Method of solid-phase cultivation of aspergillus ochraceus to obtain proteinase - protein c activator of blood plasma

FIELD: biotechnologies.

SUBSTANCE: method for obtaining proteinase - protein C activator of blood plasma provides for solid-phase cultivation of fungus strain Aspergillus ochraceus BKM F-4104D on a substrate. The substrate contains the following components at a certain ratio: a solid substance or a carrier, glucose, amylum, hydrolysate of fish flour, peptone, NaCl, KH2PO4, MgSO4·7H2O, and water. As a solid substance or a carrier, vermiculite, polyurethane foam, silica gel or wheat middlings are used. Proteinase activity is up to 300 U/ml.

EFFECT: invention allows obtaining a target product with improved activity.

5 tbl, 15 ex

 

The invention relates to the field of biotechnology, namely the method of production of proteolytic enzymes activators of protein From human blood plasma.

Activators of protein used in medicine for the diagnosis of protein C in the plasma of human blood. The lack of protein in the blood decreases the activity of its activated form, which leads to the occurrence of thromboembolic complications may be fatal [1]. In this regard, it is urgent diagnosis of protein C in the blood [2], which is carried out with the use of diagnostic drugs (Protac®company PENTHAPHARM (Switzerland) - specific proteases - activators of the protein [3].

A method of obtaining activator protein from the venom of the Copperhead snake Ukraine contortrix contortrix [4]. The disadvantages are its extreme difficulty of obtaining and limited Copperhead snake venom as a source of activator protein C.

A method of obtaining activator protein using submerged cultivation of strains of microscopic fungi Aspergillus ochraceus 1, Penicillium claviforme 2, P. granulatum 7, Alternaria sp.10, [5] and strain of Aspergillus ochraceus 513, the culture fluid which has anticoagulant properties [6].

Closest to the proposed method, which should be the closest analogue is the way of gaining the activator protein by culturing a strain of Aspergillus ochraceus BKM F-4104D or Aspergillus ochraceus VKM F-4105D, or Aspergillus ochraceus BKM F-4106D, or Aspergillus ochraceus BKM F-4107D for 2 days at 28°C on seed medium containing wort, glucose and peptone and within 2 days on the rocking chair on a fermentation medium containing (%): glucose - 3,2-3,8, starch - 0,1-0,5, hydrolyzed fish meal - 0.5 to 1.0, peptone, and 0.1 to 0.5, NaCl - 0.1 to 0.2, KH2PO4- 0,04-0,06, MgSO4·7H2O - 0,04-0,06, water to 100%, initial pH 6.5 (RF patent No. 2468081). This method allows to obtain the culture fluid containing the protease - activator protein With the activity of 79.8-89,9 u/ml, where one unit of activity is the amount of enzyme, which it 1 µmol·10-3p-nitroaniline (pNA) from pGlu-Pro-Arg-pNA for 1 min [7].

The disadvantage of the closest analogue is the low yield of the enzyme.

The present invention is to increase the output of the activator protein and the development of standard conditions for its receipt.

The problem is solved:

1) using a strain of Aspergillus ochraceus BKM F-4104D producer activator of protein C;

2) the development of a new method of obtaining activators of protein, providing for cultivation within 3-7 days producing its microscopic strain of the fungus Aspergillus ochraceus BKM F-4104D in a fermentation medium containing glucose, starch, hydrolyzed fish meal, peptone, sodium chloride, potassium dihydrophosphate and magnesium sulfate, water, characterized in that the farm is operating environment contains solid bulk substrate or solid granular media, which use wheat bran, silica gel, vermiculite or polyurethane foam.

3) by using the following ratio of components (%):

vermiculite - 14,0-20,0,

glucose - 2,5-3,0,

starch - 0,4 - 0,5,

hydrolyzed fish meal - 0,5-0,6

peptone - 0,2-0,3,

NaCl - 0,07-0,08

KH2PO4- 0,035-0,040,

MgSO4·7H2O - 0,035-0,040,

water the rest.

The initial pH of 6.5.

4) by using the following ratio of components (%):

polyurethane - 7,0-15,0,

glucose - 3,0-3,3,

starch - 0,4-0,5,

hydrolyzed fish meal - 0,6-0,7,

peptone - 0,25-0,30,

NaCl - 0,09-0,13,

KH2PO4- 0,04-0,05,

MgSO4·7H2O - 0,04-0,05,

water the rest.

