RU2321627C1 - Microorganism strain halobacterium salinarum as producer of bacteriorhodopsin - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: invention relates to microorganism strain Halobacterium salinarum VPKM B-9451 isolated by step-by-step selection of microorganism strain Halobacterium salinarum VPKM B-9025. Proposed strain is characterized by enhanced level of synthesis of bacteriorhodopsin. Synthesis of bacteriorhodopsin is estimated to be 50-112 mg/l of cultural fluid.

EFFECT: valuable properties of strain, enhanced synthesis of bacteriorhodopsin.

3 tbl, 3 ex

Description

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 topographic 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, as well as an antioxidant in medicine, pharmaceuticals, cosmetology and agriculture as a plant growth stimulator [1].

The most studied and closest to practical use is bacteriorhodopsin, a retinal-containing transmembrane protein of the purple membranes of halophilic bacteria of the genus Halobacterium. [2].

A known strain of Halobacterium halobium CMPM B-1739, capable of synthesizing bacteriorhodopsin in an amount up to 52 mg / l (2.5 μmol / l) of the culture fluid [3].

Among strains of Halobacterium salinarum, the most widely studied strain is D96N (VKPM B-9026) [5-13]. It synthesizes bacteriorhodopsin in an amount up to 47 mg / L (2.3 μmol / L) of the culture fluid and is selected as the closest analogue [4].

The task of the invention is to obtain a strain of Halobacterium salinarum with an increased level of bacteriorhodopsin synthesis.

The problem was solved by obtaining a bacterial strain Halobacterium salinarum ST 1, deposited in the All-Russian collection of industrial microorganisms (VKPM) as Halobacterium salinarum VKPM B-9451 and capable of synthesizing bacteriorhodopsin in an amount of 50-112 mg / l of culture fluid. The inventive strain was obtained by multistage selection of a halophilic bacteria strain Halobacterium salinarum VKPM B-9025, without preliminary treatment with chemical reagents.

The strain VKPM B-9451 has the following characteristics:

Cultural and morphological characters.

The size of the cells of a three-day culture is 2-5 × 0.5-0.9 μm, single sticks, sometimes 2-3 sticks are combined.

Moved, maximum mobility on 4 days of growth. It does not form a dispute; it does not contain gas vacuoles.

Colonies are translucent, smooth with a smooth edge, dark red in color, do not grow into the environment, are easily removed by a loop. The culture is an extreme halophile - it requires the presence of 25% NaCl in the medium. When the content of sodium chloride is below 20%, the cells lose their color, and when they enter the medium with a NaCl content of less than 15%, the cells are very quickly lysed.

It grows well at 36-40 ° C, in a salt liquid medium having the 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. as well as salt agar medium having the composition (wt.%): peptone - 1, yeast extract - 0.5, NaCl - 25, MgSO ₄ - 2, KCl - 0.2, sodium citrate - 0.3, CaCl ₂ - 0.02, agar - 1.5, water - the rest, the pH of the medium is 7.2-7.4.

Relation to temperature: grows well at 36-40 ° C, the optimum temperature is 38 ° C.

Relation to pH: grows well at 7.2-7.4, optimum pH is 7.3.

Physiological and biochemical characteristics.

Optional aerob, prototroph, sucrose, maltose, lactose, fructose does not acidify, does not grow on potatoes. It does not dilute gelatin. No antagonistic properties were found.

Like all representatives of the genus Halobacterium, the claimed strain is non-pathogenic and can be assigned to safety group No. 1 (based on the conclusion of the ATCC Collection). Working with it does not require special precautions.

It is not a genetically modified strain.

Strain of the strain: It is well stored on an agarized medium in back prints and on the jambs in a refrigerator for a year. For long-term storage of the strain, lyophilization of fresh biomass is used.

The invention is illustrated by the following examples.

EXAMPLE 1. The productivity of the strain of Halobacterium salinarum VKPM B-9451 when grown in flasks.

The inventive strain VKPM B-9451, as well as the control strains VKPM B-9025, VKPM B-9026 and TsMPM V-1739 were grown in 750 ml flasks in a volume of 300 ml growth medium under fluorescent light (L 18 W / 530, Osram, Germany) to a stationary growth stage (6 days) on a circular rocking chair at 37 ° C and shaking 100 rpm. 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.

The precipitate was separated by centrifugation at 7000 g for 10 min and the resulting biomass was weighed.

Then, distilled water was added to the precipitate in an amount equal to the volume of the growth medium, and incubated with shaking at room temperature for 3 hours.

