RU2558232C1 - STRAIN OF BACTERIA Halobacterium salinarum - PRODUCER OF BACTERIORHODOPSIN - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of bacteria Halobacterium salinarum RNCIM B-11850 - producer of bacteriorhodopsin is described. The strain of Halobacterium salinarum ST 3 is obtained by multistage selection of halophilic bacteria strain Halobacterium salinarum RNCIM B-10425 without preliminary treatment with chemicals and is deposited under the number RNCIM B-11850.

EFFECT: strain Halobacterium salinarum RNCIM B-11850 exceeds the prototype RNCIM B-10425 in productivity.

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Description

The invention relates to biotechnology, namely to the production of physiologically active compounds, and relates to a producer strain of bacteriorhodopsin. The Halobacterium salinarum ST 3 strain was obtained by multistage selection of the halophilic bacteria strain Halobacterium salinarum VKPM B-10425 without preliminary treatment with chemicals and deposited in VKPM under registration number B-11850. The strain of Halobacterium salinarum VKPM B-11850 is superior in productivity (biomass and bacteriorhodopsin) to the VKPM B-10425 prototype.

The known bacterial strain Halobacterium salinarum VKPM B-10425 is the producer of bacteriorhodopsin, which we have chosen as a prototype, significantly superior in productivity (biomass and bacteriorhodopsin) bacterial strains Halobacterium salinarum VKPM B-9451 and Halobacterium salinarum VKPM B-9025 [1].

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

The problem was solved by obtaining a bacterial strain Halobacterium salinarum ST 3 deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) as Halobacterium salinarum VKPM B-11850 and capable of synthesizing bacteriorhodopsin 30% more than VKPM B-10425 under various cultivation conditions. The inventive strain was obtained by multistage selection of a halophilic bacteria strain Halobacterium salinarum VKPM B-10425, without preliminary treatment with chemical reagents.

The strain VKPM B-11850 has the following characteristics:

Cultural and morphological features

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 gallophil; 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, pH of the medium - 7.2-7.4.

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

Relation to the pH of the medium: grows well at 7.2-7.4, the 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 (99.9%).

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 Halobacterium salinarum VKPM B-11850 when grown in flasks.

The inventive strain VKPM B-11850, and also used as a control strain VKPM B-10425 (prototype) 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 _solution · V _cuv. · / E _br · V _tests,

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. - the volume of the solution of bacteriorhodopsin in a spectrophotometric cell;

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) The amount of bacteriorhodopsin (mg) VKPM B-10425 6 twenty VKPM B-11850 8 27

From the results shown in table 1, it can be seen that the inventive strain B-11850 exceeds the prototype VKPM B-10425 both in the amount of biomass and in the concentration of bacteriorhodopsin after growing in flasks.

EXAMPLE 2. The productivity of the strain of Halobacterium salinarum VKPM B-11850 when grown in a fermenter.

The inventive strain VKPM B-11850, and also used as a control strain VKPM B-10425 (prototype) was grown in a BioFlo 110 fermenter (New Brunswick Scientific, USA), in a volume of culture medium of 10,000 ml. The medium used was used as the cultivation medium, as in Example 1. The fermenter was kept at a constant temperature of 37 ° C, stirring: for the first 24 hours 400 rpm, for the second day 600 rpm, for 3 days 800 rpm for 7 days 1000 rpm Aeration during fermentation - 0.5 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 2.

table 2 The studied strains Biomass Weight (g) The amount of bacteriorhodopsin (mg) VKPM B-10425 45 144 VKPM B-11850 57 170

As follows from the results shown in tables 1 and 2, the inventive strain VKPM B-11850 exceeds the prototype VKPM B-10425 in the number of biomass and bacteriorhodopsin synthesized under various cultivation conditions.

Information sources

1. RF patent No. 2416634.

Claims (1)

The bacterial strain Halobacterium salinarum VKPM B-11850 is a producer of bacteriorhodopsin.