Strain of fungus aspergillus fumigatus as producer of indole alkaloids and method for their preparing

FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: the strain-producer is isolated from soft coral Sinularia sp. relating to species Aspergillus fumigatus and deposited in Collection of Marine Microorganisms of Pacific institute of bioorganic chemistry DVO RAN (KMM TIBOKH) at № 4631. Method for preparing indole alkaloids involves surface solid-phase culturing the indicated strain on nutrient medium, milling mycelium and medium, two-fold extraction of prepared mixture with system chloroform - ethanol (2:1), concentrating extract, its chromatography on column with silica gel and the following separation of indole alkaloids by HPLC method. Method based on applying this strain provides enhancing yield of indole alkaloids. Invention can be used in preparing indole alkaloids eliciting an anti-tumor activity.

EFFECT: improved method for preparing, valuable medicinal properties of indole alkaloids.

4 cl, 1 tbl, 2 ex

 

The invention relates to biotechnology and can be used to obtain an indole alkaloid with antitumor activity.

It is known that microscopic fungi of the genus Aspergillus are producers of a number of biologically active substances, including organic acids, enzymes, protein and polysaccharide antigens, antibiotics.

Among the fungi species Aspergillus fumigatus is known and producers of indole alkaloids.

So it is known that the strain of the fungus Aspergillus fumigatus DSM 790, isolated from soil, is the producer 12, 13-dihydro-fumitremorgin C. Obtaining 12,13-dihydro-fumitremorgin requires pre-growing producer in the medium of the following composition: 1% glucose, 1% peptone, 2% extract maltose and 0.3% yeast extract. The cultivation is carried out for 48 hours at 27° and the stirring speed of 100 rpm Obtained mycelium is filtered, washed twice Tris-buffer (0.05 M, pH 7.0) and again suspendered 200 ml of the same buffer containing 0.5% glucose. After 10 days of culturing mycelium is separated by filtration, the mycelium and culture filtrate extracted three times with ethyl acetate. Extract pariveda, and the remainder is applied on RP-8 column in the H2O-Meon, 30:70. The output of 12,13-dihydro-fumitremorgin With is only 1.5 mg/l [Arfmann, H. - A. 12,13-dihydroxy-fumitremorgin From Aspergilus fumigatus. Phytochemistry 1990, 29, 1025-1026].

The disadvantages of the known strains of the fungus and the method of obtaining a low yield of the target product.

The closest to the claimed strain and process for the preparation of indole alkaloids is a strain of the fungus Aspergillus fumigatus BM 939 isolated from marine sediments of the Japan sea from a depth of 760 m, producing cyclopentadiene a, b, C and D, and the retrieval method. To obtain these alkaloids provides for the cultivation of microscopic fungus at 28° C for 28 hours method of submerged culture medium of the following composition: glucose 3%, soluble starch 2%, soy flour 2%, K2NRA40.5% MgSO4·7H2O of 0.05, with continuous stirring at 350 rpm, and aeration of 200 l/min, and subsequent extraction of the product from the culture fluid. For this purpose, the mycelium is separated by filtration, extracted with 90%aqueous acetone extract pariveda to the aqueous solution. Aqueous solution and the culture filtrate is extracted with ethyl acetate, and the combined extract pariveda to an oily residue. The residue twice is distributed between chloroform and hexane, and chloroform extract is purified on a column of silica gel. Selected alkaloids are separated by high performance liquid chromatography (HPLC) columns CAPCELL CANCER C-18. The output of Cyclops the statin And - 2.5 ág/l of culture fluid, cyclopentadiene - 11 µg/l, the outputs of cyclopentamine C and D minor [Cui, C.-B., Kakeya, H., Osada, H. Novel Mammalian Cell Cycle Inhibitors, Cycloprostatins A-D, Produced by Aspergillus fumigatus, Which Inhibit the Mammalian Cell Cycle at G2/M Phase. Tetrahedron 1997, 53, 59-72].

