Strain aspergillus ochraceus - human blood plasma protein c activator proteinase producer

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

 

The invention relates to the field of biotechnology, getting proteolytic enzyme activators of protein From human blood plasma.

Activators of protein With proteases, involved the formation of vitamin K-dependent proferment of the protein To its active form of activated protein C in the blood of mammals, including humans. Activated protein C is a key enzyme of the hemostatic system, which inactivates coagulation factors Va and VIIIa, thereby preventing excessive thrombus formation. The lack of protein in the blood decreases the activity of activated protein C, which leads to the occurrence of thromboembolic complications may be fatal [1, 2]. In this regard, the actual diagnosis is its content in the blood, which can be done using specific proteases - activators of the protein [3].

It is known that activators of protein are present in the venom of some species of snakes [4]. Activators of protein from the venom of the Copperhead snake Ukraine contortrix contortrix used for the production of diagnostic products (Protac®company PENTHAPHARM (Switzerland)) [5, 6]. The disadvantages of obtaining activators of protein from snake venom are extreme difficulty of getting the serpent and the limitations of this source.

Because of the importance of activator protein for the diagnosis of a heart condition is about-vascular diseases are searching for alternative organisms to obtain, among them the most accessible and promising for large-scale production can be microorganisms. Microscopic fungi are some of the objects such as their culture liquid of the detected complexes extracellular proteases possessing not only fibrinolytic, and anticoagulant properties. Known strains of Aspergillus ochraceus 1, Penicillium claviforme 2, P. granulatum 7, Alternaria sp. 10 [7] and Aspergillus ochraceus 513 [8], the culture fluid which has anticoagulant properties - effect-type activator of protein C. Antikoagulyantna activity, by type of activator protein With a defined elongation of activated partial thromboplastin time of human blood plasma [9], Aspergillus ochraceus 1 is 854%, Penicillium claviforme 1 - 690%, P. granulatum 7 - 500%, Alternaria sp. 10 - 500% [7] and in A. ochraceus 513 - 65,7% [8].

The closest analogue should take the microscopic strain of the fungus Aspergillus ochraceus I, which is known strains of the most high anticoagulant activity of activator protein - 854% [7].

Disadvantages closest analogue Aspergillus ochraceus 1 are long term cultivation of strain (7 days) and low activity derived activator of protein C.

The present invention is the finding of a new strain of microscopic fungus Aspergillus ochraceus, producing high activity Proteus is azu - activator protein From blood plasma.

The problem is solved using the newly selected by the authors of the strain Aspergillus ochraceus, producer activator protein of blood plasma, as described below.

A new strain of Aspergillus ochraceus designed for proteases allocated Avecom of the samples Chernozem soils on carbonate rocks, which were selected in the steppe zone of the Voronezh region. The strain selected for the bait - filter paper soaked in butter from water and soil suspension during incubation for 7 days at 49°C and then at 20°C and Parisian on wort agar.

A strain of Aspergillus ochraceus deposited in the all-Russian Collection of Microorganisms under the number VKM F-4106D.

A strain of Aspergillus ochraceus VKM F-4106D refers to Microsporum mushrooms - anamorphism Ascomycetes of affinity: the genus Emericella, family Trichocomaceae, order Eurotiales, class Eurotiomycetes, phylum Ascomycota.

Cultural and morphological characteristics.

A strain of Aspergillus ochraceus VKM F-4106D is typical for this kind of cultural-morphological characteristics (Bilai, Koval, 1988; Raper, Fennel, 1949) [10, 11]. Colonies on media of čapek and wort-agar, on average 3.5 cm in diameter in 10 days, flat, can bear radial folding, silky, buff, dark ochre to brown. The reverse side of brown tones may appear reddish tone, the edge of the colony is light-colored. Conidial heads globose, ageing split into 2 compact speakers pale Buffy-brown. Condenser bunk, across the vesicles with tightly spaced matulane (15-20×5-6 μm) and Felidae size (7-13×2-3 microns). Conidiophores 300-1500 mm, a diameter of 9 µm, smooth, slightly rough, unpainted to yellowish, light brown tones, the expansion of canadianese rounded, conidia rounded, smooth to weakly melkosherohovatuyu, 3.5 to 4.0 microns.

