RU2229520C1 - Cyclodecapeptide antibiotic laterocidin with broad spectrum of antagonistic effect, strain of bacterium brevibacillus laterosporus eliciting broad spectrum of antagonistic effect as producer of antibiotic laterocidin - Google Patents

Abstract

FIELD: antibiotics, biotechnology, microbiology. SUBSTANCE: invention relates to manufacturing the novel antibiotic laterocidin. Laterocidin shows antagonistic effect against bacteria, cyanobacteria, phytopathogenic fungi and protozoa. Laterocidin is a cyclodecapeptide antibiotic and represents white amorphous substance of the formula: [cyclo-(L-Val-L-Om-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Leu)] and molecular mass 1224.4 Da. Producer of laterocidin is the strain of bacterium Brevibacillus laterosporus that is obtained from the natural strain of the same species as result of multi-step selection. The strain is deposited at number VKPEM B-8287. The strain shows activity against series of Gr⁺ and Gr^- microorganisms, phytopathogenic fungi and protozoa microorganisms. Application of the strain and laterocidin provides expanding the assortment of antagonists with broad spectrum of effect. EFFECT: valuable medicinal properties of antibiotic. 3 cl, 4 tbl, 8 ex

Description

The invention relates to microbiology, biotechnology and, in particular, to the production of means of bacterial origin for combating diseases of plants and animals.

Currently, chemical fungicides are mainly used as a means of combating plant pathogens. Despite its high efficiency, a number of shortcomings of chemical plant protection products (the emergence of resistance in pathogens, the lack of a selective effect, toxicity to warm-blooded animals) necessitate the search for new compounds. As alternative plant protection products, bacterial fungicidal preparations can be used, which include strains of microorganisms that exhibit antagonistic activity against various biological objects.

The widespread use of the so-called “classic” antibiotics has led to the emergence of resistant forms of pathogens, which creates serious social and medical problems. One way to solve this problem is to search for new generation antibiotics, which include peptide antibiotics. Interest in them is due to the mechanism of their action, which causes physical disturbances in the cell membranes of microorganisms. It is assumed that such a mechanism of action to a certain extent reduces the likelihood of the emergence of resistance of pathogenic bacteria.

Currently, a number of bacillary strains are known that are capable of producing peptide antibiotics having antibacterial and / or fungicidal activity. Characteristic features of bacillary peptide antibiotics and fungicides are their non-ribosomal synthesis and the content of unusual (ornithine, etc.) and D-amino acids. Thus, strains of Bacillus licheniformis are described that synthesize low molecular weight (molecular weight 1400-3200) polypeptides and lipopeptides with a wide spectrum of antagonistic action (1, 2). Strains of Bacillus subtilis produce a number of antimicrobial factors. Mycobacillin is an anionic 13-membered cyclic polypeptide with mol. weighing ~ 1800, has a wide spectrum of action against bacteria, protozoa, and fungi (3). A new class of lipopeptide fungicides combines the active peptides of the families of iturin, surfactin and fengicin (4). Iturins (4) and bacillomycin (5) are neutral or anionic cycloheptapeptides with mol. weighing ~ 1000, combined with a fatty acid or an ester bond with the formation of a lactone cycle (iturins), or a peptide bond in the cycle (bacillomycin) (6). Iturins and bacillomycin have limited antibacterial activity and a wide range of activity against fungi and yeast. Surfactin is a cyclic lipopeptide containing seven hydrophobic amino acid residues connected to β-hydroxy fatty acid (7). This is a powerful surface-active compound, also known as an antibacterial and fungicidal agent. Fengicin (8) and plipastatin (9) are lipopeptides containing ten amino acids and a lipid part located at the N-terminus of the molecule. They differ from iturin and surfactin in the presence of such unusual amino acids as ornithine and allo-threonine. A known group of cyclodecapeptides (gramicidin S and thyrocidins) produced by strains of Bacillus brevis, which has antibacterial and weak fungicidal activity (10). There is evidence of strains of Bacillus laterosporus that inhibit the growth of bacteria and fungi (11, 19, 20). Loloatins A, B, C, D were isolated from the Bacillus laterosporus MK-PNG-276A strain, a group of cyclic decapeptides of the thyrocidic family that exhibit antagonistic activity against gram-positive bacteria Staphylococcus aureus, Staphylococcus ssp., Streptococcus spp., Enterococcus funecum faecum funecida but not active against gram-negative bacteria Pseudomonas mendocina and Pseudomonas marginata (12, 13). There is no information on the antagonistic activity of these substances against protozoa, cyanobacteria, Saccharomyces cerevisiae yeast, and phytopathogenic fungi. The strain producing Lacatoins Bacillus laterosporus MK-PNG-276A is selected as the closest analogue of the claimed strain with a wide spectrum of antagonistic action.

