RU2475541C1 - METHOD TO PRODUCE DEINOXANTHINE-CAROTENOID OF MICROORGANISM Deinococcus radiodurans - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention may be used to extract carotenoids, in particular, deinoxanthine, which is used to develop new antioxidant and radioprotector preparations to increase adaptation capabilities of humans and aminals, prevention and treatment of diseases. The method provides for extraction of carotenoids from the bacterial mass Deinococcus radiodurans by the mixture acetone : ethanol (at the 1:1 ratio). Separation of carotenoids with extraction of deinoxanthine on preparative columns of a liquid low-pressure chromatograph, where the sorbent is hydroxyapatite, and the eluent - ethanol.

EFFECT: invention makes it possible to increase quantity and quality of extracted deinoxanthine.

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Description

FIELD OF THE INVENTION

The invention relates to the field of carotenoid compounds produced by microbes and is directed to the isolation of carotenoids, in particular deinoxanthin, which can be used to develop new antioxidant and radioprotective drugs to enhance the adaptive capabilities of humans and animals, prevention and treatment of diseases, from the microorganism Deinococcus radiodurans.

State of the art

A known method of isolating a crystalline carotenoid compound from a microbial carotenoid-containing biomass (RU 2235783) (1), in which the microbial cell walls are destroyed in a microbial carotenoid-containing biomass, cell debris is separated in a microbial carotenoid-containing biomass from a carotenoid-containing lipid-containing solvent to remove a carotenoid-containing lipid-containing suspending the resulting carotenoid-containing residue in water to float the carotenoid compound , Separation of the crystalline carotenoid compound and further purification of the crystalline carotenoid compound. This method is intended for the isolation of carotene crystals from microorganisms containing carotenoids inside the cells in the form of crystals, but is unsuitable for the isolation of carotenoids contained in cells in a dissolved state.

The non-pathogenic microorganism Deinococcus radiodurans is known to be resistant to radiation doses that are ten times higher than lethal for the microorganisms studied to date. One of the characteristic biochemical features of this microorganism is the synthesis of carotenoids, in particular deinoxanthin, which gives the colonies a characteristic pink-orange color. This substance has a uniquely high antioxidant activity. Deinoxanthin is capable of significantly more effective than β-carotene and α-tocopherol in intercepting singlet oxygen and hydroxyl radical (the most dangerous types of reactive oxygen species). This carotenoid is of particular interest from the point of view of developing new antioxidant and radioprotective drugs to increase the adaptive capabilities of humans and animals, and the prevention and treatment of diseases. In order to obtain the drug deinoxanthin it is necessary to solve a number of problems. First of all, it is the development of inexpensive methods for the separation of carotenoids Deinococcus radiodurans.

For the first time, deinoxanthin was isolated from acetone-methanol extracts of the microbacteria Deinococcus radiodurans (Lemee L, Peuchant E. and Clerc M. Deinoxanthin: A New Carotenoid Isolated from Deinococcus radiodurans // Tetrahedron. 1997. V.53. T. 3. 9260) (2). The method involves centrifugation and extraction of the bacterial mass of Deinococcus radiodurans with acetone-methanol mixture, vacuum evaporation of the solvent, re-extraction of the dry residue with ethyl acetate, followed by washing the ethyl acetate phase with water and drying it over magnesium sulfate. The primary extract is separated by flash chromatography on silica gel (liquid phase: acetone - petroleum ether - triethylamine; 20: 80: 1). The obtained fractions are checked by thin layer chromatography (TLC) and reverse phase (HPLC). The fraction containing the target component is then separated using semi-preparative reverse phase chromatography. Final purification of the drug is carried out using conventional semi-preparative HPLC. The disadvantages of this method are the complexity, duration, multi-stage, which entails the loss of carotenoids, the need to acquire sophisticated expensive equipment, the use of large quantities of expensive reagents and, as a consequence, uneconomical.