The initial pH of 6.5.

5) by using the following ratio of components (%):

silica gel - 35,0-45,0,

glucose - 1,9-2,3,

starch - 0,3-0,4,

hydrolyzed fish meal - 0,40-0,50,

peptone - 0,20-0,30,

NaCl - 0,08-0,10,

KH2PO4- 0,03-0,04,

MgSO4·7H2O - 0,03-0,04,

water the rest.

The initial pH of 6.5.

6) by using the following ratio of components (%):

wheat bran - 22,0,-28,0,

glucose - 2,5-2,8,

starch - 0,4-0,5,

hydrolyzed fish meal - 0,5-0,6,

peptone - 0,2-0,3,

NaCl - 0,07-0,08

KH2PO4- 0,035-0,040,

MgSO4·7H2O - 0,035-0,040,

water the rest.

The initial pH of 6.5.

P the completion of the cultivation of 0.05 M Tris-HCl buffer (pH 8,2) the extraction is carried out activators of protein from the medium on an orbital shaker. The liquid fraction is separated from the biomass and residues of components of the environment by filtration (centrifugation).

The secretion of extracellular enzymes with an activator for protein With the action of the liquid fraction of conduct known methods by precipitation of proteins with ammonium sulfate to the degree of saturation of 80%, by dissolving in a minimum volume of distilled water. The formed precipitation centrifuged, dissolved in 0.005 M Tris-HCl buffer (pH 8,2) or distilled water to remove the insoluble portion of the precipitation and removal of ammonium salts solution cialiswhat at 4°C against the same buffer or hold gel-filtration on a column of Sephadex G-25 [5]. The resulting solution of protease - activator protein stored in a refrigerator at 4°C or lyophilizer for long-term storage.

A strain of Aspergillus ochraceus BKM F-4104D selected A. C. Karakulam deposited in the all-Russian Collection of Microorganisms and is an active producer of extracellular proteinases of activators of protein [8].

Example 1.

Seed Aspergillus ochraceus BKM F-4104D obtained by cultivating in vitro the beveled wort-agar 4-5° Balinga. To kasparovchess culture 6-8 day old add sterile 0,0005% solution of tween-80 and by flushing prepare spore suspension of a producer. I spend 1 ml of suspension in a conical flask of 250 ml with 30.0 g is fermentational medium of the following composition (in%):

vermiculite - 17,0

glucose - 2,6,

starch - 0,4,

hydrolyzed fish meal - 0,4,

peptone and 0.2,

NaCl is 0.07,

KH2PO4- 0,035,

MgSO4·7H2O - 0,035,

water the rest.

The initial pH of 6.5.

Vermiculite (5.0 g) and liquid medium (25,0 ml) sterilized separately and then mixed in flasks.

Solid-phase cultivation is carried out in stationary conditions at 28°C.

After 7 days of cultivation carried out the extraction of the enzyme activators of protein C. In flasks add 35 ml of 0.05 M Tris-HCl buffer (pH 8,2) and placed on an orbital shaker (200 rpm) for 40-60 minutes Then hold the separation of the liquid fraction from the biomass by filtration (centrifugation).

The resulting filtrate (supernatant) define an activator for protein C activity by the method of [9] using the chromogenic peptide substrate pGlu-Pro-Arg-pNA (pyroglutamyl-shed-arginyl-para-nitroanilide) [10, 11]. Activated protein C plasma specific it p-nitroaniline (pNA) from pGlu-Pro-Arg-pNA. Per unit activity of activator protein With take that quantity of enzyme which it 1 µmol·10-3p-nitroaniline (pNA) from pGlu-Pro-Arg-pNA for 1 min in 1 ml at a pH of 8.2 and 37°C.

The activity of activator protein From A. ochraceus BKM F-4104D 300 u/ml of fermentation medium (without taking into account the weight of vermiculite).

