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

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

 

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 2 - 690%, P. granulatum 7 - 500%, Alternaria sp. 10 - 500% [7] and A.ochraceus 513 - 65,7% [8].

The closest analogue should take the microscopic strain of the fungus Aspergillus ochraceus 1, which shows from the 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 of the protein is zu - 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 strain of Aspergillus ochraceus BKM F-4107D selected Avecom of the sample gray (Turkmenistan) on Wednesday of Hutchinson with microcrystalline cellulose during incubation of Petri dishes for 7 days at 54°C, and then for 7 days at 20°C.

A strain of Aspergillus ochraceus deposited in the all-Russian Collection of Microorganisms under the number BKM F-4107D.

A strain of Aspergillus ochraceus BKM F-4107D refers to Microsporum mushrooms - anamorphism Ascomycetes of affinity: the genus Emericella, family Trichocomaceae, order Eurotiales, class Ewotiomycetes, phylum Ascomycota.

Cultural morphological traits

A strain of Aspergillus ochraceus BKM F-4107D is typical for this kind of cultural-morphological characteristics [10, 11]. Colonies on the medium čapek velvety, richly paronomasia, ochre and dark buff, 3.5 cm in diameter, flat and wrinkled, dark buff, back Orangevale brown, may have reddish tones. Forms colonies with a lighter mycelium with less sporification diameter up to 4.5 cm and light brown underside. Conidial heads globose, ageing split into 2-3 compact column. Conidia rounded, smooth, diameter is rum 3.5 µm. Young condenser with a diameter of 4-5 μm, with age, stained, smooth, with a diameter of 10 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, abundantly paronomasia colony forming to 10 days. The strain possesses proteolytic (caseinolytic) and cellulolytic activity. The strain is stable, can withstand heating for 1 h at 100°C, grows at temperatures up to 43°C. 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 with reseeding after 1-3 months, when stored at 5°C with reseeding after 6-12 months.

A strain of Aspergillus ochraceus BKM F-4107D 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 the minimum volume of the e distilled water. The formed precipitation centrifuged, dissolved in buffer (pH 8,2) or distilled water to remove the insoluble portion of the precipitation and removal of ammonium salts solution cialiswhat or hold gel-filtration on a column of Sephadex G-25 [6] the resulting solution was stored in a refrigerator at 4°C or lyophilizer for long-term storage.

Example 1.

Anticoagulant activity of activator protein With Aspersillus ochraceus BKM F-4107D.

As inoculum using a two-day culture obtained on the medium containing wort, glucose and peptone. A strain of Aspergillus ochraceus BKM F-4107D grown in submerged cultivation in 100 ml of culture medium in katalozhnyh flasks 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

Water water (1 l)

pH 5.5.

The mycelium after 2 days of coltivirus the Deposit 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 inactivates 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 the 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 time of swertia the Oia plasma in the presence of the test solution proteases mushroom (s), In the clotting time of plasma in the control (C). Repetition - 3x.

Anticoagulant activity of a strain of Aspergillus ochraceus BKM F-4107D on APTT is 955%.

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-4105D 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. 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 at pH of 8.2 and 37°C

In culture liquid of A. ochraceus BKM F-4107D find the activator of protein C activity is 76.1 units/ml.

Example 2

Comparison of the anticoagulant activity of activators of protein From a strain of Aspergillus ochraceus BKM F-4107D and the nearest equivalent strain of Aspergillus ochraceus 1.

Cultivation of a strain of A. ochraceus BKM F-4107D carried out as in example 1. The activity of activators of protein in terms of relative APTT in A. ochraceus BKM F-4107D (955%) far exceeds that in strains of A. ochraceus 1 (854% (table)). Time of cultivation of the proposed strain to achieve such an activity of activator protein To reduce the W 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-4104D and A. ochraceus 1
OptionTime of cultivation, daysAPTT**, %
Prototype - A. ochraceus 1*7854
A. ochraceus BKM F-4107D2955
* - [7]; ** - the coefficient of variation of the data does not exceed 3%.

A strain of Aspergillus ochraceus BKM F-4107D producing the protease - activator protein From human blood plasma.



 

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