Closest to the proposed invention is a method for the isolation of deinoxanthin (Lysenko BC et al. Separation and mass spectrometric identification of carotenoids of radioresistant bacteria Deinococcus radiodurans. - Mass Spectrometry Journal, 2010, v. 7 No. 4, p. 277-273) (3) adopted by the authors of the present invention as a prototype in which a suspension of Deinococcus radiodurans microbacteria is extracted with hexane, then additionally extracted with acetonitrile, the biphasic mixture is settled, the upper hexane layer having a weak yellow color is discarded, the acetonitrile phase is evaporated in a stream of nitrogen to obtain a dry residue, which is re-extracted with anhydrous acetone. Samples are applied to preparative plates for thin layer chromatography, coated with a layer of silica gel with 2% starch. As a mobile liquid phase for thin-layer chromatography, a mixture of petroleum ether-chloroform-methanol (3: 1: 1) is used. After chromatography, the colored sections of the sorbent are separated from the plate, extracted with acetonitrile and analyzed on a mass spectrometer. The disadvantage of this method is the toxicity of the solvents used in the extraction and separation of carotenoids Deinococcus radiodurans, which entails undesirable consequences when using deinoxanthin as a pharmacological drug and as a dye for food production. To achieve the desired purity, the subsequent additional steps for purification of deinoxanthin are required, which can lead to losses and also significantly increase the cost of obtaining deinoxanthin. On the other hand, disposal of solvents leads to significant losses of deinoxanthin. In addition, thin-layer chromatography used in the known method, does not allow you to take for the separation of substances in preparative quantities. Typical amounts of separable substances for thin-layer chromatography are 10 ^-7 -10 ^{-2 -2} g, which is not enough for its practical use. Moreover, preparative plates for thin-layer chromatography are disposable, which makes the process more expensive.

Disclosure of invention

The objective of the invention is to improve the quality and quantity of deinoxanthin obtained, as well as reducing its losses during the isolation of microbacteria from biomass, by selecting a less toxic solvent, an optimal carrier and a more efficient chromatographic separation mode.

The technical result of the invention is to reduce the cost of the selected deinoxanthin, increase its quality and the possibility of obtaining in an amount sufficient for practical use.

The specified technical result is achieved by the fact that in the known method for the production of deinoxanthin - a carotenoid of the microorganism Deinococcus radiodurans, including extraction of carotenoids from a suspension of microbacteria Deinococcus radiodurans, separation of carotenoids on a sorbent and elution with the release of deinoxanthin, according to the invention, the extraction is carried out with the separation of carotenoide-ethanol with acetone-ethanol preparative columns of a low pressure liquid chromatograph, where hydroxyappatite is used as an adsorbent, and this is used as an eluent zero

The use of the low-toxic extractant and eluent in the proposed method improves the quality of the isolated deinoxanthin and reduces its losses in the production process.

Separation of carotenoids on preparative columns of a low-pressure liquid chromatograph allows one to take substances in larger quantities than in the prototype, which will significantly increase the amount of deinoxanthin isolated. (Typical amounts of separable substances for low pressure liquid chromatography are 10 ^-4 -10 ² g). In addition, preparative columns are reusable, which reduces the cost of the method.

It is not known to use acetone-ethanol and ethanol as solvents for the isolation of deinoxanthin from a suspension of Deinococcus radiodurans microbacteria, and the separation of carotenoids using preparative low-pressure liquid chromatography, where hydroxyappatite is used as a sorbent, which are the hallmarks of the proposed method.

Brief Description of the Drawings

Figure 1 shows a graph of the chromatographic profile of carotenoids Deinococcus radiodurans obtained by the claimed method (carrier - hydroxyappatite, eluent - ethanol).

Figure 2 shows a graph of the chromatographic profile of carotenoids Deinococcus radiodurans obtained by HPLC in the experiments of LeMee et al. (1997) (carrier — silica gel, eluent — acetone — petroleum ether — triethylamine; 20: 80: 1).

The implementation of the invention

The method is as follows:

The experiments were carried out on a low-pressure chromatograph <BioLogic LP Chromatographic System> of the company <Bio-Rad>. To obtain the bacterial mass of Deinococcus radiodurans for the extraction of carotenoids and their further separation, the non-pathogenic strain Deinococcus radiodurans VKPM B-8209, acquired at the All-Russian Collection of Industrial Microorganisms FSUE GosNII Genetika, was used. The culture of Deinococcus radiodurans VKPM 8209 was grown in 250 ml Erlenmeyer wide-necked flasks with 50 ml of TGY medium of the following composition (g / l): tryptone - 5.0; glucose - 1.0; yeast extract - 3.0; distilled water - 1.0 l. Flasks with crops were incubated on a circular shaker at 30 ° C and 150 rpm for 48 hours. The bacterial suspension was extracted with a mixture of acetone-ethanol (1: 1) - 3-6 ml per 1 g of fresh weight of bacteria with the release of carotenoids. Then, carotenoids were separated and eluted with a preparative version of deinoxanthin on a low-pressure chromatograph. Hydroxyappatite was used as a sorbent, 96% ethanol was used as an eluent. A series of experiments were conducted to select the optimal and least toxic solvents. Evaluation of the effectiveness of the solvent system was determined by the two dominant peaks of the chromatogram of hexane extract D. radiodurans as Rf1-Rf2, where Rf are the retention indices of the main (dominant) and second peak, respectively. The obtained dependence on the type of solvent is shown in Table 1.

