SU960256A1 - Strain erwina carototora ml-1 producer of trans-eliminase - Google Patents

Description

The invention relates to the microbiological industry, and in particular to strains - producers of pektolytic enzymes, and can find application in agriculture in the field of plant tissue maceration.

Known strains producing pectolytic enzymes, for example; fungi of the genus Rhizopus £ l J.

However, such strains secrete pectolytic enzymes with a low transeliminase activity.

Known strain of Bacilluspolymyxa ATCC 21551, forming pectolytic enzymes with transeliminase activity of 2100-19000 units / mg [2).

The disadvantages of this strain are expensive and complex nutrient medium and low growth rate (50-70 hours).

Also known is a strain of Cldstridium feIsineum-22, a producer of pectolytic enzymes, including pectate-trans-aluminase with an activity in the culture fluid of 360 units / ml, and in the enzyme preparation of 700028000 units / g [3].

The disadvantages of the known producer are the low activity of trans ‘eliminase and prolonged fermentation time (48 h).

The invention aims polu_ chenie new strain with high MSE ³ Height growth in a cheap medium, synthesizing pectolytic enzymes with high activity.

The proposed strain of Erwinii caroto; .Q voga ML-1 is isolated from forest litter in the vicinity of Minsk. The culture is stored on potato agar at 4-5 ^r C in the Museum of Cultures of the Institute of Microbiology of the Academy of Sciences of the BSSR and in the Museum of Cultures of Industrial Microorganisms * · 15 mov of the Institute ^ VNIIGenetika, collection number TsMIM V-2031. The bacterial strain Erwiniacarotovora ML-1 has the following morphological and physiological-biochemical characteristics.

Morphological signs.

The colonies are colorless, convex, pike-like toward the center with wavy edges. Cells are single, straighter than 25 'sticks 0.5-0,' 7X1.5-1.9 microns, motile, with peritrichous flagella. (about 8), they do not have a pronounced shell. After a 5-day growth on potato-glucose medium, colonies 30 reach 12-16 mm in diameter.

Physiological and biochemical characteristics. Aerob, optimum growth at 29 ° C. specific growth rate 0.35 ~ ⁴ , optimal pH 7.0-7.2.

Relation to carbon sources. | In the process of growth, it intensively ferments glucose, sucrose. . lactose., maltose, arabinose J assimilates glycerin, mannitol. Ethyl alcohol does not ferment, starch does not hydrolyze. When growing on potatoes, it forms colonies in the form of a brilliant wet coating.

Relation to nitrogen sources. It utilizes nitrates, mineral and organic salts, peptone, asparagine ,. urea

It dilutes gelatin.

Milk peptones.

The content of HC in DNA is 51 mol.%. Acetoin does not form.

. Does not release H _t . '

The maximum growth temperature is 40 ° C.

Example 1. Sowing material is grown by the deep method on a shaker at 31 ° C and pH 7.0-7.2 for 16 hours on a nutrient medium of the following composition, g / l:

K _g NR0 ₄ KN2.RO4

M ^ SO ₄ -7H.iO (NH4) iS0 ₄ Beet pulp

For growing crops, seed is introduced into the nutrient medium of the specified composition in an amount of 0.5%. The cultivation conditions are the same as for the seed.

The enzyme preparation is obtained from the culture fluid by precipitation with ethyl alcohol in a ratio of 1: 3.5, followed by drying in a vacuum desiccator or by spray drying.

The activity of trans-eliminase in the enzyme preparation is up to 7200 units / mg.

Per unit. quantity

7.0

2.0

0,2, 0

11.25 takes an enzyme activity, causing an increase in the optical density of the reaction mixture by 0.1 in 10 minutes at a wavelength of 235 nm. The composition of the reaction mixture: 0.6 ml of pectate) 0.05 ml of 0.01 M CaCl ^, '2.25 ml of Tris buffer, pH 8.5. The reaction mixture in an amount of 2.9 ml is introduced with 0.1 ml of culture liquid into a 10-mm quartz cuvette, the mixture is incubated at room temperature for 1 min.

