RU2096451C1 - Strain of bacterium pseudomonas species able to oxidize l-proline specifically - Google Patents

Abstract

FIELD: biotechnology, microbiological industry. SUBSTANCE: new strain, Pseudomonas species VKPM B-4787. Bacterial cells obtained after culturing the strain on medium containing L-proline as a single source of carbon and nitrogen oxidize specifically L-proline but do not oxidize practically other amino acids and certain abundant sugars. The proposed strain was obtained as a result of selection of bacterium colonies isolated from soil and culturing on media containing 0.05-0.45% L-proline. Strain Pseudomonas species can be used for quantitative determination of L-proline in different media in microbiological industry, clinical and laboratory investigations. EFFECT: strain indicated above. 4 tbl

Description

 The invention relates to the microbiological industry, in particular for the isolation of a new strain of Pseudomonas species Ш-22. These cells, after cultivation on a medium containing L-proline as the sole source of carbon and nitrogen, specifically oxidize L-proline and practically do not oxidize other amino acids and some common sugars.

 The strain Pseudomonas species Ш-22 is of interest for the development of a method for the quantitative determination of L-proline in various environments and may find application in the microbiological industry, clinical and laboratory studies.

It is known that representatives of such genera of microorganisms as, for example, Escherichia, Salmonella and Pseudomonas possess the ability to oxidize L-proline. Moreover, it was shown that this property is most pronounced in representatives of the genera Escherichia and Pseudomonas [1, 2]
Wild and mutant Salmonella typhimurium 15 59 strains with proline oxidizing activity have been described [3] Deep cultivation of the cells of both strains in a medium containing L-proline leads to the activation of their proline oxidizing activity 25 and 10 times, respectively. However, activated cells of both wild and mutant strains retain the ability to oxidize certain sugars (e.g., glucose and maltose) and amino acids (e.g., histidine) at almost the same rate as L-proline. Thus, the main disadvantage of these strains is the low substrate specificity.

 Closest to the proposed strain are the wild strain E. coli-B and auxotrophic E. coli-B4 described in [4] The deep cultivation of cells of both strains in a medium containing L-proline leads to the activation of their proline-oxidizing activity in 14 and 10 times respectively. However, activated cells of both wild and auxotrophic strains retained the ability to oxidize many amino acids at a rate of 20 to 50% of the oxidation rate of L-proline. The disadvantage of this strain is the low substrate specificity.

 The present invention relates to the isolation of a new wild strain of Pseudomonas species Ш-22 having specific proline-oxidizing activity.

 The proposed strain was obtained by selective selection of colonies of bacteria isolated from the soil, and their subsequent cultivation on media containing 0.05 0.45% L-proline.

 The strain Pseudomonas species Ш-22 is stored in the All-Union Collection of Industrial Microorganisms of the Institute "VNIIGenetika" (Moscow), N B-4877.

 The strain has the following characteristics.

 Morphological features of the strain. The cells are straight, rod-shaped, 0.1 - 0.2 x 0.9 1.0 μm, are found in long and short chains, motile, gram-negative, do not form spores.

Cultural signs. When growing on agarized nutrient media (meat peptone agar, LB medium) after 24 hours at 25 ^o C colonies are formed, light yellow, convex, shiny, smooth, the edge is slightly wavy, reaching a diameter of 1.5 mm. The growth in injection in MPA is plentiful, mainly on the surface of the medium. Abundant growth is also observed in liquid nutrient media.

 It grows well on mineral media of simple composition supplemented with L-proline. It does not need to add growth factors and other amino acids. When cultured on a proline-containing medium, synthesis of an extracellular yellow-green pigment is observed.

Physiological and biochemical characteristics. Relative to oxygen, an obligate aerob. The maximum growth temperature is 37 ^o C, the minimum 15 ^o C, the optimal 25 30 ^o C, the pH of the medium in the range of 6.8 to 7.5.

 Nitrogen is disposed of in the form of ammonium salts, urea, nitrates.

 Pigments. It forms a yellow-green water-soluble fluorescent pigment on a proline-containing synthetic medium. It does not form intracellular pigments. The reaction to oxidase and catalase is positive. The Voges-Proskauer reaction is negative. Indole does not form. Anaerobic arginine dehydrogenase test is positive. The reaction to anaerobic denitrification is negative. Lecithinase activity is absent.

