EA032524B1 - Halobacterium salinarum bacterial strain - a bacteriorhodopsin producer - Google Patents

Abstract

The invention relates to biotechnology, and specifically to the production of physiologically active compounds, and concerns a bacteriorhodopsin producer strain. The Halobacterium salinarum ST 3 strain is produced by multi-stage selection of a halophilous Halobacterium salinarum RNCIM B-10425 bacterial strain without pretreatment with chemical reagents and is deposited in the RNCIM under number B-11850. The Halobacterium salinarum RNCIM B-11850 strain exceeds the productivity of RNCIM B-10425 (in biomass and bacteriorhodopsin).

Description

Technical field

The invention relates to biotechnology, namely to the production of physiologically active compounds, and relates to a producer strain of bacteriorhodopsin. The strain Na1bac1etst kaipatite 8T 3 was obtained by multistage selection of a strain of halophilic bacteria Na1bac1etst kaipatite VKPM B-10425 without preliminary treatment with chemicals and deposited in VKPM under registration number B-11850. The strain Na1bac1etst kaipatite VKPM B-11850 is superior in productivity (biomass and bacteriorhodopsin) to the prototype VKPM B-10425.

State of the art

The bacterial strain Bactobacterium kaipatite VKPM B-9451, a producer of bacteriorhodopsin, is disclosed in the patent KI 2321627, published on 04/10/2008.

The disadvantages of this strain are low parameters of microbiological productivity, such as the amount of biomass and bacteriorhodopsin.

Closest to the claimed invention is a bacterial strain Na1oBac1etst kaipatite VKPM B-10425 - a producer of bacteriorhodopsin disclosed in patent KI 2416634 published on 04/20/2011, significantly superior in productivity (biomass and bacteriorhodopsin) of the bacterial strains Na1bac1beta kapatitkbtbt1b1b1b1b1b1b1bb1b1b1b1b1b1b1b1b1b1b1b1b1b1b1b1b1b1bb1b2bcb. B-9025.

The disadvantages of this strain are low parameters of microbiological growth according to the main indicators, such as the amount of biomass and bacteriorhodopsin.

Disclosure of invention

The problem to which the claimed invention is directed is to obtain a Na1Obac1etst kaipatite strain with an increased level of biomass and bacteriorhodopsin synthesis.

The problem was solved by obtaining a bacterial strain Na1bac1etst kaipatite 8T 3 deposited in the All-Russian collection of industrial microorganisms (VKPM) as Na1bac1etst kaipatite 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 strain of halophilic bacteria Na1Obac1etst kaipatit 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 2-5x0.5-0.9 microns, 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. Culture is an extreme halophile - it requires the presence of 25% No. 1C1 in the environment. When the content of sodium chloride is below 20%, the cells lose their color, and when they enter the medium with a content of No. 1C1 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, Md8O 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 in a salt agar medium having the composition (wt.%.): peptone

- 1, yeast extract - 0.5, NaCl - 25, Md8O ₄ - 2, KCl - 0.2, sodium citrate - 0.3, CaCl ₂ - 0.02, agar - 1.5, water - the rest, pH Wednesday - 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 8 rRNA, the strain is the closest to the Na1bac1etst kaipatite species (99.9%).

Like all representatives of the genus Na1bac1etst, 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 storage:

It is well stored on an agarized medium in back prints and on 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 Na1Obac1etst kaypitit VKLM B-11850 when grown in flasks

The inventive strain VKPM B-11850, as well as the strain VKPM B10425 used as a control (prototype) was grown in flasks with a capacity of 750 ml in a volume of a growth medium of 300 ml when illuminated with fluorescent lamps (daylight 18 ν / 530, Okgat, Oettapu) to a stationary one hundred

- 1,032524 growth diy (6 days) on a circular rocking chair at 37 ° С and shaking 100 rpm.

For growth, we used a medium of the following composition (wt.%): Peptone - 1, yeast extract 0.5, No. 1C1 - 25, Mg8O ₄ - 2, KC1 - 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 d 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 h.

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

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

M _g - molecular weight of bacteriorhodopsin (26700); ν _ρ-ρα 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. one.

Table 1

Test strains Biomass Weight (g) The amount of bacteriorhodopsin (mg) VKPM B-10425 6 20 VKPM B-11850 eight 27

From the above table. From the results of 1 it can be seen that the inventive strain B-11850 exceeds 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 Ia1bac1epish zapagish VKPM B-11850 when grown in a fermenter

The inventive strain VKPM B-11850, as well as the strain VKPM B10425 used as a control (prototype) was grown in a fermenter ΒΐοΕ1ο 110 (Νΐ '\ ν Vgshyichsk 8s1epIys, IL8L), in a volume of culture medium of 10,000 ml. The medium used as the cultivation medium was as in Example 1. A constant temperature of 37 ° C was maintained in the fermenter, 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) amount bacteriorhodopsin (mg) VKPM B-10425 45 144 VKPM B-11850 57 170

As follows from the results given in table. 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.

Claims (1)

CLAIM The bacterial strain Ia1bac1epish zapagish VKPM B-11850 is a producer of bacteriorhodopsin. <5 Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2