DE2024251B2 - Method for isolating a fibrinolytic enzyme and that enzyme - Google Patents

Description

The invention relates to a method for isolating a fibrinolytic enzyme in highly purified form Shape and this enzyme.

In addition to the use of streptokinase, based on the activation of the body's own plasminogen to plasmin, increasing attempts are being made to thrombolytic therapy enriched by an application of fibrinolytic proteolytic enzymes. Already minor Quantities of such a protease are able to completely dissolve arterial and venous thrombi. One therapeutically usable protease must be in a highly purified form and under physiological conditions Conditions without toxic side effects be fibrinolytically active.

It is known that certain molds form mixtures of proteolytic enzymes which are also fibrinolytic contain active proteases.

It is already known that Aspergillus oryzae can produce a fibrinolytically active protease called Can isolate aspergillin O [Proc. Soc. exp. Biol., N.Y., 99,54 (1958)].

By direct precipitation from culture filtrates with organic solvents or by precipitation with organic solvents after pre-cleaning over synthetic resin exchanger IRA 400 became the protease enriched. Subsequently, the precipitates obtained are resuspended, filtered, and dietary fiber precipitated by adding copper, zinc or cadmium sulfate solution. After dialysis it will the active ingredient-containing supernatant lyophilized [Lancet 2, 443 (1959)].

A fibrinolytically active protease from Aspergillus oryzae, Aspergillus flavus or Absidia coerulea is obtained by AP Truant and FG Norstrom [USP 3 256157] by adding twice the volume of ethanol cooled to -20 ° C. to the ^{culture filtrate at 0 ° C.} precipitated active material is suctioned off or the culture filtrate is treated with tannin solution and the active ingredient-containing precipitate is treated with acetone after standing for 48 hours at + 44 ° C. by stirring, the acetone-insoluble active ingredient remaining. The active ingredient, dried in vacuo, is then suspended in water, the insoluble fraction is centrifuged off, and the active ingredient-containing solution is dialyzed against water and lyophilized.

Furthermore, in Canadian patent 671648 there is a method for the production of highly active fibrinolytic Agents from Aspergillus oryzae described, which is characterized in that raw preparations after dissolving in water and after adjusting to pH 3.5 to 5.5 by means of hydrochloric acid chromatography Subjected to Sephadex G-50 via cross-linked dextran and then after development with hydrochloric acid Water, the active ingredient-containing fractions are lyophilized.

B. A. Kudrjaschov [(Ber. D. Akd. D. Wiss. D. USSR 153,4,39 (1963)] found except in Aspergillusoryzae strains Also in Aspergillus awamori a fibrinolytically active substance, which he has described above Method of Stefanini [Proc. Soc. exp. Biol., N.Y., 99, 504 (1958)].

By A. A. Imschenetzki and colleagues. [Ber. d. Akad. D. Knowledge of the USSR 163, 3, 737 (1965)] a method for obtaining a fibrinolytically active protease from Aspergillus terricola is given. It is obtained by using culture filtrate with ammonium sulfate to precipitate the active ingredient or acetone is added. After centrifugation, the precipitate is dissolved in water, insoluble Fractions are filtered off, and the filtrate is filtered through a Sephadex G-25 column to remove ammonium sulfate and to separate pigments. The active eluate is lyophilized.

In US Patent 3,281,331 there is a method of extraction fibrinolytically active proteases described in which the active ingredients from the solution of a crude enzyme mixture of Aspergillus oryzae, Aspergillus flavus or Absidia coerulea at pH 5.5 on CM cellulose or CM-Sephadex adsorbed, with buffer from

pH 7.0 and then lyophilized after dialysis or precipitated with tannin or adsorbed on DEAE-Celluolse at pH 6.0 and gradually Increase the salt concentration to be eluted with buffer.

