Method of producing tartrate of epoxyagroclavine-1 and quinocitrinines

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to bioengineering. Penicillium citrinum VKM F-4043 D fungus is cultured in a mineral medium of the following composition (g/l): sucrose - 50.0-100, urea - 2.14-4.28, KH2PO4 -1.00-2.00, MgSO4·7H2O - 0.30-0.60, ZnSO4·7H2O -0.0044-0.0088 and distilled water - up to 1l. After separation of the biomass, epoxyagroclavine-1 is separated from the alkaline extract of the culture fluid through extraction with chloroform at acidic and alkaline pH values with subsequent dehydration and concentration. The obtained alkaline extract is dissolved in acetone and while stirring, a solution of tartaric acid in methanol is gradually added. Quinocitrinines are separated from the acidic extract of the culture fluid through column chromatography (with silica gel, ratio of the height to the diametre of the column equal to 1:1.8-2.0), with chloroform first and then with a mixture of chloroform and methanol in ratio 9:1.

EFFECT: method increases content of epoxyagroclavine-1 in the culture fluid, simplification of methods of purifying epoxyagroclavine-1 and quinocitrinines, reduced cost of the medium, longer period of storing epoxyagroclavine-1.

3 ex

 

The invention relates to biotechnology and Microbiology and concerns a method for obtaining tartrate etoxiacroleine-I and khinotsitrininov.

Epoxyacrylate-I - 6,8 dimethyl-8,9-amoxillin is clavinovas Arguello having unusual stereochemistry of the 5', 10S - configuration ergalieva kernel. Earlier studies of pharmacological activity etoxiacroleine-I showed that the connection has neurotropic activity, has a moderate hypotensive effect, slows heart rate, reduces the reactivity of the vascular system on norepinephrine (Kozlovsky A.G. investigation of the alkaloids saprophytic fungi: biosynthesis, structure, properties, chemical and microbiological synthesis. The dissertation on competition of a scientific degree of the doctor of biological Sciences. App. Pushchino, ibfm wounds. 1987. P.35-40).

Identified effects testified to the fact that the test compound has useful therapeutic properties and is of interest to more extensive pharmacological studies and possible implementation in practical medicine. In addition, epoxyacrylate-I is a convenient object for obtaining derivatives ergalieva alkaloids. The presence of epoxypropyl in position 8,9 in the structure etoxiacroleine-I as the reaction center is exceptional in the EPEC for almost any derivatives, the biological activity of which can be varied by changing the nature of substituents in ring D of Ergoline.

When heated etoxiacroleine-I in aqueous solution of hydrochloric acid was found the addition products of HCl on apachegroup 8-Cl and 9-HE-agroclavine-I and 8 HE, 9-Cl - agroclavine - I. When heated solutions etoxiacroleine-I in ethanol in the presence of a catalyst leads to the formation of products of accession of ethyl alcohol by apachegroup. Education glycol-8,9-deoxyinosine-I aqueous solution of H2SO4. When interacting etoxiacroleine-I with acetic acid formed oxoacetate and diacetate agroclavine-I.

Epoxyacrylate-I is produced by a limited number of strains penicillin. First epoxyacrylate-I was isolated from the fungus Penicillium corylophilum ibfm F-152, later overridden in R. kapusciskii (=P. janczewskii) (Kozlovsky A.G., Soloviev THUS, Sakharov VG, Adanin V.M. ergot alkaloids agroclavine-I and epoxyacrylate-I - metabolites of Penicillium corylophilum. Applied biochemistry and Microbiology. 1982. Vol.18. No. 4. S-541). This invention was issued avts SU955693, SR 17/00, 1984.04.23.

The strain of the fungus Penicillium corylophilum ibfm F-152 grow deep way at t=24°C under stirring flasks 750 ml with 150 ml of medium composition (g/l):

Mannitol 50,0
Succinic acidof 5.40
MgSO4·7H2O0,30
KH2PO41,00
25%NH4OHTo a pH of 5.4

Duration of cultivation 14-16 days (5-7 days - growing seed, 9 days - the main fermentation).

Epoxyacrylate-I allocate extraction of the filtrate culture liquid of chloroform at alkaline pH values with subsequent separation by the method of column chromatography and planting from chloroform-hexane. From 1 l of culture liquid after separation and purification using column chromatography receive 90 mg etoxiacroleine-I.

The disadvantage of this method is the high cost environment, the duration of the isolation and purification etoxiacroleine-I from the culture fluid, which occupies more than a day, and the instability of the compound during long-term storage, as epoxyacrylate-I easily oxidized.

In the literature there is information about the synthesis etoxiacroleine-I strains of Penicillium fellutanum BKM FW-1073 (=P. sizovae), isolated from soil of Syria in 1969 (Kozlovsky A.G., Varecka I.G., Gayazova NB Alkalotolerant the fungus Penicillium sizovae. Applied biochemistry and microbial the Gia. 1986. THH. V.2. P.205-210). The strain was grown in-depth way at t=24°C for 17 days (4 days - growing seed, 13 days - the main fermentation) on the medium composition, g/l:

Mannitol50,0
Succinic acidof 5.4
MgSO4·7H2About0,3,
KH2PO41,0,
25%NH4OHTo a pH of 5.4

Concentration etoxiacroleine-I in the culture fluid is 75 mg/l according to UV-spectroscopy after preparative TLC on silica gel.

