RU2468081C2 - Method of obtaining proteinase-activator of protein c in blood plasma - Google Patents

Abstract

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

Description

The invention relates to the field of biotechnology, and in particular to a method for producing proteolytic enzymes - activators of protein C of human blood plasma.

Protein C activators are used in medicine for the diagnosis of protein C in human blood plasma. With a lack of protein C in the blood, the activity of its activated form decreases, which causes thromboembolic complications with a possible fatal outcome [1]. In this regard, it is important to diagnose the protein C content in the blood [2], which is carried out using specific proteinases - activators of protein C in diagnostic preparations (Protac ^® from PENTHAPHARM (Switzerland)) [3].

A known method of obtaining a protein C activator from the venom of the thyroid muzzle Agkistrodon contortrix contortrix [4]. Its disadvantage is the extreme difficulty of obtaining and the limited poison of the muzzle as a source of protein C activator.

A known method of producing a protein C activator using strains of microscopic fungi Aspergillus ochraceus 1, Penicillium claviforme 2, P. granulatum 7, Alternaria sp.10, the culture fluid of which has anticoagulant properties [5].

A known method of obtaining a protein C activator using a strain of Aspergillus ochraceus 513 [6].

Closest to the proposed method, which should be taken as the closest analogue, is a method for producing a protein C activator by culturing for 7 days a strain of Aspergillus ochraceus 1 - the most active producer of protein C activator in a seed medium containing wort, glucose and peptone, and a fermentation medium containing (in%): glucose - 3.5, starch - 1.0, soy flour - 2.0, meat extract - 0.5, peptone - 0.5, NaCl - 0.2, KH ₂ PO ₄ - 0,05, MgSO ₄ · 7H ₂ O - 0,05, initial pH 5.5, allows to obtain a culture broth containing protease - activator C roteina with anticoagulant activity 854% [5], defined by lengthening of activated partial thromboplastin time of human plasma [7].

The disadvantages of the closest analogue are the low yield of the enzyme and the long fermentation time (7 days).

The objective of the present invention is to increase the yield of protein C activator, reduce fermentation time and develop standard conditions for its production.

The problem is solved:

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

2) using the strain Aspergillus ochraceus BKM F-4105D, a producer of the protein C activator;

3) using the strain Aspergillus ochraceus BKM F-4106D, a producer of the protein C activator;

4) using the strain Aspergillus ochraceus BKM F-4107D, a producer of the protein C activator;

5) the development of a new method for producing extracellular proteinases - activators of protein C, which provides a two-stage cultivation of the strains of the microscopic fungus Aspergillus ochraceus producing it - on a seed medium containing glucose and peptone must, and a fermentation medium containing glucose, starch, peptone, sodium chloride, potassium dihydrogen phosphate and magnesium sulfate, characterized in that in a fermentation medium soy flour and meat extract are replaced with a hydrolyzate of fish meal, and a higher initial pH of the medium is set , with the following ratio of components (%):

glucose 3.2-3.8 starch 0.1-1.0 fish meal hydrolyzate 0.5-1.0 peptone 0.1-0.5 NaCl 0.1-0.2 KN ₂ PO ₄ 0.04-0.06 MgSO ₄ · 7H ₂ O 0.04-0.06 Water Rest

The initial pH is 6.5.

Isolation of extracellular enzymes with activator to protein C by action from the culture fluid is carried out by known methods by precipitation of proteins with ammonium sulfate to a degree of saturation of 80%, dissolving in a minimum volume of distilled water. The precipitates formed are centrifuged, dissolved in 0.005 M Tris-HCl buffer (pH 8.2) or distilled water, the insoluble part of the precipitates are removed, and the solution is dialyzed at 4 ° C against the same buffer to remove ammonium salts or gel filtration is performed on columns with Sephadex G-25 [5]. The resulting solution of proteinase - activator of protein C is stored in a refrigerator at 4 ° C or lyophilized for long-term storage.

The strain Aspergillus ochraceus BKM F-4104D allocated Kurakov A.V. on Wednesday Chapek-agar, from samples of buried soil taken in the Krasnodar Territory (v. Sennoy, excavation of the ancient Greek city of Fanagoria).

