RU1806194C - Strain of fungus aspergillus awamori - a producer of extracellular @@@-glucosidase - Google Patents

Abstract

Usage: biotechnology, can be used in the pharmaceutical and essential oil industry. SUMMARY OF THE INVENTION: Aspergillus awamorl 18 strain is grown in test tubes with beveled wort agar for 5 days at a temperature of 30 ° C and 7 days at room temperature. The obtained spore material is seeded with a nutrient fermentation medium of the following composition,%: bagasse 30; malt sprouts 1.0; (0.7; MgS04 0.05; KNARO 0.1, natural pH. The microorganisms are cultured in 750 ml flasks on a circular shaker - 180 rpm at a temperature of 30 ° C for 3 days. Activity / 3-glucosidase 5.0 u / ml culture fluid, Activity in the dry preparation 3600 u / h (on p-nitrophenyl glycoside). 1 table.

Description

The invention relates to biotechnology.

The ability to synthesize / glucosidase is observed in many microscopic fungi.

The purpose of the invention is to obtain a strain of β-glucosidase more active than the prototype, which catalyzes the hydrolysis of / 2-glycosidic bonds in the structure of glycosides, precursors of drug substances. In our country, until now, due to the lack of a producer, there is no industrial production of this enzyme.

The strain was obtained by induced selection of strain Aspergillus awamorl 16, a producer of pectinase under superficial conditions, containing insignificant amounts of glucosidase. The rptd strain Asp awamori VKPM F-590 is different from FIR .-; TNA. high productivity producers / glucosidazg Strain is cultured in deep conditions on a liquid nutrient medium. Β-glucosidase activity was distributed on a standard substrate p-nitrophenyl-O-glucopyranoide with a molecular weight of 301.3. A modification of the method was proposed by the All-Russian Research Institute of Biotechnology, /.

The unit of activity is the amount of glucose released in µmol. within 1 mich. under optimal hydrolka conditions. (RF 4.7, temperature 406C). Culture-in signs. On wort agar on the 5th day of growth at 30 ° C, the diameter of the colonies is 7.0-7.5 cm. Colony

00 O O

Yu

with

round, with regular even edges. Two distinct zones are distinguished: a wide gray zone and an adjacent narrow white zone. A small tubercle dotted with brown conidia is observed in the center of the colonies. Radial folding is observed on the back of the colony, especially in the center

CRLONIA.

On Chapek’s agarized medium, the colony diameter on the 7th day of growth is 2.5-3.0 cm. Colony with uneven edges, the surface of the colony is fluffy, in the center is brown, and a white-yellow stripe is edged along the edges of the colony. Large folding with a radius is observed. On the back of the colony there is a pronounced folding.

On Waxman’s agarized medium, colony size 5.0-6.0 cm, fluffy, brown center. The peripheral part of the colony is white-yellow. Folding is absent on both sides of the surface. The edge of the colony is irregular in shape. Morphological signs. The mycelium is highly branched, tortuous, has the appearance of thin separated hyphae. The cells are short, thick. The diameter of the conidiophores is 7-17 microns.

Carrot agar: colonies of light color, with smooth edges, s. strong radial folding. The diameter of the colony is 4.0 - 6.0 cm.

Synthetic agar medium: small colonies of light color. The diameter of the colony is 2.0-3.0 cm. Folding is absent.

Nitrate restores. Temperature limits of development: at 20 ° С - slow growth; at 30 ° C - good growth; at 40 ° C, growth is weak; at 50 ° C - growth is very weak. Physiological and biochemical characteristics. Growth in milk: milk peptones. It dilutes gelatin.

Growth on potato slices: plentiful, with good sporulation. Dextrins, starch, sucrose, lactose, maltose, glucose, mannitol, glycerin consume

Example 1. The strain Asp. awamorl VKPM F-590 is grown in test tubes with beveled wort agar for 5 days. at a temperature of 30 ° C and 7 days. at room temperature. The obtained spore material is seeded with a nutrient enzymatic medium of the following composition,%: bagasse 3.0; malt sprouts 1.0: (0.7;

MgSCM 0.05; KN.RSm 0.1, natural pH. The cultivation of microorganisms is carried out in flasks with a capacity of 750 ml on a circular shaker - 180 rpm, at a temperature of 30 ° C for 3 days. The activity of / 8-glucosidase is 5.0 u / ml of culture fluid.

The table shows a comparative characteristic of enzymatic activity.

the proposed strain Asp. awamori VKPM F-590 and famous. The strain Asp. awamori VKPM F-590 is highly productive and its use is cost-effective for industry.

EXAMPLE 2. A culture fluid is prepared as described in Example 1. An enzyme preparation, glucosidase, is prepared from the culture fluid by precipitation. On 1 volume of culture

3.5 volumes of alcohol (96%) are consumed in liquid. The drug is dried in air at room temperature. The yield of dry enzyme preparation is 0.6%. The activity in the dry preparation is 3600 u / g (on p-nitrophenyl glycoside). An enzyme preparation solution was prepared at a rate of 5 mg / ml.

Example 3. The same as in example 2. but using lanatoside A, obtained from a thimble (deacetylated form) as a substrate, is waste from the production of celanide. The concentration of the substrate solution is 1 mg / ml. Hydrolysis is carried out at pH 4.7, at a temperature of 40 ° C. The duration of hydrolysis is 22 hours. The activity was determined by the glucose oxidant method. The amount of enzyme preparation required to produce 1 µM glucose in 1 min was taken per unit of activity. at the indicated hydrolysis parameters. The activity on this substrate is 0.40 units / g. Hydrolysis products with a pharmaceutical effect (eglikons) were determined by thin layer chromatography on Sllufol plates (F. Kavalier.

Czechoslovakia). The hydrolysis of deacetylated lanthroside A went all the way to the final products: digitoxin, digoxin and gitoxin, which, after appropriate purification, are

drugs - cardiac glycosides.

Claims (1)

SUMMARY OF THE INVENTION Asperglllus awamori VKPM 55 F-590 fungal strain is an extracellular / -glucosidase producer.