NZ-casein (manufactured Humko Sheffield Chemical Co.), 0.2% sodium chloride, 0.1% calcium carbonate, and 1.6% agar (manufactured by WAKO Pure Chemical Industries, Ltd.)) strain AB-1896 was inoculable in the test tube 65 ml, containing 5 ml of seed medium (a 2.0% soluble starch, 1.0% glucose, 0.5% polypath (manufactured by Nihon Pharmaceutical Co., Ltd.), 0.5% of yeast extract (manufactured Oriental Yeast Co., Ltd.) and 0.1% calcium carbonate) and cultivated at 28°within ten days on a rotary shaker to obtain a seed culture broth. Then 0.1 ml of this seed culture broth of InocuLAN is whether the test probirka 65 ml, containing 5 ml of a production culture medium (2.0% of soluble starch, 1.0% glucose, 0.5% polypath (manufactured by Nihon Pharmaceutical Co., Ltd.), 0.5% of yeast extract (manufactured Oriental Yeast Co., Ltd.) and 0.1% calcium carbonate) and cultivated at 28°C for three days on a rotary shaker.

Preparing a solution of 40 mg/ml substrate substance W in ethanol, and 0.05 ml of this solution was added to the obtained culture broth (5 ml/65 ml test tube). Once added, the test tube was shaken at 28°within 24 hours for the reaction of hydroxylation. 3 ml of the obtained culture broth was collected separately, was added 2 ml of 1-butanol was shaken and then centrifuged at 3000 rpm for 10 minutes. The obtained supernatant was removed to obtain 2 ml of methanolic solution of this residue. He was subjected to HPLC analysis under the following analytical conditions (e) and (f) to confirm that the 11107D substance formed in this reaction mixture.

Conditions analytical HPLC (e)

Column: UNISON UK-C18, ϕ4.6 mm × 50 mm (manufactured Imtakt)

Temperature: 40°

The flow rate: 2 ml/min

Detection: 240 nm

Eluent: acetonitrile/0.01% of triperoxonane acid (2:8 to 5:5.about., 0-10 minutes, linear gradient)

Retention time: the 11107D substance of 6.1 minutes

The analytical conditions In the LC (f)

Column: UNISON UK-C18, ϕ4.6 mm × 50 mm (manufactured Imtakt)

Temperature: 40°

The flow rate: 2 ml/min

Detection: 240 nm

Eluent: methanol/0.01% of triperoxonane acid (4:6 - 7:3 vol./about., 0-10 minutes, linear gradient)

Retention time: the 11107D substance of 6.1 minutes

1. A method of obtaining a macrolide compound 11107D represented by the formula (II)

where macrolide compound 11107D derived from macrolide compounds V represented by the formula (I)

by way of biological transformation, which involves the following processes (a) and (b):

(A) incubation process macrolide compounds V represented by the formula (I), in the presence of strain, having the ability to conduct the above mentioned biological transformations and belonging to the genus Mortierella, the genus Streptomyces or the family Micromonosporaceae, or preparation of its cultivated mycelium; and

(B) the process of gathering the macrolide compound 11107D represented by the formula (II), from inkubiruemykh solution obtained in stage (A).

2. The method of receiving according to claim 1, where the by Tamm, belonging to the genus Mortierella, is a strain of Mortierella sp. FERM BP-8547 or strain Mortierella sp. FERM BP-8548.

3. The method of receiving according to claim 1, where the strain belonging to the genus Streptomyces, is a strain of Streptomyces sp. FERM BP-8551, the strain Streptomyces sp. FERM BP-8446 or strain Streptomyces sp. FERM BP-8447.

4. The method of receiving according to claim 1, where the strain belonging to the family Micromonosporaceae, is the strain FERM BP-8550.

5. Strain actinomycete Streptomyces sp. FERM BP-8551, with the ability to turn macrolide compound B represented by the formula (I), macrolide compound 11107D represented by the formula (II).

6. The strain of the fungus Mortierella sp. FERM BP-8547, with the ability to turn macrolide compound B represented by the formula (I), macrolide compound 11107D represented by the formula (II).

7. The strain of the fungus Mortierella sp. FERM BP-8548 with the ability to turn macrolide compound B represented by the formula (I), macrolide compound 11107D represented by the formula (II).

8. Strain, belonging to the family Micromonosporaceae, FERM BP-8550, possessing the ability to turn macrolide compound B represented by the formula (I), macrolide compound 11107D represented by the formula (II).