The disadvantage is the low concentration etoxiacroleine-I in the culture fluid, not described allocation of the target product.

Described getting etoxiacroleine-I using the fungus Penicillium citrinum BKM FW-800 (Kozlovsky A.G., Selifonova VP, Adanin V.M., Antipova T.V., Ozerskaya S.M., Kochkina G.A., U. Graefe Fungus Penicillium citrinum Thorn 1910 BKM FW-800, selected from ancient permafrost sediments, as producer of ergot alkaloids agroclavine-I and etoxiacroleine-I. Microbiology. 2003. T. No. 6. S-821). The strain grown on the medium composition (g/l): mannitol - 50, antiracist - 5,4, MgSO4·7H2O - 0,3 KH2PO4to 1.0, 25%NH4OH to a pH of 5.4 at t=24°C for 27 days (14 days - growing seed, 12 days - the main fermentation). The cultural filtrate liquid is acidified with 2%tartaric acid to pH 3.5-4.0 and extracted three times with chloroform.

Then the aqueous phase is alkalinized with concentrated ammonia to pH 8.0-9.0 and also extracted with chloroform. The alkaline extract is used to highlight etoxiacroleine-I using the method of column chromatography on silica gel (Silicagel 60, 0,040-0,063 mm, Merck, Germany), the weight of the sorbent - 60 g, the column diameter 20 mm, eluent - solvent system chloroform: methanol: NH4OH in the ratio 90:10:0.1 to. The volume fractions of 5 ml Of 1 l of culture fluid gain of 30.4 mg etoxiacroleine-I.

The disadvantage of this method is the high cost environment, the duration of the allocation, occupying more than a day, low concentration in the culture fluid and the instability of the compound during long-term storage.

Minocycline And (TRANS-isomer) and B (CIS-isomer) are secondary metabolites related to quinoline alkaloids. They are effective against gram-positive, gram-negative bacteria, yeast and fungi. Moreover, antibacterial and antifungal activity minociclina a and B was the same. Minocycline also possess cytotoxicity for puhalovich cells. And genocidenin And more effective compared to minociclina B. Thus, the inhibitory concentration (LC50µg/ml) to cells fibroblasts of mice with L-929 was 33.1; 18,6, for cells of human leukemia K-562 line - 19,5; 7,8 and HeLa>50; >50 for minociclina a and B, respectively.

A known method of producing khinotsitrininov using the fungus Penicillium citrmum BKM FW-800 (Kozlovsky, A.G., Zhelifonova, V.P., Antipova T.V., Adanin, V.M., Ozerskaya, S.M., Kochkina, G.A., Schlegel, W., Dahse, H.M., Gollmick, F.A., and Grafe, U., Qumocitrinines A and B, new quinoline alkaloids from Penicillium citrmum VKM FW-800, a permafrost fungus. J.Antibiotics. 2003. V.56. No. 5. P.488-491). Producer grow deep way in flasks with a volume of 750 ml with 150 ml of medium composition (g/l): mannitol - 50, succinic acid and 5.4, MgSO4·7H2About to 0.3, KH2PO4to 1.0, 25%NH4OH to a pH of 5.2 at t=24°C.

Cultivation spend 11-13 days. Minocycline separated from the culture fluid. To do this, the cultural filtrate liquid is acidified with 2%-tartaric acid to pH 3~4 and extracted three times with chloroform. Further purification is carried out using column chromatography on silica gel (Silicagel 60, 0,063-0.1 mm, Merck, Germany). The first elution is carried out in the solvent system chloroform: methanol: NH4OH in the ratio 90:10:0.1 to, and then in the ratio of 80:20:0,2.

The disadvantage of this method is the high cost environment, duration of cleanup khinotsitrininov, occupying more than a day. Not indicated is ANO content khinotsitrininov in the culture fluid.

Closest to the proposed combination of essential features for a way to get khinotsitrininov and ergot alkaloids, including etoxiacroleine-I, using Penicillium citrmum BKM FW-800 (Kozlovsky A.G., Selifonova VP, Antipova VP Penicillium citrmum isolated from permafrost deposits as producer of ergot alkaloids and new quinoline alkaloids of khinotsitrininov. Go active. biochemistry and Microbiology. 2005. V.41. No. 5. S-572).

According to this method, the strain is cultivated in flasks with a volume of 750 ml with 150 ml of medium composition (g/l);

Mannitol50,0
Succinic acidof 5.4
MgSO4-7H2O0,3
KH2PO41,0
ZnSO4·7H2O0,0044
25%NH4HETo a pH of 5.4

at t=24°C for 27 days (14 days - growing seed, 13 days - the main fermentation).

Minocycline separated from the filtrate of the culture fluid at acidic pH values, and epoxyacrylate-I at alkaline pH values.

Contents argollo the s and khinotsitrininov determine UV spectrophotometric after preparative TLC. In the culture fluid concentration etoxiacroleine-I - 72 mg/l, khinotsitrininov - 38 mg/L. the Selection of target products from the culture fluid is not described.