Physiological and biochemical characteristics.

The strain grows well on standard media: wort-agar, the medium čapek, environment Hutchinson, glucose-peptone medium at 25°C. Sporulation strain begins 4-5 days, mobilesphonestore colony forming to 10 days. Strain thermotolerant, able to grow in the temperature range 8-46°C. Spores remain viable when applied in the soil for several hours at 60°C. On nutrient media grows in a wide range of pH, including alkaline region (pH 7-10). The strain possesses proteolytic (caseinolytic), cellulolytic and xylanase (hydrolyzes cellulose, hemicellulose) and lipolytic activity. Produces alkanolamine lipase.

The strain is supported on the posts with wort-agar 4-5°B with an initial pH of 6.0 to 6.2 or standard environment čapek-agar at room temperature is round with reseeding after 1-3 months, and during storage at 5°C with reseeding after 6-12 months.

The strain is non-pathogenic and non-toxic microorganism. When intravenous infection of rabbits does not show zoopathogenic properties. When applying the extract of the culture of strain on the skin of the rabbit does not cause toxic reactions.

Strain when cultivated in a liquid nutrient medium produces extracellular enzyme activator to a protein With the activity, the selection of which of the culture liquid 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 buffer (pH 8,2) or distilled water, 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 [7]. The resulting solution was stored in a refrigerator at 4°C or lyophilizer for long-term storage.

Example 1.

Anticoagulant activity of activator protein From Aspergillus, ochraceus BKM F-4106D.

As inoculum using a two-day culture obtained on the medium containing wort, glucose and peptone. A strain of Aspergillus ochraceus BKM F-4106D grown in submerged cultivation in 100 ml of culture medium in katalozhnyh to the Bach 750 ml at the traffic units (200 rpm) at 28°C. Further cultivation of the strain is carried out under the same conditions on a complex medium containing glucose, starch, soy flour, meat extract, peptone, sodium chloride, potassium dihydrophosphate, hydrated magnesium sulfate in the following ratio of components, g/l (in terms of crystallohydrate form of salts):

Glucose - 35,0

Starch - 10,0

Soy flour - 20,0

Meat extract - 5,0

Peptone - 5,0

Sodium chloride NaCl - 2,0

Potassium dihydrophosphate KH2PO4- 0,5

Magnesium sulfate MgSO4×7H2O - 0,5

Tap water up to 1 l

pH 5.5.

The mycelium after 2 days of cultivation is separated from the environment by filtering through a paper filter or by centrifugation) and get the culture fluid.

Proteins of the culture fluid is precipitated by ammonium sulfate at 80%saturation in the cold. After the formation of precipitation them separated by centrifugation at 15 thousand Rev/min the precipitated protein was dissolved in 0.005 M Tris-HCl buffer (pH 8,2), remove the insoluble part of the precipitate, the solution is free from ammonium ions by dialysis.

Anticoagulant activity of the resulting protein solution (preparation of enzymes from the culture fluid is determined by the prolongation of the activated partial thromboplastin time (APTT) of human blood plasma. The test is used to assess the activation of protein C in the form, which is activates factors Va and VIIIa blood clotting. The lengthening of the APTT is determined according to the method recommended by the Institute of Haematology and blood transfusion (gwada) the following modifications [7, 9]. Prepare a mixture of 0.1 ml of standard plasma, 0.1 ml of the preparation of enzymes of the fungus in physiological solution, 0.1 ml of a suspension of kaolin. The mixture is incubated for 2 min in a water bath at 37°C with constant stirring, then add 0.1 ml of an aqueous solution of calcium chloride (0,277%). The clotting time is measured with a stopwatch In control instead of the test solution using an equivalent amount of saline solution (0,86%solution of sodium chloride). Relative APTT calculated according to the formula (a-b)×100%/B, where a is the clotting time of plasma in the presence of the test solution proteases mushroom (s), the clotting time of plasma in the control (s). Repetition - 3x.

