RU2558230C1 - STRAIN OF BACTERIA Halobacterium salinarum - PRODUCER OF BACTERIORHODOPSIN - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of bacteria Halobacterium salinarum D96N RNCIM B-11953 - producer of bacteriorhodopsin, significantly surpassing in productivity (biomass and bacteriorhodopsin) as the closest analogue, as well as others selected as controls strains under different conditions of culturing.

EFFECT: increased properties of the strain.

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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 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].

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].

Known strain of Halobacterium salinarum D96N (VKPM B-9026) [5-13]. It synthesizes bacteriorhodopsin in an amount of up to 47 mg / L (2.3 μmol / L) of the culture fluid and was selected by us as the parent strain [3].

A known bacterial strain Halobacterium salinarum VKPM B-10425 is the producer of bacteriorhodopsin, which we have chosen as a prototype, significantly superior in bacteriorhodopsin productivity to the bacterial strains Halobacterium salinarum VKPM B-9451 and Halobacterium salinarum VKPM B-9025 [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 deposited in the All-Russian collection of industrial microorganisms (VKPM) as Halobacterium salinarum VKPM B-11953.

The inventive strain was obtained by multistage selection of a halophilic bacteria strain Halobacterium salinarum D96N VKPM B-9026, without preliminary treatment with chemical reagents.

The strain VKPM B-11953 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 a composition (wt.%.): Peptone - 1, yeast extract - 0.5, NaCl - 25, MgSO ₄ - 2, KCl - 0.2, citrate sodium - 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.

According to the results of the analysis of sequencing of variable regions of genes encoding 16 S rRNA, the strain is closest to Halobacterium salinarum species (98%).

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-11953 when grown in flasks

The inventive strain VKPM B-11953, as well as the parent strain VKPM B-9026 and the prototype VKPM B-10425 were grown in flasks with a capacity of 750 ml in the volume of the growth medium 300 ml when illuminated with fluorescent lamps (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:

C = D · M _r · V _p-pa · V _cuv. / E _br · V _samples

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) Bacteriorhodopsin concentration
(mg / l) VKPM B-11953 17 55 VKPM B-9026 four twenty VKPM B-10425 10 35

From the results shown in table 1, it can be seen that the inventive strain VKPM B-11953 significantly exceeds the parent strain VKPM B-9026 and the prototype VKPM B-10425 in the amount of biomass and in the concentration of bacteriorhodopsin in the culture medium.

EXAMPLE 2. The productivity of the strain of Halobacterium sulinarum VKPM B-11953 when grown in a flat cuvette

The inventive strain VKPM B-11953, as well as the parent strain VKPM B-9026 and the prototype VKPM B-10425 were grown in flat cuvettes with a volume of 15 l with 10 liters 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 the growth 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-11953 strain significantly exceeds the parent VKPM B-9026 strain and the VKPM B-10425 prototype in bacteriorhodopsin concentration in the culture medium and biomass of the grown culture.

table 2 The studied strains Biomass Weight (g / L) Bacteriorhodopsin concentration
(mg / l) VKPM B-11953 thirty 91 VKPM B-9026 13 33 VKPM B-10425 17 fifty

EXAMPLE 3. The productivity of the strain of Halobacterium salinarum VKPM B-11953 when grown in a fermenter

The inventive strain VKPM B-11953, as well as the parent strain VKPM B-9026 and the prototype VKPM B-10425 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 min 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) Bacteriorhodopsin concentration
(mg / l) VKPM B-11953 40 115 VKPM B-9026 23 53 VKPM B-10425 fourteen 65

As follows from the results shown in tables 1, 2, and 3, the claimed VKPM B-11953 strain significantly exceeds the parent VKPM B-9026 strain and the VKPM B-10425 prototype 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. Mironova Ε.V. “Biosynthetic preparation of bacteriorhodopsin analogues”, dissertation abstract for the degree of candidate of chemical sciences, Moscow, 2002.

4. RF patent No. 2416634.

5. USA patent No. 16616964.

6. Miller Α., Oesterhelt D., BBA, 1020 (1990), pp. 57-64.

7. Otto H., Marti T., Holz M., Mogi T., Lindau M., Khorana H.G. and Heyn M.P., http: //Proc.Natl.Acad.Sci., USA, 86 (1989), pp. 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), pp. 9133-9142.

9. US Patent No. 4927180

10. USA Patent No. 5518858

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13. US Patent No. 59920058

Claims (1)

The bacterial strain Halobacterium salinarum VKPM B-11953 is a producer of bacteriorhodopsin.