Example 2.

olivinovye strain of Aspergillus ochraceus BKM F-4104D, receiving filtrate and the determination of the activity of activator protein carried out as in example 1, but using a nutrient medium of the following composition (in %):

vermiculite - 20,0.

glucose - 2,8,

starch - 0,5,

hydrolyzed fish meal - 0,6,

peptone - 0.3, and

NaCl and 0.08

KH2PO4- 0,040,

MgSO4·7H2O - 0,035-0,040.

water the rest.

The initial pH of 6.5.

To do this in conical flasks of 250 ml mixing 4.0 g of vermiculite and 16.0 ml of a liquid medium.

The activity of activator protein With the cultivation of A. ochraceus BKM F-4104D on the environment is 290 u/ml of fermentation medium.

Example 3.

Cultivation of a strain of Aspergillus ochraceus BKM F-4104D, receiving the filtrate and the determination of the activity of activator protein carried out as in example 1, but using a nutrient medium of the following composition (in %):

vermiculite - 14,0.

glucose - 2,8,

starch - 0,5,

hydrolyzed fish meal - 0,6,

peptone - 0.3, and

NaCl and 0.08

KH2PO4- 0,040,

MgSO4·7H2O - 0,035-0,040,

water the rest.

The initial pH of 6.5.

To do this in conical flasks of 250 ml mixing 4.0 g of vermiculite and 24.0 ml of fermentation medium.

The activity of activator protein From A. ochraceus BKM F-4104D is 235 u/ml of fermentation medium.

Example 4.

Cultivation of a strain of Aspergillus ochraceus BKM F-4104D, receiving the filtrate and the determination of the activity and is of tuatara protein With the conduct of example 1, but the cultivation is conducted for 5 days and use a nutrient medium with polyurethane foam of the following composition (in %):

polyurethane foam -11,0,

glucose - 3,2,

starch - 0,4,

hydrolyzed fish meal - 0,6,

peptone - 0,25,

NaCl - 0,11,

KH2PO4- 0,04,

MgSO4·7H2O - 0,04,

water the rest.

The initial pH of 6.5.

The activity of activator protein From A. ochraceus BKM F-4104D is 131,8 u/ml of fermentation medium.

Example 5.

Cultivation of a strain of Aspergillus ochraceus BKM F-4104D, receiving the filtrate and the determination of the activity of the activator protein is performed according to the example 4, but using a nutrient medium with polyurethane foam of the following composition (in %):

polyurethane - 15,0,

glucose - 3,3,

starch - 0,5,

hydrolyzed fish meal - 0,7,

peptone - 0,30,

NaCl - 0,13,

KH2PO4to 0.05,

MgSO4·7H2O - 0.05,

water the rest.

The initial pH of 6.5.

The activity of activator protein From A. ochraceus BKM F-4104D makes 125.5 u/ml of fermentation medium.

Example 6.

Cultivation of a strain of Aspergillus ochraceus BKM F-4104D, receiving the filtrate and the determination of the activity of the activator protein is performed according to the example 4, but using a nutrient medium with polyurethane foam of the following composition (in %):

polyurethane - 7,0,

glucose - 3,0,

starch - 0,4,

hydrolyzed fish meal - 0,6,

peptone - 0,25,

NaCl - 0,09,

KH2 PO4- 0,04,

MgSO4·7H2O - 0,04,

water the rest.

The initial pH of 6.5.

The activity of activator protein From A. ochraceus BKM F-4104D is 126,5 u/ml of fermentation medium.

Example 7.

Cultivation of a strain of Aspergillus ochraceus BKM F-4104D, receiving the filtrate and the determination of the activity of activator protein carried out as in example 1, but growing lead 4 days and use a nutrient medium of the following composition (in %):

silica gel - 35,0,

glucose - 1.9,

starch - 0.3, and

hydrolyzed fish meal - 0,40,

peptone - 0,20,

NaCl and 0.08,

KH2PO4- 0,03,

MgSO4·7H2O - 0,03,

water the rest.

The initial pH of 6.5.

The activity of activator protein From A. ochraceus BKM F-4104D is 185,5 u/ml of fermentation medium.

Example 8.

Cultivation of a strain of Aspergillus ochraceus BKM F-4104D, receiving the filtrate and the determination of the activity of activator protein carried out as in example 1, but growing lead 4 days and use a nutrient medium of the following composition (in %):

silica gel - 45,0,

glucose - 2,3,

starch - 0,4,

hydrolyzed fish meal - 0,50,

peptone - 0,30,

NaCl - 0,10,

KH2PO4- 0,04,

MgSO4·7H2O - 0,04,

water the rest.