The isolation and separation processes known from Swedish patents 193288 and 328 534 of proteolytic enzymes from mold cultures, etc. from Aspergillus strains can cannot be generalized, since molds are always a mixture of extracellular proteases and others Form hydrolases, which differ in their molecular properties (e.g. molecular weight, electrical Charge, structure) as well as their substrate specificity (see Y. Na kagawa: Alkaline Proteinase from Aspergillus, in S. P. Colowick - N. O. Kaplan, Methods in Enzymology, Vol. XIX, p. 581, Acad. Press New York and London, 1970).

In CA 54,17549 d a procedure is reported according to which a bran culture of Aspergilius ochraceus is extracted with 0.75% NaCl solution. From it gets after multi-stage precipitation and purification steps that are not industrially applicable, a crystalline one proteolytic enzyme, the fibrinolytic effect of which has not been demonstrated.

In CA 56, 5103 b, inter alia. describes the purification of a protease preparation from Aspergillus ochraceus, whose fibrinolytic effect has not been proven. The cleaning operations used there are not used in accordance with the invention.

From CA 65.4548 e there is only one fermentation using an Aspergillus ochraceus strain known, the decrease in proteolytic activity being measured during the culture period will. Isolation of proteases is not described here.

Other works such as CA 55, 22484 a (1961) and CA 60, 16143 g (1964) do not concern isolation methods, but only investigate the proteolytic effect of various microbial proteases Casein (without information about their fibrinolytic activity) or compare the effect of a Proteinase from Aspergillus ochraceus on oxidized insulin with the behavior of pepsin, trypsin and Chymotrypsin.

All known methods relate to the isolation of fibrinolytically active protease from Aspergillus oryzae, Aspergillus terricola, Aspergillus flavus, Aspergillus awamori or Absidia coerulea.

The work-up procedures described are due to the different molecular structure and the resulting specific physicochemical properties of the proteases - only for the Isolation of fibrinolytically active enzymes from the specified molds can be used.

Some work-up procedures also lead to only partially purified protease preparations, which means their therapeutic uses are limited.

The purpose of the invention was to develop a method for the rational isolation of a sub-physiological Conditions for optimally effective fibrinolytic, highly purified protease from Aspergillus ochraceus, which are applied externally or parenterally in therapy without any toxic side effects can.

The invention is based on the object of producing one from crude enzyme mixtures from Aspergillus ochraceus to isolate highly purified fibrinolytically active protease. Most of all it required a procedure set up, with the help of which the separation of fibrinolytically inactive proteases, ballast proteins and interfering Dyes succeed in a rational way.

According to the invention it was found that a fibrinolytically effective and without toxic side effects therapeutically applicable protease can be produced if one from Aspergillus ochraceus cultures isolated crude enzyme mixture extracted with aqueous buffer solution, the active ingredient-containing extract on the anion exchanger known under the trade name DEAE-Sephadex on the filing date or DEAE cellulose filtered and then from the active ingredient-containing filtrate, the active ingredient through water-miscible organic solvents is precipitated. After drying in vacuo, the precipitate which has been separated off and which contains the active substance is dissolved in water dissolved and subjected to gel chromatography under the trade name Sephadex G-75 or Sephadex G-100 subjected to known cross-linked dextran gels. The protease-containing fractions are freeze-dried, and thus the highly purified fibrinolytically active protease is obtained.

The extraction of the raw enzyme mixture takes place in such a way that this raw preparation - with a proteolytic Activity of approx. 1 CE / mg - with 0.05 to 0.1 M Tris-HCl buffer pH 6.5 to 7.5 or 0.05 to 0.1 M ammonium acetate buffer from pH 6.5 to 7.5, stirred several times at room temperature to bring the active ingredient completely into solution. Centrifugation at 15,000 g becomes clear, dark in color Get active ingredient-containing solution and separated the insoluble inactive portion.

