RU2187553C1 - Strain of microorganism pseudomonas species as producer of chitinolytic enzymes - Google Patents

Abstract

FIELD: biotechnology, agriculture microbiology. SUBSTANCE: strain is isolated from sandy loam soil. The isolated strain is stored in collection of Institute of biology UNTS RAN at number 182. The strain of microorganism Pseudomonas species 182 inhibits the growth of different species of phytopathogenic fungi causing root rots in plants. The strain does not induce opportunistic infections in animals or parasite in animals and humans. The strain shows the more high hydrolytic activity with respect to chitin. EFFECT: strain indicated above, enhanced chitin-hydrolyzing activity. 1 tbl

Description

 The invention relates to the field of biotechnology and microbiological production and may find application for the production of a chitinolytic enzyme preparation.

 Known strains of Serratia marcescens - producers of chitinase [1]. However, the known strains are conditionally pathogenic for humans and warm-blooded animals and therefore their industrial use is unsafe for maintenance personnel.

 The closest to the claimed strain (prototype) is a bacterial strain Pseudomonas maltophilia CR-332D with chitinolytic activity [2]. However, the effectiveness of the known strain as a producer of chitinolytic enzymes is relatively low.

 The purpose of the invention is the expansion of the range of available producers and the selection of a natural producer strain of chitinolytic enzymes, characterized by high efficiency of chitin hydrolysis.

 The new strain was isolated from the sandy loam soil of the Iglinsky district of the Republic of Bashkortostan and is stored in the microorganism collection of the Institute of Biology of the Ufa Scientific Center of the Russian Academy of Sciences under number 182.

 The strain is characterized by the following features. Rod-shaped cells, motile, gram-negative, non-spore-forming. Colonies on meat-peptone agar (MPA) are round, with smooth edges, slightly convex, opaque, grayish-white. The pigment is yellow-green, diffusing into the medium, non-fluorescent.

Physiological and biochemical characteristics. It does not need growth factors. In relation to oxygen - aerob. The oxidase reaction is positive. Gelatin does not thin. Hydrolyzes lecithin, starch, produces catalase, does not reduce nitrates to nitrites. It forms arginine dihydrolase. Accumulates poly-β-hydroxybutyrate. Grows at 4 ^o C and at 41 ^o C, grows on medium with 10 mg / ml Ba ²⁺ . It uses glucose, xylose, rhamnose, sucrose, galactose, ribose, trehalose, L-valine, β-alanine, L-arginine, malonate as a carbon source. Does not absorb arabinose, maltose.

 The strain is resistant to ampicillin (500 μg / ml), streptomycin (50 μg / ml), erythromycin (500 μg / ml) and is sensitive to gentamicin (100 μg / ml) and kanamycin (100 μg / ml). The strain Pseudomonas species 182 is active in suppressing the activity of phytopathogenic fungi Bipolaris sorokiniana, Fusarium solani, F. culmorum, Altemaria altemata.

 The strain is stored on the jambs of MPA in the refrigerator with reseeding to fresh jambs after 2-4 months.

 According to physiological and biochemical characteristics, the strain does not correspond to any of the bacteria described in the determinant (Bergey, Vol. 1: Translated from English / Edited by J. Holet, N. Krieg, P. Spit, J. Staley, S. Williams .-M .: Mir, 1997.-432c.) And identified as Pseudomonas species 182.

 The strain Pseudomonas species 182 is not a causative agent of opportunistic infections of animals, a parasite of animals and humans.

 The use of bacteria Pseudomonas species as a producer of chitinolytic enzymes has not yet been described.

Example 1. Determine the chitinolytic activity of a new strain of Pseudomonas species 182. For this, the strain is grown on a medium with 1.5% agar of the following composition, g / l of distilled water:
Yeast extract - 0.5
(NH ₄ ) ₂ SO ₄ - 1
MgSO ₄ • 7H ₂ O - 0.3
KN ₂ PO ₄ - 1.36
Colloidal chitin is introduced into the medium as a carbon source. Chitin is added to the medium in an amount of 0.5%. The strain is cultivated on Petri dishes at 28 ^o C.

 The efficiency of chitin hydrolysis was determined by the ratio of the diameter of the clarification zone of the turbid medium around the colony to the diameter of the colony.

 The results are presented in the table and indicate the presence of chitinolytic activity in the strain Ps. species 182, and the efficiency of chitin hydrolysis in the new strain is higher than that of the prototype strain (approximately 15%).

Example 2. To obtain chitinolytic enzymes strain Ps. species 182 is grown in liquid culture on the medium prepared according to example 1, with the addition of 0.1% peptone. Incubated at 28 ^o C for 5-7 days under aeration conditions. Next, the biomass is separated by centrifugation. The culture fluid supernatant is a crude chitinase preparation.

To determine the activity of the drug, the agar medium from Example 1 was poured into Petri dishes with the addition of 0.5% chitin. After solidification of the medium using a sterile cork drill, holes were cut into it, into which the test drug was introduced. Petri dishes were incubated at 28 ^o C. The activity of the drug was evaluated by the diameter of the agar clearing zones formed around the wells, the results did not differ from those shown in the table.

Literature
1. Moreal J., Relse ET The chitinase of Serratia marcescens // Can. J. Microbiology.-1969.-v. 15, p. 689-696.

 2. Patent 2031119, Russia, cl. C 12 N 9/42, 1/20, 1995, BI 8.

Claims (1)

 The bacterial strain Pseudomonas species 182 collection of the UC RAS is a producer of chitinolytic enzymes.

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