EXAMPLE 2. The cultivation conditions are the same as in Example 1. Nutrient medium of the same composition. Beet pulp is taken 5 g / l. The activity of trans-eliminases in the enzyme preparation is up to 3000 u / mg.

Example 3. The cultivation conditions are the same as in example 1. A nutrient medium of the same composition. Beet pulp is taken 15 g / l. Activity of trans-eliminase in enzyme preparation ”up to J1000. u / mg The enzyme preparation macerates flax stalks, potato tubers __ and carrots for 24 hours.

Thus, the implementation of the invention allows to obtain a highly active strain, producing trans (Eliminase, having a high growth rate on a cheap nutrient 40 'medium.

Claims (3)

Physiological and biochemical signs. Aerobe. growth optimum at a specific growth rate of 0 ,, the optimum pH is 7.0-7.2. Relation to carbon sources) In the process of growth, it intensively ferments glucose, sucrose, lactose, maltose, arabinose | absorbs rjw cerin, mannitol. Ethyl alcohol does not ferment; starch e hydrolyzes. When grown on potatoes, it forms colonies in the form of a brilliant, moist coating. Relation to nitrogen sources. Utilizes nitrates, mineral and organic salts, peptone, asparagine,. urea . Gelatinu liquefies. Peptonized milk. The content of HZ in the DNA 51 mol.%. Acetoin does not form. / Does not release N. Maximum growth temperature 40 ° C. Example 1. The seed is grown in the deepest way on a rocking chair at pH 7.0-7.2 for 16 hours in a nutrient medium of the following composition, g / l K HPO47.0 KNg.P04.0 MgS047HijO d, 2 (NH4) iS04 1, 0 Beet pulp11.25 For the cultivation of a culture, the seed is introduced into the culture medium of the indicated composition in the amount of 0.5%. The cultivation conditions are the same as for the inoculum. The enzyme preparation is obtained from a culture liquid by precipitation with ethyl alcohol in a ratio of 1: 3.5, followed by drying in a vacuum desiccator or by spray drying. Trans-eliminase activity in the enzyme preparation up to 7200 units / mg. Per unit of activity, takes with, the amount of enzyme causing an increase in the optical density of the reaction mixture by 0.1 per 10 min at a wavelength of 235 nm. The composition of the reaction mixture is 0.6 ml of pectate; 0.05 ml of 0.01 M CaClji; 2.25 ml of tris buffer, pH 8.5. The reaction mixture in the amount of 2.9 ml is introduced with 0.1 ml of the culture fluid into a 10-mm quartz cell, and the mixture is incubated at room temperature for 1 minute. PRI me R 2. The cultivation conditions are the same as in Example 1. The nutrient medium of the same composition. Beet pulp is taken 5 g / l. The activity of trans-eliminase in the enzyme preparation is up to 3000 units / mg. EXAMPLE 3. The cultivation conditions are the same as in Example 1. The nutrient medium is of the same composition. Beet pulp is taken 15 g / l. Trans-eliminase activity in fer; ment drug to 000. units / mg. Enzyme preparation macerates flax stalks, potato tubers and carrots for 24 hours. Thus, the implementation of the invention allows to obtain a highly active strain, a producer of transeliminase, which has a high growth rate on a cheap nutrient medium. Claim of the invention Strain Erwinia cafotovora ML-1 (Museum of cultures of industrial microorganisms of the Institute of VNI genetics, collection number TsMPMV-2031) produces trans-eliminases Sources of information taken into account during the examination 1. USSR author's certificate 535347, cl. C 12 N 9/14, published 1976. 2 Patent of the Federal Republic of Germany 2054948, cl. C 12 D 13/10. publish 1972. 3. USSR author's certificate 622838, cl. C 12 N 9/00, 1977.