This active ingredient-containing extract is, preferably at 0 to 4 ° C, over that with the anion exchanger DEAE-Sephadex A-50 or the anion exchanger DEAE-Cellulose, capacity 0.5 to 0.9 mVal / g, loaded column, which has been equilibrated with the extraction buffer, filtered. The ion exchanger is then used washed with the buffer used for the extraction.

As a result of the specified anion exchanger treatment, the active ingredient-containing extract becomes both fiber and proteases unsuitable for fibrinolytic therapy as well as the largest Part of the dyes is removed, whereas the fibrinolytically active protease is not adsorbed.

The active ingredient solution obtained in this way becomes the active ingredient by adding threefold Amount of water-miscible organic solvent pre-cooled to -20 ° C, preferably with ethanol, precipitated and dried in vacuo.

The re-dissolved in as little water as active substance-containing substance is subjected to gel chromatography in the manner that the solution - is preferably added at 0 to +4 ⁰ C to a charged with Sephadex G-75 or Sephadex G-100 gel column. It is then eluted with water. The proteolytic activity is tested in the fractions obtained during the elution. The active fractions are freeze-dried and result in a white dry powder of a pure fibrinolytically active protease with a proteolytic activity of approx. 15 CE / mg.

The process according to the invention makes it possible to use raw enzyme mixtures obtained from Aspergillus ochraceus, a highly purified fibrinr> lytically active under physiological conditions Isolate protease with simple technical tools.

Parenteral application of the enzyme was used in the clinic at a dosage of approx. 250 to 500 mg per patient and treatment (daily doses 20 to 120 mg) venous thromboses, such as B. Pelvic vein, einvein and axillary vein thrombosis as well as postihrombotic syndrome, arterial embolism and chronic arterial occlusive disease treated, resulting in a cure or amelioration through resulted in complete or partial recanalization of the blood vessels.

In the course of the clinical investigations it could be confirmed that the invention from Aspergillus ochraceus isolated protease is an effective thrombolytic agent.

A particular advantage of this protease is its lack of antigenicity and its applicability also in streptokina-resistant cases.

The fibrinolytically active protease leads with external or parenteral application under therapeutic Conditions to no toxic side effects.

The proteolytic activity, expressed in caseinolytic Units (CE), one of Kunitz [j. Gene. Physiol. 30 (1947) 291] specified test method at pH 8.0.

The fibrinolytically active alkaline protease has been characterized biochemically and can be found on the basis of Molecular weight parameters and the detection of a disulfide bridge from other Aspergillus proteases differentiate. The molecular weight of the fibrinolytically active alkaline protease is 25,000 isoelectric point is between pH 6 and 7. The protease has opposite natural substrates Casein, gelatin, hemoglobin and fibrin have a broad pH optimum, which is roughly from pH 7.0 to pH 8.0 extends. In the fibrin plate test, the protease was about 5 times more active than trypsin. (Acta biol. med. germ. 26, 1111-1115 [1971], (Folia Haematol., Leipzig 101/1 [1975], 91-98).

Embodiments:
example 1

50 g of a crude enzyme mixture isolated from Aspergillus ochraceus cultures with a proteolytic Total activity of 1.4 CE / mg is measured 4 times with 200 ml of 0.05 M Tris-HCl buffer of pH 7.2 each time extracted with mechanical stirring for 15 minutes. The extraction mixture is centrifuged until clear at 15,000 g. The clear, dark brown solutions containing the active ingredient are combined and give a Volume of 750 ml.

The entire solution is at 0 to 4 ° C through a with the anion exchanger DEAE-Sephadex A-50 charged column (diameter 10 cm, filling height 18 cm) filtered. Then with 0.05 M Tris-HCl buffer, pH 7.2, rewashed.

To produce the exchanger bed, 50 g DEAE-Sephadex A-50 with 0.05 M Tris-HCl buffer are used pH 7.2 equilibrated.

