Thrombolytic and fibrinolytic enzyme from streptomyces
United States Patent 1191 Belloc et a1.  
 l l THROMBOLYTIC AND FIBRINOLYTIC ENZYME FROM STREPTOMYCES [75] Inventors: Andre Belloc, Vanves; Jean Florent;  
 Jean Lunel, both of Paris; Denise Mancy, Charenton; Jean Verrier, Boulogne-sur-Seine, all of France [731 Assignee: Rhone-Poulenc S.A., Paris, France [22] Filed: July 25, 1972 [21] App]. No: 275,091  
 [30] Foreign Application Priority Data Apr. 1,1975  
 $331,751 7/1967 Reusser i. 195/62 3.657.416 4/1972 Reid et a1. 195/62 X FOREIGN PATENTS OR APPLICATIONS 7,646 2/1970 France OTHER PUBLICATIONS Trop et al.. The Specificity of Proteinases from Streptomyces griseus (Pronase), Biochem. J. 1970, Vol. 11c (Pp. 19-25), QP501.B47.  
 Primary E.raminerDavid M. Naff Attorney, Agent, or Firm-Stevens, Davis, Miller &amp; Mosher [5 7] ABSTRACT A thrombolytic and fibrinolytic proteolytic enzyme is produced by aerobically culturing in an aqueous medium a newly discovered microorganism Srrepromcres venerus DS 24,288 (NRRL 3987) and isolating the enzyme after culturing.  
 10 Claims, 3 Drawing Figures RHENIEUAPR H975 3.875.005  
  u p l u o PATENTEU H975 3.8751305 sumzufia F/GZ Activity +tryps/n chymolrypsin Substrate ATEE BA EE gelline mdn dog rabbit azo-ccsein cattle casein 1 THROMBOLYTIC AND FIBRINOLYTIC ENZYME FROM STREPTOMYCES This invention relates to a new enzyme, denoted hereinafter by the number 22,750 RP, to a process for its preparation and to pharmaceutical compositions containing it.  
  The new enzyme is of particular value as a thrombolytic agent and is characterised by a general proteolytic activity in vitro.  
  The enzyme 22, 750 RP is obtained by culture, in suitable media, of a microorganism of the Streptomyces genus, identified more completely hereinafter and denoted by the name Slrepwmyces venerus, DS 24, 288.  
  A specimen of the strain has been deposited with the United States Department of Agriculture, Northern Regional Research Laboratory, at Peoria, 111., U.S.A. and has been given the number NRRL 3987&#39;, a sample of the microorganism can be obtained from the aforementioned Research Laboratory.  
  The enzyme 22,750 RP according to the invention is characterised by the following physico-chemical properties:  
  It is very soluble in water, soluble in aqueousalcoholic and aqueous-acetone mixtures, very slightly soluble in concentrated solutions of neutral salts [e.g. NaCl; (NH,),SO,] or of polyethylene glycol and insoluble in anhydrous alcohols, acetone, hexane, ethyl acetate, diethyl ether and chlorinated solvents.  
  It is a protein which gives the conventional reactions of proteins (biuret reaction, Folin reaction and staining with Ponceau Red, Amide Black 12 BN or Coomassie Blue).  
  The enzyme contains carbon, hydrogen, oxygen, nitrogen and sulphur, the relative proportions being approximately:  
 C 50.4% H 7.35% N 17% S 0.50%  
  According to its chromatographic behaviour on a dextran gel or on a polyacrylamide gel, 22, 750 RP has a molecular weight of 40,000 i 5,000.  
  Acid hydrolysis of the enzyme 22,750 RP demonstrates the presence of the following amino-acids, for which the content in millimol per g. of product is given: aspartic acid (1 1.5), threonine (8.5), serine (7), glutamic acid (3.5), proline (2.5), glycine (13), alanine (7), valine (4.5), methionine l isoleucine (2.5), leucine (4), tyrosine (5 phenylalanine (2), lysine (3), arginine (2.5) and histidine (2).  
  The enzyme 22,750 RP is characterised by the physical properties given below:  
 Appearance: white powder (after lyophilisation) Ultra-violet spectrum: (determined with a 0.03%  
 strength solution in water) Absorption at 210 nni E, 187 Absorption minimum at 250 nm F. 4.35 Absorption maximum at 278 nm E, 13.55  
 Infra-red spectrum: (determined with tablets of a mixture with KBr) This spectrum is shown in FIG. 1 of the accompanying drawings, in which the abscissae give the wavelengths expressed in microns (upper scale) and the wave numbers in cm, (lower scale) and the ordinate gives the optical density.  
  The principal infra-red absorption bands of 22,750 RP, expressed as wave numbers (cm), are given in Table l which follows:  
 vw very weak w weak m medium v5 very strong sh. shoulder Optical rotation:(determined with a 0.5% strength solution in water) The enzyme 22,750 RP can be characterised by its electrophoretic behaviour on a cellulose acetate gel (Celloge1&#34; N.D. Chemetron), using different buffers, for example as follows:  
 Citric acid/disodium phosphate pH 2.2 *p. 0.09 Acetic acid/sodium acetate pH 4.0 p 0.1 5,5-Diethyl-barbituric acid (barbital) pH 8.6 u 0.075 Sodium hydroxide/glycine pH 9.6 u 0.1  
 &#34;p ionic strength The enzyme can be detected by colourimetric methods or by measurement of proteolytic activity.  
  For example, at pH 4.0, the active factor migrates towards the cathode at a rate of 5 mm/2 hours under a constant potential of 250 volts (intensity varying from 6 to 15 milliamperes).  
  The enzymatic activity of 22,750 RP is manifested on a large number of proteinaceous substrates, and in particular on casein, haemoglobin and fibrin. It is manifested only very weakly on the ethyl ester of benzoylarginine (BAEE) and is not manifested at all on the ethyl ester of acetyltyrosine (ATEE).  