 

Same patents:

FIELD: bioengineering.

SUBSTANCE: method for revealing micobacteria provides the preparation of diagnostic material. The prepared diagnostic material is seeded into the solid medium which contains the mixture of the standard meat infusion agar and vaseline oil. The micobacteria are incubated in thermostat. After the incubation, the results are recorded in 24-48-72-96 hours.

EFFECT: timelines for revealing micobacteria are reduced

1 tbl

FIELD: chemistry, organic, biotechnologies.

SUBSTANCE: invention relates to biotechnology. Comma bacillus cellular mass will be obtained, the proteins will be removed from the surface with orbital rotation of the bacterial mass with the velocity of 2500 r.p.m. during 2 minutes in the vibration mixer. The cellular mass will be fractioned with low velocity centrifuging at 3-4 thousand r.p.m. The supernatant will be fractioned with high velocity centrifuging at 18-20 thousand r.p.m. Then the sediment will be selectively extracted for obtaining supernatant containing purified proteins TCAP and OmpU with the solution 125 mM of ethanolamine (pH 10.5) at 4°C in the shaker. From the supernatant, proteins of toxin-coregulated pili adhesine and OmpU will be isolated.

EFFECT: isolation of proteins of toxin-coregulated pili adhesine and OmpU from supernatant.

3 cl, 3 dwg, 4 ex

FIELD: chemistry, organic.

SUBSTANCE: invention relates to biotechnology and may be used for production of enzyme preparations of thermostable α-amylase. The invention enables one to increase output of thermostable α-amylase.

EFFECT: increase of thermostable α-amylase output.

1 tbl, 4 ex

FIELD: production methods.

SUBSTANCE: stamp of bacterium Bacillus macerans ARC V-2419 D - producer of pectaltliase, pectilase and polygalactturonase and complex of alkaline carbohydrase, contains csilanse, β-glucanese, galactanese, arabinase and amylase can be used at micro-biological, food, textile, paper production, and also at food additions.

EFFECT: it is increased the output of acid protease and complex of carbohydrase.

2 ex

FIELD: veterinary virology.

SUBSTANCE: invention proposes virulent strain of virus Bovine herpes virus type 1 isolated from sperm of latently infected bull-sire deposited in NII "Collection of microorganisms" GNTS VB "Vector" at № V-337. This strain promotes to investigation of clinics of infectious rhinotracheitis virus in cattle calves.

EFFECT: valuable biological and veterinary properties of virus strain.

2 tbl, 4 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes a nutrient medium comprising potassium hydrogen phosphate, magnesium sulfate, L-asparagine, glycerol, citric acid, ferrous ammonium citrate, agar, humivite and distilled water. Invention provides enhancing growth properties of the nutrient medium.

EFFECT: valuable properties of nutrient medium.

7 tbl, 6 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes a nutrient medium comprising potassium hydrogen phosphate, magnesium sulfate, L-asparagine, glycerol, citric acid, ferrous ammonium citrate, agar, humivite and distilled water. Invention provides enhancing growth properties of the nutrient medium.

EFFECT: valuable properties of nutrient medium.

7 tbl, 6 ex

FIELD: biotechnology, genetic engineering, biochemistry.

SUBSTANCE: invention proposes the strain Bacillus simplex 23 isolated from soil and providing preparing site-specific endonuclease. This enzyme is able for recognizing and cleaving both chain in nucleotide sequence of DNA comprising at least one C5-methylcytosine base in the recognition site 5'-GCNGC-3' to form 3'-prominent ends. The novel strain can be used for isolation of the novel site-specific endonuclease that can be used for detection and cleavage of methylated sites in DNA.

EFFECT: valuable biological and biochemical properties of strain and endonuclease.

4 dwg, 4 ex

FIELD: biotechnology, microbiological industry.

SUBSTANCE: invention relates to a novel culture of microorganism producing high-molecular exopolysaccharide. Invention proposes the strain of microorganism Paracoccus denitrificans VKPM B-8617 that produces exopolysaccharide possessing cross-linking properties in aqueous and water-containing hydrocarbon systems. Exopolysaccharide is formed by residues of glucose, galactose, mannose and rhamnose in the ratio = 52:4:1, respectively, and comprises glucuronic and pyruvic acids, and acyl groups also and has molecular mass (0.5 x 106)-(2 x 107) Da. This exopolysaccharide is able to form pseudoplastic and thixotropic highly viscous solutions showing stable values of dynamic viscosity in the range of temperature from 20°C to 90°C and unstratifying emulsions. Proposed exopolysaccharide can be used in building, paper, textile, perfume-cosmetic, food, chemical, oil- and gas-extracting industry, agriculture, and in pharmaceutics and medicine.

