RU2563349C9 - Bacteriorhodopsin obtaining method - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: bacteriorhodopsin obtaining method provides for growth in a fermenter of Halobacterium salinarum VKPM V-11953 strain during six days with further separation of biomass by centrifugation and its destruction at incubation in distilled water during one night at constant stirring. The deposit is separated by centrifugation so that a supernatant is obtained. The obtained supernatant is centrifuged and rejected. Distilled water is added to the obtained deposit in the same volume; the deposit is suspended in water for 3-4 times. After that, the deposit is applied onto a chromatographic column with silica gel based on 200 optic units in an application specimen at wave length of 280 nm per 100 ml of silica gel. After elution is completed, specimens are taken with the ratio of optic density of the solution at wave length of 280 nm to optic density of the solution at wave length of 570 nm (D280\D570) of less than 2.5.

EFFECT: invention allows increasing yield of bacteriorhodopsin.

1 tbl, 1 ex

Description

Technical field

The invention relates to the microbiological industry, microbiology and biotechnology, in particular to the production of physiologically active compounds, and for the production of protein, bacteriorhodopsin, by microbiological synthesis.

The area of possible use of bacteriorhodopsin is striking in its diversity. It includes double-sided holographic memory, ultra-fast random access memory, spatial light modulation, nonlinear optical filters, recognition systems, high-contrast displays, optical switches and picosecond detectors. Bacteriorhodopsin is used in the production of materials that protect securities from counterfeiting, and also as an antioxidant in medicine, pharmaceuticals, cosmetology and agriculture as a plant growth stimulator [1-10].

State of the art

The prior art method for producing bacteriorhodopsin by growing the bacterial strain Halobacterium salinarum VKPM B-9025 for 6 days, after which the biomass is separated by centrifugation at 7000 g and incubated in distilled water, the precipitate is discarded, and the resulting supernatant is centrifuged at 50,000 g, the supernatant is discarded, and distilled water is poured into the obtained precipitate in the same volume, the precipitate is suspended several times until the ratio of the optical density of the solution is restored in the suspended precipitate with a length waves of 280 nm to the optical density of the solution at a wavelength of 570 nm (D ₂₈₀ / D ₅₇₀ ) less than 2.5, then the supernatant is standardized to a concentration of bacteriorhodopsin from 10 ^-9 M to 10 ^-6 M [1]. The disadvantage of this method is the relatively low yield of bacteriorhodopsin.

Closest to the claimed invention in terms of essential features is a method for producing a preparation based on the biomass of bacteria Halobacterium salinarum, in which a strain of halophilic bacteria Halobacterium salinarum VKPM B-9025 is grown in flasks with a capacity of 750 ml in a volume of a growth medium of 300 ml when illuminated with fluorescent lamps up to daylight stationary growth stage on a circular shaker at 37 ° C and shaking 100 rpm. After 6 days, the precipitate was separated by centrifugation at 7000 g for 10 min and the resulting biomass was weighed.

Then, distilled water in a volume of 300 ml was added to the precipitate (20 g) and incubated with stirring at room temperature for 16 hours. After centrifugation at 7000 g for 10 min, the resulting preparation is standardized by the content of bacteriorhodopsin in the supernatant, the main component of cell membranes, the amount of which is determined at a wavelength of 570 nm [2].

The disadvantage of this method is the low yield of bacteriorhodopsin.

Disclosure of invention

The problem to which the invention is directed is the creation of a method that allows to obtain a high yield of bacteriorhodopsin.

The technical result achieved by the implementation of the claimed invention is to increase the yield of bacteriorhodopsin.

The specified technical result is achieved due to the fact that the method of producing bacteriorhodopsin includes growing in a 10 liter fermenter for 6 days a strain of Halobacterium salinarum VKPM B-11953, separating the biomass by centrifugation at 7000 g and destroying it by incubation in distilled water overnight and constant stirring, separating the precipitate by centrifugation at 10,000 g for 15 minutes, centrifuging the resulting supernatant at 50,000 g for 55 minutes, treating the resulting precipitate with distilled water in the same volume 3-4 times and chromatographing ue to silica gel column rate of 200 absorbance units in the sample application at a wavelength of 280 nm to 100 ml of silica gel, elution and sampling with a solution absorbance ratio at 280 nm to the optical density of the solution at a wavelength of 570 nm (D ₂₈₀ / D ₅₇₀ ) less than 2.5.

The implementation of the method for producing bacteriorhodopsin is illustrated by the following example.

Example 1

The strain VKPM B-11953, as well as the strain VKPM B-9025 (prototype) used as a control, were grown in a BioFlo 110 fermenter (New Brunswick Scientific, USA) in a volume of a 10 l culture medium. For growth, we used a medium of the following composition (wt.%): Peptone - 1, yeast extract - 0.5, NaCl - 25, MgSO ₄ - 2, KCl - 0.2, sodium citrate - 0.3, glycerol - 0.1 , CaCl ₂ - 0.02, water - the rest, the pH of the medium is 7.2-7.4. In the fermenter, the temperature was maintained at 36.8-37.2 ° C. Air consumption for aeration 2.5 dm ³ / min. The studied strains were grown under fluorescent daylight (L 18 W / 530, Osram, PRC) for 6 days.

After fermentation, after 6 days, the separation and destruction of biomass was carried out similarly to the method described in [1], namely, the precipitate was separated by centrifugation at 7000 g for 10 min and the resulting biomass was weighed. Then, distilled water in a volume of 300 ml was added to the precipitate per 20 g and incubated with stirring at room temperature for 16 hours.