The strain Aspergillus ochraceus is deposited in the All-Russian Collection of Microorganisms under the number BKM F-4104D.

The strain Aspergillus ochraceus BKM F-4104D belongs to mitospore fungi - anamorphs of ascomycetes affinity: genus Emericella, family Trichocomaceae, order Eurotiales, class Eurotiomycetes, Ascomycota department.

Cultural and morphological features

The strain Aspergillus ochraceus BKM F-4104D has cultural and morphological characters typical of this species [8, 9]. Colonies on Chapek and wort-agar media reach an average of 3.5 cm in diameter for 10 days, flat, velvety, bright ocher in color, with small drops of exudate. Reverse side of the reddish-brown tones. The conidial heads are spherical, with aging, they decompose into 2-4 compact columns of light ocher-brown color. The conidiophore is two-tiered with densely located throughout the vesicle metula (15-20 × 5-6 microns) and phialids with a size (7-13 × 2-3) microns. Conidiophores with a diameter of 7-9 microns, smooth, unpainted to yellowish tones, the expansion of the conidiophores is round, the conidia are round, smooth, 3.0-3.5 microns.

Physiological and biochemical characteristics

The strain grows well on standard media: wort agar, Chapek's medium, Getchinson's medium, glucose-peptone medium at a temperature of 25 ° C. Sporulation of the strain begins at 4-5 days, forms copiously spore-forming colonies by 10 days. The strain is thermo-tolerant, able to grow in the temperature range of 8 ° C-42 ° C. Spores remain viable when heated in the soil for several hours at a temperature of 60 ° C. On nutrient media it grows in a wide range of pH, including acidic (up to pH 2.5-3.0) and slightly alkaline region (pH 7-9). The strain has proteolytic (caseinolytic), cellulolytic and xylanase (hydrolyzes cellulose, hemicellulose) and lipolytic activity.

The strain is maintained on jambs with wort agar 4-5 ° B with an initial pH of 6.0-6.2 or standard Chapek-agar medium at room temperature with reseeding after 1-3 months, and when stored at 5 ° C with reseeding through 6-12 months.

The strain Aspergillus ochraceus BKM F-4104D is a non-pathogenic and non-toxic microorganism. With intravenous infection of rabbits does not show zoopathogenic properties. When applying the strain culture extract to rabbit skin, it does not cause a toxic reaction.

The strain Aspergillus ochraceus BKM F-4105D was isolated by A.V. Kurakov, isolated from samples of plant residues on wort agar.

The strain Aspergillus ochraceus is deposited in the All-Russian Collection of Microorganisms under the number BKM F-4105D.

The strain Aspergillus ochraceus BKM F-4105D belongs to mitospore fungi - anamorphs of the ascomycetes affinity: the genus Emericella, the Trichocomaceae family, order Eurotiales, class Ewotiomycetes, Ascomycota department.

Cultural and morphological features

The strain Aspergillus ochraceus BKM F-4105D has typical cultural and morphological characters [8, 9]. The colonies on the Chapek and wort-agar media reach 2.5-3.5 cm in diameter for 10 days, flat, velvety, buffy in color with small drops of exudate. Reverse side of brownish-reddish tones. Forms spherical sclerotia of a reddish color. The conidial heads are spherical, with aging, they decompose into 2 compact columns of light ocher-brown color. The conidiophore is two-tiered; throughout the vesicle, metulae (15-20 × 5-6 μm) and phialids (7-13 × 2-3) μm are densely located. Conidiophores are smooth, unpainted to yellowish tones, the expansion of the conidiophores is round, the conidia are round, smooth, 3.5-4.0 microns.

Physiological and biochemical characteristics

The strain grows well on standard media: wort agar, Chapek's medium, Getchinson's medium, glucose-peptone medium at a temperature of 25 ° C. Sporulation of the strain begins at 4-5 days, forms copiously spore-forming colonies by 10 days. The strain is thermo-tolerant, able to grow in the temperature range of 8 ° C-42 ° C. The strain has proteolytic (caseinolytic) and cellulolytic activity. The strain is maintained on jambs with wort agar 4-5 ° B with an initial pH of 6.0-6.2 or standard Chapek-agar medium at room temperature with reseeding after 1-3 months, and when stored at 5 ° C with reseeding through 6-12 months.