The disadvantage of this method is the high cost environment, low concentration etoxiacroleine-I in the culture fluid and instability etoxiacroleine-I during long-term storage.

The task of the invention is to develop a new microbiological method of obtaining etoxiacroleine-I and khinotsitrininov.

The technical result, which can be obtained by carrying out the invention, is to increase the content etoxiacroleine-I in the culture fluid, simplifying cleaning methods etoxiacroleine-I and khinotsitrininov, lower cost environment, increase retention epoxyoctadecane-I due to the more stable salt - tartrate epoxyoctadecane-I.

The essence of the proposed method lies in the fact that the fungus Penicillium citrinum BKM F-4043 D (formerly Penicillium citrmum BKM FW-800) were cultured in mineral medium of the following composition, g/l:

Sucrose50,00-100,00
Urea2,14-4,28
KH2PO4MgSO4×7H2O0,30-0,60
ZnSO4×7H2O0,0044-0,0088
Distilled waterTo 1 l

Epoxyacrylate-I isolated from the culture fluid using chloroform extraction at acidic and alkaline pH values. The obtained chloroform extracts dehydrated and concentrated in vacuo.

Content etoxiacroleine-I in the culture fluid is determined by means of UV-spectroscopy, a sample of the alkaline extract was dissolved in methanol and measured the optical density at λ=282 nm on a spectrophotometer (Kozlovsky A.G., Selifonova VP, Adanin V.M., Antipova T.V., Ozerskaya S.M., Kochkina G.A., U. Graefe Fungus Penicillium citrinum Thorn 1910 BKM FW-800, selected from ancient permafrost sediments, as producer of ergot alkaloids agroclavine-1 and etoxiacroleine-I. Microbiology. 2003. T. No. 6. S-821).

To obtain tartrate etoxiacroleine-I alkaline extract was dissolved in minimum amount of acetone (solution 1). Prepare a saturated solution of tartaric acid in methanol (solution 2). The amount of tartaric acid is calculated from data for the quantitative determination etoxiacroleine-I in the extract of the culture liquid is ti (by means of UV-spectroscopy). 1 mol etoxiacroleine-I requires 1 mol of tartaric acid. In solution 1 added gradually with stirring, a solution of 2. Almost immediately falls light brown precipitate. The mother liquor is separated by decantation. The precipitate is washed with acetone. Wash the acetone decanted. The resulting friable light brown precipitate is tartrate etoxiacroleine-I.

The obtained acidic extract is used to highlight khinotsitrininov. To determine the content of khinotsitrininov a portion of the acidic extract was dissolved in methanol. The record Silufol mark a line at a distance of 1.5 cm from the bottom edge of the plate and the point at a distance of 1 cm from the lateral line, the obtained methanol solution is applied by using the pipettor on the length of the line. In point cause standard solution khinotsitrininov. The vinyl coated samples are placed in a chromatographic chamber with a solvent system and chromatographic. The plate is dried in the air, say under UV light absorbing area corresponding to minociclina, on the basis of comparison with Rf standard sample. The area cut out and elute with the solvent system (80:20:0.2). The sample is evaporated on a rotary evaporator, dissolved in methanol and measured the optical density at λ=314 nm on a spectrophotometer (Kozlovsky A.G., Selifonova VP, Antipova VP Penicillium citrmum, vydelennyi permafrost sediments, as the producer of ergot alkaloids and new quinoline alkaloids of khinotsitrininov. Go active. biochemistry and Microbiology. 2005. V.41. No. 5. S-572).

The proposed method obtained by concentration of the chloroform extract of the precipitate, containing minocycline, can be refined by the method of column chromatography in a special way ("pillow"). Use a column with a ratio of height and diameter equal to 1:1,8-2,0, and solvents.

Use, in particular, silica gel 60 for column chromatography, 0,040-0,063 mm, Merck, Germany.

For example, one third of the pure silica gel is distributed evenly across the diameter of the column. The chloroform extract was dissolved in 5 ml of chloroform, mixed with the second third of silica gel and evaporated on a rotary evaporator. The sample is adsorbed on silica gel (yellow), distributed evenly along the diameter of the column. On top of it fill the remainder of the pure silica gel (obtained three layers).

Elution starting with chloroform. The volume fraction - 600 ml. Next eluent system: chloroform: methanol (9:1, vol/vol.), the first two fractions of 100 ml, the remaining 5 fractions of 50 ml. At the final step, the elution system chloroform-methanol (1:1, vol/vol.), 50 ml.

The elution time is 2 h, which is an order of magnitude faster than when using the known method column chrome is adopted, usually lasts at least one day.

The fractions are checked by TLC on plates Silufol UV-254 in the system chloroform: methanol: 25% ammonia (80:20:0,2) with the standards of khinotsitrininov. The fractions containing minocycline, evaporated in vacuum on a rotary evaporator. Further cleaning is performed before planting khinotsitrininov from acetone-hexane.

This allocation method dozens of times reduces the duration of cleanup khinotsitrininov compared with the known method of column chromatography.