  The activity on casein is determined according to a technique suggested by that of Kunitz, developed for the determination of trypsin (M. Kunitz, .1. Gen. PhysioL, 30, 291 1947 )1. The peptides set free during the hydrolysis, which are soluble in trichloroacetic acid, are determined by spectrophotometry (measurement of the optical density at 280 nm). The enzymatic activity can be expressed in Kunitz units (K.U.): according to the definition of Kunitz, one unit is the amount of enzyme which sets free sufficient soluble peptides for the optical density at 280 nm of trichloroacetic acid filtrate to increase by 1.000 in one minute.  
  The results which have been obtained with the enzyme 22,7 50 RP compared with two crystalline proteolytic enzyme, namely trypsin and chymotrypsin, are given in Table 11.  
  FIG. 2 gives these results in diagrammatic form. Reaction conditions are given below.  
 tinue their investigation are removed and transplanted onto nutrient agar slopes in order to produce more abundant cultures.  
  Streptomyces venetus DS 24,288 forms oval spores, measuring 0.4 to 0.6 u/l to 1.2 pt. [ts sporophores comprise spiral sporiferous filaments, the coil of which most frequently possesses live to eight turns, but can sometimes contain a few more; the latter are quite often inserted singly on the filaments which support them, but it is also possible to observe sporophores in clusters of a few elements. According to its method of sporulation, this strain is classified in the Spira section of the Pridham classification.  
  Streptomyces venerus DS 24,288 develops well at C. and at 37C., but not at 50C. It possesses the property ofproducing black melanin pigment on a suitable medium containing tyrosine, produces H 5,  
 till  
  Reaction at pH 8.0 at 3 concentration of substrate 5 g./] (b) 7.5 at 37C. concentration of substrate 12.5 g./l (cl 7.5 at 37C. on a gelutine film (d) 7.4 at 37C. concentration of fibrinogcn: 10 g./] (e] 7.5 at 37C concentration of plasma: 400 cell (1&#39;) 7.4 at 37C. concentration of plasma: 200 cell (g) 7.4 at 37C. concentration of plasma: 200 cc/l l 7.7 at 25C. concentration of substrate: 5 X 10&#34; M (1) 7.0 at 25C. concentration of substrate: 5 X 10 M The thrombolytic and fibrinolytic activity of 22,750 RP has been confirmed on laboratory animals.  
  In the rabbit, at a dose of 0.5 mg/kg. animal body weight, administered intravenously, the enzyme provides protection to a highly significant extent, against intravascular coagulation induced by passing a nylon thread impregnated with collagen into the jugular vein [technique suggested by that of]. L. David et coll, CR. Soc. BioL, 162, 1763 (1968)]. Acceleration of fibrinolysis can be demonstrated, in rabbits, from a dose of 0.05 mg/kg., administered intravenously.  
  The toxicity of the enzyme 22,750 RP has been studied in several animal species. In mice, the 50% lethal dose (LD determined by intravenous administration, is 5 mg/kg. animal body weight; it is the same after repeated administration over the course of 5 days as after a single administration. In rabbits, the LD determined by intravenous administration, is 1.5 mg/kg. animal body weight.  
  These properties make it possible to use the product according to the invention in human therapy in the prevention and the treatment of venous thromboses, pulmonary embolisms and thromboses of the coronary arteries, the arteries supplying the extremities and the cerebral arteries.  
  The organism which produces the enzyme 22,750 RP, viz. Streptomyces venetus, DS 24,288 (NRRL 3,987), has been isolated from a sample of earth taken in India.  
 The isolation of Streptomyces venetus, DS 24,288 was carried out by following the general method which consists of suspending a small amount of earth in sterile distilled water, diluting the suspension to different concentrations and spreading a small volume of each dilution on the surface of Petri dishes containing a nutrient agar medium. After incubation for several days at 26C., which allows the microorganism to develop, the colonies which it is desired to isolate in order to constrongly reduces nitrates to nitrites, hydrolyses starch, liquefies gelatine, peptonises milk without first coagulating it and utilises cellulose. The colouration of its vegetative mycelium ranges, depending on the media, from yellow to brown-yellow, yellow-brown or dark brown; the soluble pigments which it produces are generally of a darker or lighter yellow-brown tone depending on the media, and they can, in certain cases, be blackish-brown. lts aerial mycelium is coloured blue when sporulation occurs.  
  The cultural characteristics and the biochemical properties of Streptomyces venelus DS 24,288 are given in Table 111 which follows. Unless otherwise stated they are those of cultures which have reached a good stage of development, that is to say, about 3 weeks at 25C. These characteristics have been observed on nutrient agars and broths usually employed to determine the morphological characteristics of Streptomyces strains, the cultures on agar media being carried out on agar slopes. A certain number of the culture media used were prepared in accordance with the fonnulations indicated in Actinomycetes, S. A. Waksman, p. 193-197, Chronica Botanica Company, Waltham, Mass, U.S.A., 1950&#39;, in this case, they are indicated by the letter W followed by the number given them in The Actinomycetes.&#34; The references or compositions of the other culture media are as follows:  
 Ref.A Hickey and Tresners Agar&#34; T. G. Pridham et coll Antibiotics Annual, 1956-1957, p. 950  
 Ref.B K. L. Jones Journal of Bacteriology, 57,  
 Ref.C Formulation W-23, to which 2% of agar has been added.  
