DE2024251A1 - Method for isolating a fibrinolytic enzyme - Google Patents

Description

ARZNEIWinav / ERX DRSSDEH -.

Method for isolating a fibrinolytic enzyme

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

In addition to the use of streptokinase, based on a Activation of the body's own plasminogen ^ to plasmin, attempts are increasingly being made to reduce the thrombolytic Enrich therapy with the application of fibrinolytic proteolytic enzymes · Already done | Small amounts of such a protease are able to completely dissolve arterial and venous thrombi. One therapeutic Usable protease must be in a highly purified form and without toxic under physiological conditions Side effects be fibrinolytically active

It is known that certain molds are more proteolytic in mixtures Form enzymes that are also fibrinolytically active proteases contain"

It is already known that a fibrinolytically active protease with the designation Aspergillus ory zae can be isolated from Aspergillus ory zae with the designation Asper- * gillin 0 · ^ "Proc.Soceip.Biol., N, Y., 99. 504 (195827

The protease was enriched by direct precipitation from culture filtrates with organic solvents or by precipitation with organic solvents after pre-cleaning via synthetic resin exchange IBA 400. The precipitates obtained are then resuspended, filtered, and dietary fiber is precipitated by adding copper, zinc or Oadmium sulfate solution · After dialysis, the active ingredient-containing supernatant is lyophilized. ^ Lancet g ± 443 (195927

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BY AP THJAKT & FGNORSTROli ^ fU.SaP. 3 256 152? a fibrinolytically active protease from Aspergillus Aspergillus flavus or Absidla coerulea is obtained by adding twice the volume of ^{ethanol cooled to -20 0 G to the culture filtrate at O 0} C and sucking off the precipitated active material or riveting the culture filtrate with lianni Active precipitate is treated with acetone after 48 hours standing at 4-4 ⁰ O by stirring ο with the active ingredient remaining. Then the active ingredient dried in the Vakiam® is suspended in water, the insoluble fraction is centrifuged off, the solution containing the active ingredient is dialyzed against water and lyophilized *

Furthermore, in Caaadi patent 671,648 there is a method for the production of high-active fibrinolytic agents Aspergillus oryzae described, which is characterized is that Hohpräparat® after dissolving in water and after adjusting to pH 3 »5 - 5j? 5 by means of hydrochloric acid © chromatography via cross-linked as Bösbran Sepfoadex G-50 under- "■ thrown and then after, development with hydrochloric water the active ingredient-containing fractions are lyophilized

BAEUDHJASOHOV /~(Ber.d.Akad.d ₉ Wiss. Der USSH 153 «4, 939 (1963 ^ 7 found not only in Aspergillus oryzae strains, but also in Aspergillus awamori a fibrinolytisoti-active substance, which he obtained according to the above-described method by STEFANINI ^ ■ proc.Soo.exp.Biöl ·, Ν · ϊ ·, ^ L · 5 ⁰ ^ (1958i7isolierte.

By AA # IMSCHENETZKI & Mitarbt «^" * Ber.d »Akad.d« Wiss · der USSR 163. 3, 737 (1965 ^ 7 a method for obtaining a fibrinolytically active protease from. Aspergillus terricola is stated «The production takes place in that, for the precipitation of the active ingredient, culture filtrate with ammonium sulphate

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or acetone is added * After centrifugation, the Precipitate is dissolved in water, insolubles are filtered off, and the filtrate is passed over a Sephadex G-25 column filtered to separate ammonium sulfate and pigments. The active eluate is lyophilized.

