A fibrinolytic enzyme obtained from B. subtilis was purified, using DEAE-cellulose column chromatography, and gel filtration on Sephadex G-100. The preparation was homogeneous as tested by gel filtration on Sephadex G-200, and disc electrophoresis. The molecular weight of this enzyme was 29.400 estimated by gel filtration on Sephadex G-100. The optimum pH for enzyme activity was 7.2 Copper ions significantly increased enzyme activity, while Zn++ and Mn++ caused marked inhibition.

A potent extracellular fibrinolytic enzyme was obtained from cultures of the imperfect fungus Fusarium semitectum under certain growth conditions. Nitrate addition to cultures increased enzyme production. The enzyme showed a versatile proteolytic activity against several protein substrates including casein, gelatin, haemoglobin, bovine serum albumin, and fibrin from both buffalo and human sources.Optimal fibrinolysis occurred at p H values around 7.0. The fibrinolytic activity exhibited marked heat stability in enzyme samples heated to 60 "C, and retained more than 40% of its activity i n samples heated to 100 "C for 10 min. Fibrinolysis proceeded optimally in the temperature range between 60-60 "C. Copper ions significantly activated the enzyme. Other biochemical properties are also reported.Lately, there has been great interest in the search for new thrombolytic agents from various origins with particular reference to microbial sources. This interest was stimulated by the discovery by TILLETT and GARNER (1933) of a bacterial enzyme capable of thrombus dissolution. Subsequent investigations reviewed by TAYLOR and TOMAR (1970) demonstrated the value of the enzyme, streptokinase, in the therapy of thrombosis in man. GRINBERG and MIKHAILETS (1972) studied a fibrinolytic enzyme, terrilytin, obtained from Aspergillus terricola and demonstrated its effectivity in preventing thrombus forination in vitro. From Yenicillium lilacinurn EGOROV et al. (1972) isolated a strong thrombolytic enzyme which was active against fibrin, haemoglobin, and casein substrates. An active fibrinolytic enzyme formed in submerged cultures of Tnchothecium roseum was reported by ANDREENKO et al. (1973). This enzyme, tricliolysin, was formed by the fungus during the entire growth period and its production was favoured by the presence of high concentrations of carbon compounds and protein substrates in the medium. Recently, FODA and FAYEK (1975) obtained potent fibrinolytic preparations from cultures of Bacillus subtilis.Although several microbial enzymes were shown to possess thrombolytic activities in vitro, for various reasons only one enzyme, namely streptokinase, has thus far, been used in the treatment of thrombosis in man. This necessitates further search for new sources of this vitally important group of enzymes. The present paper reports the production physiology and properties of a promising fibrinolytic activity from the imperfect fungus Fusarium semitectum. Materials and methodsOrganism used and growth conditions: A strain of Fusarium semitectum obtained from the NRC collection was used throughout this study. This fungus was selected based on an initial screening for proteolytic enzyme producers. The medium used for fungal growth contained: glucose lyo, potassium dihydrogen phosphate 0.5%, magnesium sulfate 0.05y0, and traces of ferrous sulfate. Enzyme production: The fungus was grown in 300 ml conical flasks each containing 50 ml aliquots of the liquid medium. These flasks were originally inoculated from an exponentially growing cul...

Proteolytic activity of a fibrinolytic enzyme isolated from B. subtilis was tested on fibrin plates plasminogen-free and plasminogen-rich. Results showed that the enzyme is not a plasminogen activator. Inhibitory effect of human serum and plasma was also tested. The plasma and not serum showed a slight inhibitory effect on proteolytic activity of this enzyme.

Some properties of two purified fibrinolytic enzymes from Bacillus subtilis and B. polymyxa K . I . FAYEK and SANAA T. EL-SAYED (Eingegangen a m 6. 8.1979)Two fibrinolytic enzymes isolated from B. subtilis and from B. polymyxa were purified using a five step method. The pH optimum for the enzyme from B. subtilis was 7.2 and for the enzyme from B. polymyxa was 7.0. Both enzymes were activated by Cu++.The molecular weight of the first enzyme was 29,400 and that for the second enzyme was 18,000 on the basis of gel filtration on Sephadex G-100.The enzyme from B. aubtilis has higher affinity to buffalo fibrin than towards human fibrin. The enzyme from B. polymyxa has higher affinity to human fibrin than towards buffalo fibrin.

A fibrinolytic enzyme obtained from B. subtilis was purified, using DEAE-cellulose column chromatography, and gel filtration on Sephadex G-100. The preparation was homogeneous as tested by gel filtration on Sephadex G-200, and disc electrophoresis.The molecular weight of this enzyme was 29.400 estimated by gel filtration on Sephadex G-100. The optimum pH for enzyme activity was 7.2. Copper ions significantly increased enzyme activity, while Zn++ and Mn++ caused marked inhibition.Microbial proteases are well-known enzymes, several investigators have explored this field and studied the production of these enzymes. A review about characterization and methods for their production and isolation was presented by KEAY (1971).Proteolytic enzymes produced by bacteria, e.g. by B. I n a previous study we reported the production of a fibrinolytic enzyme from a strain of Bacillus subtilis (FODA and FAYEK 1975). For further characterization it was necessary to have this enzyme in a pure form. This study presents a method for purification and some properties of the enzyme. Materials and methodsOrganism and growth conditions: A strain of Bacillus subtilis from the NRC collection was used throughout this study. Details of growth medium and enzyme production were as described by FODA and FAYEK (1975). At the end of the growth period, bacterial cells were removed by centrifugation and the cell free supernatant was dialyzed against 0.03 M phosphate buffer a t 4 "C.Enzyme concentration : Small aliquots of crude enzyme preparation after dialysis were concentrated by dialysis against sucrose. The concentrated enzyme solution was than dialyzed against cold 0.1 M phosphate buffer pH 7.0 to eliminate sucrose. DEAE-cellulose column chromatography : DEAE-cellulose was pretreated as recommended by PETERSON and SOBER (1962) and equilibrated with 0.025 M phosphate buffer pH 7.0. After packing in a column (50 x 1.7 om), the concentrated enzyme was placed on the top, and eluted with a stepwise gradient ranging from 0.0-1.0 M sodium chloride in 0.025 M phosphate buffer, p H 7.0. The flow rate was 30 ml/hour. The effluent was collected in samples of 5 ml, transmittance a t 260 and 280 nm was recorded. The protein concentration in each fraction was then calculated using the nomograph developed by ADAMS (1942). Gel filtration on Sephadex G-100: The gel was allowed to swell for 3 days in excess water, and then equilibrated for another 2 days a t 4 "C in 0.05 M phosphate buffer pH 7.0, after which it was packed in a column (1.5 x 60 cm). Collected active fractions from the DEAE-cellulose column were concentrated against sucrose before applying to the top of the Sephadex column, the flow rate was 25 ml/hour. and the effluent has collected in 5 ml samples. The transmittance at 260 and 280 nm was recorded. Test of purity and homogeneity: A) Pooled active fractions irom the Sephadex 6-100 column were placed on the top of a Sephadex G-200 column (50 x 0.8 om), equilibrated and developed