The task: to expand the arsenal of products based on cyclic decapeptides used to combat pathogens of plants and animals.

The problem is solved by:

- isolation of a cyclodecapeptide antibiotic of a wide spectrum of antagonistic action of laterocidine;

- obtaining a strain of bacteria Brevibacillus laterosporus VKPM B-8287, which produces the antibiotic laterocidin and has a wide spectrum of antagonistic action, including due to the antibiotic laterocidin.

The inventive cyclodecapeptidium antibiotic, called us laterocidin, is not known from sources of information. Laterocidin has an antagonistic effect against bacteria, cyanobacteria, phytopathogenic fungi and protozoa (table 1). Laterocidin is a white amorphous solid having the formula [cyclo- (L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp- L-Leu)] and a molecular weight of 1224.4. Laterocidin is soluble in methanol, ethanol, other lower alcohols, not soluble in water.

The inventive strain was isolated by us as a result of multistage selection from a natural strain of Brevibacillus laterosporus. It is deposited in the All-Russian Collection of Industrial Microorganisms under the number VKPM V-8287.

The strain of Brevibacillus laterosporus VKPM B-8287 is claimed

- as a producer of the antibiotic laterocidine and

- as a strain with a wide spectrum of antagonistic action.

The inventive strain of Brevibacillus laterosporus VKPM B-8287 has the following characteristics:

1. Cultural and morphological characteristics.

Aerobic gram-positive movable sticks with a size of 0.5-0.7 × 2.5-5.0 μm do not form a capsule. Vegetative cells - sticks with flagella, peritrichi. The spores are oval, with canoe-like inclusions attached to one side of the spores. The size of the spores is 0.9-1.2 × 1.5-1.7 microns. After 24 hours of growth on rich agarized media [NBY g / L: nutrient broth -8; yeast extract (yeast extract) - 3, agar-agar - 20; DPS g / l: yeast - 30, corn flour - 15, agar-agar - 20; GD g / l: hydrolysis yeast - 45, agar-agar 20] strain forms round colonies 3-4 mm in diameter with a smooth cream-colored surface and an uneven edge.

2. Cultivation of the strain.

When grown in liquid nutrient medium [NBY (g / l): nutrient broth (nutrient broth) - 8; yeast extract (yeast extract) - 3] at 28-30 ° C with shaking (250 rpm) uniform growth of the culture is observed throughout the volume. By 96 hours of cultivation, mainly 90-95% of spores and 5-10% of vegetative cells are observed. The culture titer after 96 hours of cultivation is ~ 3.0 × 10 ⁹ ml.

3. Physiological and biochemical characteristics.

The strain ferments glucose, maltose, fructose. Does not ferment sucrose and lactose. Forms lecithinase; does not hydrolyze starch; hydrolyzes casein and gelatin. Urea does not break down. Forms a twin esterase.

Based on morphological (the presence of a canoe-like structure attached to the spore) and physiological and biochemical characteristics, the isolated strain VKPM B-8287 is assigned to Bacillus laterosporus (14). According to the new classification, to Brevibacillus laterosporus (15).

4. Antagonistic activity.

Strain synthesizes antibiotic laterotsidin and shows antagonistic activity against Gram-positive bacteria: Bacillus thuringiensis, Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Bacillus sphaericus, Staphylococcus ssp, Staphylococcus aureus, Streptococcus ssp, Enterococcus ssp, Enterococcus faecium, Sarcina flava (... table 2);

gram-negative bacteria: Acinetobacter calcoaceticus, Flavobacterium aquatile, Pseudomonas marginata, Pseudomonas mendocina, Xanthomonas ssp., Erwinia herbicola, Erwinia carotovora, Erwinia atroseptica. Vibrio nereis (table 2);

cyanobacteria: Nostoc ssp. 29411 and Anabaena ssp. 5781 (table 3);

phytopathogenic fungi: Fusarium graminearum, Fusarium nivale, Fusarium solani, Rhizoctonia solani, Sclerotinia sclerotiorum, Phomopsis helianthi, Phoma solanicola, Alternaria tenuis, Aspergillus niger (table 4);

yeast: Saccharomyces cerevisiae (table 4);

protozoa: Tetrahymena pyriformis u Entamoeba moshkovskii (example 8).

The invention is illustrated by the following examples.