The initial pH of 6.5.

The activity of activator protein From A. ochraceus BKM F-4104D is 191,1 u/ml of fermentation medium.

Example 9.

Cultivation of a strain of Aspergillus ochraceus BK F-4104D, receiving filtrate and the determination of the activity of activator protein carried out as in example 1, but growing lead 6 days and use a nutrient medium of the following composition (in %):

wheat bran - 22,0,

glucose is 2.5,

starch - 0,4,

hydrolyzed fish meal - 0,5,

peptone and 0.2,

NaCl is 0.07,

KH2PO4- 0,035,

MgSO4·7H2O - 0,035,

water the rest.

The initial pH of 6.5.

The activity of activator protein From A. ochraceus BKM F-4104D is 100,9 u/ml of fermentation medium.

Example 10.

Cultivation of a strain of Aspergillus ochraceus BKM F-4104D, receiving the filtrate and the determination of the activity of activator protein carried out as in example 1, but growing lead 6 days and use a nutrient medium of the following composition (in %):

wheat bran - 28,0,

glucose - 2,8,

starch - 0,5,

hydrolyzed fish meal - 0,6,

peptone - 0.3, and

NaCl and 0.08

KH2PO4- 0,040,

MgSO4·7H2O - 0,040,

water the rest.

The initial pH of 6.5.

The activity of activator protein From A. ochraceus BKM F-4104D is 99.1 u/ml of fermentation medium.

Example 11.

Solid-phase cultivation of a strain of Aspergillus ochraceus BKM F-4104D spend with vermiculite as a carrier of example 1. The activity of activator protein To determine daily from 2 to 8 days (table.1).

Table 1
MediaAn activator for protein C activity, u/ml
2 days3 days4 days5 days6 days7 days8 days
Vermiculite98,1104,7110,4148,2180,3300,0256,9
the coefficient of variation of the data is 4%

The activity of activator protein From A. ochraceus BKM F-4104D grows from 98,1 u /ml (2nd day) and reaches the highest values, 148,2-300 u/ml of fermentation medium for 5-7 days, and decreases with longer cultivation (256,9 u /ml for 8 days).

Example 12.

Solid-phase cultivation of a strain of Aspergillus ochraceus BKM F-4104D spend with polyurethane foam as the carrier according to example 4, but the duration of cultivation is 2, 3, 4, 5 and 6 days. The activity of activator protein reaches its highest values in the period of 3-5 days, 111,0-131,8 u/ml of fermentation medium (table.2).

Table 2
MediaAn activator for protein C activity, u/ml
2 days3 days4 days5 days6 days
Polyurethane foam88,7111,0118,4131,894,6
the coefficient of variation of the data - 2%

Example 13.

Solid-phase cultivation of a strain of Aspergillus ochraceus BKM F-4104D carried out with silica gel as the carrier according to example 7, but growing lead for 2, 3, 4, 5, 6 and 7 days and use the medium of the following composition (in %):

silica gel - 40,0

glucose - 2,1,

starch - 0,35,

hydrolyzed fish meal - 0.45 and

peptone - 0,25,

NaCl - 0,09,

KH2PO4- 0,035,

MgSO4·7H2O - 0,035,

water the rest.

The initial pH of 6.5.

The activity of activator protein reaches its highest values in the period of 3-5 days with a maximum of 200.0 u /ml of fermentation medium for 4 days (table.3).

Table 3
MediaAn activator for protein C activity, u/ml
2 days3 days4 days5 days6 days7 days
Silica gel148,3152,7200,0156, 3mm146,3135,4
the coefficient of variation of the data is 3%

Example 14.

Solid-phase cultivation of a strain of Aspergillus ochraceus BKM F-4104D spend with wheat bran in example 9, but growing lead for 3, 4, 5, 6 and 7 days and use the medium of the following composition (in %):

wheat bran - 25,0,

glucose - 2,7,

starch - 0.45 and

hydrolyzed fish meal is 0.55,

peptone - 0,25,

NaCl - 0,07

KH2PO4- 0,035,

MgSO4·7H2O - 0,035,

water the rest.

