SU1252336A1 - Aspergillus terreus at-490 strain - producer of cellulases - Google Patents

Abstract

The strain Aspergillus terreusAT-490 (collection of the Central Museum of Industrial Microorganisms All-Union Scientific Research Institute of Genetics, collection number CMPM F-232) is a producer of cellulose. (L

Description

The invention relates to the microbiological industry, in particular to the production of cellulolytic enzymes of microbial origin, used in the process of saccharification of cellulose.

The purpose of the invention is a strain of microscopic fungus producing thermostable highly active cellulases catalyzing the conversion of cellulose to glucose.

The proposed strain Aspergillus terreus AT-490 was obtained by ultraviolet irradiation of the original strain Aspergillus terreus 17P with a dose of 6400 J / m.

Chamm is deposited in the Central Museum of Industrial Microorganisms of the Institute of All-Russia Scientific Research Institute of Genetics, collection number TsMPM F-232.

The proposed strain has the following characteristic.

Cultural and morphological features.

The culture is well developed on Chapek-Doksa medium, modified Chapek-Doks medium with the addition of corn extract and filter paper strips on the agar surface, wort agar, and also on natural nutrient substrates.

Wednesday of čapek-doksa. On the 5th day, the colonies are yellowish, gradually transitioning into a beige color, velvety, flat. The reverse side is colorless. Mycelium of the fungus has thin hyphae, weakly septate or not at all septate. The average thickness of hyphae is 3-4 microns, Conidiophores are smooth, colorless, up to I00 microns in length and 6 to 8 microns in width. Hemispherical bubbles 15-18 microns in diameter. Their upper part is covered with sterigmas. Sterigms are double-stranded, usually closely adjacent, steric-sh the first rus (3-4) 2 microns, the second rus (5-6) -2 microns. Conidia are elliptical, smooth, met separately or joined in chains. The diameter of the conidia 2-3 m.

Physiological and biochemical signs.

The strain absorbs well lactose, glucose, xylose, maltose, sucrose and cellulose. It absorbs arabinose, galactose, juahmal and mannitol. Does not absorb ramiosis, raffinose, mannose and sorbitol. From nitrogen sources it is well absorbed by organic and inorganic nitrogen compounds. The most intensive growth is observed on nitrates. Assimilates a variety of phosphorus compounds, however more

1Intensively mono- and disubstituted potassium phosphate. A high endoglucanase strain (p-1, 4-1 -glucan-4-β-glucan hydrolase) and cellobiasis activity are found within the pH range.

4.5 - 4.6. Optimum temperature for growth and biosynthesis. Strain hydrolyzes cellulose (different cellulosic substrates). High endoglucanase and cellobiasis are observed.

activity on a medium containing hay as the sole carbon source.

The cellulase of the proposed thermostable strain — after maintaining the enzymatic reaction at 70 ° C for one hour, the activity is almost completely preserved.

A modified agar medium is used to propagate the strain.

Capek-Doksa composition,%: NaNOg 0.3; , 0,2; MgSO-yHjO 0.05; corn extract 1,5; agar-agar 2.0, a strip of filter paper on the surface of the agar, pH 6.3.

The inoculum is a suspension of a 20-day culture of a strain grown on a modified Chapek-Doks medium. A liquid nutrient medium is used to produce the illuminase.

composition,%: NaNO, 0.30; KN, PO 0.2; MgSO, 0.05; hay 2; corn extract 1.5, pH 4.5.

150 ml of nutrient medium in conical flasks with a capacity of 250 mp sterilize 40 min at 0.7 atm. The maximum activity of cellulases is observed when the strain is grown on a thermostatic shaker, which makes 270 rpm at 40 ° C for 96 hours and is equal to:

endoglucanase activity at CMC

20 units / mp, filter paper activity 0.5 units / mp, cellobiase, 0.5 units / ml.