Example 1

The selection of deinoxanthin from a suspension of bacteria Deinococcus radiodurans was carried out by the method described in the prototype. The bacterial suspension was centrifuged for 10 min at 2000 g, the precipitate was extracted with acetone - 10 ml per 1 g of wet weight of bacteria. The extract was mixed with saturated NaCl to an acetone concentration of 50 vol. %, 2 ml of hexane was added and mixed vigorously. The upper hexane layer separated after settling was removed from the mixture and centrifuged for 10 min at 3000 g. Further processing of the obtained hexane extract was carried out as follows. To 10 ml of the obtained hexane extract was added 2 ml of acetonitrile. The resulting two-phase mixture was shaken, transferred to a separatory funnel and left to stand for 30 minutes. The bottom layer (acetonitrile), painted intensely red, was separated and used for further analysis and purification. The upper hexane layer, having a faint yellow color, was discarded. The acetonitrile phase, containing a small admixture of water, was evaporated in a stream of nitrogen to obtain a dry residue, which was re-extracted with 50-100 μl of anhydrous acetone. The sample was applied to preparative plates coated with a layer of 0.5 mm thick silica gel with 2% starch. As the mobile liquid phase, a mixture of petroleum ether-chloroform-methanol (3: 1: 1) was used. After chromatography and drying, the plates were photographed. The resulting digital image files in JPEG format were analyzed using the ImageJ software package. The image was converted to the 8-bit BMP format, inverted by brightness, and the image area of interest was highlighted (the band corresponding to the chromatogram of an individual sample). A chromatographic profile and the corresponding numerical data file (* .xsl) were obtained. Then, the individual shared components were quantified by the peak area of the chromatographic profile using the previously obtained numerical data file and the Origin 8.0 software package. The data are given in table 1.

Example 2

Analogously to example 1, an experiment was carried out in which extraction with the release of carotenoids, separation of carotenoids and estimation of the relative yield of fractions was carried out in the same way as in example 1. Hexane extract was used, silica gel with 2% starch was used as a carrier, and the other eluent was PE chloroform-ethanol 3: 1: 1. The data are shown in table 1.

From table 1 it is seen that when the media is silica gel with 2% starch, the maximum separation efficiency was revealed in Example 1, where the PE-chloroform-methanol 3: 1: 1 system was used as a solvent. However, the solvents used in Example 1 are toxic, which requires additional purification steps to achieve the desired purity of deinoxanthin. It is uneconomical since requires additional costs. In addition, deinoxanthin loss is inevitable.

Example 3

Analogously to example 1, an experiment was conducted with an inexpensive and non-toxic solvent - 96% ethanol. Hexane extract was used. Silica gel with 2% starch was used as a carrier. Extraction with the release of carotenoids, separation of the carotenoids and estimation of the relative yield of fractions was carried out in the same way as in Example 1. The data obtained are shown in Table 1. The experimental results showed that the separation of D. radiodurans carotenoids did not occur. On the plate, all D. radiodurans carotenoids are recorded as a single colored spot. Therefore, deinoxanthin was not isolated. That is, on silica gel, using ethanol as an eluent, separation of the extract and isolation of deinoxanthin does not occur.