The initial pH of 6.5.

The activity of the activator protein is highest in the period of 4-6 days, 98,3-102,0 u /ml of fermentation medium (table.4).

Table 4
Solid with Strat An activator for protein C activity, u/ml
3 days4 days5 days6 days7 days
Wheat bran84,498,3116,3102,080,4
the coefficient of variation of the data - 2%

Example 15. Comparison of the proposed method and the nearest analogue

Comparison of an activator for protein C activity in the culture fluid of strain of A. ochraceus BKM F-4104D under cultivation 7 days of the proposed method in a medium containing (in %):

vermiculite - 17,0

glucose - 2,6,

starch - 0,4,

hydrolyzed fish meal - 0,4,

peptone and 0.2,

NaCl is 0.07,

KH2PO4- 0,035,

MgSO4·7H2O - 0,035,

water the rest,

initial pH 6.5;

the cultivation of A. ochraceus BKM F-4104D 4 days in a medium containing (in %):

silica gel - 40,0

glucose - 2,1,

starch - 0,35,

hydrolyzed fish meal - 0.45 and

peptone - 0,25,

NaCl - 0,09,

KH2PO4- 0,035,

MgSO4·7H2O - 0,035,

water the rest,

initial pH 6.5;

the cultivation of A. ochraceus BKM-4104D 5 days on the environment, contains (in %):

polyurethane foam is 11.0

glucose - 3,2,

starch - 0,4,

hydrolyzed fish meal - 0,6,

peptone - 0,25,

NaCl - 0.11,

KH2PO4- 0,04,

MgSO4·7H2O - 0,04,

water the rest,

the initial pH of 6.5.

the cultivation of A. ochraceus BKM F-4104D 4 days in a medium containing (in %):

wheat bran - 25,0,

glucose - 2,7,

starch - 0.45 and

hydrolyzed fish meal is 0.55,

peptone - 0,25,

NaCl - 0,07

KH2PO4- 0,035,

MgSO4·7H2O - 0,035,

water the rest,

the initial pH of 6.5.

and the cultivation of A. ochraceus BKM F-4104D the nearest analogue to the fermentation medium with an initial pH 6.5, containing (in %):

glucose - 3,5,

starch - 0,1,

hydrolyzed fish meal - 1,0,

peptone - 0,1,

NaCl to 0.2,

KH2PO4to 0.05,

MgSO4·7H2O - 0,05,

water to 100%,

initial pH 6.5;

shows that the proposed method of solid-phase cultivation have higher activator for protein C activity (13-250%) depending on a solid substrate or substrate in a fermentation medium (table.5).

Table 5
OptionAn activator for protein C activity, u/ml
Way - the closest analogue of the underlying cultivation of A. ochraceus BKM F-4104D86,1
The proposed method of solid-phase cultivation of A ochraceus BKM F-4104D with vermiculite in a fermentation medium300,0
The proposed method of solid-phase cultivation of A ochraceus BKM F-4104D with silica gel in a fermentation medium200.0
The proposed method of solid-phase cultivation of A. ochraceus BKM F-4104D with polyurethane foam in a fermentation medium131,8
The proposed method of solid-phase cultivation of A. ochraceus BKM F-4104D with wheat bran in a fermentation medium116,3

Thus, the proposed method allows to obtain the culture fluid with a higher activity of protease - activator of protein C.

Literature

1. Kogan, A. E., Strukova, S. M. 1993. Protein: mechanism of activation and anticoagulant action. Biochemistry, 58, 6, 827-844.

2. Gempeler-Messina P. M., Volz, K., Buhler C., Muller, S. 2001. Protein With activators from snake venoms and their diagnostic use. Haemostasis, 31, 266-272.

3. Martinoli J. L., Stoker K. 1986. Fast functional protein With assay using Protac®, a novel protein With activator. Thromb. Res., 43, 253-256.

4. Stoker, K., Fisher, H., Meier J., Brogli M., Svedsen L., 1987. Characterization of the protein With activator Protac® from the venom of the southern copperhead (Ukraine contortrix) snake. Toxicon, 25, 3, 239-252.

