The method of cultivation of bacteria serratia marcescens, producer chitinases

 

The invention relates to biotechnology. The method of cultivation of bacteria Serratia marcescens includes the cultivation of these bacteria on a nutrient medium containing the yeast Saccharomyces cerevisiae 50-150 g/l, buffer salt 1.5 g/l water to 1 L. the Cultivation is carried out at a temperature of 28 to 30°C and pH of 8.0-8.4 and aeration within 3-5 days. The invention allows to simplify the composition of the nutrient medium, to reduce the duration of culturing bacteria, to increase the output chitinases, to reduce the cost of media for culturing bacteria. 1 table, 3 Il.

The invention relates to Microbiology and biotechnology and relates to a method of culturing strains of bacteria Serratia marcescens, producers of chitinase.

Chitinases (EC 3.2.1.14) - enzymes that catalyze the hydrolysis-1,4 glycosidic bonds in chitin is a polysaccharide that is part pathogenic fungi and arthropods. In this regard, the use of chitinases promising for various applications in crop production and animal health related to the protection of animals and plants from fungal diseases and insect pests, as well as in the fishing industry for recycling contain the organisms, including bacteria, fungi, plants, arthropods and vertebrates, but the most promising sources of these enzymes are microorganisms.

Among microorganisms capable of producing a chitinase, enterobacteria Serratia marcescens is one of the most effective [2]. Different strains of S. marcescens, including artificially altered genomes, have the ability to secrete extracellular space complexes of chitinolytic enzymes. However, these enzymes are induced and the level of production depends on the culture conditions and nutrient medium composition [2, 3]. In this regard, the development of methods of cultivation of S. marcescens high yield chitinases is an important task.

There is a method of culturing a mutant strain of S. marcescens IMR-1E1 - superproduct chitinases [4]. The strain is cultivated in a nutrient medium containing as the main components of colloidal chitin (1.5%) and yeast extract (0.5 g/l) at 30° C for 5 days. The output of chitinases in the culture fluid is 0.36 U/ml

Significant disadvantages of the known method of cultivation is the instability of productivity of chitinase associated with the genetic nature of the mutation in strain

Closest to the claimed method of cultivation - the prototype is the method of cultivation of a stable mutant strain of S. marcescens In-10 M-1 (VKPM B-3237) - superproduct chitinases [5] the nutrient medium of the following composition, g/l:

Chitin powder 10-30

Yeast extract 5,0

Ammonium sulfate 0,1

Magnesium sulfate 0,3

Water 1.0 l

the pH of the medium is brought to a value of 8.0-8.4 and by adding a buffer mixture of one - and disubstituted phosphates potassium (1.5 g/l). The cultivation is carried out on a rocking chair with shaking at a temperature of 28 to 30° C With aeration for 5-7 days. The output of chitinases in the culture fluid is 0.3-0.5 U/ml

Significant disadvantages of the known method of cultivation is the duration of cultivation, inadequate production of chitinases, as well as the complexity of nutrient factors, including expensive powdered chitin and yeast extract and ammonium sulphate and magnesium.

An object of the invention is to reduce the duration of cultivation, while increasing the yield chitinases, as well as the simplification of the composition of the nutrient medium.

The goal of the project is achieved by the proposed method, Zack what do the following composition, g/l (dry matter):

The yeast Saccharomyces cerevisiae 50-150

Water 1.0 l

the pH of the medium is brought to a value of 8.0-8.4 and

adding a buffer mixture of one - and disubstituted phosphates potassium (1.5 g/l). The cultivation is carried out on a rocking chair with shaking at a temperature of 28 to 30° C With aeration in 3-5 days. The output of chitinases is 0.77-1,02 E/ml.

The concentration of viable cells of S. marcescens in the culture fluid is determined by the method of seeding in a Cup with mesopartner agar.