US Pat. No. 3,281,331 describes a process for obtaining fibrinolytically active proteases, in which the active ingredients are adsorbed 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, eluted with buffer of pH 70 and then lyophilized after dialysis or precipitated with tannin or at pH 6.0 to ~ 2

DEAE cellulose is adsorbed and, by gradually increasing the Salt concentration to be eluted with buffer

All known methods relate to the * isolation fibrinolytically active protease from Aspergillus oryzae, Aspergillus terricola, Aspergillus flavus, Aspergillus awamori or Absidia tfoerulea

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

Some work-up methods also lead to only partially purified protease preparations, which makes them therapeutic Possible uses are limited

The purpose of the invention was to develop a method for rational isolation of an under physiological conditions

fibrinolytically optimally effective, highly purified protease from Auspergillus ochraceus, which is applied externally or parenterally in therapy without any toxic side effects can be · ■

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The invention is based on the object, from crude enzyme mixtures from Aspergillus ochraceus a highly purified fibrinolytically to isolate effective protease with the help of which the separation of fibrinolytically inactive proteases, ballast proteins and interfering dyes in a rational way Wise succeed

According to the invention it was found that a fibrinolytic Effective and therapeutically applicable protease without toxic side effects can be produced if a protease is obtained from Aspergillus ochraceus cultures isolated crude enzyme mixture extracted with aqueous buffer solution, the active ingredient-containing Extract via the anion exchange ^ DEAE-Sephadex or DEAE-Cellulose filtered and then from the active ingredient-containing Eiltrat the active ingredient with water-miscible organic Solvents are precipitated · After drying in vacuo, the separated active ingredient-containing precipitate is in water to be dissolved and subjected to gel chromatography on cross-linked dextran gels Sephadex G-75 or Sephadex G-10Ö » The fractions containing proteas are freeze-dried, and this gives the highly purified fibrinolytically effective protease «

The extraction of the crude enzyme mixture takes place in the manner that this raw preparation - with a proteolytic activity of approx. 1 CE / mg - with 0.05-0.1 M Tris-HOl buffer pH 6.5 - 7 »5 or 0.05 - 0.1 M ammonium acetate buffer from pH 6.5 - 7i5 »is stirred several times at tree temperature, in order to bring the active substance completely into solution · By centrifuging at 1500 g a clear, dark colored drug-containing solution obtained and the insoluble inactive Share separated "

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This drug-containing Exbrakt, preferably at 0 4 ⁰ O through which the Anionenaust from rather EEAE-Sephadex Λ-50 or the anion exchanger DEAE-Gellulose, capacity from 0.5 to 0.9 meq / g "packed column, with the Extraction buffer has been equilibrated, filtered «The ion exchanger is then washed with the buffer used for the extraction.

The specified anion exchanger treatment will result in the extract, which contains active ingredients, contains both dietary fiber and fibrinolytic therapy removes unsuitable proteases as well as most of the dyes, whereas the fibrinolytic active protease is not adsorbed · |

From the active ingredient solution obtained in this way, the active ingredient is ^{precipitated by adding three times the amount of pre-cooled, water-miscible organic solvent to -20 0} G, preferably with Atihanoi, and dried in vacuo.

The active ingredient-containing substance dissolved in as little water as possible is subjected to gel chromatography in such a way that the solution - preferably at 0 to +4 ⁰ O - is placed on a column loaded with Sephadex 6-76 or Sephadex GMOO-Gel eluted with water λ. 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 fibrinolytic protease with a proteolytic activity of approx. 15 CE / mg,

The process according to the invention makes it possible, from raw enzyme mixtures obtained from Aepergillus oohraoeua , to obtain a high-quality brinolytic effect that is active under philological conditions.

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The fibrinolytisoh active protease results in external or parenteral application under therapeutic conditions with no toxic side effects

The proteolytic activity, expressed in oaseinolytic units (035), was determined by a modified method of a test method given by KfJNITZ ^ "j.Gen.Physiol. JQ (1W) 2917 at pH 8.0.

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Execution example © t

Example 1:

50 g of a crude enzyme mixture isolated from Aspergillus ochraceus cultures with a total proteolytic activity of 1 _t 4-0U / mg expand 4 times with 200 ml each of 0.05 M Tris-HOl buffer of pt 7 »2 for 15 minutes mechanical drilling extracted. The extraction mixture is centrifuged until clear at 15,000 g. The clear, dark brown solutions containing the active substance are combined and give a volume of 750 ml.