In early work on search of alkaloids from filamentous fungi in nutrient media was proposed to use two sources of carbon and energy - carbohydrate and acid of the Krebs cycle, in particular succinic acid (Abe M., Yamatodami S., Jamano T., Kozu, J., Jamada S. Production of alkaloids and related substances by fungi. Examination of filamentous fungi for their ability of producing ergot alkaloids. J. Agr. Chem. Soc. Japan. 1967. V.41. No. 2. Page 67-73). The majority of the studied fungi of the genera Claviceps, Penicillium this combination was good for biosynthesis of secondary metabolites, including for biosynthesis etoxiacroleine-I and khinotsitrininov (Kozlovsky A.G., Selifonova VP, Antipova VP Fungus Penicillium citrinum, isolated from permafrost deposits as producer of ergot alkaloids and new quinoline alkaloids of khinotsitrininov. Go active. biochemistry and Microbiology. 2005. V.41. No. 5. S-572). However, the requirements of mushrooms-producers to timelinea source of carbon and energy for biosynthesis of alkaloids determined by physiological and biochemical characteristics of each individual strain.

It was investigated the influence of different combinations of carbohydrates and acids of the Krebs cycle in the biosynthesis etoxiacroleine-I and agroclavine-I producers .kapuscinski and .sizovae (Kozlovsky A.G., Soloviev TF, the Influence of cultivation conditions on the biosynthesis of alkaloids Penicillium kapuskinskii. Microbiology. 1986. T. No. 1. P.34-40. 1986; Kozlowski A.G., Varecka I.G. Effect of carbon sources on the biosynthesis of ergot alkaloids and activity of enzymes of carbon metabolism in Penicillium sizovae. Microbiology. 1987. T. 4. S-592). The fungus .kapuscinski as a source of carbohydrate used mannitol, sucrose, sorbitol, xylose, maltose, xylose, lactose, glucose, and .sizovae - mannitol, sorbitol and glucose. Both producers maximum accumulation of alkaloids occurred on medium containing mannitol. However, the best source of organic acids for biosynthesis of alkaloids was different.

The .kapuscinski maximum accumulation of alkaloids was observed on media with succinic and malic acids, .sizovae - α-Ketoglutarate acid. Growth in these environments characterized by diauxie associated with the transition from one carbon source to another, often accompanied by a fall in the concentration of alkaloids.

In the proposed environment is used only carbohydrate - sucrose and no acid TCA. Urea is used as the nitrogen source. Mushrooms urea usually decomposes the I to ammonia and carbon dioxide (J..Slaughter. Nitrogen metabolism. Discrimination of industrial fungi. Ed. Berry D.R., Oxford: Blackwell scientific publications, 1988. P.58-76). Growth in this environment is not tauxigny character.

The fungus Penicillium citrinum BKM FW-800, selected from ancient, Dating back to the age of 1.8-3.0 million years, permafrost deposits in the Arctic, currently stored in the all-Russian collection of microorganisms IBPM RAS number BKM F-4043 D.

Culture is supported by the lyophilization method.

Identification of the strain was performed according to macro - and micromorphological features, the 7-day culture grown on three different nutrient media at different temperatures.

In the process of identification of the strain .citrinum BKM FW-800 was shown by the offset of the temperature optimum growth compared to the known sample and reference strains. In the rest of the micromorphology of the studied culture was typical for the species .citrinum Thorn 1910. Macromorphology colonies on different media corresponded to the description of the species (Kozlowski A.G., Selifonova VP, Adanin V.M., Antipova T.V., Ozerskaya S.M., Kochkina G.A., U. Graefe Fungus Penicillium citrinum Thorn 1910 BKM FW-800, selected from ancient permafrost sediments, as producer of ergot alkaloids agroclavine-1 and etoxiacroleine-I. Microbiology. 2003. So 72. No. 6. S-821).

Cultural and morphological characteristics of the fungus Penicillium citrinum BKM F-4043 D.

The strain forms aerial mycelium and gives conidial sporulation. videonasty away from the substrate or aerial hyphae, 50-200×2,2-3 µm, sometimes bearing one or a few sprigs 25-35 µm, smooth. Metulla 3-4 in the apical whorl, 12-20×2,2-3 μm. Sterigma 6-10 in the beam, 8-11×2-2,8 μm. Conidia are spherical and hemispherical, more or less smooth, but sometimes grainy 2,2-3,0 µm in diameter.

Colonies on wort agar for 14 days of growth reach 25-30 mm in diameter. Colonies restricted growth, typically radially striated, smooth, bluish-green conditonal area. Exudate rich in the form of droplets straw color. There is a mushroom smell.

View Penicillium citrinum belong to the subgenus Furcatum section Furcatum. This species occupies a constant position in the taxonomy of penicillin despite the existence of some objective criteria. This species is widely distributed in soils and is known as contaminants of food and feed, as well as active biodestruction various polymers. Some strains of the species .citrinum known as mycotoxin producers O-heterocyclic nature - citrinin. Others have found clavinova ergot alkaloids - proclain, timedelay, chanoclavine-I, ethanolamin-I, cataclasis and epicatechin.

The possibility of using the invention is illustrated by examples which do not limit the scope and nature of claims.