 Ref.D Yeast Extract Agar T. G. Pridham et coll Antibiotics Annual, 1956-1957, p. 950  
 Ref.E Tomato Paste Oatmeal Agar&#34; T. G. Pridham et coll Antibiotics Annual, l9561957, p. 950  
 Ref.F Melanin formation medium&#34; The Actinomycetes, vol. 2. p. 333, No. 42 S. A. Waksman The Williams and Wilkins Company, Baltimore, 1961 Ref.L corresponds to the formulation W-lS, with 30 g. of sucrose replaced by 15 g. of glucose Ref.M corresponds to the formulation W48. with the sucrose removed and replaced by small strips of Ref.G W. E. Grundy et coll Antibiotics and 5 finer Paper Pamally lmmersed the hqu&#39;d Chem. 2, 40l. 1952 Ref.N Manual of Methods for Pure Culture Study of Bacetria&#34; of the Societ of American Bacteriol- Ref.l-l Inorganic Salts Starch Agar&#34; T. G. y  
  Pridham et coll Antibiotics Annual 19564957 ogsts Geneva 95] Ref.P Plain gelatin prepared in accordance I 10 with the instructions ofManual of Methods for Pure Ref.l Substrat l mrt mmerahscher Sttckstoff- Culture study of Bactena of the soclety of queue 14 cause et coll ZUR KLAS&#39; American Bacteriologists Geneva, N.Y. -18 SIFIZIERUNG DER ACTINOMYCETEN VEB Ref.G Commerrcally available skimmed milk pow- GUSTAV FlSCHER VERLAG&#39; jENAlgss der. reconstituted 1n accordance with the manufac- RefJ corresponds to the formulation W-l, with 30 (5 [uyef&#39;s i nqi 0f Sucrose replaced y 15 of glucose Ref.R Medium indicated for the research of the pro- Ref.l( corresponds to the formulation W-l, with 30 duction of H 5 by H. D. Tresner and F. Danga g. of sucrose replaced by 15 g. of glycerme Journal of Bacteriology, 76, 239-244. i958.  
 TABLE III Culture medium Degree of Vegetative myeelium Aerial structure Soluble Observations and development (V.m.) or Underside (comprisin the pigment biochemical of the culture serial myce ium properties and sporulation combination Hickey and Very good Undcrside blackish- Light greyish- Dark brown. Oval spores Tresner agar brown blue. ranging towards measuring 0.4 to (Ref. A) Well developed. 0.6/1 to 1.2 a.  
 blackish-brown Spiral sporophores.  
 unbranched or in short clusters Bennett agar Good Underside yellowbrown Whitish to light Yellow brown (Ref. B) blue Emerson agar Good V.m. thick and Whitishin trace Yellow-brown (Ref. C] wrinkled. yellow-brown amounts Pridham yeast Good V.m. thick and Whitish to light Orange-brown extract agar wrinkled. yellowblue (Ref. D] brown Pridham oatmeal Good V.m. thick and Whitish to light Yellow-brown and tomato extract wrinkled. yellow blue brown agar (Ref. E) G ucose- Quite good V.m. thick and Greyish-whitc. Yellow-brown peptone agar wrinkled. yellow- Very poorly (W .6) brown developed Nutrient agar Average V.m. yellow-brown None Yellow-brown l Tyrosine-yeast Moderate Underside blackish Blue-grey Black Fonnation of melaextract agar for brown nin: sitive the formation of (rea ings carried melanin out in accordance (Ref. F) with the recom&#39; mendations of the author) Krainsky Moderate V.m. lemon yellow Whitish. None Solubilisation of calcium ln trace amounts the malate: malate agar positive. good (Ref. Gl Ovalbumin agar Poor V.m. colourless to Whitish. Brownish-yellow (W-lZ) brownish. poorly in trace amounts weak developed Glucose- Quite good V.m. thick and Whitish to yellow-brown asparagine wrinkled. hrownbluish. Rather agar yellow to yellowpoorly developed (W-Z brown Glyeerinc- Quite good V.m. thick. yellow- Whitish to Yellow-brown asparagine brown bluish. Very agar poorly developed Pridham starch- Quite good Undcrside yellow- Light grcyish None Oval spores inorganic salts brown blue measuring 0.4 to war 0.6/l to 1.2 a. cl. Hl Spiral sporophores.  
 unhranched or in short clusters. Hydrolysis of the starch: positive Starch- Moderate L&#39;ndcrsidc yellow- Whitish to light Yellow-brown Hydrolysis of the nitrate agar brown blue starch: positive lW-lll) Gausc stareh- Good V.m. thick and Light grayish Dark inorganic wrinkled. yellowblue yellow-hrown. nitrogen agar brown ranging towards (Ref. l| blackish (zapek sucrose Good V.m. thick and Whitish. Very Yellowbrown synthetic agar wrinkled. yellowpoorly developed (W4 brown TABLE III Cnt|nued Culture medium Degree of Vegetative mycelium Aerial structure Soluble Observations and development (\&#34;.m.) or Underside (comprising the pigment biochemical of the culture serial mycelium properties and sporulation combination) Czapek glucose Good -\/.m. thick and Whitish. In Li in synthetic agar wrinkled, browm trace amounts ye low-brown (Ref. .ll yellow (Zapek glyccrinc Average V.m. light brownish- None Light synthetic agar yellow hrt wnishycllovv (Ref. K)  
 Starch-nitrate Good Thick velum. White Brownish-yellow Production of broth Undcrsidc brownnitrites:  
 (W-l9] yellow to yellowstrongly positive brown Czapek Moderate Ycllowish-white None None or slow, Production of sucrose broth velum weak yellow nitritcs:  
 (W48) strongly positive Clapek Moderate Yellowish-whitc None None or slow, Production of glucose broth velum weak yellow nitrites:  
 (Ref. L) strongly positive Czapek cellulose Moderate Whitish velum. Light greyish- None Production of broth Moderately blue. nitrites:  
 (Ref. Ml developed. Moderately strongly positive developed on the Use of the cellulose: velum and on the positive paper emerging from the broth Nitrate nutrient Good Well developed ring Whitish. Brownish Production of broth yellowish in trace amounts nitrites:  
 (Ref. NI strongly positive Potato culture Good V.m. very thick and Whitish. Black (W-27l very wrinkled, In trace amounts dark yellow-brown 12% strength Good Thick central colony Whitish. Brown-yellow to Rapid liquefaction pure gelatine at the surface. Poorly yellow-brown of the gelatine (Ref. P) Van. brown-yellow developed.  
 Skimmed milk Good Light yellow-brown None Peptonisation l) at C. ring without R f, 0 coagulation.  
 pH going from 6.2 to (1.6 in 1 month 2) at 37C. Good Dark brown ring None Pcptonisation without coagulation. pH going from 6.2 to 6.8 in 1 month Tresner and Good V.m blackish-brown None Black, abundant Production of Dan at agar H 8: positive (Re R) (readings carried out in accordance with the recommendations of the authors].  