The activity of chitinases (HA) is determined as follows. After cultivation separate cells and insoluble components by centrifugation at 5000 rpm for 30 minutes, the Culture fluid is harvested. To 0.3 ml of a suspension of colloidal chitin (10 mg/ml) add 0.1 ml of 0.5 M sodium acetate - acetic acid (pH 5.5), an aliquot of the analyzed sample the culture fluid, water to a final volume of 1.0 ml and incubated at a temperature of 37° C for 15 minutes then the reaction mixture was centrifuged and the supernatant determine the concentration of N-acetylglucosamine using dinitrosalicylic reagent [6]. Per unit HA take that amount of enzyme, which in the described reaction conditions causes primost cell growth of S. marcescens In-10 M-1 (columns) and output chitinases (line) from the concentration in the nutrient medium Baker's yeast Saccharomyces cerevisiae. Time of cultivation for 3 days. On the X - axis is the concentration of yeast biomass production (dry weight) in a nutrient medium, g/l On the left Y - axis hitimana activity in the culture liquid, E/ml. On the right Y - axis the concentration of cells of S. marcescens, 10 cells/ml.

It is evident from Fig.1 shows that the maximum yield of chitinases is observed in the concentration range of yeast 50-150 g/l with a maximum HA=0.8 u/ml at 90-150 g/l When the concentration of yeast below 50 g/l output chitinases reduced by more than 25% of the maximum. Increasing the concentration of yeast 150 g/l is impractical because it does not lead to an additional increase in the yield of chitinases.

In Fig.2 shows the dependence of the yield of chitinases from time cultivation of S. marcescens In-10 M-1 when used as a nutritional factors Baker's yeast (curve 1) or specified in the prototype powdered chitin, yeast extract and sulfates of magnesium and ammonium (curve 2). The concentration of yeast 90 g/l, chitin 20 g/l On the x - axis, time of cultivation, days. Y - axis hitimana activity in the culture liquid, E/ml.

It is evident from Fig.2 shows that the cultivation of the inventive method, in comparison with the prototype leads to increased production of chitinase and reduce the duration of cultivation is investing in powdered chitin. After 5 days of growth, the accumulation of HA slows down, so the longer cultivation is not meaningful.

In Fig.3 presents comparative data on HA in the culture fluid of three natural strains of S. marcescens In the 10, HY+ and 209 under cultivation for 3 days in media containing 20 g/l of chitin (gray columns) and 90 g/l of Baker's yeast (hatched columns). On the X - axis is the name of the investigated strains of S. marcescens. Y - axis hitimana activity in the culture fluid, IU/ml

It is evident from Fig.3 shows that compared with the prototype of the yeast in 5-30 times stronger stimulate the production of chitinases by cells of different natural strains of S. marcescens. These results confirm that the use of the proposed method of cultivation universal and applicable to increase the production of chitinase by various strains of S. marcescens as natural and mutant.

The table presents comparative data on HA in the culture fluid mutant strain of S. marcescens In-10 M-1 when grown for 3 days in media containing 20 g/l of chitin and biomass of different variants of the yeast Saccharomyces cerevisiae in concentrations equivalent to 90 g/l (dry weight). Additional studied factor Malinowski products chitinases are the properties of different strains of yeast Saccharomyces cerevisiae. Inducing effect have as dried cells, and wet pressed biomass. The table also shows that when using whole cells of the yeast need to add yeast extract disappears. Yeast extract is a common component of the nutrient medium during the cultivation of various microorganisms containing water-soluble amino acids, peptides, vitamins and carbohydrates in yeast cells. However, insoluble elements of yeast cell walls were removed. Probably used in the present method of whole yeast cells contain all necessary for the growth of a culture of S. marcescens nutritional factors, and direct inducer of the production of chitinases are chitin molecules included in the cell walls of the yeast Saccharomyces cerevisiae [7]. Confirmation of this assumption are presented in the table data that is comparable levels of production of chitinases cause as a whole yeast cells and isolated cell walls. However, in contrast to whole cells, cell walls, as well as powdered chitin, are inducing effect only in the presence of yeast extract.