Example 1. The fungus Penicillium citrinum BKM F-4043 D grown in flasks with a volume of 750 ml with 150 ml of medium status is VA (g/l):

Sucrose50,00
Urea2,14
KH2PO41,00
MgSO4×7H2O0,30
ZnSO4×7H2O0,0044
Distilled waterTo 1 l

Bulbs planted environment placed on a rocking chair (220 rpm) and grown for 7 days at 24±1°C. At the end of the fermentation culture liquid FilterOutputStream cultural liquid acidified with 2% tartaric acid to a pH of 3.0-3.5 and extracted with chloroform in a ratio of 1:1 (vol./about.) 2-3 times. Then the filtrate is alkalinized 25% ammonia to pH 8-9 and extracted with chloroform in a similar way. Acidic and alkaline extracts of dehydrated sodium sulfate and concentrated in vacuo on a rotary evaporator at a temperature not exceeding 40°C. the Contents in the obtained extract of the culture fluid etoxiacroleine-I - 70 mg/l, and khinotsitrininov - 3 mg/l (method UV-spectroscopy).

To obtain tartrate etoxiacroleine-I alkaline extract was dissolved in minimum amount of acetone (solution 1). Prepare a feast upon the config solution of tartaric acid in methanol (solution 2). The amount of tartaric acid is calculated from data for the quantitative determination etoxiacroleine-I in the extract of the culture fluid. 1 mol etoxiacroleine-I requires 1 mol of tartaric acid. In solution 1 added gradually with stirring, a solution of 2. Almost immediately falls light brown precipitate. The mother liquor is separated by decantation. The precipitate is washed with acetone. Wash the acetone decanted.

The resulting friable light brown precipitate is tartrate etoxiacroleine-I.

To highlight khinotsitrininov, acidic extract was purified from impurities by the method of column chromatography in a special way ("pillow"). Use a column with a ratio of height and diameter equal to 1:1,8-2,0, silica gel 60 for column chromatography, 0,040-0,063 mm, Merck, Germany and solvents. One third of the pure silica gel is distributed evenly across the diameter of the column. The chloroform extract was dissolved in 5 ml of chloroform, mixed with the second third of silica gel and evaporated on a rotary evaporator. The sample is adsorbed on silica gel (yellow), distributed evenly along the diameter of the column. On top of it fill the remainder of the pure silica gel (obtained three layers). Elution starting with chloroform. The volume fraction - 600 ml. Next eluent system: chloroform: methanol (9:1, vol/on the Sabbath..), the first two fractions of 100 ml, the remaining 5 fractions of 50 ml. At the final step, the elution system chloroform-methanol (1:1, vol/vol.), 50 ml of the elution Time is 2 PM fractions are controlled by TLC on plates Silufol UV-254 in the system chloroform: methanol: 25% ammonia (80:20:0,2) with the standards of khinotsitrininov. The fractions containing minocycline as the basic substance, evaporated in vacuum on a rotary evaporator.

From 1 l of culture liquid after extraction and purification obtain 110 mg of tartrate etoxiacroleine-I and 3 mg khinotsitrininov.

Example 2. The fungus Penicillium citrinum BKM F-4043 D grown as described in Example 1, but the duration of cultivation is 5 days.

The resulting culture used for inoculation of the flasks primary fermentation in the amount of 10% by volume environment. For basic use fermentation medium of the following composition (g/l):

Sucrose75,00
Urea2,14
KH2PO41,00
MgSO4·7H2O0,30
ZnSO4·7H2O0,0044
Distilled waterTo 1 l

Bulbs planted environment placed on a rocking chair (220 rpm) at 24°C. the Duration of the cultivation of P. citrinum BKM F-4043 D is 12 days. The content in the extract of the culture fluid etoxiacroleine-I - 170 mg/l, and khinotsitrininov - 14 mg/L.

The separation, purification of khinotsitrininov and getting tartrate etoxiacroleine-I were carried out as described in example 1.

From 1 l of culture liquid get 270 mg tartrate etoxiacroleine-I and 14 mg khinotsitrininov.

Example 3. The method is carried out as described in Example 2, but for basic use fermentation medium of the following composition (g/l):

Sucrose100,0
Urea4,28
KH2PO42,00
MgSO4·7H2O0,60
ZnSO4·7H2O0,0088
Distilled waterTo 1 l

Method and duration of cultivation of P. citrinum BKM F-4043 D carried out as in example 2. The content in the extract of the culture fluid Epoque is agroclavine-I - 170 mg/l, and khinotsitrininov - 14 mg/L. the separation, purification of khinotsitrininov and getting tartrate etoxiacroleine-I were carried out as described in example 1.

From 1 l of culture liquid receive 348 mg tartrate etoxiacroleine-I and 8 mg khinotsitrininov.

The proposed structure of the environment had not been used for producers of secondary metabolites of fungi of the genus Penicillium.