  Strepromyces venems DS 24,288 possesses a combination of properties which do not coincide exactly with any of those of the strains of Streptomyces previously described.  
  Considering the species described by S. A. Waksman in The Actinomycetes (volume 2, The Williams and Wilkins Company, Baltimore, l96l S. venerus Ds 24,288 would be placed in the Viridochromogenes Series described on page 149 of this work, in view of its production of melanin pigment, its blue sporulated aerial mycelium and light brown to dark brown vegetative mycelium, formation of soluble pigments ranging from brown-yellow to yellow-brown or very dark brown, depending on the particular case, on synthetic or organic media, and its spiral sporophores. However, it cannot be likened to any of the three species mentioned by S. A. Waksman as forming this series, namely Srrepmmyces viridochromogenes, Strepromyces charrreusis and Srreplumyces cyaneus.  
 It cannot be identified with S. cyaneus which, unlike S. venetus, forms a blue-coloured vegetative mycelium on agar media; furthermore, unlike S. venerus Ds 24,288, S. cyaneus does not use cellulose and does not reduce nitrates to nitrites.  
  It cannot be likened to S. charrreusis which gives a green-yellow to black soluble pigment on gelatinc and does not give a soluble pigment on nutrient agar or on potato, whilst S. venetus DS 24,288 gives a yellow brown soluble pigment on gelatine as well as on nutrient agar and a black soluble pigment on potato.  
 does not reduce nitrates to nitrites, whilst S. venetus DS 24,288 does so in a particularly strong and rapid manner, both on synthetic media and on organic media.  
  S. A. Waksman also includes a number of strains de scribed by Gause et coll (zur Klassifizierung der Actinomyceten, G. Fischer, Jena i958) in the Viridochromogenes group&#34;: when comparing 5. venetus DS 24,288 with this group, which forms the largest part of the Coerulescens Series&#34; of Gause, it would be situated in the latter group which comprises the strains of which the vegetative mycelium on starch-inorganic nitrogen agar of Gause is of a dark colour and which contains only two species, namely Acrinomyces (Streptomyces) viridochromogenes and Actinomyces (Streptomyces) coeruleofuscus.  
  S. venetus DS 24,288 differs from the species S. viridochromogenes essentially in the dark green colouration which the vegetative mycelium of the latter can assume on a number of media, which S. A. Waksman has mentioned in his description. In addition in the description given by Gause e! coll, S. viridochmmogenes also produces a vegetative mycelim which assumes a dark green shade on milk, on starch agar, on potato and on the starch-inorganic nitrogen agar of Gause and does not produce any soluble pigment on the latter medium; in comparison, S. venelus DS 24,288 does not give a dark green vegetative mycelium on these media and produces a dark yellow-brown soluble pigment on the starch-inorganic nitrogen agar of Gause. The formation of a dark green soluble pigment on gelatine by S. viridoc/zmmogenes, mentioned by Gause, does not occur in the case of S. venerus DS 24,288.  
  S. venetus DS 24,288 cannot be identified with .5. ceruleofuscus which, in contrast to it, does not reduce nitrates and does not use cellulose; furthermore, s. c0- emleofuscus does not give a soluble pigment on an organic agar medium or on potato, whilst S. venetus 24,288 consistently gives a yellow-brown soluble pigment on all the organic media tested, and a black soluble pigment on potato.  
  The ability of S. venetus DS 24,288 to use various sources of carbon and nitrogen to ensure its development was determined according to the principle of the Pridham and Gottlieb method (J. of Bact., 56 l07-l l4, (1948). The degree of development was observed on the base medium indicated by the authors, either replacing the glucose by the various sources of carbon tested respectively, or (NH SO by the various sources of nitrogen tested respectively. The results are given in Table IV.  
 TABLE lV-Continued Sources of carbon Use Sources of nitrogen Use tested tested Starch Positive DL-Threonine Positive Glycogen Positive Taurine Negative Glycerol Positive DL-Phenylalanine Positive E thritol Negative L-T rosine Positive A onitol Positive DL- roline Positive Dulcitol Negative L-Hydroxyprolinc Positive D-Mannitol Positive L-Histidine Positive D-Sorbitol Negative L-Tryptophane Positive lnositol Positive Betain Positive Salicine Positive but slow According to a feature of the invention, the enzyme 22,750 RP is produced by aerobically cultivating Strepromyces venems DS 24,288 (NRRL 3987), or a mutant thereof capable of producing the enzyme, using an aqueous medium containing assimilable sources of carbon, nitrogen and inorganic substances and isolating from the medium 22,750 RP formed during the culture.  
  The culture of Srrepmmyces venetus DS 24,288 can be carried out by any of the known aerobic surface or submerged culture methods, the latter being preferred because they are more convenient. Conventional types of apparatus currently employed in the fermentation industry may be used. In particular, the following sequence of operations may be adopted:  
 Srreptomyces venetui DS 24,288 stock culture on agar culture in an agitated flask inoculum culture in a fermenter production culture in a fermenter The fermentation medium must contain a source of assimilable carbon and a source of assimilable nitrogen and inorganic substances and, optionally, growthpromoting factors and thickners; all these ingredients may be supplied as well-defined products or complex mixtures, such as those found in biological products of various origins.  
  As sources of assimilable carbon, there may be used carbohydrates such as glucose, sucrose, lactose, dextrins, starch, molasses or other carbon-, hydrogenand oxygen-containing substances such as sugar alcohols, e.g. glycerol or mannitol, or certain organic acids eg. lactic acid, citric acid and tartaric acid. Certain animal or vegetable oils such as lard oil or soya bean oil may be advantageously used instead of, or in admixture with the aformentioned substances.  
  The suitable sources of assimilable nitrogen are extremely varied. They may be very simple chemical compounds such as nitrates, inorganic or organic ammonium salts, urea and certain amino acids. They may also be complex substances, containing principally nitrogen in a protein form, e.g. casein, lactalbumin, gluten and their hydrolysates, soya bean flour, peanut meal, fish meal, meat extract, yeast extract, distillers solubles and corn-steep liquor.  