The strain Aspergillus ochraceus BKM F-4105D is a non-pathogenic and non-toxic microorganism. With intravenous infection of rabbits does not show zoopathogenic properties. When applying the strain culture extract to rabbit skin, it does not cause a toxic reaction.

The strain Aspergillus ochraceus BKM F-4106D was isolated by A.V. Kurakov from samples of chernozem soil on carbonate rocks that were selected by him in the steppe zone of the Voronezh region. The strain was isolated on bait - filter paper, impregnated with butter from a water-soil suspension during incubation for 7 days at 49 ° C, and then at 20 ° C and transferred to wort agar.

The strain Aspergillus ochraceus is deposited in the All-Russian Collection of Microorganisms under the number BKM F-4106D.

The strain Aspergillus ochraceus BKM F-4106D belongs to mitospore fungi - anamorphs of ascomycetes affinity: genus Emericella, family Trichocomaceae, order Eurotiales, class Eurotiomycetes, Ascomycota department.

Cultural and morphological features

The strain Aspergillus ochraceus BKM F-4106D has typical cultural and morphological characters [8, 9]. Colonies on Chapek and wort-agar media reach an average of 3.5 cm in diameter for 10 days, flat, can have radial folding, velvety, ocher, dark ocher to brown in color. The reverse side of brown tones, reddish tones may appear, the edge of the colony is light-colored. The conidial heads are spherical, with aging, they decompose into 2 compact columns of light ocher-brown color. The conidiophore is two-tier, along the entire vesicle with densely spaced mules (15-20 × 5-6 μm) and phialids with a size (7-13 × 2-3) μm. Conidiophores 300-1500 microns, 9 microns in diameter, smooth, slightly rough, unpainted to yellowish, light brownish tones, conidiophores are rounded, conidia are rounded, smooth to slightly finely roughened, 3.5-4.0 microns,

Physiological and biochemical characteristics

The strain grows well on standard media: wort agar, Chapek's medium, Getchinson's medium, glucose-peptone medium at a temperature of 25 ° C. Sporulation of the strain begins at 4-5 days, forms copiously spore-forming colonies by 10 days. The strain is thermo-tolerant, able to grow in the temperature range of 8 ° C-46 ° C. Spores remain viable when heated in the soil for several hours at a temperature of 60 ° C. On nutrient media it grows in a wide range of pH, including the alkaline region (pH 7-10). The strain has proteolytic (caseinolytic), cellulolytic and xylanase (hydrolyzes cellulose, hemicellulose) and lipolytic activity. Produces alkalostable lipases.

The strain is maintained on jambs with wort agar 4-5 ° B with an initial pH of 6.0-6.2 or standard Chapek-agar medium at room temperature with reseeding after 1-3 months, and when stored at 5 ° C with reseeding through 6-12 months.

The strain Aspergillus ochraceus BKM F-4106D is a non-pathogenic and non-toxic microorganism. With intravenous infection of rabbits does not show zoopathogenic properties. When applying the strain culture extract to rabbit skin, it does not cause a toxic reaction.

The strain Aspergillus ochraceus BKM F-4107D was isolated by A.V. Kurakov from serozem samples (Turkmenistan) on Hetchinson's medium with microcrystalline cellulose during incubation of Petri dishes for 7 days at 54 ° C and then for 7 days at 20 ° C.

The strain Aspergillus ochraceus is deposited in the All-Russian Collection of Microorganisms under the number BKM F-4107D.

The strain Aspergillus ochraceus BKM F-4107D belongs to mitospore fungi - anamorphs of the ascetic affinity: the genus Emericella, the Trichocomaceae family, order Eurotiales, class Eurotiomycetes, Ascomycota department.