The precipitate was separated by centrifugation, the supernatant was used to determine the amount of bacteriorhodopsin 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.

Table 1. The studied strains. Biomass Weight (g / L) The concentration of bacteriorhodopsin (mg / l) VKPM B-9451 16 fifty VKPM B-9025 6 eighteen VKPM B-9026 3 22 TsMPM V-1739 9 thirty

From the results shown in table 1, it can be seen that the inventive strain VKPM B-9451 significantly exceeds the parent strain VKPM B-9025, the closest analogue of VKPM B-9026 and the control CMPM B-1739 both in the amount of biomass and in the concentration of bacteriorhodopsin in the culture medium.

EXAMPLE 2. The productivity of the strain of Halobacterium salinarum VKPM B-9451 when grown in a flat cuvette.

The VKPM strain V-9451 and the VKPM V-9025, VKPM V-9026 and TsMPM V-173 9 control strains were grown in 15-liter flat cuvettes with 10 l filling under illumination with fluorescent lamps of daylight, continuous air supply to the cuvettes (air flow rate 1.5 l / min) for aeration and mixing to a stationary stage of growth in a growing medium as in example 1.

Next, the precipitate was separated by centrifugation at 7000 g for 10 min, and distilled water was added to the precipitate in an amount equal to the volume of the growth medium, incubated, and the bacteriorhodopsin content was determined as in Example 1.

The data presented in table 2 allow us to conclude that, when grown in flat cuvettes, the claimed VKPM B-9451 strain significantly exceeds the parent VKPM B-9025 strain, the closest analogue of VKPM B-9026 and the control TsMPM B-173 9 in bacteriorhodopsin concentration in culture medium and biomass of the grown culture.

Table 2. The studied strains. Biomass Weight (g / L) The concentration of bacteriorhodopsin (mg / l) VKPM B-9451 28 88 VKPM B-9025 eleven 32 VKPM B-9026 fifteen 46 TsMPM V-1739 17 55

EXAMPLE 3. The productivity of the strain of Halobacterium salinarum VKPM B-9451 when grown in a fermenter.

The inventive strain VKPM B-9451, the control (initial, comparison, and prototype) strains VKPM B-9025, VKPM B-9026 and CMPM B-1739 were grown in an Anglicon fermenter (Anglicon, USA) in a volume of a growth medium of 1500 ml. As the growth medium, we used the medium as in Example 1. The fermenter maintained a constant temperature of 37 ° C, stirring: the first 24 hours 400 rpm, on the second day 600 rpm, on the 3rd day 800 rpm, on the 7th day 1000 rpm Aeration during fermentation - 0.75 l / l · min. The studied strains were grown to a stationary growth stage. Next, we determined the weight of the biomass and the content of bacteriorhodopsin in the growing medium as in example 1. The data obtained are presented in table 3.

Table 3. The studied strains. Biomass Weight (g / L) The concentration of bacteriorhodopsin (mg / l) VKPM B-9451 35 112 VKPM B-9025 twenty fifty VKPM B-9026 12 64 TsMPM V-1739 25 70

As follows from the results shown in tables 1, 2 and 3, the claimed strain VKPM B-9451 significantly exceeds the closest analogue, as well as other strains selected as controls, in the amount of bacteriorhodopsin and biomass synthesized under various cultivation conditions.

Information sources

1. Application for patent of the Russian Federation No. 2005129525 of September 23, 2005.

2. Rosa Margesin, Franz Schinner, Extremophiles, 5, (2001), pp. 73-83.

3. RF patent No. 770184.

4. Mironova E.V. "Biosynthetic preparation of bacteriorhodopsin analogues." Abstract of dissertation for the degree of candidate of chemical sciences, Moscow, 2002.

5. USA patent No. 16616964.

6. Miller A., Oesterhelt D., BBA, 1020 (1990), p. 57-64.

7. Otto H., Marti T., Holz M., Mogi T., Lindau M., Khorana H.G. and Heyn M.P., Proc. Natl. Acad. Sci., USA, 86 (1989), p. 9228-9232.

8. Thorgeirsson T.E., Milder S.J., Milreke L.J.W., Betlach M.C., Shand R.F., Stroud R.M. and Kliger D.S., Biochemistry, 30 (1991), p. 9133-9142.

9. USA patent No. 4927180.

10. USA patent No. 5518858.

11. USA Patent No. 5807625.

12. USA patent No. 5872648.

13. United States Patent No. 5920058.

Claims (1)

The bacterial strain Halobacterium salinarum VPKM B-9451 is a producer of bacteriorhodopsin.