  Amongst the inorganic substances added, some may have a buffering or neutralisng effect, such as the alkali metal or alkaline earth metal phosphates, or the carbonates of calcium or magnesium. Others contribute to the ionic equilibrium necessary for the development of Streptomyces venetus DS 24,288 and for the production of the enzyme 22,750 RP; examples of these are the chlorides and sulphates of the alkali metals and alkaline earth metals. Finally, some of them act more especially as activators of the metabolism of Streptomyces venetus DS 24,288: to these belong the salts of iron and cobalt.  
  Amongst the thickeners, the most usually employed are starch, carboxymethylcellulose and agar.  
  The pH of the fermentation medium at the start of the culture should be between 6.0 and 7.8, and preferably between 6.4 and 7.5. The optimum fermentation temperature is 2528C., but satisfactory production is achieved at temperatures between 23 and 40C. The rate of aeration of the fermentation broth can vary within quite wide limits, but it has been found that aeration rates of 0.3 to 2 litres of air per litre of broth per minute are particularly suitable. The maximum yield of the enzyme 22,750 RP is obtained after 1 to 7 days culture, but this period depends predominantly on the medium used.  
  The enzyme 22,750 RP can be extracted from the fermentation broth and purified by the usual methods for isolating and fractionating protein materials, the activity and the purity of the product being measured by suitable methods such as the determination of the activity on casein and the electrophoresis.  
  The enxyme 22,750 RP can be isolated from the fermentation broths by filtering the broth in the presence of a filtration aid, concentrating the filtrate under reduced pressure, acidifying the filtrate to a pH of about 4, filtering and then dialysing the filtrate against a stream of distilled water and then precipitating the enzyme 22,750 RP by adding acetone to the dialysate.  
  The enzyme 22,750 RP thus obtained can then be purified by the usual physicochemical methods. The preferred method of purification used consists of dissolving the enzyme in distilled water and then precipitating the enzyme by adding agents, such as neutral salts, e.g. sodium chloride or ammonium sulphate, in concentrated aqueous solutions of which the enzyme is very sparingly soluble.  
  The enzyme 22,750 RP can also be purified by chro&#39; matography on various supports such as alumina, cellulose powder and dextran or polyacrylamide gels, by dialysis or by preparative electrophoresis.  
 The following Examples illustrate the invention.  
 EXAMPLE I A Fermentation A 170 litre fermenter is charged with:  
 peptone 600 g. meat extract 600 g. hydrated glucose l,200 g. soya bean oil I cc. sodium chloride 600 g. tap water. sufficient to make up to 1 l0 litres The pH is adjusted to 7.50 by adding ION sodium hydroxide solution (120 cc. The medium is sterilised by bubbling steam at l22C. through it for 40 minutes. After cooling, the volume of the broth is 120 litres due to condensation of the steam during the sterilisation and the pH is 7.0. The medium is inoculated with a cul ture of Slrepromyces venetus DS 24,288 (200 cc.), produced in an agitated Erlenmeyer flask. The culture is developed at 26C. for 28 hours with agitation and aeration with sterilised air; it is then suitable for inoculation of the production culture.  
  The production culture is carried out in an 800 litre fermenter charged with the following substances:  
 corn steep liquor (50% solids content) 8 kg. soya bean oil 6 litres ammonium sulphate 0.8 kg. cobalt chloride hexahydrate 0.008 kg. tap water, sufficient to make up to 360 litres The pH is adjusted to 6.40 by adding lON sodium hydroxide solution (450 cc.), calcium carbonate (2 kg.) is added, and then the medium is sterilised by bubbling steam at 122C. through it for 40 minutes. After cooling, the volume of the broth is 390 litres due to condensation of the steam during the sterilisation. It is made up to 400 litres by adding a sterile aqueous solution 10 litres) containing hydrated glucose (4 kg.). The pH is then 6.60. It is inoculated with the inoculum culture (40 litres) from the litre fermenter described above. The production culture is carried out at 26C., for 1 l6 hours, with agitation using a stirrer rotating at 260 revolutions/minute and aeration with a volume of sterilised air of 35 cu.m.lhr. The final pH of the medium is 6.80 and the volume of the broth is 380 litres. The proteolytic strength of the broth is 0.4 K.U./cc. B Extraction The fermentation broth (360 litres) obtained as described above and having a strength of 0.4 K.U./cc. is placed in a vat equipped with a stirrer. A filtration aid (l8 kg.) is added to it and the mixture is filtered on a filter press and the filter cake is washed with water (I00 litres). A filtrate (400 litres), of proteolytic strength 0.35 K.U./cc., is thus obtained.  
  The filtrate is concentrated under reduced pressure at a temperature of 35C. A concentrate (30 litres), of strength 3.77 K.U./cc., is obtained. The pH of the concentrate is adjusted to 4.0 by adding 6N hydrochloric acid (450 cc.) and then a filtration aid (300 g.) is added. The mixture is filtered and the filter cake is washed with water (2 litres). The clarified concentrate is cooled to 4C. and dialysed through a membrane of regenerated cellulose for 7 hours against a stream of distilled water at the same temperature.  
  Acetone (l l 1 litres), previously cooled to l0C., is added, with stirring, to the dialysate obtained (37 iitres), kept at 4C., and stirring is continued for 10 to 15 minutes after the end of the addition of the acetone.  
  The active precipitate produced is collected by filtration, washed with cold acetone and dried under reduced pressure (20 mm. Hg) for 24 hours at a temperature of about 30C.  
  A product (997 g.), of strength 97 K.U./g., is thus obtained.  
 C Purification stage 1 Crude product (1,915 g.), prepared as described above, of strength 97 K.U./g. is dissolved in distilled water (40 litres).  