EFFECT: valuable properties of strain and exopolysaccharide.

2 cl, 9 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to the microorganism strain Klebsiella pneumoniae GISK № 278 isolated from a patent feces suffering from intestine dysbacteriosis. The strain is used for preparing an agent for producing the lysozyme inhibitor. The level of activity of lysozyme inhibitor produced by this strain is 1.64-2.16 mcg/ml of *OD value.

EFFECT: valuable properties of strain.

1 tbl, 2 ex

FIELD: bioengineering.

SUBSTANCE: medium contains, in mass percent: 9.0 of glucose, 3.0 of corn-steep extract, 0.1 of the twice-substituted ammonium phosphate, 0.05-0.2 of cystine, 50.0-85.0 of the water extract from soybean prepared on basis of 7% suspension of the fat-free soybean flour as a nutritious substrate, up to 10.0 of water.

EFFECT: provides increased efficiency of coproporphyrin III biosynthesis.

3 ex

FIELD: biotechnology, biochemistry, enzymes, microbiology.

SUBSTANCE: invention proposes a method for microbiological oxidation of N-, O- or S-heterocyclic mono- or multinuclear aromatic compounds. The method involves culturing the recombinant microorganism that expresses cytochrome P-450-dependent monooxygenase BM-3 from Bacillus megaterium with amino acid sequence represented in SEQ ID NO:2 that comprises at least one functional mutation in region 86-88 and, if necessary, at least one functional mutation in one of regions 73-82, 172-224, 39-43, 48-52, 67-170, 300-335 and 352-356. The prepared oxidation product is isolated from medium, Invention provides carrying out oxidation of organic compounds with enhanced degree of effectiveness.

EFFECT: enhanced effectiveness of enzymes.

17 cl, 1 tbl, 7 ex

-d-ribofuranosyl-1,2,4-triazole-3 - carboxamide (ribavirin)" target="_blank">

The invention relates to biotechnology, in particular to the production of antiviral compounds

The invention relates to the chemistry of biologically active compounds and is used in photodynamic therapy of cancer

The invention relates to methods of obtaining porphyrins by microbiological synthesis

FIELD: bioengineering.

SUBSTANCE: probiotic is preliminary grown in the Soton liquid medium at 37°C within 5 days and the spores and the spores are couched in two cycles at 37°C within 18-20 hours with the thermal treatment at 132°C within 30 min at the beginning of the first and at the end of the first and the second cycles. 1 ml sterile suspension of the studied probiotic strains is layered on the Levenstein-Jensen solid medium, incubated at 37°C within 24 hours. Then, the tracer strain is intercropped and grown at the temperature and time optimum, with the following determination of the growth blocking index.

EFFECT: provides impartial evaluation of antagonistic activity of spore bacteria against micobacteria.

1 tbl, 4 ex

FIELD: bioengineering.

SUBSTANCE: method for revealing micobacteria provides the preparation of diagnostic material. The prepared diagnostic material is seeded into the solid medium which contains the mixture of the standard meat infusion agar and vaseline oil. The micobacteria are incubated in thermostat. After the incubation, the results are recorded in 24-48-72-96 hours.

EFFECT: timelines for revealing micobacteria are reduced

1 tbl

FIELD: agricultural microbiology; geochemistry.

SUBSTANCE: strain Bacillus megaterium var. phosphaticum "ВКМ В-2357 Д" is capable to leach phosphorus and silicon from objects of lithosphere and it is resistant to poly (hexamethylene guanidine).

EFFECT: strain is perspective for reception of the bacterial preparation raising a survival in soil and productivity of crops, for recultivation of technogenic polluted earths, and also in biotechnological works in the field of geochemistry.

15 dwg, 1 ex, 2 tbl

FIELD: technological processes.

SUBSTANCE: strain of bacteria Vibrio cholerae, which is deposited in State collection of pathogenic bacteria of "ФГУЗ РосНИПЧИ" "Microbe" under number "КМ234" produces choleraic toxin of II type. Strain of V.cholerae possesses high production of choleraic toxin of II type, which forms antitoxic immunity in case of cholera.

EFFECT: preparation of choleraic chemical vaccines for prophylactics of cholera.

3 ex

Up!