Bacterial peptide antibiotic laterocin for inhibiting growth of microalgae

FIELD: chemistry.

SUBSTANCE: described is a novel cyclodecapeptide antibiotic laterocin, produced by the Brevibacillus laterosporus VKPM V-10531 strain. Laterocin is a white amorphous solid substance of formula [cyclo-(L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Met-)] and molecular weight 1240.6 Da. Laterocin has algicidal (antagonistic) effect on various types of microalgae, particularly blue-green algae Nostoc, Anabaena, Microcystis aeruginosa.

EFFECT: biologically active compound laterocin can be used as an agent for inhibiting growth of blue-green microalgae.

3 tbl, 8 ex

 

The invention relates to Microbiology and biochemistry, in particular to the selection of biologically active compounds - antibiotics to fight microscopic algae.

Microscopic algae are under intensive development pose serious economic and social problems. Economic aspects associated with the pollution of natural waters and reservoirs that provide drinking water to the population. A consequence of the development of energy and industry in recent decades has been the creation of a large number of artificial and technological reservoirs (reservoirs and ponds), and water supply systems of industrial enterprises. The massive increase in the number of algae in reservoirs leads to adverse consequences such as "blooming" water [1] and overgrowing algae water tanks used in the metallurgical industry and energy, as well as fouling of the process equipment in water tanks.

Social aspects caused by the release of certain types of microscopic algae in the course of their life connections with fetid odor, in particular of geosmin, which greatly reduces the quality of drinking water and recreational attractiveness of natural water bodies [2]. Microscopic algae, particularly blue-green in the Doros (cyanobacteria), produce toxins that, when you die, cyanobacteria are released into the aquatic environment. Describes several types of toxins - toxins, neurotoxins, hepatotoxins. The level of toxicity of these substances is extremely high. So, microcystin LR toxicity of cyanide in 200 time [3]. Neurotoxins block the nervous and muscular system and can lead to changes in the respiratory system. The hepatotoxins poison the liver and other internal organs, which often leads to death. Toxins can be produced and stored in water for several weeks. They can also accumulate in fish, molluscs, crustaceans. The concentration of toxins that causes 50%death of animals, ranging from 15 mg/kg to 150 mg/kg of animal weight. Antidotes to the toxins of cyanobacteria does not currently identified. Blue-green bacteria cause a number of allergic diseases. Therefore, at high level "blooming" water is an undesirable use of water bodies for recreational purposes, i.e. you need to a minimum to limit the time spent by humans and domestic animals in such water. The concentration of toxin microcystin in recreational water should not exceed 20 ág/l

Excessive development of cyanobacteria is accompanied by the release into water of a number of toxic compounds (phenols, indole, skatole), and sensory manifestations is niania, in particular the products of geosmin. A high number of cells of algae in their death can lead to hypoxia and death of many aquatic organisms.

Serious economic and medico-social problems caused by algae, determine the need for research and development of methods for the suppression of the development of microscopic algae and forecasting the condition of aquatic ecosystems.

Currently, there are various physical, chemical and biological means of suppressing the development of microalgae. Despite the effectiveness of chemical algaecide compounds in laboratory conditions, the mass use of their in vivo is limited by Toxicological and hygienic requirements. In addition, chemicals are not selective action and have a number of adverse biological effects (lethal, mutagenic, teratogenic, and so on) on plant and animal organisms. The use of chemicals for treatment of water bodies is limited by health and safety standards in connection with the danger to humans and animals and also adversely affects the habitability of other aquatic (aquatic organisms).

Used physico-chemical methods of pest algae - water releases from reservoirs with subsequent mechanical deletion is of the biomass, aeration huge bodies of water, use of chemicals and substances coagulants, the use of ultraviolet irradiation and ultrasound is ineffective and is associated with high financial costs. Physical control methods of microalgae is aimed at creating conditions or preventing the development of algae or destroy already formed community of algae, the so-called mats. Ultrasonic treatment of "blooming" water though and is quite effective uliginosa action, leads to undesirable consequences. Processing "blooming" water ultrasound reduces the pH of the water and the amount of total nitrogen and phosphorus in the water, raise the water temperature. Alternative chemical and physical means to suppress the development of algae in the aquatic environment is the use of biological means on the basis of bacteria and their metabolites with algicidal properties. Known strains of bacteria that suppress the development of microalgae [4-11].