  Crystalline ammonium sulphate (9.6 kg.) (amount calculated to obtain a solution containing 38% of that required for a saturated salt solution) is added slowly, with stirring, to the solution thus prepared. Stirring is continued for 20 minutes after the end of the addition of the salt and then the mixture is centrifuged at L800 G. The precipitate obtained is removed.  
  Crystalline ammonium sulphate (5 kg.) (amount calculated to obtain a solution containing 52% of that required for a saturated salt solution) is added slowly, with stirring, to the supernatant liquid. Stirring is continued for 15 minutes after the end of the addition of the salt, the mixture is left to stand for 1 hour and then centrifuged at 1,800 G.  
  The active precipitate obtained is collected and taken up in distilled water l litre) and the solution obtained is dialysed through a membrane of regenerated cellulose for 24 hours at 4C. against distilled water (40 litres).  
  The dialysate is lyophilised; a product (70 g.), of strength 1,200 K.U./g., is obtained. According to electrophoretic analysis, this product contains about 13% of active factor.  
 D Purification stage 2 The product (102 g.) prepared as described above and of strength 1,200 l(.U./g. is dissolved in distilled water (4,100 cc.) and the pH of the solution is adjusted to 8.0 by adding N sodium hydroxide solution (66 cc.).  
  Polyethylene glycol 1,500 1.25 kg.) is added slowly, with stirring, to the solution thus obtained. Stirring is continued for 15 minutes after the end of the addition of the precipitating agent. The mixture is left to stand for 15 minutes and then centrifuged for 15 minutes at 12,000 G at 4C.  
  The active precipitate obtained is dissolved in distilled water (700 cc.) and the solution produced (800 cc.) is cooled to 4C. lsopropanol (4 litres), cooled to 60C., is slowly added to it, with stirring.  
  The active precipitate produced is collected by centrifuging for 15 minutes at 3,000 O at 10C. and is then dried under reduced pressure (0.2 mm. Hg) in the presence of P at a temperature of about 20C.  
  A product (30.7 g.), of strength 1,600 K.U./g. and containing, according to electrophoretic analysis, approximately 20% of active factor, is thus obtained.  
 E PUrification stage 3 The product g.) prepared as described above and of strength 1,600 K.U./g. is dissolved in distilled water (400 cc.) and the pH of the solution is adjusted to 8.0 by adding N sodium hydroxide solution (3 cc.).  
  A 6% strength aqueous solution of 6,9-diaminO-Z-ethoxy-acridine lactate (26.6 cc.) is added slowly, with stirring, to the solution thus obtained. After minutes of additional stirring followed by 1 hours standing at 40C., the mixture is centrifuged for 10 minutes at 12,000 G at 4C.  
  The inactive precipitate obtained is removed. The supernatant solution is concentrated under reduced pressure (0.2 mm. Hg) at 25C. to a volume of 25 cc. It is then cooled to 4C. and isopropanol (250 cc.), cooled to -60C., is slowly added to it, with stirring.  
 The active precipitate produced is collected by centrifuging for 15 minutes at 12,000 G at 10C. and then dried under reduced pressure (0.2 mm. Hg) in the pres ence of P 0 at a temperature of about 20C.  
  A product (4.7 g.), of strength 2,240 K.U./g. and containing, according to electrophoretic analysis, about 37% of active factor, is thus obtained.  
 F Purification stage 4 The product (200 mg.), prepared as described above and of strength 2,240 K.U./g., is dissolved in a 0.01 M phosphate buffer (3 cc.) at pH 6.5 and isopropanol (3 cc.) and cellulose powder (4 g.) for chromatography are added.  
  The paste thus obtained is placed in an even thickness at the top of a cellulose column. The column is 3 cm. in diameter and contains cellulose (80 g.) in a volume for volume mixture of isopropanol and 0.01 M  
 phosphate buffer of pH 6.5 (which represents a height of 39 cm.). It is eluted with a phosphate bufferlisopropanol mixture, increasing the proportion of buffer in the mixture, in a linear manner, from 50 to by volume (total volume of eluent with a composition gradient: 1.5 1.); the flow rate is 35 cc./hour. The eluate is collected in fractions of approximately 20 cc. and its optical density is measured continuously by a UVl- CORD L.K.B.&#34; analyser at 280 nm. A suitable plot of the results obtained shows a first very asymmetric peak and then, well separated, a second symmetric peak corresponding to a buffer concentration of between 78 and 92%. Electrophoretic analysis shows that only the second peak contains the active factor.  
  The fractions corresponding to the second peak (fractions 53 to 70, total volume 415 cc.) are combined, the alcohol is removed by evaporation under reduced pressure (2 mm. Hg) and then the solution is dialysed across a membrane of regenerated cellulose against distilled water (10 litres) for 24 hours at 4C. and is then lyophilised.  
  A product (43 mg.) of strength 6,400 K.U./g. and containing, according to electrophoretic analysis, approximately 55% of active factor, is thus obtained.  
 EXAMPLE 2 The product (3 g.), of strength 1,600 K.U./g., prepared as described in Example lA-D, is dissolved in a barbital buffer, pH 8.6, p. =0.0375 (40 cc.) and this solution is dialysed through a membrane of regenerated cellulose for 48 hours against the same buffer (10 lltres) at 4C.  
  The solution is then clarified by centrifuging for 15 minutes at 12,000 G at 4C. and then placed at the top of a L.K.B. column, of the PORATH type, containing cellulose (1.3 kg.) in barbital buffer, pH 8.6, p. 0.075 (which represents a height of 66 cm.), for preparative electrophoresis.  
  A potential of 500 volts (anode at the top) is then applied to the terminals of the apparatus; the current is about 0.46 A; the temperature inside the column is kept at about 20C. by continuously circulating a cooling liquid at 4C. in the double wall. From the first hour of the electrophoresis, coloured impurities migrate into the anode compartment. The coloured buffer is removed and is replaced by fresh buffer, repeating this operation as often as is necessary; the removal of these pigments is almost complete after 6 hours of electrophoresis. The potential is then reduced to 400 volts (current 0.32 A) and a counter-current elution is started in the foot of the column, with a flow rate of 250 cc./hour; the eluate is collected in fractions of about 50 cc. and its optical density is measured continuously by a UVlCORD L.K.B. analyser at 280 nm. After 15 hours under these conditions, the potential is increased again to 500 volts (current 0.40 A) whilst bringing the counter-current elution flow rate to cc./hour. After 10 hours, Le. 31 hours after the start of the operation, the electrophoresis is stopped and direct elution is carried out from the top downwards with barbital buffer at a flow rate of 210 cc./h0ur.  