Example 1

Microbiological synthesis of laterocidine with a strain of Brevibacillus laterosporus VKPM B-8287, isolation and purification of laterocidine.

, 20 мкм (19×300 мм. Waters) при скорости потока 2 мл/мин в линейном градиенте (от 0 до 100%) раствора Б (метанол - ацетонитрил - вода, 75:22,5:2,5) в растворе А (метанол - ацетонитрил - вода, 60:5:35) за 100 мин и затем в растворе Б в течение 20 мин. На колонку наносили по 10 мкл раствора. Полученные фракции, проявляющие антагонистическую активность, объединяли, упаривали досуха и растворяли в этаноле. Далее раствор (по 10 мкл) наносили на обращенно-фазовую колонку Vydac С-18, 300

, 5 мкм (4,6×250 мм, Vydac). Элюцию осуществляли при скорости потока 0,5 мл/мин в линейном градиенте (от 10 до 100%) раствора Б в растворе А за 60 мин и затем в растворе Б в течение 20 мин. Фракции, проявляющие антибактериальную активность, объединяли и упаривали досуха. Полученный гомогенный антибиотик представлял собой аморфное вещество белого цвета.The strain Brevibacillus laterosporus VKPM B-8287 was cultured in NBY liquid nutrient medium for 72 hours at 30 ° C on a circular shaker (250 rpm). The culture fluid was centrifuged at 10,000 g for 15 min, the resulting supernatant was discarded, and absolute ethanol was added to the triply washed water by centrifugation. Alcohol extraction was continued for 20 hours at room temperature. The resulting alcoholic extract was separated by centrifugation (15 min at 10000 g), the resulting solution was evaporated at 56 ° C to obtain yellow-brown crystals. The alcohol solution of the crystals was fractionated by HPLC on a Delta Pak C-18, 300 reverse phase column

, 20 μm (19 × 300 mm. Waters) at a flow rate of 2 ml / min in a linear gradient (from 0 to 100%) of solution B (methanol - acetonitrile - water, 75: 22.5: 2.5) in solution A (methanol - acetonitrile - water, 60: 5: 35) for 100 minutes and then in solution B for 20 minutes. 10 μl of solution was applied to the column. The obtained fractions exhibiting antagonistic activity were combined, evaporated to dryness and dissolved in ethanol. Next, the solution (10 μl each) was applied to a Vydac C-18, 300 reversed-phase column

5 μm (4.6 × 250 mm, Vydac). Elution was carried out at a flow rate of 0.5 ml / min in a linear gradient (from 10 to 100%) of solution B in solution A for 60 minutes and then in solution B for 20 minutes. Fractions exhibiting antibacterial activity were combined and evaporated to dryness. The resulting homogeneous antibiotic was an amorphous white substance.

Example 2

Determination of the primary structure of laterocidine.

NMR spectroscopy was used to determine the structure of the isolated antibiotic. ¹ H-NMR spectrum of a peptide antibiotic dissolved in methanol (1.7 mg / 600 μl, pH = 8.4; 30 ° C) was obtained on a Varian Unity 600 spectrometer with an operating frequency of 600 MHz. Based on the analysis of the NMR spectrum of COSY, TOCSY and ROESY using the standard procedure (17), the proton signals of the test sample were completely assigned, which corresponds to the amino acid sequence [cyclo- (L-Val-L-Orn-L-Leu-D-Tyr- L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Leu)]. After analyzing the SRS database, it was revealed that the peptide with the above sequence has no analogues. This peptide, called us laterocidin, belongs to the family of thyrocidin antibiotics.

Example 3

Determination of the molecular weight of laterocidine.

Mass spectrometric analysis was used to determine the molecular weight of laterocidine.

MALDI (matrix-assisted lazer desorption ionization) peptide mass spectrometry was performed on a VISION 2000 mass spectrometer (ThermoBioAnalysis, OK). As a matrix used 0.15 M 2,5-dihydroxybenzoic acid (DHB) in a mixture containing 25% methanol and 0.1% trifluoroacetic acid. The sample was irradiated with a UV laser with a wavelength of 337 nm and a maximum energy of 250 μJ in a pulsed mode with a frequency of 3 ns. During mass spectrometric analysis of the peptide (laser power - 57%, the number of laser strokes - 20), a peak was obtained with m / z 1224.4; corresponding to the molecular ion of the new antibiotic laterocidine.

Example 4

Determination of the minimum inhibitory concentration of laterocidine.