Thus mainly the output chitinases. An additional advantage is that Baker's yeast are easily available and inexpensive component of the nutrient medium. The cost of compressed yeast biomass production for food use is about 0.02 rubles per gram, which corresponds to 5-10 rubles per 1 liter of nutrient medium. For comparison powdered chitin, suitable for use as a component of the nutrient medium is made in limited quantities and cost of different manufacturers varies from 5 to 50 US dollars per gram, which corresponds to the cost 3000-50000 roubles per 1 litre of medium. Another advantage of the proposed method is that its application allows to exclude from media for culturing yeast extract and ammonium sulphate and magnesium, which further simplifies and reduces the cost of the method of cultivation of S. marcescens.

Since the proposed method for the cultivation of S. marcescens offered for the first time and have never been used to highlight chitinases, it is possible to draw a conclusion on the conformity of the proposed method criteria invention of "novelty" and "inventive step".

The invention is illustrated by the following examples of specific performance.

Example 1

Cultivation ..... >the ode 1.0 l

the pH of the medium is brought to a value of 8.2 by adding a buffer mixture of one - and disubstituted phosphates potassium (1.5 g/l). The cultivation is carried out on a rocking chair with shaking at a temperature of 29° C With aeration for 5 days. The output of chitinases is 1.02 U/ml

Example 2

Cultivation of a strain of Serratia marcescens In-10 M-1 carried out analogously to example 1, except that the contents of the yeast in the environment is 50 g/l and culturing duration - 3 days at a temperature of 28° C and pH 8.0. The output of chitinases is 0.60 U/ml

Example 3

Cultivation of a strain of Serratia marcescens In-10 M-1 carried out analogously to example 1, except that the contents of the yeast in the environment is 150 g/l and culturing duration is 4 days at a temperature of 30° C and a pH of 8.4. The output of chitinases is 0.92 U/ml

Thus, a method of culturing bacteria Serratia marcescens, producers of chitinase has the following advantages compared with the known method:

- reduced cultivation (2 times).

- higher the chitinase (more than 2 times);

- reduced cost environment for cultivation (more than 10 times).

The application of the new to spite cost.

SOURCES of INFORMATION

1. Patil R. S., V. Ghormade, Deshpande M. V. Chitinolytic enzymes: an exploration / Enzyme and microbial technology. 2000. V. 26. P473-483.

2. Monreal J., Reese, E. T. The chitinase of Serratia marcescens II Canadian Journal of Microbiology. 1969. V. 15. P. 689-696.

3. Watanabe T., Kimura K., T. Sumiya, Nikaidou, N., Suzuki K., Suzuki M., Taiyoji M., Ferrer, S., Regue, M. Genetic analysis of the chitinase system of Serratia marcescens 2170 //Journal of Bacteriology. 1997. V. 179. R. 7111-7117.

4. Reid J. D., D. M. Ogrydziak Chitinase-overproducing mutant of Serratia marcescens II Applied and Environmental Microbiology. 1981. V. 41. P. 664-669.

5. Panfilov H. I., Dujac A. B., Salganik R. I. bacterial Strain Serratia marcescens - producer chitinases: Patent 2026348 RF //B. I. No. 1, 1995, S. 174.

6. Miller G. L. Use of dinitrosalicilic acid reagent for determination of reducing sugar// Anal. Chem. 1959. V. 31. P. 426-428.

7. Lipke, P., R. Ovalle Yeast cell walls: new structures, new challenges //J. Bacteriol. 1998. V. 180. P. 3735-3740.

Claims

The method of cultivation of bacteria Serratia marcescens, producer chitinases, which includes the cultivation of bacteria in a nutrient medium containing nutrient factors, buffer salts and water at a temperature of 28 to 30°C and pH 8.0-8.4 and aeration, characterized in that as nutritional factors use the yeast Saccharomyces cerevisiae at a concentration of 50-150 g/l, and the cultivation is carried out in 3-5 days.



 

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