RU2026348C1 - Strain of bacterium serratia marcencsens - a producer of chitinase - Google Patents

Abstract

FIELD: microbiology. SUBSTANCE: known strain Serratia marcescens (B. prodigiosum) B-10 M-1 (VKPM B-3273) is proposed for chitinase production. Strain grows on the available cheap medium, it stable at storing. Chitinase yield is 0.3-0.5 U/ml cultural fluid. EFFECT: broadened assortment of chitinase strain-producers. 1 tbl

Description

 The invention relates to microbiology and biotechnology and relates to a strain producing a chitinase enzyme. Chitinolytic enzymes, especially chitinase, have the ability to break down chitin in pathogenic fungi and arthropods. Chitinases are potential antimycotic and entomopathogenic drugs and can be widely used in healthcare, veterinary medicine and crop production. The search for new affordable and stable strains - producers of chitinases, is an urgent national economic task.

Known strain of Streptomuces sp. WAK-83 - producer of chitinase [1]. This strain is grown in a medium containing yeast extract (0.3%), polypeptone (0.3%), glucose (0.2%) and colloidal chitin (0.4%) at ²⁸ ° C for 3-4 days. Then the culture fluid is clarified by centrifugation and used as a crude preparation of chitinase with an activity of 0.01 units / ml.

 A disadvantage of the known strain is the low activity of the chitinase produced by it in the preparation.

Also known is a strain of Serratia marcescens IMR-IET, a producer of chitinase [2]. The known strain was obtained by stepwise selection from S.marcescens strain QM 131466 after mutagen treatment with UV rays, followed by exposure to ethyl methanesulfonate. The strain is grown in a nutrient medium containing as main components the colloidal chitin (1.5%) or crystalline (2%) of chitin and yeast extract (0.5 g / l) at 30 ^{° C} for 5 days. The yield of chitinase in the culture fluid is 0.36 u / ml.

 A significant disadvantage of the known strain is its instability associated with the genetic nature of the mutation, as well as inaccessibility for widespread use. An object of the invention is the creation of a stable and affordable strain that provides a high yield of chitinase.

 The task is achieved by using as a producer strain the chitinase strain of Serratia marcescens B-10 M-1.

This strain is known as an endonuclease producer [3] and is obtained from the original museum strain of Serratia marcescens B 10 under the action of nitrosomethylurea. The strain of Serratia marcescens B10 M-1 is characterized by the following features:
Morphological: cells are short rods of 0.8 / 3 0.6-0.8 microns in size, elongated, single, paired or in chains, motile, gram-negative.

Cultures: on nutrient agar after 48 hours of growth at ³⁰ ° C to form round, colorless, shiny colonies with smooth edges. The profile of the colonies is convex.

Physiological and biochemical: The strain is an auxotroph. The optimum growth temperature is 28-30 ^o C, the pH of the medium is 6-8.

 Attitude to carbohydrates: ferments glucose, sucrose, maltose, mannitol before fermentation, does not absorb lactose.

 It dilutes gelatin.

 Milk coagulates, but does not peptone.

 Starch hydrolyzes very weakly.

 Fiber does not decompose.

 Nitrates reduces to nitrites.

 The strain Serratia marcescens B-10 M-1 is stored in the VKPM All-Russian Research Institute of Genetics collection under registration number B-3273.

For the cultivation of S. marcescens B-10 M-1 in order to obtain chitinase, a nutrient medium of the following composition is used, g / l of water: Crystal chitin 1.0-3.0 Yeast extract 0.5 Ammonium sulfate 0.1 Magnesium sulfate 0.3 Water Else
Cultivation is carried out on a rocking chair with shaking at 28-30 ^about C, pH 8.0-8.4, with aeration.

 The yield of chitinase is 0.32-0.46 u / ml of culture fluid.

Chitinase activity is determined as follows. To 0.3 ml of a suspension of colloidal chitin (10 mg / ml) add 0.1 ml of 0.5 m tris-acetic acid (pH 6.5), an aliquot of the analyzed chitinase preparation, water to a final volume of 1.0 ml and incubate at 37 ^about C for 30 '. After that, the reaction mixture is centrifuged, and the concentration of N-acetylglycosamine is determined in the supernatant using a dinitrosalicylic reagent (Miller GL Use of dinirtosalicilic acid reagent for determination of reducing sugar // Anal. Chem. - 1959. - V.31, N 3. - P.426-428). A unit of HA is taken to be such an amount of an enzyme that under the described conditions causes an increase in the concentration of N-acetylglycosamine 1.0 μmol ˙ min ^-1 ˙ ml ^-1 .

 The table shows the dependence of the yield of chitinase on the time of growing the producer on a nutrient medium containing 2% crystalline chitin.

 The table shows that the strain proposed as a producer of chitinase is not inferior to the known strain in yield of the target product, and the most optimal is to grow the producer for 5-7 days. The advantage of the proposed strain as a producer of chitinase is its stability during cultivation, availability and high yield of the target enzyme.

 The well-known chitinase superproducer - strain S.marcescens IMK-IEI is extremely unstable due to its genetic nature beyond productivity - tandem duplication. At the end of fermentation, when grown on a medium with chitin, revertants with chitinase activity similar to the activity of the initial strain often prevail in the cell population. The proposed strain S.marcescens B-10 - M-1 is characterized by genetic stability in relation to chitinase productivity. The stability of the latter lies in the genetic nature of the mutation — the overproduction of a point pleiotropic mutation, the frequency of which is reversed to the original genotype is very low. All tested clones of the proposed strain retained the characteristics of the mutant genotype and did not change productivity in relation to chitinase.

 Currently, there are no affordable, high-performance strains producing chitinases.

PRI me R 1. The cultivation of the strain S.mercescens B-10 M-1 is carried out on a nutrient medium of the following composition, g / l of water: Chitin crystalline 3.0 Yeast extract 5.0 Ammonium sulfate 1.0 Magnesium sulfate 0, 3 Water up to 1 liter pH 8.5
Shaking incubation is carried out at 30 ^about C for 7 days. The culture fluid is separated by centrifugation. The yield of chitinase is 0.46 U / ml in the culture fluid (QOL).

PRI me R 2. Obtaining QOL is carried out analogously to example 1. For additional purification of chitinase, ammonium sulfate is added to 100 ml of QL to a concentration of 80% saturation. Formed precipitate was collected by centrifugation, dissolved in 20 ml water and dialysed against 5 L of water for 5 days at -8 ° ^C. The thus obtained title product Chitinase activity (HA), which is 1.76 au / ml (five times the cleaning protein relative to the original QOL).

 PRI me R 3. Obtaining QOL is carried out analogously to example 1. Chitinase from QL is isolated by concentration on cells with hollow porous fibers: for this, 150 ml of QL were concentrated to 18 ml on a cell with a transmittance of 15 KDa. The CA of the target enzyme was 2.8 ea / ml.

 Using the proposed strain will expand the assortment of strains - producers of chitinase.

 The proposed strain has advantages compared with the known, namely it is affordable, stable, provides a high yield of chitinase.

Claims (1)

 STRAIN OF BACTERIA SERRATIA MARCESCENS - CHITINASE PRODUCER.  The use of the bacterial strain Serratia marcescens VKPM B-3273 as a producer of chitinase.

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