Quantitative assessment of the antagonistic action of laterocidine was carried out by determining its minimum inhibitory concentration (MIC). For this, the obtained laterocidin was dissolved in a minimum volume of absolute ethanol and a stock solution was prepared containing 200 μg / ml of 5% ethanol. The minimum inhibitory concentration of laterocidine was determined by the standard method of serial two-fold dilution of the initial antibiotic solution (18) against each test microorganism (table 1).

From the data given in table 1, it follows that laterocidine has a wide spectrum of antagonistic action, it shows the highest level of antagonistic activity against phytopathogenic fungi of the genus Fusarium, protozoa, cyanobacteria, pathogenic bacteria of streptococcus, staphylococcus and enterococci groups.

Example 5

Determination of the spectrum of antibacterial action of the strain VKPM B-8287 and its comparison with the closest analogue.

The antibacterial effect of the strain culture was detected by the hole method. In 25 ml of agarized NBY medium, 1 ml of a 4-hour culture of test bacteria was added, and 50 μl of 72-hour QOL of strain B-8287 were added to cut holes in a diameter of 8 mm. Petri dishes were kept for 2 hours at + 5 ° C and then incubated at 30 ° C. The antagonistic effect of the strain was fixed after 24 hours by measuring zones of inhibition of growth of test microorganisms. The results are shown in table 2.

Example 6

Determination of the cyanolytic activity of the strain VKPM B-8287.

The cyanolytic activity of the strain was detected by mixed cultivation. The culture fluid of the 72-hour culture VKPM B-8287 was introduced into a suspension of a 14-day culture of cyanobacteria Nostoc ssp. 29411 or Anabaena ssp. 5781 (16). Measured the optical density (at a wavelength of 590 nm) of the mixture at zero time and after incubation at 25 ° C and round-the-clock illumination. Cyanolytic activity was evaluated by the decrease in optical density after 24 hours of incubation. Strain VKPM B-8287 caused a drop in optical density by 3-4 times, which indicated its lytic effect on cyanobacteria (table 3). Lysis of cyanobacterial cells was also detected by optical microscopy.

No information on the cyanolytic activity of the strain, the closest analogue, was found in the information sources.

Example 7

Determination of the fungicidal activity of a strain of Brevibacillus laterosporus VKPM B-8287.

To identify the fungicidal activity of the strain against fungi and yeast, the hole method was used. The culture of the test microorganism was placed in the center of the Petri dish with potato agar (bacto-agar - 20 g, glucose - 15 g, potato broth - up to 1 l). The culture fluid of the 72-hour B-8287 culture was added to the wells. The plates were incubated in the dark at room temperature. After 48 hours, the presence or absence of fungal growth inhibition zones was assessed.

The inventive strain inhibited the growth of the mycelium of the following microorganisms: Fusarium graminearum, Fusarium nivale, Fusarium solani, Rhizoctonia solani, Sclerotinia sclerotiorum, Phomopsis helianthi, Phoma solanicola, Alternaria tenuis, Aspergillus niger and Saccharomyce cerebra 4, Candice unlike the closest analogue.

Example 8

Determination of the activity of the strain VKPM B-8287 against protozoa.

The activity of the VKPM B-8287 strain against protozoa was determined by mixed cultivation of the strain and protozoa. The 72-hour culture fluid of the strain was added to a suspension of protozoan cells. A 4-day culture of Tetrahymena pyriformis ciliates was used (15-20 × 10, the number of ciliates was counted microscopically) or a 7-day culture of Entamoeba moshkovskii amoeba (5-8 × 10 ³ , the number of amoebas was counted microscopically). After 24 hours of incubation, the death of the protozoa was 100%.

Information on the antagonistic activity of the nearest analogue against protozoa is absent.

Thus, we obtained a strain of bacteria Brevibacillus laterosporus VKPM B-8287 with a wide spectrum of antagonistic action, showing antibiotic activity against a number of gram-positive and gram-negative bacteria, cyanobacteria, phytopathogenic fungi, yeasts and protozoa. He is the producer of the first isolated cyclodecapeptide antibiotic, which we called laterocidin, which also has a wide, but slightly different from that of the producer strain, spectrum of antagonistic action. Both of these objects can be independently used as antibacterial, fungicidal and antiprotozoal agents for controlling pathogens in plants and animals.

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Claims (2)

1. Cyclodecapeptide antibiotic with a wide spectrum of antagonistic action of laterocidin having a primary structure: [cyclo- (L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn- L-Asp-L-Leu)] and a molecular weight of 1224.4 Da. 2. The bacterial strain Brevibacillus laterosporus VKPM B-8287, which has a wide spectrum of antagonistic action, is the producer of the antibiotic laterocidine.