As the closest analogue of the claimed object is considered tionalities (algaecide) connection elemicin And synthesized by the bacterium Sphingomonas sp. M-17 [12]. Elemicin And causes suppression of photosynthesis.

The objectives of the claimed invention:

provide new tionalities connection, producere the CSOs strain of bacteria Brevibacillus laterosporus In-10531;

characterisation of the chemical structure and physical properties of new tionalities connection.

Declare us tionalities connection, called by us lateration, is not known from the information sources. Literacy suppresses the development of blue-green algae. Literacy 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-Met-)] and the molecular weight 1240,6 Yes. Literacy soluble in methanol, ethanol and other lower alcohols, insoluble in water.

Declare tionalities compound isolated from natural strain of Brevibacillus laterosporus obtained by multistage selection and deposited in Russian national collection of industrial microorganisms under the number VKPM B-10531. The strain forms a rounded colonies with a diameter of 3-5 mm with a smooth surface light cream color and rough edges.

Biological connection laterals claimed as a factor having algaecide (antagonistic) effect on different species of microscopic algae.

The invention is illustrated by the following examples:

Example 1. Cultivation of the strain VKPM B-10531 producer literocia

For cultivation of strain In-10531 use environment NBY [13]. The medium prepared with distilled water, adjusted pH to 7.0; poured in a flask of 50 ml and female sterilization is t at a pressure of 0.8 ATM, the temperature of 120°C for 30 minutes After sterilization, the flasks contained in the medium is cooled to 20°C. Sterility and pH of the medium is controlled by selection of appropriate samples. As inoculum using 5 ml of culture In PMBC-10531, obtained by cultivation in medium NBY 16 hours at 30°C with shaking 250 rpm No extraneous microflora controlled microscopically. The productivity of the strain is determined by the serial dilution method followed by plating on agar medium.

Example 2. Cultivation of microscopic algae

Use strains of nitrogen-fixing filamentous species of microscopic algae cyanobacteria: Anabaena sp. 5781 - obtained from the Microbiology Department of the Leningrad state University; Nostoc sp. A-10 is obtained from the collection of IMET, Jena, GDR. The strains do not fix nitrogen unicellular cyanobacteria Microcystis aeruginosa 562 and 905 obtained from the Institute of Hydrobiology of the Chinese Academy of Sciences (Hubei province, Wuhan).

The strains of the cyanobacteria Anabaena and Nostoc are grown in modified medium BG-11 [14]. Strains of Microcystis aeruginosa 905 562 and grow in the environment-12

[15]. Microalgae cultured in flat-bottomed glass flasks with a capacity of 250 ml per 100 ml of medium without aeration at a temperature of 20°C and round-the-clock coverage for two weeks. Use fluorescent lights fluorescent what about the white light the LIGHT FOREHEAD-30, which provide illumination of 40 µmol quanta·m-2·with-1in the field of LIGHT (photosynthetically active radiation).

Example 3. Isolation and purification of literocia from the strain of Brevibacillus laterosporus In-10531