Endoglucanase activity is determined by the action of the enzyme on Na CMC as follows, I ml of a 1.5% solution of the substrate in 0.05 M acetate buffer (pH 4.7) and 1 ml of culture liquid are incubated in

for 30 minutes at 60 C. Per unit of activity, take the amount of the enzyme that forms 1 micromole of reconstituted sugars at 1% concentration

substrate centration per minute. The amount of reducing sucrose is determined by the Somoji-Nelson method.

Filter paper activity is determined as follows. 50 mg of Whatman filter paper (16 cm) in 1 ml of 0.05 acetate buffer (pH 4.7) and 1 ml of culture liquid are incubated for one hour at 55 ° C. Per unit of activity take the amount of ferment forming 1 µmol of reducing Sugars per minute. The amount of reducing sucrose is determined by the Somoji-Nelson method.

Cellobiasis activity is determined as follows. 0.2 ml mol of cellobiase, 1.6 ml of 0.05 M acetate buffer (pH 4.7) and 0.2 ml of the culture fluid solution are incubated for

10 min at 55 C. Per unit of activity, take the amount of the enzyme that forms 2 µmol glucose per I min at a concentration of cellobiase 2 10 mol. Determination of glucose is carried out by the gluco-oxidase method.

In addition to highly active cellulases, the proposed producer forms a protein-rich (35-40%) biomass.

Example 1. The strain Aspergillus terreus AT-490 is grown on agar medium containing,%: corn extract 1.5; NaNO 0.3; 0.2; MgSO-7H20 0,05, pH and a strip of filter paper on the surface of the agar, pH 6.3. Cultivation is carried out for 72 hours at 40 ° C. A suspension of spores obtained by flushing from this medium is inoculated with 250 ml flasks containing 150 ml of medium of composition,%: hay 2.0, corn extract 1.5; NaNO-j 0.3; KH-jP04 0.2; MgSO 7H20 0.05, pH 4.5. Growing the culture is carried out on a circular rocking chair (270 rpm) for 72 hours at

s 0 5

0

five

0

five

0

five

70 С

40 ° C. The mycelium with the remains of hay is separated, and the cellulase activities are determined in the culture fluid by the indicated methods. The activity of endoglucanase on Na CMC is 14.5 U / MP, the activity on filter paper is 0.32 U / ml; cellobiose 0.35 u / ml

Example 2. The strain Aspergillus terreus AT-490 is grown according to Example 1 for 96 hours. The activity of the cellulases is determined as in Example 1.

The enzyme activities in the culture fluid are, u / ml: endogluconase by Na CMC 20; activity on filter paper 0.5; cotton 0.9-1.0; cellobiase 0.5 u / ml; xylanase 7-8.

When keeping the drug at

within an hour, the activity is almost completely preserved.

Mutant Asp. terreus AT-490 possesses not only a set of all enzymes of the cellulase complex, but also high xylanase activity. In preparations of cellulase xylanase provides a more complete splitting of agricultural waste to glucose. The xylanases hydrolyze hemicelgapo, which is found in plant and natural substrates, such as straw, wood, etc.

Example 3. Strain Asp. terreus AT-490 is grown in 250-ml Erlenmeyer flasks containing 100 ml of medium of composition, g / l of tap water: sawdust crushed in a ball mill to the size of 0.2-1.0 mm 20.0; NaNO 3.0; 2.0; MgS0.7H ,, 0 0.5, pH „, 4.2–4.6, for 96 h, on a circular rocking chair (230 rpm) at 40 C.

The culture fluid is separated from the biomass by centrifugation and the activity is determined by the indicated methods.

The activity of enzymes is presented in the table.

Claims (2)

STRAIN A5REKS1YL15 TEKNEIZ AT490 - PRODUCER OF CELLULASES Strain AaregegPiz IeggeizAT490 (collection of the Central Museum of Industrial Microorganisms VNIIgenetika, collection number TsMPM R-232) - producer of cellulose. "about 00 00 > 1252336 2