 Does not hydrolyze starch, gelatin, tween-80.

 It uses many sugars, amino acids and organic acids as carbon sources.

 The strain Pseudomonas species Ш-22 is non-pathogenic for humans and animals.

 In accordance with the noted morphological, physiological and cultural-biochemical characteristics, the strain was identified as Pseudomonas species.

In determining the systematic position, the Bergey determinant was used:
Bergey's Manual of Determination Bacteriology. 1974. 8th ed. Baltimore. The William Wilkins Co.

 Example 1

Cells of Pseudomonas species Ш-22 are grown on synthetic medium containing 0.05% L-proline, 0.2% K ₂ HPO ₄ • 3H ₂ O, 0.05% KH ₂ PO ₄ , 0.02% MgSO ₄ • 7H ₂ O, pH 7.2 7.5, at 30 ^o C in 750 ml conical flasks (volume of fermentation medium 200 ml) on a circular shaker (180 rpm) for 20 hours. The biomass is separated by centrifugation (10000 rpm, 15 min), suspended in 50 mM potassium phosphate buffer pH 7.2. Proline-oxidizing activity is determined amperometrically. For this, 50 mM potassium phosphate buffer pH 7.2 is placed in a measuring cell (5 ml volume) equipped with a stirrer. 0.05 ml of a cell suspension is introduced into the cell, the initial concentration of which is 0.5 0.1 g / ml and 0.05 ml of a 5 mM L-Proline solution. The change in the oxygen content in the measuring cell is recorded using a Clark membrane oxygen electrode. The proline-oxidizing activity of cells is calculated by their oxygen consumption rate in the presence of L-proline and expressed as the amount of oxygen (μmol) consumed by 1 mg of cells (wet weight) in 1 min [5]
For a given L-proline concentration, the oxygen consumption rate was 3.00 μmol / min • mg of cells.

 Example 2

Cell cultivation and measurements were carried out as in Example 1, only 0.45% L-proline was added to the growth medium instead of 0.05%. The proline-oxidizing activity of the cells was 4.50 µmol O ₂ / min • mg of cells.

 Example 3

 Cell cultivation and measurements were also performed as described in example 2, only 0.05 ml of 5 mM solutions of various amino acids were sequentially introduced into the cell (Table 1). Cells of Pseudomonas species Ш-22 grown on a medium containing L-proline practically did not oxidize other amino acids.

 Example 4

 Cell cultivation and measurements were also performed as described in example 2, only instead of L-proline, 0.05 ml of 5 mM solutions of glucose, fructose, galactose, lactose and sucrose were sequentially introduced into the measuring cell. Cells do not oxidize sugar data.

 Example 5

 Cells of Pseudomonas species Ш-22 were grown on a medium containing 1% peptone, 0.5% yeast extract, 1% sodium chloride (pH 7.2). The cultivation conditions and the determination of activity are the same as in example 2. The proline-oxidizing activity of the cells was 0.5% of the activity of cells grown on medium with proline. In addition, the cells showed a high affinity for other amino acids and sugars (Table 2).

 Example 6

 Cell cultivation and measurements were also performed as described in Example 2, but instead of L-proline, L-lysine was added to the culture medium as the sole source of carbon and nitrogen. The proline-oxidizing activity of cells grown on L-lysine was 0.5% of the activity of cells grown on medium with L-proline. In addition, the cells showed a high affinity for other amino acids and sugars (Table 3).

 Example 7

 The culture medium and the conditions for determining proline-oxidizing activity, as in example 2, only instead of cells of Pseudomonas species Ш-22, the strain Pseudomonas putida VKM B-1458 was used. Proline-oxidizing activity of this strain was 10% of the activity of Pseudomonas species Ш-22. In addition to L-proline, Pseudomonas putida VKM 1458 cells oxidized a number of amino acids and sugars (Table 4).

 The proposed strain Pseudomonas species Ш-22 differs from E. coli B and E. coli B4 in that after cultivation on a medium containing L-proline as the sole source of carbon and nitrogen, it specifically oxidizes L-proline and practically does not oxidize other amino acids and some common sugars.

 Based on this strain, a method for quantitative determination of proline can be developed, which is of interest for the microbiological industry, clinical and laboratory studies.

Claims (1)

 The bacterial strain Pseudomonas species VKPM B-4877, capable of specifically oxidizing L-proline.

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