The strain of Brevibacillus laterosporus In-10531 cultivated in a liquid nutrient medium NBY 90 hours, as described above. After culturing cells bacteria Brevibacillus laterosporus separated by centrifugation at high speed centrifuge with a speed of 10,000 rpm for 50 min at 4°C. To the precipitate was added to a cold (5°C) absolute alcohol from a rate of 6 ml/g of biomass and leave for days in the refrigerator. Then alcohol extract is separated from the biomass with a Buechner funnel, using a filter with a pore size of 0.2 μm, and subjected to fractionation using reversed-phase high-performance liquid chromatography on a column of Luna 5u C5, 250×10 mm, equilibrated with solution A (5% acetonitrile, 60% methanol)in a linear gradient of solution B (22,5% acetonitrile, 75% methanol) from 0 to 100% over 40 min (2,5% min-1) at a flow rate of 1.6 ml/min Detection is carried out at a wavelength of 214 nm, applied to the column at 0.5 ml alcoholic extract. Aliquots of the obtained fractions are tested for tionalities activity by the method of joint incubation. In the chromatographic separation on a column of Luna 5u C5 obtain an active fraction, which is corresponds to the main peak, extending from the column with acetonitrile 17.5% and methanol to 70.5%. The active fraction is twice subjected to rechromatography on the same column, equilibrated with solution A. the First rechromatography carried out in a linear gradient of solution B from 30% to 100% within 40 minutes (1,75% min-1) at a flow rate of 1.6 ml/min Peak, containing the active fraction goes from the column with acetonitrile 15.5% acetonitrile and methanol 69%. The second rechromatography carried out in isocratic mode on a column equilibrated with a mixture of solutions a and B in the ratio of 40:60 by volume. The rate of elution of 1.6 ml/min Peptide literacy with tionalities activity eluted from the column on the 15th minute. The individuality of the selected peptide literocia confirmed by mass spectrometry and NMR spectroscopy.

Example 4. The structure definition of literocia

To define the structure of the selected antibiotic used method of NMR spectroscopy. 1.0 mg of peptide literocia dissolved in 450 μl of methanol (pH of 6.2). The NMR spectra of peptide receive at a temperature of 33°C on a Bruker spectrometer AvanceIII-600 (Germany) with an operating frequency of 600 MHz. For the measurement of NMR spectra using the sensor triple resonance (1H,13C,15N) with cryogenic cooled1N coil (Bruker, Germany). Based on the analysis of two-dimensional1H-NMR spectra COY, TOCSY, NOESY and ROESY using standard procedures [16] carried out a full assignment of the signals of nuclei1N sample. Based on the analysis of two-dimensional13C-NMR HSQC spectra and NMFS spend a full assignment of the signals13With the nuclei of the sample. Received the assignment corresponds to the amino acid sequence of literocia: [cyclo-(L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Met-)].

After an analysis of databases of amino acid sequences revealed that the above structure is not described previously, and allocated us cyclodepsipeptide is a new antibiotic. This peptide, called by us lateration and with tionalities activity, belongs to the family tyrocidine antibiotics.

Example 5. Determination of the molecular mass of the peptide literocia and confirmation of its structure by the method of mass spectrometry

Mass spectra get on a hybrid mass spectrometer LTQ FT (Thermo Electron, Germany), which consists of a linear quadrupole ion trap mass spectrometer ion cyclotron resonance Fourier transform (DDI PF). As an ion source using a universal source of ionization Finnigan Ion Max Source (Thermo Electron, Germany) in mode elektrorazpredelenie. For peptide literocia see a singly charged ion with m/z 1241,61 corresponding to the protonated molecular ion (MH) +and monoisotopic molecular mass of the peptide 1240,60±0,01 Yes. The resulting mass with regard to the accuracy of measurement coincides with the calculated monoisotopic molecular mass of the peptide lateritia (1240,60 Yes). Mass spectrometric analysis confirms the claimed structure of the peptide literocia.

Example 6. The definition of the spectrum tionalities activity literocia

Tionalities activity literocia identify when mixed incubation of the peptide with a two-week cultures of microalgae. Measure the optical density of the mixture on the spectrophotometer at a wavelength of 590 nm at zero time and after incubation at room temperature, and round-the-clock coverage. Tionalities activity judged by the drop in optical density after 24 hours of incubation. Determine the residual optical density according to the formula: (ODAbout), as (soT/OPN)×100, where ODN- the initial OP, OPTOP after incubation for T hours, respectively. Literacy causes a drop in optical density in the incubation mixtures (table 1). When optical microscopy revealed lysis of the cells of cyanobacteria.