Cultural and morphological features

The strain Aspergillus ochraceus BKM F-4107D has typical cultural and morphological characters [8, 9]. The colonies on Chapek’s environment are velvety, plentifully bearing, ocher and dark ocher in color, 3.5 cm in diameter, flat and with folds, dark ocher in color, the reverse side is orange-brown in color, may have reddish hues. Forms colonies with zones of lighter mycelium with less sporulation with a diameter of up to 4.5 cm and a light brown reverse side. Conidial heads are spherical, with aging, they decompose into 2-3 compact columns. Conidia are round, smooth, with a diameter of 3.5 microns. Young conidiophore with a diameter of 4-5 microns, with age colored, smooth, with a diameter of 10 microns.

Physiological and biochemical characteristics

The strain grows well on standard media: wort agar, Chapek's medium, Getchinson's medium, glucose-peptone medium at a temperature of 25 ° C. Sporulation of the strain begins at 4-5 days, forms copiously spore-forming colonies by 10 days. The strain has proteolytic (caseinolytic) and cellulolytic activity. The strain is thermostable, can withstand heating for 1 hour at 100 ° C, grows at temperatures up to 43 ° C. The strain is maintained on jambs with wort agar 4-5 ° B with an initial pH of 6.0-6.2 or standard Chapek-agar medium at room temperature with reseeding after 1-3 months, and when stored at 5 ° C with reseeding through 6-12 months.

The strain Aspergillus ochraceus BKM F-4107D is a non-pathogenic and non-toxic microorganism. With intravenous infection of rabbits does not show zoopathogenic properties. When applying the strain culture extract to rabbit skin, it does not cause a toxic reaction.

Example 1

Inoculum Aspergillus ochraceus BKM F-4104D is grown in test tubes of beveled mash agar 4-5 ° Baling. Inoculation is made with fungal spores, a well-debated culture of 6-8 days old is used. Spores are washed off the surface of the agar with a sterile seed medium containing wort, glucose and peptone into the same medium. Inoculated flasks are grown on a shaker with 200 rpm at 28 ° C. After 2 days, part of the grown material is transferred into 750 ml flasks with 100 ml of fermentation medium of the following composition (in%):

glucose 3,5 starch 1,0 fish meal hydrolyzate 0.5 peptone 0.5 NaCl 0.2 KN ₂ PO ₄ 0.05 MgSO ₄ -7H ₂ O 0.05 Water Rest

The initial pH is 6.5.

After 2 days of cultivation, the mycelium is separated from the medium by filtration through a paper filter.

Next, the precipitation of the enzymes of the culture fluid with ammonium sulfate to a degree of saturation of 80%. The precipitates formed are centrifuged, dissolved in 0.005 M Tris-Hcl buffer (pH 8.2) and dialyzed at 4 ° C against the same buffer.

The anticoagulant activity of the obtained protein solution of the culture fluid is determined by the lengthening of the activated partial thromboplastin time (APTT) of human blood plasma. The test is used to evaluate the activation of protein C in a form that inactivates coagulation factors Va and VIIIa. The lengthening of the APTT is determined by the method recommended by the Research Institute of Hematology and Blood Transfusion (Vyatka) in the following modification [5, 7]. Prepare a mixture of 0.1 ml of standard blood plasma, 0.1 ml of the preparation of fungal enzymes in physiological saline, 0.1 ml of a suspension of kaolin. The mixture was incubated for 2 min in a water bath at 37 ° C with constant stirring, after which 0.1 ml of an aqueous solution of calcium chloride (0.277%) was added. Coagulation time is measured by a stopwatch. In the control, instead of the test solution, an equivalent amount of physiological saline (0.86% sodium chloride solution) is used. Relative APTT is calculated by the formula (AB) × 100% / B, where A is the plasma coagulation time in the presence of the tested fungal protease solution (sec), B is the plasma coagulation time in the control (sec). Repeatability is 3 times.

The anticoagulant activity of the protein solution of the culture fluid of the strain Aspergillus ochraceus BKM F-4104D by APTT is 1030%.