The entire solution is A-50 packed column (diameter 10 cm, filling level 18 cm) at 0 to 4 ⁰ O a "anion exchanger with the anilines DEAE-Sephadex filtered. Subsequently, with 0.05 M Tris-HOl buffer, pH 7.2, rewashed

50 g DEAE-Sephadex are used to produce the exchanger bed A-50 equilibrated with 0.05 M Tris-HOl buffer pH 7> 2

The active ingredient is precipitated from the filtrate (approx. 3500 ml), which is now only slightly colored, with the active ingredient-containing filtrate in 3 times the volume of ethanol cooled ^{to -20 0 O.} For complete flocculation of the drug is allowed to stand, the precipitation overnight at -20 ⁰ O. The precipitate is separated off by centrifugation, washed with ethanol and acetone and dried in vacuo.

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

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

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The clear, brownish active ingredient-containing extract is ^{filtered at 0 to 4 °} C. through a column (diameter 2.5 cm, filling height 95 cm) charged with Sephadex G 75 gel swollen in water and washed with distilled water. Water eluted · The eluate coming from the column is captured in 20 ml fractions aiii.

After an inactive forerun of 220 ml, the Active ingredient in fractions 12 to 18, dyes and proteins with a lower molecular weight remain on the Pillar·

The active ingredient solution (140 ml) is subjected to freeze-drying and gives 616 mg of a white, easily soluble dry powder with a specific proteolytic activity of 15ι7 GE per mg.

Example 2:

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

The clear, dark-colored solution containing the active ingredient obtained (290 ml) is over a DEAE cellulose with the anion exchanger loaded column (diameter 4.5 ov, filling height 15 cm), which equilibrates with 0.05 M Tris-HCl buffer pH 7.2 has been filtered. Then with 0.05 M Tris-HCl buffer pH 7 »2 rewashed

The active ingredient-containing filtrate (approx. 400 ml) becomes the Active ingredient, as described in Example 1, precipitated, with 3 »19 g of an enriched in fibrinolytically active protease Product with 4.5 GE / mg are obtained, which is used to obtain the highly purified active ingredient according to Example 1 to be subjected to gel chromatography "

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Claims (4)

- -9 - ■ '■ Patent claims * 1 · Method of Isolating a Fibrinolytic Enzyme from mold cultures, characterized by this! that a crude enzyme mixture from Aspergillus ochraoeus is extracted with aqueous buffer solution, containing the active ingredient Extract via an anion exchanger DEAE-Sephadex or DEAE cellulose filtered from the obtained active ingredient liltrat the active ingredient precipitates with water-miscible organic solvents, the Niederechlagnaoh Drying in vacuo with water and gel chromatography on cross-linked Sextrangelen Sephadex G-75 or Sephadex G-100 subjects and finally the freeze-dried protease-containing erections 2. The method according to claim 1, characterized in that the crude enzyme mixture from Aspergillus oohraoeus with 0.05 M to 0.1 M Eris-HOl buffer or 0.05 M to 0.1 M ammonium acetate buffer of pH 6.5 up to 7 »5» preferably 7.0 to 7.2, is extracted · 3 * Method according to claim 1 to 2, characterized in that the active ingredient-containing extract is fed via a column with the anion exchanger DEAE-Sephadex A-50 or DEAE-oil, capacity of 0.5 to 0.9 meq / g , equilibrated with the buffer used for extraltion, preferably at 0 to 4 ⁰ O, filtered, the exchanger washed with paffer and the active ingredient from the liltrate with; Water with measurable organic ingredients, prepared with ^{ethanol pre-cooled to -20 0} O, in a ratio of 1: 3 is precipitated. 009887/1803 4. The method according to claim 1 to 3, characterized in that the active ingredient-containing substance obtained after the anion exchanger treatment is redissolved in as little water as possible, on a column charged with Sephadex G-75 or Sephadex G-100, preferably at 0 to 4 ⁰ G, subjected to gel chromatography, eluted with water and the eluate is collected in fractions and the fractions containing protease are freeze-dried » 5 · Suitable for therapeutic purposes, highly purified Aspergillus ochraceus protease produced according to claims 1-4. v 4 »γ 009887/1803