A known strain of Aspergillus fumigatus BM 939 also, under certain conditions of cultivation produces 12,13-dihydro-fumitremorgin C. For this purpose, the cultivation is carried out in a fermenter (30 l), containing 18 liters of the culture liquid medium of the following composition: glucose 3%, soluble starch 2%, soy flour 2%, K2NRA40,5%, MgSO4·7H2O 0,05. The cultivation is carried out at 28° within 72 hours if the stirring speed 350 rpm, and the speed of aeration 7 l/min Extraction is carried out as well as when receiving cyclopentadien. The output of 12,13-dihydro-fumitremorgin With - 1.8 mg/l [Cui, C.-B., Kakeya, H., Okada, G., Onose, R., Osada, H. Novel Mammalian Cell Cycle Inhibitors, Tryptostatins a, b and Other Diketopiperazines Produced by Aspergillus fumigatus. J. Antibiotics 1996, 49, 527-533].

The disadvantages of the known strains of Aspergillus fumigatus BM 939 can be attributed to the low productivity in respect of indole alkaloids.

The method of obtaining, providing for the cultivation of strain on liquid nutrient medium requires large energy consumption for mixing and aeration, as growing producers is carried out by the method of deep cultivation. For the next isolation and purification of target products are characterized by the duration and complexity, as include several stages of extraction of mycelium of different solvents, and a fairly complex process of purification of alkaloids.

Due to the fact that indole alkaloids such as cyclopentadiene and 12,13-dihydroxy-fumitremorgin, fumitremorgin With and verruculogen, possess antitumor activity and therefore are of considerable interest as a promising anticancer drugs, the task of producing products with a high yield.

The problem is solved by the identification of a new strain of the fungus Aspergillus fumigatus, with the ability to produce indole alkaloids, and the development of a new method of producing indole alkaloids, involving the cultivation of a producer on a nutrient medium, the extraction of the mycelium and the environment organic solvent and the selection of target products by chromatographic purification on silica gel and separation by HPLC, in which the producer used a strain of the fungus Aspergillus fumigatus KMM 4631, and cultivation is carried out on solid nutrient medium.

In the method using nutrient media RM or MM (Monaghan R.L, Can. J. Bot. 1995. V. 73. Supl. I.Sect. E-H. R. 925-931), modified by the authors, in which instead of fresh water as a source of mineral salts use natural sea water. These environments authors identified as RMM or MMM. Pirateland RMM characterized by the following composition: rice - 1.0 kg, natural sea water 2.0 l, yeast extract 1.0 g, sodium tartrate - 0.5 g, KH2RHO4to 0.5, the culture medium MMM characterized by the following composition: wheat - 1.5 kg, natural sea water 2.0 l, sodium tartrate - 0.5 g, KN2RHO40.1 g MgSO4·7H2O 0.1 g FeSO4·7H2O - 0,01 g

The optimum temperature for growing the fungus on these media 20-22° C.

Extraction of the mycelium and the environment carried out with a mixture of chloroform-ethanol 2:1. The separation of the target products perform consistently on silica gel Silsor Si (12 μm, column 4× 250 mm, producer company ELSICO, HPLC&LC COMPANY, Moscow) and reversed-phase silica gel Diaster-110-C18 (6 μm, column 4,0× 250 mm, manufacturer JSC Biochimmach, , Moscow).

A new strain of Aspergillus fumigatus isolated from the soft coral Sinularia sp., collected at a depth of 52 m near coast of Kunashir island (Kuril Islands).

Strain Aspergillus fumigatus is stored in the Collection of Marine Microorganisms Pacific Institute of Bioorganic chemistry RAS (CMY, tibah) under No. 4631.

A strain of Aspergillus fumigatus KMM 4631 characterized by the following properties.

Cultural and morphological characteristics.