To establish that the anticoagulant activity is due to the action of a proteinase — an activator of protein C, the activity of an activator of protein C of A. ochraceus BKM F-4106D in the culture fluid is determined according to the procedure [10] using the chromogenic peptide substrate pGlu-Pro-Arg-pNA (pyroglutamyl- prolyl-arginyl-para-nitroanilide) [11, 12]. Activated plasma protein C specifically cleaves p-nitroaniline (pNA) from pGlu-Pro-Arg-pNA. The unit of activity of the protein C activator is taken as the amount of the enzyme that cleaves 1 μmol · 10 ^-3 p-nitroaniline (pNA) from pGlu-Pro-Arg-pNA for 1 min in 1 ml at pH 8.2 and 37 ° C.

The activity of the protein C activator A. ochraceus BKM F-4104D in the culture fluid is 83.2 u / ml.

Example 2

The culture fluid of the strain Aspergillus ochraceus BKM F-4104D obtained in example 1, but using a nutrient medium of the following composition (in%):

glucose 3,5 starch 0.1 fish meal hydrolyzate 0.75 peptone 0.3 NaCl 0.1 KN ₂ PO ₄ 0.05 MgSO ₄ · 7H ₂ O 0.05 Water Rest

The initial pH is 6.5.

Protein C activating activity of 85.3 u / ml was detected in the culture fluid.

Example 3

The culture fluid of the strain Aspergillus ochraceus BKM F-4104D obtained in example 1, but using a nutrient medium of the following composition (in%):

glucose 3,5 starch 0.1 fish meal hydrolyzate 1,0 peptone 0.1 NaCl 0.2 KN ₂ PO ₄ 0.05 MgSO ₄ · 7H ₂ O 0.05 Water Rest

The initial pH is 6.5.

Protein C activator activity of 86.6 u / ml was detected in the culture fluid.

Example 4

The culture fluid of the strain Aspergillus ochraceus BKM F-4104D obtained in example 1, but using a nutrient medium of the following composition (in%):

glucose 3,5 starch 0.1 fish meal hydrolyzate 1,0 peptone 0.5 NaCl 0.2 KH ₂ PO ₄ 0.05 MgSO ₄ · 7H2O 0.05 Water Rest

The initial pH is 6.5.

Protein C activator activity of 86.0 units / ml was detected in the culture fluid.

Table 1 shows data on the protein C activator activity of the culture fluid Aspergillus ochraceus BKM F-4104D at various concentrations of components in the fermentation medium.

Table 1 The effect of the concentration of medium components (%) on protein C activator activity of Aspergillus ochraceus BKM F-4104D culture fluid glucose starch Hydrolyzed Fish Meal peptone Sodium chloride Potassium dihydrogen phosphate Magnesium sulfate u / ml 3,5 1,0 0.5 0.1 0.2 0.05 0.05 81.3 3,5 0.1 0.5 0.3 0.1 0.06 0.04 83.0 3.8 1,0 0.5 0.3 0.2 0.06 0.06 83.1 3.2 1,0 0.5 0.5 0.2 0.04 0.04 82.8 3.2 0.1 0.75 0.5 0.2 0.05 0.05 86.1 3,5 1,0 0.75 0.5 0.1 0.04 0.06 85,4 3.8 1.0 0.75 0.5 0.2 0.05 0.05 86.0 3.2 0.1 1,0 0.1 0.2 0.05 0.05 85.1 3.8 0.1 1,0 0.1 0.2 0.05 0.05 79.8 3,5 0.1 1,0 0.3 0.2 0.04 0.04 86.4 3,5 0.1 1,0 0.5 0.2 0.05 0.05 85.9 - coefficient of variation of data - 3%.

Example 5

The strain Aspergillus ochraceus BKM F-4105D is grown and receive the culture fluid and the enzyme preparation of the protein activator C of example 1. The activity of the activator of protein C in the culture fluid of strain BKM F-4105D according to the standard recommended method described in example 1 [10] reaches 85.0 units ./ml. The activity of the protein C activator of strain BKM F-4105D, determined by APTT, is 1020%.

Example 6

The strain Aspergillus ochraceus BKM F-4106D is grown and receive the culture fluid and the enzyme preparation of the protein activator of Example 1. The activity of the activator of protein C in the culture fluid of strain BKM F-4106D according to the standard recommended method described in Example 1 [10] reaches 89.9 units ./ml. The activity of the protein C activator of strain BKM F-4106D, determined by APTT, is 1032%.