5. La is Dow N.S., Kurkov A. C., Kulikova O. M., Batmunkh Bpts, Strukova, S. M., Egorov, N. With.1998. Extracellular proteases of micromycetes with fibrinolytic and anticoagulative properties. Microbiology, 67, 2, 215-220.

6. Batmunkh B. P., N. Egorov.With.2001. Isolation, purification and separation of the complex preparation of extracellular proteases of Aspergillus ochraceus 513 with fibrinolytic and anticoagulant properties. Microbiology. 70, 5, 602-606.

7. RF patent №2468081.

8. RF patent №2461614.

9. Osmolovskaya, A. A., Creier Century, Kurkov A. C., Baranov N. A., Egorov N. With. 2012. The phytopathogenic fungi Aspergillus ochraceus - producers of extracellular proteases - activators of protein From blood plasma. Applied biochemistry and Microbiology, 48, 5, 537-542.

10. Sakata T., Hatsuyama H., Kitamura T., Hekida K., J. Katayama, T. Matsumata Study of chromogenic substrate on protein With the activity assay in patients treated with narfarin. Rinsho Byori. 1990. V. 38, No. 8, P. 937-941.

11. Dang Q. D., Cera E. D., 1997. Chromogenic Substrates selective for activated protein C. Blood, 89, 2220-2221.

The method of obtaining the protease - activator protein of blood plasma, by cultivation of a strain of microscopic fungus Aspergillus ochraceus BKM F-4104D, characterized in that conduct solid-phase cultivation in fermentation medium with the following composition, %:
vermiculite - 14,0-20,0,
glucose - 2,5-3,0,
starch - 0,4-0,5,
hydrolyzed fish meal - 0,5-0,6,
peptone - 0,2-0,3,
NaCl - 0,07-0,08
KH2PO4- 0,035-0,040,
MgSO4·7H2O - 0,035-0,040,
in the a - up to 100%;
or in the following ratio of components, %:
polyurethane - 7,0-15,0,
glucose - 3,0-3,3,
starch - 0,4-0,5,
hydrolyzed fish meal - 0,6-0,7,
peptone - 0,25-0,30,
NaCl - 0,09-0,13,
KH2PO4- 0,04-0,05,
MgSO4·7H2O - 0,04-0,05,
water up to 100%;
or in the following ratio of components, %:
silica gel - 35,0-45,0,
glucose - 1,9-2,3,
starch - 0,3-0,4,
hydrolyzed fish meal - 0,40-0,50,
peptone - 0,20-0,30,
NaCl - 0,08-0,10,
KH2PO4- 0,03-0,04,
MgSO4·7H2O - 0,03-0,04,
water up to 100%;
or in the following ratio of components, %:
wheat bran - 22,0,-28,0,
glucose - 2,5-2,8,
starch - 0,4-0,5,
hydrolyzed fish meal - 0,5-0,6,
peptone - 0,2-0,3,
NaCl - 0,07-0,08
KH2PO4- 0,035-0,040,
MgSO4·7H2O - 0,035-0,040,
water up to 100%.



 

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EFFECT: invention provides normalising the intestinal microbiocenosis and increasing the animal's body weight.

1 tbl

FIELD: biotechnology.

SUBSTANCE: carrier material for biomass for filtration of oil-contaminated waste waters is proposed. The carrier comprises a filtering material with immobilised cells of oil-oxidising microorganism Rhodotorula sp. VKM Y-2993D with the titer of cells - 106 CFU/cm3. The filtering material is used as basalt fibre STBFconst preliminary modified with cationic starch - oxyamil OPV-1.

EFFECT: proposed carrier material has a high retention capacity of suspended particles and oil products and is used for filling the filters for cleaning oil-contaminated waste waters.

1 tbl, 1 ex

FIELD: biotechnology.

SUBSTANCE: proposed strain Pseudomonas migulae is deposited in the Russian National Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms under the number of VKM B-2761D. The strain is isolated from samples of bottom soil contaminated with oil of freshwater lake of Usinsky district of the Komi Republic in the zone of chronic oil pollution.

EFFECT: strain has high oil-oxidising activity against naphthenic hydrocarbons, is capable of reducing the concentration of hydrocarbons dissolved in water and accelerates the destruction of oil in soils and bottom sediments of water reservoirs.