It is shown that the cultivation of the producer on this environment allows to increase the content etoxiacroleine-I to 220 mg/l, which is more than 2 times the content etoxiacroleine-I in the culture fluid in a known way (Kozlovsky A.G., Selifonova VP, Adanin V.M., Antipova T.V., Ozerskaya S.M., Kochkina G.A., U. Graefe Fungus Penicillium citrinum Thorn 1910 BKM FW-800, selected from ancient permafrost sediments, as producer of ergot alkaloids agroclavine-1 and etoxiacroleine-I. Microbiology. 2003. T. No. 6. S-821).

Beforehand it was impossible to assume that the proposed environment so will significantly increase the content of etoxiacroleine-I in the culture fluid.

The advantage of this method is the exception to the stage of purification etoxiacroleine-I method, column chromatography, so as the selection is made directly from the alkaline extract of the culture liquid.

This is ten times faster, the time of receipt of the finished product. In addition to t the th, getting etoxiacroleine-I in the form of salts of tartaric acid can increase its shelf life several times.

Has not previously been described getting tartrate etoxiacroleine-I.

Thus, a method of providing content etoxiacroleine-I in the culture fluid is 2-3 times higher in comparison with the known methods and which allows to obtain tartt etoxiacroleine-I, which is stable during storage, directly from the alkaline extract of the culture liquid.

Simplified stage selection khinotsitrininov.

The method of obtaining tartrate etoxiacroleine-I and khinotsitrininov, characterized in that cultivate the fungus Penicillium citrinum BKM F-4043 D in mineral medium of the following composition, g/l:

sucrose50,0-100,0
urea2,14-4,28
KH2PO41,00-2,00
MgSO4·7H2O0,30-0,60
ZnSO4·7H2O0,0044-0,0088
distilled waterup to 1 l

separate biomass, produce target products from the cult of the Central liquid, however, the selection etoxiacroleine-I spend directly from the alkaline extract of the culture liquid extraction with chloroform at acidic and alkaline pH values, followed by dehydration and concentration, then the alkaline extract is dissolved in acetone, the solution to obtain tartrate etoxiacroleine-I added gradually with stirring, a solution of tartaric acid in methanol, the selection of khinotsitrininov conduct of the acidic extract of the culture liquid column chromatography, which is carried on a column of silica gel at a ratio of height and diameter of the column, equal to 1:1.8 to 2.0, where in column 3 layers, the first and the third of which contains pure silica gel, the middle layer is a silica gel with adsorbed on the acidic substances of the extract of the culture fluid, the elution of khinotsitrininov spend the first chloroform, then with a mixture of chloroform : methanol 9:1.



 

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25 cl, 34 dwg, 5 tbl, 57 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention refers to genetic engineering and biotechnology and can be used in food industry. Enzyme chosen from gamma glutamyl hydrolase, GTP cyclohydrolase, dihydroneopterin aldolase and 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase is superexpressed in lactobacillus used in enzyme method for production of monoglutamylpholate and in composition of food, including dairy product.

EFFECT: higher pholate content in bioavailable form.

9 cl, 5 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: invention pertains to new compounds with general formula (I) , and their salts used in pharmacology, and their hydra as well as others, where W represents or and R3, R7, R16, R17, R20, R21 and R21 are identical or different and each of them represents a hydrogen atom or assumes other values, given in the formula of invention. The invention also relates to pharmaceutical compositions and medicinal preparations based on these compounds, cultures, used for obtaining them, methods of inhibition and treatment and use.

EFFECT: formula (I) can be used as medicinal preparations for curing solid malignant tumours.

43 cl, 4 tbl, 60 ex

FIELD: chemistry.

SUBSTANCE: invention pertains to new compounds with general formula (I) , and their salts used in pharmacology, and their hydra as well as others, where W represents or and R3, R7, R16, R17, R20, R21 and R21 are identical or different and each of them represents a hydrogen atom or assumes other values, given in the formula of invention. The invention also relates to pharmaceutical compositions and medicinal preparations based on these compounds, cultures, used for obtaining them, methods of inhibition and treatment and use.

EFFECT: formula (I) can be used as medicinal preparations for curing solid malignant tumours.

43 cl, 4 tbl, 60 ex

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

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: organic chemistry, biotechnology.

SUBSTANCE: invention refers to biotechnology and can be applied in food industry for sweeteners production. Method of indole-3-pyruvic acid production includes indole-3-pyruvic acid production with enzyme catalyzing transformation of tryptophane to indole-3-pyruvic acid and deaerating treatment or oxygen-removing treatment in acid conditions. And enzyme made of microorganism characterized with amino-acid oxidase activity and catalase activity is used. 4-(indole-3-ilmethyl)-4-hydroxy-2-oxoglutaric acid is made of indole-3-pyruvic acid and oxaloacetic acid and pyruvic acid. Methods are easy realized and enable to produce high yield products.

EFFECT: production of indole-3-pyruvic acid.

15 cl, 17 tbl, 4 ex

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention proposes a method for purifying macrolide compounds. Invention describes a method for isolation of macrolide in a purified state that involves the following steps: (a) treatment of an unpurified macrolide or crude macrolide with a solvent not mixing with water; (b) not obligatory concentrating the mixture; (c) treatment with gaseous ammonia to precipitate impurities; (d) separation of impurities; (e) not obligatory concentration of macrolide-containing phase; (f) carrying out chromatography on silica gel column in not obligatory reversed-phase, or with preliminary treatment with silver and elution of macrolide; (g) preparing macrolide in the essential purified form; (h) not obligatory repeat of steps (f) and (g) for preparing macrolide in the essential purified form. Macrolide is represented preferably as tacrolimus, immunomycin or syrolimus. Proposed method provides preparing macrolides of high purity degree by simplified technology.