On the environment of čapek colony of 3.7-4.2 cm in diameter for two weeks of growth, widely spreading, smooth, velvety, in the Central part of the felt, first the white, as the formation of conidia become gray-green to gray-bluish-green in the center of the colonies. Conidial heads Kalinkovichi, compact, varying in size from 40 to 400 microns in length. Conidiophores depart directly from submerged hyphae or as a very short sprigs of aerial mycelium, or as short, 300-400× 5-8 microns, gradually widening towards the apex, smooth coat greens, especially intense in the upper part. The apical part of canadianese bottle-shaped, 20-30 µm in diameter. Sterigma of a single-layer, are formed only in the center of the apical part, taking no more than 2/3 of its surface, dense, arranged on an axis parallel to the axis of canadianese; 6-8× 2-4 μm. Conidia globose, 2.5 to 3 μm, in the mass of dark green, smooth, rough or unclear. Reversum yellow, the aging of the culture becomes red-brown.

On the environment wort-agar colony 3,8-4,6 cm in diameter for two weeks of growth, widely spreading, smooth, tomentose to pubescent, at first white, as the formation of conidia become gray-bluish-green. Microscopic characteristics are the same as when grown in the medium of čapek. Reversum unpainted.

On medium potato-dextrose agar colony of 3.2-4.0 cm in diameter for two weeks of growth, widely spreading, smooth, velvety, first white, as shaped who I am conidia become dark gray-green, to dark olive-gray in the center of the colonies. Microscopic characteristics are identical to the characteristics of the strain grown on the above-mentioned environments. Reversum unpainted or yellow.

A new strain of Aspergillus fumigatus produces indole alkaloids: cyclopentamine a and b output 115,0 mg per 1 kg of nutrient medium RMM, fumitremorgin With access to 42.0 mg/kg nutrient medium RMM and 102,0 mg/kg nutrient MMM, and produces 12,13-dihydro-fumitremorgin With output 98,0 mg/kg nutrient medium RMM and 125,0 mg/kg nutrient MMM and verruculogen with the release of 70.0 mg/kg nutrient medium RMM and 100.0 mg/kg nutrient MMM.

The inventive method of obtaining indole alkaloids: cyclopentadien a and b (1 and 2), fumitremorgin With (3), 12,13-dihydro-fumitremorgin With (4) and verruculogen (5), differs from the known method by using a new, more productive strains of Aspergillus fumigatus KMM 4631, which is grown on a nutrient medium by means of surface of solid-phase cultivation, and less time-consuming and laborious procedures of extraction and purification of target products. The outputs of indole alkaloids: 12,13-dihydro-fumitremorgin With, cyclopentamine a and b in the present method exceed outputs in ways analogous to 60-25000 time. In addition, the cost of aeration culture and costly sugar, which reduces the cost of obtaining the products is that 10-20 times, in comparison with obtaining product on liquid nutrient media in the known methods.

The technical result of the strain Aspergillus fumigatus KMM 4631 grown on solid nutrient media, is its high productivity. The new strain can be used for industrial production of indole alkaloids with anticancer activity.

The authors of the present invention, a comparative study of the amount produced by a strain of the fungus KMM 4631 indole alkaloids in liquid and solid nutrient media. Used liquid nutrient medium containing natural sea water, glucose - 30.0 g/l, peptone - 1.0 g/l, yeast extract 0.5 g/l, KN2RHO4- 1.0 g/l, MgSO4·7H2O - 0.5 g/l FeSO4·7H2O - 0.02 g/L. the Cultivation was carried out in flasks on a shaker 180-200 rpm for 168 hours at a temperature of 20° and a pH of 7.8. The culture fluid was chromatographically on a column with polychrome-1, elwira fractions successively with water and 50%ethyl alcohol. Aqueous-alcoholic eluate was evaporated, the residue was extracted with ethyl acetate. The extract was concentrated in vacuo to a minimum volume and divided by high performance liquid chromatography (HPLC) on silica gel in the system ethyl acetate-hexane, 4;1. The data are summarized in the table.