Anticoagulant activity of a strain of Aspergillus ochraceus BKM F-4106D on APTT is 960%.

To establish that the anticoagulant activity due to the action of protease - activator protein With the activity of activator protein From A. ochraceus BKM F-4106D in the culture fluid is determined by the method [12] using the chromogenic peptide substrate pGlu-Pro-Arg-pNA (pyroglutamyl-shed-arginyl-para-nitroanilide) [13, 14]. Activated protein C plasma specific it p-nitroaniline (pNA) from pGlu-Pro-Arg-pNA. C is 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 at pH of 8.2 and 37°C.

In culture liquid of A. ochraceus BKM F-4106D find the activator of protein C activity - 77,9 IU/ml

Example 2

Comparison of the anticoagulant activity of activators of protein From a strain of Aspergillus ochraceus BKM F-4106D and the nearest equivalent strain of Aspergillus ochraceus 1.

Cultivation of a strain of A. ochraceus BKM F-4106D carried out as in example 1. The activity of activators of protein in terms of relative APTT in A. ochraceus BKM F-4106D (950%), much higher than that of the strain - the closest analogue - A. ochraceus 1 (854% (table)). Time of cultivation of the proposed strain to achieve such an activity of activator protein C is reduced from 7 to 2 days less than 5 days.

Table
The time of culturing in a fermentation medium and the activity of activators of protein From Aspergillus ochraceus BKM F-4106D and A. ochraceus I
OptionTime of cultivation, daysAPTT**, %
The closest analogue - A. ochraceus I*7854
A. ochraceus BKM F-4106D2 960
* - [7]; ** - the coefficient of variation of the data does not exceed 3%.

Claims

A strain of Aspergillus ochraceus BKM F-4106D producing the protease - activator protein From human blood plasma.

Literature

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

2. Dahlbäck Century, B.O. Villoutreix, 2005. Regulation of blood coagulation by the protein With anticoagulant pathway: novel insights into structure-function relationships and molecular recognition. Arterioscler. Thromb. Vasc. Biol, 25, 1311-1320.

3. Gempeler-Messina P.M., K. Volz, Bühler C., Müller C., 2001. Protein With activators from snake venoms and their diagnostic use. Haemostasis, 31, 266-272.

4. Stocker K., Fisher H., Meier J., Brogli M., Svendsen L., 1986. Protein With activators in snake venoms. Behring. Inst. Mitt., 79, 7-47.

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

6. 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.

7. Landau NS, Kurakov AV, Golikova O.M., Batmunkh BP, Strukova S.M., Smith NS, 1998. Extracellular proteases of micromycetes with fibrinolytic and anticoagulant properties. Microbiology, 67, 2, 215-220.

8. Batmunkh BP, Egorov NS 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./p>

9. Strukova S.M., Cohen AE, Tara A., Avissar A.A. 1989. Antithrombotic effect of activator protein from snake venom. The matters. The honey. Chemistry. 35, 5,115-119.

10. Bilai VI, Koval AS 1988. Aspergilli. The determinant. Kiev: Naukova Dumka, 203 S.

11. Raper K.B., D.J. Fennell, The genus Aspergillus. USA, Baltimore, Williams a. Wilkins Co., 1965, 686 R.

12. Kogan AU, A.N. Makarov, Bobrushkin I.D., Strukova S.M. 1991. Comparative study of protein With activators from the Ukraine snake venoms. Trombosis research, V.62. P.775-780.

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

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

A strain of Aspergillus ochraceus BKM F-4106D producing the protease - activator protein From human blood plasma.



 

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