The precipitate was separated by centrifugation at 10,000 g for 15 minutes and discarded, and the obtained supernatant was centrifuged at 50,000 g for 55 minutes, the supernatant was discarded, distilled water was added to the same precipitate in the same volume, the precipitate was suspended 3-4 times, after which chromatography was performed on a column with silica gel balanced with 0.01 M phosphate buffer pH 7.0. The precipitate was deposited on a chromatographic column with silica gel at the rate of 200 optical units in the sample at a wavelength of 280 nm per 100 ml of silica gel [11]. Elution was performed with the same buffer. Before chromatography, 0.1 M phosphate buffer pH 7.0 was added to the final sample to a final concentration in the sample of 0.01 M.

After elution, samples were taken with a ratio of the optical density of the solution at a wavelength of 280 nm to the optical density of the solution at a wavelength of 570 nm (D ₂₈₀ / D ₅₇₀ ) less than 2.5.

The ratio of optical densities at wavelengths of 280 and 570 is independent of protein concentration and serves as a criterion for its purity. Depending on the method of purification, as well as the purpose of using the drug, this ratio should be within 1.9-2.4 according to the source of information RUSSIAN CHEMICAL MAGAZINE (Journal of the Russian Chemical Society named after DI Mendeleev), 2006, v. 1, No. 5, p. 25-36. Grebennikov E.P. "Bacteriorhodopsin is a biological light energy converter with unique technological capabilities."

In the selected samples, the amount of bacteriorhodopsin was determined at a wavelength of 570 nm. The concentration of bacteriorhodopsin was determined by the following formula:

Where

D is the optical density of the solution at a wavelength of 570 nm;

M _r is the molecular weight of bacteriorhodopsin (26700);

V _p-pa is the total volume of the bacteriorhodopsin solution;

V _cuv is the volume of a solution of bacteriorhodopsin in a spectrophotometric cuvette;

E _br - the molar absorption coefficient of bacteriorhodopsin (63000 M ^-1 cm ^-1 );

V _sample - the sample volume of bacteriorhodopsin in a spectrophotometric cell.

The data obtained are presented in table 1.

From the results shown in table 1, it can be seen that the inventive method for producing bacteriorhodopsin using the bacterial strain Halobacterium salinarum VKPM B-11953 gives a higher level of bacteriorhodopsin compared to the method based on the use of the bacterial strain Halobacterium salinarum VKPM B-9025.

INFORMATION SOURCES

1. RF patent N 2416634, published on 04/20/2011.

2. RF patent N 2307506, published on 10/10/2007.

3. Skladnev D., Tyurin S., Gricevich J. "Plant-growth-promoting effects of biomass lysate of selected agronomically important strain Halobacterium salinarum." Abst. XI International Congress PHYTOPHARM-2007 "Actual problems of creation of new medicinal preparations of natural origin" Leiden, The Netherlands, 2007. - P. 18.

4. Tyurin C.A., Skladnev D.A., Khodonov A.A. “The retinal of bacteriorhodopsin as an immobilized phytohormone.” Poster abstracts. III Russian Symposium "Proteins and Peptides." - Pushchino. - 2007 .-- S. 94.

5. Tyurin S.A., Khodonov A.A., Skladnev D.A. "Unfolded bacteriorhodopsin become a phytohormone" Abstracts 3rd International Meeting "Molecular Simulation Studies in Material and Biological Sciences", September 10-12, JINR, Dubna, Russia, 2008. - P. 36, 37.

6. S.A. Tyurin, Yu.G. Gritsevich, D.A. Skladnev, A.A. Hodonov. "Bacteriorhodopsin as a stimulator of plant growth and development." Agrochemistry. - 2009. - No. 6. - S. 32-39.

7. Korotkov A.V., Prusakova L.D., Fesenko A.N., Gritsevich Yu.G., Tyurin S.A. "A method of growing buckwheat." Joint Scientific Journal. - 2010. - No. 3. - S. 66-69.

8. Alexander KOROTKOV, Alexey FESENKO, Sergey TYURIN, Yuliy GRITSEVICH, Lidiya PRUSAKOVA. “Effect of Bacteriorhodopsin on the growth and development of buckwheat” Advances in buckwheat research. Proceedings of the 11th International Symposium on Buckwheat. Orel, Russia, 2010. P. 670-674.

9. A.V. Korotkov, L.D. Prusakova, S.L. Belopukhov, A.N. Fesenko, S.A. Tyurin, Yu.G. Gritsevich. "The effect of lurastim and bacteriorhodopsin on the yield and quality of buckwheat grain." Proceedings of the TSHA, 2011, issue 1, p. 118-123.

10. Osterman L.A. "Chromatography of proteins and nucleic acids." Moscow, Nauka Publishing House, 1985, p. 58-62.

Claims (1)

A method for producing bacteriorhodopsin, including growing in a 10-liter fermenter for 6 days a strain of Halobacterium salinarum VKPM B-11953, separating the biomass by centrifugation at 7000 g and destroying it by incubation in distilled water overnight and stirring continuously, separating the precipitate by centrifugation at 10000 g 15 min, centrifugation of the obtained supernatant at 50,000 g 55 min, processing of the obtained precipitate with distilled water in the same volume 3-4 times, column chromatography on silica gel based on 200 optical units per application sample at a wavelength of 280 nm per 100 ml of silica gel, elution and sampling with a ratio of the optical density of the solution at a wavelength of 280 nm to the optical density of the solution at a wavelength of 570 nm (D ₂₈₀ / D ₅₇₀ ) less than 2.5.