3 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: inventions relate to a strain of the fungus Penicillium verruculosum B10 EGII and a method of production of the fodder complex enzyme preparation. The presented strain is a producer of endo-1.3/1.4-β-glucanase, cellulase, β-glucosidase and xylanase. It is obtained on the basis of a strain Penicillium verruculosum RNCM F-3764D, by transforming with its plasmids pSTA10 and pPrCBHI-EGII. In the plasmid pPrCBHI-EGII the nucleotide sequence of the structural gene of endo-1.3/1.4-β-glucanase of Penicillium verruculosum is aligned with nucleotide sequences of the promoter region, the signal peptide and the terminator of gene cbhI of cellobiohydrolase I Penicillium verruculosum. The method of production of the preparation containing endo-1.3/1.4-β-glucanase, cellulase, β-glucosidase and xylanase comprises cultivation of the strain of fungus Penicillium verruculosum B10 EGII on the optimised nutrient medium in the fermenter and filtration, ultrafiltration and lyophilisation of the culture liquid.

EFFECT: inventions can be used to produce fodder complex enzyme preparation enriched with endoglucanase.

2 cl, 1 dwg, 2 tbl, 5 ex

FIELD: food industry.

SUBSTANCE: additive is produced from wheat offal fermented with mould fungi. The additive agents are at least one ferment and a mixture of nutritional ingredients for yeast: ergosterol, N-acetylglucosamine, vitamins, nucleic acids and amino acids. The said additive is also used for activation of alcohol fermentation or pre-fermentation intended for preparation of yeast under aerobic conditions.

EFFECT: reduction of the simultaneous saccharification-fermentation stage time in the process of ethanol production, increase of yeast growth and efficiency.

21 cl, 2 dwg, 4 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: method of obtaining proteinase-activator of protein C in blood plasma includes cultivation of strains of Aspergillus ochraceus VKM F-4104D or Aspergillus ochraceus VKM F-4105D or Aspergillus ochraceus VKM F-4106D or Aspergillus ochraceus VKM F-4107D on nutritional medium at initial value pH 6.5. Nutritional medium contains (%): glucose 3.2-3.8, starch 0.1-1.0, fish flour hydrolysate 0.5-1.0, peptone 0.1-0.5, NaCl 0.1-0.2, KH2PO4 0.04-0.06, MgSO4-7H2O 0.04-0.06, water to 100%. Anticoagulant activity of solution of proteins of culture liquid of strains on elongation of activated partial thromboplastin time (CPTT) constitutes 1020-1032%. Activity of protein C activator in culture liquid of strains constitutes 79.8-89.9 units/ml.

EFFECT: increased activator activity.

2 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: Aspergillus ochraceus strain is recovered from carbonate chernozem samples and deposited in Russian National Collection of Microorganisms, No. F-4106D. The strain produces proteinase being a human blood plasma protein C activator into a culture fluid while grown on a medium containing carbohydrate and protein compounds, and mineral salts. Anticoagulant activity of the strain to prolong activated partial thromboplastin time by 2 cultivation days makes 960%. Proteinase activity of the strain Aspergillus ochraceus in the culture fluid determined with the use of chromogenic peptide substrate pGlu-Pro-Arg-pNA makes 77.9 units/ml.

EFFECT: higher proteinase activity of the strain.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: Aspergillus ochraceus strain is recovered from plant residue samples on wort agar and deposited in Russian National Collection of Microorganisms, No. F-4105D. The strain produces proteinase being a human blood plasma protein C activator into a culture fluid while grown on a medium containing carbohydrate and protein compounds, and mineral salts. Anticoagulant activity of the strain to prolong activated partial thromboplastin time by 2 cultivation days makes 920%. Proteinase activity of the strain Aspergillus ochraceus in the culture fluid determined with the use of chromogenic peptide substrate pGlu-Pro-Arg-pNA makes 72.5 units/ml.

EFFECT: higher proteinase activity of the strain.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: Aspergillus ochraceus strain is recovered from soil sampled in Krasnodar Territory and deposited in Russian National Collection of Microorganisms, No. F-4104D. The strain produces proteinase being a human blood plasma protein C activator into a culture fluid while grown on a medium containing carbohydrate and protein compounds, and mineral salts. Anticoagulant activity of the strain to prolong activated partial thromboplastin time by 2 cultivation days makes 960%. Proteinase activity of the strain Aspergillus ochraceus in the culture fluid determined with the use of chromogenic peptide substrate pGlu-Pro-Arg-pNA makes 77.5 units/ml.