EFFECT: improved isolating and purifying method.

8 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention can be used in modifying lignin-containing materials and preparing based commercially valuable compounds, wightening the paper stock and textile, treating sewage water and soils from variety of xenobiotics, polymerising phenols and some other aromatic connections, making cosmetic products for skin wigthening and hair dyeing. The method provides growing fungus Steccherinum ochraceum LE (BIN) 1833 D on a glucose-peptone medium. The prepared culture fluid is filtered from mycelium and centrifuged. The prepared supernatant is applied on a column with DE52 cellulose neutralised with 20 mM sodium-acetate buffer (buffer A). Then it is eluted with a linear gradient of 0 - 0.5 M NaCl in the buffer A. The prepared fractions with laccase activity are unitised and concentrated to desalinate and apply on the column with an ion-exchange carrier Q-Sepharose preliminary neutralised with the buffer A. Then it is eluted with a linear gradient of 0 - 0.5 M NaCl in the buffer A. The prepared two fractions with laccase activity are concentrated and desalinated. The first fraction with laccase activity prepared is concentrated and applied on column Superdex 75. It is eluted 0.1 M NaCl in the buffer A. The prepared fractions with laccase activity unitised and concentrated to prepare laccase I. The second fraction with laccase activity chromatographied on the column with Q-Sepharose is concentrated and applied on the column with the ion-exchange carrier Resource Q. It is eluted with the linear gradient 1 M NaCl in the buffer A. The prepared two fractions with laccase activity are concentrated and desalinated. The first fraction with laccase activity chromatographied on the column with Resource Q applied on the column with Superdex 75 to elute 0.1 M NaCl in the buffer A. The prepared fractions with laccase activity are unitised and concentrated to prepare laccase II. The second fraction with laccase activity chromatographied on the column with Resource Q is applied on the column with Superdex 75 and eluted 0.1 M NaCl in the buffer A. The prepared fractions with laccase activity are unitised and concentrated to produce laccase III.

EFFECT: invention improves stability of laccase.

2 cl, 4 dwg, 2 tbl, 5 ex

FIELD: microbiology.

SUBSTANCE: agent for protection of industrial materials and building concrete, brick, wood against biodeterioration represents a cultural liquid of fungus Trichoderma lignorum 81/17, deposited in All-Union Scientific Research Institute of Agricultural Microbiology under registration number 32.

EFFECT: agent has antagonistic action at fungi-biodestructors.

4 tbl, 3 ex

FIELD: agriculture.

SUBSTANCE: biological preparations are produced by means of depth cultivation of antagonist fungi from Trichoderma or Gliocladium kinds for 20-48 hours till biomass is produced in vegetative form with further addition of substance, which assists in spore formation. Right after production the preparation has the following composition (wt %): biomass of antagonist fungi - 10-99; source of carbon and energy - not more than 85; acid up to pH of 2.0-5.0 - not more than 5, inhibitor of cell metabolism - not more than 0.05, sticker-disperser - not more than 10, or biomass of antagonist fungi - 10-90; source of carbon and energy - not more than 50, source of nitrogen - not more than 1, sorbent - 10-90, acid up to pH of 2.0-5.0 - not more than 5, sticker-disperser - not more than 10. In process of preparation storage, biomass of antagonist fungi changes from vegetative form into spore form, and as a result, preparation is preserved.

EFFECT: inventions makes it possible to produce preparations, in process of storage of which with air access activity of preparations is preserved for at least 6 months.

26 cl, 1 tbl, 29 ex

FIELD: biotechnologies.

SUBSTANCE: method of selective preparation of 7α-hydroxyandrostens with the help of floccus, includes transformation of Δ5 -androstenes with the help of mycelium Curvularia lunata VKPM F-981 separated and washed from medium. At the same time substrate is used in the form of microchips, or in the complex with cyclodextrins.

EFFECT: yield of end 7-hydroxyproducts - 60-90%.

2 cl, 5 ex

FIELD: biotechnologies.

SUBSTANCE: strain of basidium fungus Fomitopsis officinalis KKBG 24 is described, which expresses bactericide activity in relation to bacteria Yersinia pseudotuberculosis.

EFFECT: invention may be used as base for production of biological preparations and medical preparations having antibiotic activity in relation to bacteria Y pseudotuberculosis, which are pathogenic for animals and humans.