Example 4

Studies were conducted in which the low-toxic extractant acetone-ethanol (1: 1) and the eluent ethanol 96% were used, and hydroxyappatite was used as the sorbent. The bacterial suspension was centrifuged for 10 min at 2000 g, the precipitate was extracted with a mixture of acetone-ethanol (1: 1) - 3-6 ml per 1 g of fresh weight of bacteria. The extract was centrifuged for 10 min at 3000 g. Next, column chromatography was performed on a <Biologic LP> low pressure chromatograph (<Bio-Rad>). Chromatographic separation conditions: column — diameter 2 cm, length 3 cm, eluent flow — 0.7 ml / min (0.2-0.3 ml / min per 1 cm ² ). The application of the bacterial mass extract Deinococcus radiodurans on a column of this size is 3-5 ml. Chromatography was carried out in preparative form. For chromatography, hydroxyappatite was used as the sorbent, and ethanol 96% was used as the eluent.

Then, the chromatogram of the isolated carotenoids was analyzed by thin layer chromatography (TLC) in a preparative version. The results of the analysis showed that the carotenoids of Deinococcus radiodurans were divided into two fractions during chromatography - in the first, predominantly deinoxanthin, the second - the remaining carotenoids and decay products of deinoxanthin. In the first fraction, one main spot corresponding to deinoxanthin and a small amount of colored minor components are recorded on the plate; in the second fraction - a lot of colored spots containing other carotenoids, lipids and their decay products. The data obtained are shown in table 1.

Table 1 Separation efficiency of D.radiodurans carotenoids in various solvents and carriers. No. / No. Carrier Solvent Separation efficiency Rf dominant component one silica gel with 2% starch PE-chloroform-methanol 3: 1: 1 0.15 0.36 2 silica gel with 2% starch PE-chloroform-ethanol 3: 1: 1 0,07 0.27 3 silica gel with 2% starch Ethanol 0 0.18 four hydroxyappatite Ethanol 0.14 0.13 ¹ - Preparative

From table 1 it is seen that when the carrier is hydroxyappatite, the separation efficiency is approximately the same as in the PE-chloroform-methanol 3: 1: 1 system. However, in Example 4, a low-toxic estrogen - acetone-ethanol (1: 1) was used to extract a suspension of bacteria, and then 96% was used to separate the isolated carotenoids - solvent - ethanol. Deinoxanthin isolated in this way does not require additional purification from toxic substances, which reduces its losses, and also reduces the cost of its production.

After separation of the carotenoids, the column was regenerated as follows: washing - 3-5 volumes of deionized water, regeneration - 5-7 volumes of 500 mM sodium phosphate buffer, again 3-5 volumes of deionized water, then the column was balanced with 96% ethanol.

The chromatogram of carotenoids on TLC plates of Example 4 after separation on hydroxyappatite (FIG. 1) basically coincided with the chromatogram obtained by HPLC in LeMee experiments (FIG. 2).

However, the proposed method allows a simpler and cheaper way to isolate deinoxanthin from Deinococcus radiodurans.

This is due to the fact that hydroxyappatite column chromatography uses a cheap low-toxic solvent (ethanol), which allows the use of an isolated deinoxanthin preparation without further purification, which is more cost-effective.

The separation of carotenoids is carried out on preparative columns, the sizes of which can be significantly varied. Accordingly, both the cross-sectional area of the sorbent and the amount of substance passed through the column will change, which will significantly increase the amount of deinoxanthin released. Preparative columns after each use are regenerated for their further use.

The developed method for the isolation of deinoxanthin allows providing, in comparison with known methods, a more cost-effective production of preparative amounts of deinoxanthin without the use of toxic organic solvents in sufficient quantities for further pharmacological studies and its practical application.

Information sources

1. RF patent No. 2235783, IPC ⁷ С12Р 23/00, С12N 1/14, С07С 403/24.

2. Lemee L, Peuchant E. and Clerc M. Deinoxanthin: A New Carotenoid Isolated from Deinococcus radiodurans // Tetrahedron. 1997. V. 53. Number 3. P.919-9260.

3. Lysenko B.C. et al. Separation and mass spectrometric identification of carotenoids of radioresistant bacteria Deinococcus radiodurans. - Journal of Mass Spectrometry, 2010, v. 7. No. 4, p. 278-273 (prototype).

Claims (1)

A method of producing deinoxanthin - a carotenoid of the microorganism Deinococcus radiodurans, including extraction of carotenoids from the bacterial mass of Deinococcus radiodurans, separation of carotenoids on a sorbent and elution with the release of deinoxanthin, characterized in that the extraction of carotenoids is carried out with an acetone-ethanol mixture (1: 1), columns of a low pressure liquid chromatograph, in which hydroxyappatite is used as the sorbent, and ethanol is used as the eluent.

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