  A suitable plot of the results obtained shows a first small peak (about the 23rd hour of electrophoresis) followed by three imperfectly separated large peaks. Electrophoretic analysis shows that only the third peak contains the active factor. The fractions corresponding to the 3rd peak (total volume 1,425 cc.) are combined and concentrated to 400 cc. by evaporation under reduced pressure (2 mm. Hg) and then dialysed three times for 24 hours against distilled water at 4C. (50 litres each time).  
  The dialysed solution is concentrated to 10 cc. by evaporation under reduced pressure (2 mm. Hg) and then cooled to 4C. Isopropanol l cc.), cooled to 60C., is added slowly to it, with stirring. The active precipitate produced is collected by centrifuging for 10 minutes at 12,000 G at -l0C. and then dried under reduced pressure (0.2 mm. Hg) in the presence of P 0 at a temperature of about 20C.  
  A product (276 mg.) of strength 7,000 K.U./g. and containing, according to electrophoretic analysis, approximately 657c of active factor is thus obtained. This product contains carbon, hydrogen, oxygen, nitrogen, phosphorus and sulphur. Its elementary composition is approximately:  
 C 44.6% H 6.5% N 12.5% P 0.35% S Acid hydrolysis of the product shows the presence of the following amino acids for which the content, in millimol per g. of product, is as follows: aspartic acid (8.5), threonine (7), serine (6.5), glutamic acid (5), proline (2), glycine (l0.5), alanine (7.5), valine (5.5), isoleucine (2), leucine (3), tyrosine (3.5), phenylala nine (l). lysine (2), arginine (2) and histidine (L5).  
  The product possesses the physical properties given below:  
 Appearance; beige powder (after lyophilisation) Ultra-violet spectrum: (determination from a 0.02%  
 strength solution in water) absorption at 2l0 nm E 205 absorption minimum at 250 nm E 7.8 absorption maximum at 278 nm E, l4.l  
 infra-red spectrum: (determination from tablets of a mixture with KBr) This spectrum is shown in FIG. 3, in which the abscissae, give the wavelength expressed in microns (upper scale) and the wave number in cm (lower scale) and ordinate gives the optical density.  
  in the table below, the principal infra-red absorption bands for this product are indicated:  
 3.4[0 vS 1,445 m 940 m 3.300 sh 1.385 m 810 w 070 sh L330 vw 735 sh 3.0[5 sh L300 w 690 sh 2.950 m L235 m 650 sh 2,920 m Ll45 w 610 sh 2.860 vs L120 vw 550 S 2,840 sh L090 sh 480 sh 1.645 vs L070 S 460 sh l 535 v5 L045 m 420 sh \S \er strong in medium v \er weak S strong w weak sh shoulder Optical rotation: (determination from a 0.5% strength solution in water) The activity of this product on various substrates is indicated in the table below:  
  The product (22.8 g.), prepared as described in Example l, stages A to E, but of strength 2,400 K.U./g., is dissolved in distilled water (480 co), the pH of the solution is 7.4. The following operations are carried out at +4C The solution is placed at the top ofa column of cellulose, 15 cm. in diameter, containing cellulose impregnated with distilled water over a height of 17 cm. Elution is carried out with distilled water at a flow rate of 950 ccjhour. The optical density of the eluate is measured continuously by a UVICORD L.K.B. analyser at 280 nm. A suitable plot of the results obtained shows an asymmetric peak containing the enzyme, whilst the traces of 6,9-diamino-2ethoxy-acridine lactate contained in the product remain attached to the cellulose. The eluate corresponding to the ultra-violet absorbing fractions is collected (total volume 6.9 litres) and concentrated, without exceeding 25C., under reduced pressure (2mm.Hg) until the volume is 480 cc.  
  The above concentrate (475 cc.) is diluted with isopropanol (205 cc.) cooled to -l0C. and the solution obtained is placed at the top of a column of cellulose impregnated with a mixture of isopropanol and water containing 30% (by volume) of alcohol. The column is 15 cm. in diameter and contains 600 g. of dry cellulose (height of cellulose 7 cm.). Elution is carried out with the same mixture at a flow rate of 450 cc./hour, whilst continuously measuring the optical density of the elu ate at 280 nm by means of a UVICQRD L.K.B. analyser. The eluate is collected in fractions of about 20 cc. A suitable plot of the results obtained shows two asymmetric peaks. After elution of the second peak (about 10 litres of eluate), the proportion of water in the mixture is increased linearly until it is (total volume of the eluent with a composition gradient: approximately 22.5 litres) and then the elution is completed by means of distilled water until fractions are obtained which show substantially no absorption at 280 nm (total volume approximately 16 litres). The last ultraviolet absorbing peak eluted by the water consists of the fractions between 950 and L150.  
  These fractions are combined (total volume 11.6 litres), concentrated under reduced pressure (2 mm. Hg) until the volume in cc. and then lyophilised.  
  A product (4.5 g.) of strength 6,400 K.U./g. and containing, according to electrophoretic analysis, approximately 60% of active factor, is thus obtained.  
  The product (l.5 g.), prepared as described above and of strength 6,400 K.U./g. is dissolved in a 2 X l0 M solution of sodium chloride 15 cc.), cooled to +4C.  
  This solution is placed at the top of a Biogel-P.60 column, equilibrated at +4C. with the same solvent. The column has a diameter of 7.5 cm. and contains swollen gel to a height of 122 cm (which corresponds to approximately 280 g. of dry gel). Elution is carried out with the same solvent at a flow rate of 120 cc./hour. The eluate is collected in fractions of approximately 35 cc. and its optical density is measured continuously by means of a UVICORD L.K.B. analyser at 280 nm. A suitable plot of the results obtained shows a symmetric peak in its principal part with a shoulder at the start of elution which reveals the presence of an absorbing impurity.  