1 dwg, 2 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: culture medium for thread fungi of Arthrobotrys family contains malt extract in amount from 75 to 85 wt % and yeast extract in amount from 15 to 25 wt %, where specified percents represent wt % per dry mass of specified culture medium. Another culture medium for thread fungi of Arthrobotrys family contains molasses in amount from 60 to 65%, sucrose in amount from 10 to 15%, liquid prepared after soaking of corn seeds to swelling in amount from 10 to 15% and yeast extract in amount from 10 to 15%. The third culture medium for thread fungi of Arthrobotrys family contains malt extract in amount from 25 to 30 wt %, molasses in amount from 40 to 45 wt % and liquid prepared after soaking of corn grains to swelling in amount from 25 to 30%, per dry mass of medium. Method for production of thread fungi of Arthrobotrys family industrially consists in the fact that conidia of abovementioned fungi are seeded into culture medium, and culture medium is maintained at the temperature of 23-30°C for 5-10 days to estimate reproduction and growth of fungi.

EFFECT: invention makes it possible to produce dry mass of cultivated fungi of around 9-10 g with concentration of around 109 CFU/l in 7 days.

7 cl, 7 tbl, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and design of agents with immunomodulating properties. A new fungal strain Penicillium verrucosum VKPM F-984 and a new immunomodulating agent based on the strain are proposed. The strain is extracted from microflora of ginseng roots and is kept in a medium which contains mineral salts, glucose and asparagine. Fungus mycelium is extracted with a water-alcohol solution (70% ethanol solution). The advantage of this agent is its natural occurrence and effecient stimulation of adaptive capabilities of the body.

EFFECT: obtaining an extract which has stimulating action on cell and humoral immunity, improves immune status of the body.

3 cl, 2 ex

FIELD: medicine.

SUBSTANCE: water suspension of yeast cells is prepared. Endogenous enzymes of yeast cells undergo inactivation at 85-95°C for 15-20 minutes. Further deactivated yeast cells are processed with exogenous enzyme complex represented by cultural fluid of micromycete Aspergillus oryzae F-931 of All-Russian Collection of Industrial Microorganisms or concentrated enzyme preparation obtained out of cultural fluid of micromycete Aspergillus oryzae F-931 at 48-52°C and natural pH for 4 hours with constant stirring. Further exogenous enzymes are inactivated at 85-95°C for 15-20 minutes, and obtained enzymolysate is dried in dispersion dryer.

EFFECT: improved quality of yeast enzymolysate.

2 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: there is offered therapeutic agent of systemic action prepared from mycelium of fungus Fusarium sambucinum Fuckel var. Ossicolum (Berk, et Curt) Bilai "ВСБ" 917 (All-Russian collection of industrial microorganisms F-165). The agent represents a concentrated low-molecular peptide compound and defragmented DNA of diametre 1 to 50 nm. The agent contains as follows: mineral elements - about 24% to about 26%, vitamins - about 0.8% to about 0.86%, lipids - about 4.2% to about 7.6%, whole protein - about 55% to about 57%, polysaccharides - about 9.6% to about 14.6%, water - the rest. Besides, there is disclosed application of the therapeutic agent of systemic action to inhibit defective DNA-sequences and to improve replication accuracy of existing DNA-sequences, as well as application of said agent to prepare a pharmaceutical composition. There is also offered method for inhibiting cell enrichment with genetically junk DNA and improvement of defective DNA-sequences and decrease in replication accuracy of existing DNA-sequences with using said therapeutic agent.

EFFECT: agent is characterised with effective biological properties.

20 cl, 11 dwg, 8 tbl

FIELD: biotechnology.

SUBSTANCE: invention concerns biotechnology, particularly methods of obtaining preparation based on chlamydospores of nematophagous microfungus for vegetation and animal parasitic nematode control. Method involves submerged cultivation of microfungus in nutritive medium including carbon and organic nitrogen sources, salts and water, till maximum accumulation of vegetative mycelial mass, introduction of sporulation inducer, with further chlamydospore formation. Duddingtonia flagrans F-882 is used as microfungus, submerged cultivation is performed in gas vortex bioreactor until dry rate of biomass reaches at least 10.0 g/l. To obtain fluid form of preparation, water is added as sporulation inducer, diluting vegetative mycelial mass by 2-5 times, and/or glycerin is added in end concentration of 1-2 wt %, and additionally the biomass is incubated until chlamydospore accumulation in the culture reaches maximum. To obtain dry form of preparation, sterile foamed vermiculite is added to vegetative mycelial mass as sporulation inducer at the ratio of 1.0-4.0 ml of mycelial mass to 1 g of foamed vermiculite, obtained mix is stirred and additionally incubated to obtain chlamydospores.

EFFECT: enhanced efficiency of preparation.

6 cl, 10 tbl, 11 ex

FIELD: biotechnology, in particular reagent for structural protein hydrolysis.

SUBSTANCE: method for production of protheolytic reagent includes cultivation of producer strain selected from dermatophyte of species Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton equinum, Microsporum canis, Microsporum gypseum, preferably from fungus strains capable to produce complex of structural protein proteinases (scleroproteases) with total protheolytic activity at least 0.7 U/mg including collagenase 0.1-4.5 U/mg; keratinase 0.1-0.5- U/mg; elastase 0.5-1.9 U/mg. Cultivation is carried out, for example, in wort agar-agar or in wort broth for 20-24 days.

EFFECT: scleroproteases with improved specific activity; method for protheolytic cleavage of specific substrates (scleroproteins) with increased rate and depth.

2 dwg, 12 ex

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