EFFECT: higher proteinase activity of said strain.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: strain Aspergillus ochraceus is recovered from grey soil samples and deposited in Russian National Collection of Microorganisms, No. F-4107D. The strain produces proteinase being a human blood plasma protein C activator into a culture fluid while grown on a medium containing carbohydrate and protein compounds, and mineral salts. Anticoagulant activity of the strain to prolong activated partial thromboplastin time by 2 cultivation days makes 955%. Proteinase activity of the strain Aspergillus ochraceus in the culture fluid determined with the use of chromogenic peptide substrate pGlu-Pro-Arg-pNA makes 76.1 units/ml.

EFFECT: higher activity.

1 tbl, 2 ex

FIELD: food industry.

SUBSTANCE: pasteurised food product containing a proline-specific protease has water activity equal to at least 0.85. Used as the enzyme is protease extracted from Aspergillus or belonging to the S28 serine proteases family. The optimal activity of the said protease is at a pH value from 1 to 7, preferably - at a pH value from 2 to 6. Additionally proposed is a food product containing less than 1 wt % of protein or peptides. The said food products are produced by way of addition of a proline-specific protease to them.

EFFECT: such products consumption ensures gluten peptides splitting and is recommended to patients suffering from gluten intolerance.

17 cl, 5 dwg, 2 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: disclosed is isolated polypeptide being acid-proof metalloprotease isolated from Thermoascus aurantiacus. Described are strain Thermoascus aurantiacus CGMCC No 0670 for polypeptide production and method for polypeptide production using said strain. Disclosed is method for plant protein treatment to increase digestion value thereof by using said polypeptide.

EFFECT: protease of good acid resistance useful in feed production.

6 cl, 7 ex, 4 tbl

FIELD: biotechnology, microbiology, biochemistry.

SUBSTANCE: invention relates to development of a novel strain used for preparing enzyme representing a complex of acid subacid proteases. Strain is prepared by selection from the known strain Aspergillus oryzae (VKPM F-683) by multistep selection using effective methods of mutagenesis. Strain is stored as lyophilic dried culture and on slants with wort-agar in the biotechnology section of enzyme preparations in the food processing department of the State Scientific Institute VNII of food processing technology in Moscow. Invention provides preparing the strain possessing the high level of synthesis of acid and subacid proteases and high total activity of enzyme in cultural fluid exceeding activity of analogue by 2.0-2.6-fold and in reducing culturing process time.

EFFECT: improved and valuable properties of strain.

2 tbl, 5 ex

FIELD: biotechnology, microbiology, biochemistry.

SUBSTANCE: invention relates to the strain Aspergillus oryzae-12 providing the high level of synthesis of acid proteases and xylanase. The strain is obtained by multistep selection of the strain Aspergillus oryzae-387 (VKPM F-683) using effective methods of mutagenesis. The strain is deposited in collection VKPM at № F-932. Invention provides preparing the enzyme complex showing the high proteolytic and xylanolytic activity in cultural fluid wherein their level exceeds activity in analogue by 2.5-2.8-fold.

EFFECT: valuable properties of strain.

2 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology. A method of obtaining a granulated product includes growing filamentous fungi of a Monilialeae family, preferably Arthrobotrys conoides Dreschsler in a suitable liquid culture medium. The obtained culture of the filamentous fungus is mixed with at least one type of modified starch and starch flour, with the modified starch and modified flour being present in weight ratios, constituting from 30:70 to 60:40. Filling agents and in case of necessity nutritional substances are added to the obtained mixture with obtaining a paste. The granulation of the paste is carried out. The obtained granules are dried until the moisture level lower than 13% is achieved, preferably to the level constituting from 9 to 10%. The granulated product, obtained by the method described above, is used for the application in a pesticidal composition.

EFFECT: group of inventions provides obtaining the target product with high dispersability and a high percent of survival of the filamentous fungi propagules.

9 cl, 1 dwg, 4 ex

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