  The absorbing fractions, except those which correspond to the impurity eluted before the principal product, that is to say the fractions 65 to 80 (total volume 580 cc.), are combined, concentrated under reduced pressure (2 mm. Hg) to a volume of 100 cc. and lyophilised.  
  A product (387 mg), of strength 11,000 K.U./g. and containing, according to electrophoretic analysis, only one protein constituent (more than 95% of active factor), is thus obtained.  
  This product, i.e. 22,750 RP, has the physicochemical properties heretofore specified in relation to the enzyme.  
  The present invention also provides therapeutic compositions, comprising RP 22,750 RP in association with one or more pharmaceutically acceptable carriers or diluents and/or another physiologically active substance, and especially compositions suitable for intravenous or percutaneous administration. Compositions for percutaneous administration may be especially in the form of creams or emulsions. Compositions for intravenous administration may be in the form of sterile aqueous or non-aqueous solutions. As solvent or vehicle there may be used dilute aqueous solutions of propylene glycol, polyethylene-glycol or glucose. Sterilisation may be effected by a number of methods, for example by using a bacteria-retaining filter or by incorporating sterilising agents in the composition. They can also be prepared in the form of sterile compositions which may be dissolved at the time of use in sterile water or other sterile injectable medium.  
  The dosage to be used depends upon the desired therapeutic effect and the duration of treatment. It may vary between l and 50 mg. of active product per day for an adult, in one or more injections.  
  In general, the most appropriate posology will be determined by the physician taking into account the age, weight and other factors peculiar to the subject treated.  
  The following Example illustrates pharmaceutical composition according to the invention.  
 EXAMPLE 4 22,750 RP (l g.) is dissolved in water (500 cc.). The solution obtained is filtered through a bacteriaretaining filter. The sterile solution is transferred aseptically into ampoules (5 cc. per ampoule) and then lyophilised. At the time of use, the contents of the ampoule are dissolved in 5 cc of solvent suitable for intravenous injection.  
 We claim:  
  1. A proteolytic enzyme which is thrombolytic and fibrinolytic and produced by strepwmyces venems DS 24,288 (NRRL 3987), or a mutant thereof capable of producing the said enzyme, which enzyme has the following characteristics: it is a white powder which is very soluble in water, soluble in aqueous-alcoholic and aqueous-acetone mixtures, very sparingly soluble in concentrated solutions of neutral salts or of polyethylene glycol and insoluble in anhydrous alcohols, acetone, hexane, ethyl acetate, diethyl ether and chlorinated solvents; it is a protein containing carbon, hydrogen, oxygen, nitrogen and sulphur, the elementary composition of which is approximately C=50.4%, H=7.35%, N=17% and S=0.50%; its specific optical rotation is [011 14 i 05, lam 29.2 0.6&#34;, [011 53.5 i 0.8, (C=0.02; water); its ultraviolet spectrum (aqueous solution) shows absorptions at 210 nm (E 187), 250 nm (E, =4.35) and 278 nm (E 13.55); its infra-red spectrum (determined with tablets of a mixture with potassium bromide) shows principal absorption bands as follows: 3,280 very strong, 3,200 shoulder, 3,060 medium, 3,040 very weak, 2,980 shoulder, 2,960 medium, 2,935 weak, 2,880 shoulder, 2,650 shoulder, 1,650 very strong, 1,535 very strong, 1,515 medium, 1,490 shoulder, 1,465 very weak, 1,455 very weak, 1,445 medium, 1,435 shoulder, 1,418 shoulder, 1,405 weak, 1,398 shoulder, 1,385 medium, 1,375 shoulder, 1,365 shoulder, 1,330 very weak, 1,303 weak, 1,280 shoulder, 1,235 medium, 1,165 weak, 1,145 very weak, 1,110 medium, 1,075 medium, 1,055 very weak, 1,040 very weak, 927 very weak, 920 very weak, 825 very weak, 735 very weak, 695 weak, about 600 medium and wide 530 weak, 470 shoulder, 430 weak; its molecular weight, as determined by its chromatographic behaviour on a dextran or polyacrylamide gel, is 40,000 1 5,000, and acid hydrolysis demonstrates the presence of the following amino acids, for which the content is millimol per 10 g. of product is given: aspartic acid (11.5),threonine (8.5), serine (7), glutamic acid (3.5), proline (2.5), glycine (13), alanine (7), valine (4.5), methionine (l), isoleucine (2.5), leucine (4), tyrosine (5), phenylalanine (2), lysine (3), arginine (2.5) and histidine (2).  
  2. Process for the production of the enzyme of claim 1 which comprises aerobically cultivating Streptomyces venerus DS 24,288 (NRRL 3987), or a mutant thereof capable of producing the said enzyme, using an aqueous nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic substances, and isolating from the medium said enzyme formed during culturing.  
  3. Process according to claim 2 in which the culture is effected under submerged aerobic culture conditions commencing at a pH within the range 6.0 to 7.8 and at a temperature of 23 to 40C.  
  4. Process according to claim 3 in which the pH of the culture medium at the beginning of the culture is between 6.4 and 7.5.  
  5. Process according to claim 3 in which the temperature of the culture medium is 2528C.  
  6. Process according to claim 3 in which the culture medium is aerated at a rate of 0.3 to 2 litres of air per litre of medium per minute.  
  7. Process according to claim 2 in which said enzyme is isolated from the culture medium by filtering in the presence of a filtration aid, concentrating the filtrate under reduced pressure, acidifying the filtrate to a pH of about 4, filtering and then dialysing the filtrate against distilled water and then precipitating said enzyme by adding acetone to the dialysate.  
  20 phate.  
  10. Process according to claim 7 in which said enzyme is purified by chromatography on alumina, cellulose powder, dextran or polyacrylamide gel, by dialysis or by preparative electrophoresis.