From the only weakly filtered active ingredient Filtrate (approx. 3500 ml) is the active ingredient with 3 times the volume of ethanol cooled to -20 ° C failed. For complete flocculation of the active ingredient, the precipitation is left at -20 ° C. overnight stand. The precipitate is separated off by centrifugation and washed with ethanol and acetone and dried in vacuo.

4.43 g of a product enriched in fibrinolytically active protease with 11.6 CE / mg are obtained obtain.

1.5 g of this product are distilled with 8 ml. Water is added, the mixture is stirred and the insoluble residue is removed freed by centrifugation at 5000 g.

The clear, brownish active ingredient-containing extract is at 0 to 4 ° C over a swollen in water Sephadex G-75 gel loaded column (diameter 2.5 cm, filling height 95 cm) filtered and washed with dist. water eluted. The eluate coming from the column is collected in 20 ml fractions.

After an inactive forerun of 220 ml, the active ingredient is found in fractions 12 to 18, dyes and lower molecular weight proteins remain on the column.

The active ingredient solution (140 ml) is freeze-drying subjected and gives 616 mg of a white, easily soluble dry powder with a specific proteolytic activity of 15.7 CE per mg.

Example 2

20 g of a crude enzyme mixture isolated from Aspergillus ochraceus and having a total proteolytic activity of 1.0 CE / mg are extracted according to the instructions in Example 1.

The resulting clear, dark-colored solution containing active ingredient (290 ml) is mixed with the anion exchanger DEAE cellulose charged column (diameter 4.5 cm, filling height 15 cm) with 0.05 M Tris-HCl buffer pH 7.2 has been equilibrated, filtered. Then with 0.05 M Tris-HCl buffer rewashed pH 7.2.

From the active ingredient-containing filtrate (approx. 400 ml) the active ingredient is, as described in Example 1, precipitated, with 3.19 g of a more effective fibrinolytically Protease-enriched product with 4.5 CE / mg can be obtained, which is used to obtain the highly purified Active ingredient according to Example 1 is to be subjected to gel chromatography.

Claims (5)

Patent claims: 1. A method for isolating a fibrinolytic enzyme from mold cultures, thereby characterized in that one uses a crude enzyme mixture from Aspergillus ochraceus aqueous buffer solution extracted, the active ingredient-containing extract via one under the trade name DEAE-Sephadex or DEAE-Cellulose on the filing date known anion exchanger filtered from the obtained active ingredient-containing The filtrate precipitates the active ingredient with water-miscible organic solvents, the precipitate redissolved after drying in vacuo with water and subjected to gel chromatography under the trade name Sephadex G-75 or under the trade name Sephadex G-100 subjected to cross-linked dextran gels known on the filing date and finally the protease-containing fractions freeze-dried. 2. The method according to claim 1, characterized in that the crude enzyme mixture from Aspergillus ochraceus with 0.05 M to 0.1 M Tris-HCl buffer or 0.05 M to 0.1 M ammonium acetate buffer is extracted from pH 6.5 to 7.5, preferably 7.0 to 7.2. 3. The method according to claim 1 to 2, characterized in that the active ingredient-containing extract via one with the anion exchanger DEAE-Sephadex A-50 or DEAE-Cellulose, capacity from 0.5 to 0.9 meq / g charged acid equilibrated with the buffer used for extraction filtered at 0 to 4 ° C, the exchanger washed with buffer and the active ingredient from the filtrate is precipitated with ethanol pre-cooled to -20 ° C in a ratio of 1: 3. 4. The method according to claim 1 to 3, characterized in that after the anion exchanger treatment The active substance-containing substance obtained is redissolved in as little water as possible, on a column loaded with Sephadex G-75 or Sephadex G-100 at 0 to 4 ° C of the gel chromatography subjected, eluted with water and the eluate is collected in fractions and the protease-containing fractions are freeze-dried. 5. Highly purified Aspergillus ochraceus protease suitable for therapeutic purposes. manufactured according to claims 1 to 4.