Example 7

The strain Aspergillus ochraceus BKM F-4107D is grown and receive the culture fluid and the enzyme preparation of the protein activator C according to example 1.

The activity of the protein C activator in the culture fluid of VKM F-4107D strain according to the standard recommended method described in Example 1 (10) reaches 85.2 units / ml.

The activity of the protein C activator in the VKM F-4107D strain, determined by APTT, is 1024%.

Example 5. Comparison of the proposed method and the closest analogue

Comparison of protein C activator activity in the culture fluid of strains of Aspergillus ochraceus VKM F-4104D, Aspergillus ochraceus VKM F-4105D, Aspergillus ochraceus VKM F-4106D, Aspergillus ochraceus VKM F-4107D when cultured on a fermentation medium with pH 6.5 starting with 6 containing (in%)

glucose 3,5 starch 0.1 fish meal hydrolyzate 1,0 peptone 0.1 NaCl 0.2 KH ₂ PO ₄ 0.05 MgSO ₄ · 7H ₂ O 0.05 water up to 100%

and in the culture fluid Aspergillus ochraceus 1, grown on a fermentation medium with an initial pH of 5.5, containing (in%): glucose - 3.5, starch - 1.0, soy flour - 2.0, meat extract - 0.5 , peptone - 0.5, NaCl - 0.2, KH ₂ PO ₄ - 0.05, MgSO ₄ -7H ₂ O - 0.05, shows that the proposed method receive a higher activator for protein C activity (12 % and more) and for a shorter period of fermentation - for 2 days, and not for 7 days (Table 2).

table 2 Option Fermentation time, days Protein C activator activity by APTT,% Method - the closest analogue to Aspergillus ochraceus 1 7 854 The proposed method with Aspergillus ochraceus VKM F-4104D 2 1030 The proposed method with Aspergillus ochraceus VKM F-4105D 2 1020 The proposed method with Aspergillus ochraceus VKM F-4106D 2 1032 The proposed method with Aspergillus ochraceus VKM F-4107D 2 1024 Coefficient of data variation - 2%

Thus, the proposed method allows to obtain a culture fluid with a higher activity of proteinase activator of protein C and to reduce the time of fermentation.

Literature

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

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

3. Martinoli JL, Stoker K. 1986. Fast functional protein C assay using Protac ^® , a novel protein C activator. Thromb. Res., 43, 253-256.

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

5. Landau N.S., Kurakov A.V., Gulikova O.M., Batomunkueva B.P., Strukova S.M., Egorov N.S. 1998. Extracellular proteinases of micromycetes with fibrinolytic and anticoagulant properties. Microbiology, 67, 2, 215-220.

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

7. Strukova S.M., Kogan A.E., Tara A.A., Aaviksaar A.A. 1989. Antithrombotic effect of protein C activator from snake venom. Q. Honey. Chemistry. 35, 5, 115-119.

8. Bilay V.I., Koval E.Z. 1988. Aspergillus. Determinant. Kiev: Naukova Dumka, 203 p.

9. Raper K.B., Fennell D.J. The genus Aspergillus. USA, Baltimore, Williams a. Wilkins Co., 1965.686r.

10. Kogan A.E., Makarov A.N., Bobrushkin I.D., Strukova S.M. Comparative study of protein With activators from the Agkistrodon snake venoms. Thrombosis research. 1991. V.62. P.775-780.

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

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

Claims (1)

A method for the preparation of a plasma protein C protein activator activator by culturing a strain of the microscopic fungus Aspergillus ochraceus, characterized in that the strain Aspergillus ochraceus VKM F-4104D or Aspergillus ochraceus BKM F-4105D or Aspergillus ochraceus BKM F-4106DD or Aspergillus ochraceus BKM producing protein C activators, and the fermentation process is conducted on a nutrient medium at an initial pH of 6.5 and the following composition of components,%:
glucose 3.2-3.8 starch 0.1-1.0 fish meal hydrolyzate 0.5-1.0 peptone 0.1-0.5 NaCl 0.1-0.2 KN ₂ PO ₄ 0.04-0.06 MgSO ₄ · 7H ₂ O 0.04-0.06 water up to 100%