In PR the measures 1 and 2 presents data on obtaining alkaloids as a result of their production of the inventive strain on solid nutrient media and the selection of the claimed method. The data are summarized in the table.

Table
Nutrient mediumTime cultivated

of (h.)
The output of alkaloids (mg/l liquid medium and mg/kg for solid

environment)
The total number of alkaloids (mg/l liquid medium and mg/kg for

solid medium)
1 and 2345
Liquid168  2,55,07,5
Solid (example 1)504115,042,098,070,0325,0
Solid (example 2)504 102,0125,0100,0327,0

The invention is illustrated by the following examples.

Example 1. A strain of Aspergillus fumigatus KMM 4631 grown using the surface of the solid-phase cultivation at a temperature of 20° With the nutrient medium RMM following composition: rice - 1.0 kg; natural seawater - 2.0 l, yeast extract 1.0 g, sodium tartrate - 0.5 g, KN2RHO4- 0.5 g at room temperature for 504 hours. Mycelium together with the th pulverized and extracted twice with a mixture of chloroform-ethanol 2:1. The extract is evaporated, the residue chromatographic on a column of silica gel L (40/100 μm), elwira sequentially with chloroform, and the chloroform-ethanol 10:1, 5:1 and 2:1. Faction, eluruume system chloroform-ethanol 2:1 are pooled, concentrated in vacuo to a minimum volume and separated by HPLC on columns Silsor Si (ethyl acetate-hexane, 4:1) and Diester-110-C18 (55% Meon).

The output of cyclopentadiene And (65,0 mg/kg), cicloprofen In (50 mg/kg), 12,13-dihydroxy-fumitremorgin (98 mg/kg), fumitremorgin With (42 mg/kg) and verruculogen (70 mg/kg). The structure of selected alkaloids established on the basis of data of the MC-,1H and13With NMR spectroscopy.

Example 2. A strain of Aspergillus fumigatus KMM 4631 grown using the surface of the solid-phase cultivation at a temperature of 22° With the nutrient medium MMM following composition: wheat - 1.5 kg; natural seawater - 2.0 l, sodium tartrate - 0.5 g, KH2PO40.1 g MgSO4·7H2O 0.1 g FeSO4·7H2O - 0.01 g at room temperature for 504 hours. Next, the process of selection of target products carried out as described in example 1. Output 12,13-dihydroxy-fumitremorgin (125,0 mg/kg), fumitremorgin (102,0 mg/kg) and verruculogen (100.0 mg/kg). The structure of selected alkaloids established on the basis of data of the MC-,1H and13With NMR spectroscopy.

1. The strain of the fungus Aspergillus fumigatus (CMY, tibah No. 4631) - producer of indole alkaloids.

2. The method of obtaining indole alkaloids, involving the cultivation of a producer on a nutrient medium, the extraction of the mycelium and the environment organic solvent and the selection of target products by chromatographic purification on silica gel and separation by HPLC, characterized in that as a producer uses a strain of the fungus Aspergillus fumigatus KMM 4631, and cultivation is carried out on solid nutrient medium of the following composition: rice - 1.0 kg, natural sea water 2.0 l, yeast extract 1.0 g, sodium tartrate - 0.5 g, KN2RHO4to 0.5 g (RMM) or on a solid nutrient medium of the following composition: wheat - 1.5 kg, natural sea water 2.0 l, sodium tartrate - 0.5 g, KN2RHO40.1 g MgSO4·7H2O 0.1 g FeSO4·7H2O - 0,01 g (MMM).

3. The method according to claim 2, characterized in that the extraction is carried out with a mixture of chloroform-ethanol 2:1.

4. The method according to claim 2, characterized in that the separation of the target products perform consistently on columns Silsor Si and Diester - 110-C18.



 

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