Table 1.
Tionalities activity literocia.
Sample of blue-green algaeThe residual optical density after 24 hours (%)
Microalgae
NostocAnabaenaMicrocystis aeruginosa
Without adding literocia100100100
With the addition of literocia23,5028,6035,25

Example 7. The determination of minimum tionalities concentration literocia method of measuring intensities of fluorescence of chlorophyll in suspensions of microalgae

Check intensities of chlorophyll fluorescence is performed on fluorometer with amplitude modulation of the exciting light MEGA-25". The detector fluorescence in this device use a photomultiplier tube (PMT) R7400U-20 firm "HAMAMATSU". Before PMT set boundary filter COP 18, which allows to detect the chlorophyll fluorescence with a wavelength of about 680 nm. For exceptions invalid blowout PMT device provided with a shutter, automatically closing PMT when opening the camera. The fluorescence intensities of chlorine is Filla with open (F 0) and closed (Fm) reaction centers of photosystem 2 is carried out at excitation light with a power density of 0.8 µmol·m-2·with-1and 6000 µmol·m-2·with-1respectively [17, 18].

The ratio (Fm-Fo)/Fm (relative variable fluorescence) is equal to the potential efficiency of light energy RC FS 2 and is a dimensionless quantity, is capable of acquiring values from 0 to 0.84 depending on the state of the photosynthetic apparatus of plant object. The relative variable fluorescence can be defined for any plant organisms and does not depend on the content of chlorophyll in the sample under investigation.

To assess the minimum tionalities concentration literocia use a method of measuring the efficiency of use of light energy RC FS 2 chlorophyll-fluorescence. Measuring intensities of fluorescence of chlorophyll spend on fluorometer with amplitude modulation of the exciting light.

Fluorescence was determined by the photoelectric multiplier through the edge filter that transmits light with a wavelength of more than 680 nm. The intensity of chlorophyll fluorescence with open (F0) and closed (Fm) reaction centers of photosystem 2 was performed with excitation light with a power density of 0.8 µmol · m-2· with-1and 6000 µmol · m2 · with-1respectively.

Table 2.
The influence of literocia on the energy efficiency of light RC FS 2 on the suspension of cyanobacteria Nostoc, as measured by the value of the variable chlorophyll fluorescence (Fm-Fo)/Fm.
Concentration
tion literocia, ug/ml
The time of incubation, h
00,250,6611,663524
Control Nostoc sp.0,510,50,510,50,50,510,50,53
40,510,50,50,460,50,50,50,54
10 0,510,490,480,480,450,470,440,48
400,490,470,440,430,370,350,30,35
1000,460,460,390,360,280,210,220,15

From table 2 it follows that literacy inhibits the photosynthesis of cyanobacteria in concentrations higher than 10 µg/ml.

Example 8. The determination of minimum tionalities concentration literocia when the registration of the absorption spectra of suspensions of microalgae

The registration of the absorption spectra of suspensions of cyanobacteria is carried out in the range from 350 to 850 nm for single-beam spectrophotometer with integrating sphere-based spectrometer (USB 2000 (Ocean Optics, USA) in quartz cuvettes with optical path length of 1 cm [19]. The destruction of phycobilins cyanobacteria is determined by the ratio of the absorption at 630 nm, the corresponding maximum absorption of phycocyanin, the absorption at 680 nm corresponding to the absorption of chlorophyll.

Table 3.
The influence of literocia on the destruction of phycobilins suspension cyanobacteria Nostoc, as measured by the ratio of optical density at 630 nm and 680 nm.
The concentration of literocia, ug/mlThe time of incubation, h
01,3335
Control
Nostoc
1,1631,1631,1631,163
41,163n/a1,1521,150
101,1631,1351,1211,094
401,1631,0320,9680,902
1001,1630,9620,8540,747

From table 3 it follows that the action of literocia leads to the destruction of phycobilins cyanobacteria in concentrations higher than 10 µg/ml.

Cyclodepsipeptides antibiotic literacy isolated from a strain of Brevibacillus laterosporus In-10531 with tionalities activity against blue-green microalgae and having the primary structure of [cyclo-(L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Met-)] and the molecular weight 1240,60 Yes.



 

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