Streptokinase (peptidolytic), mixt. with streptococcal deoxyribonuclease

Strain culture The β-hemolytic streptococcus was inoculated into 5ml of beef broth culture medium containing 2% sheep serum, adjusted to pH 7.1-7.2, and cultured at 370C for about 8 hours. When the culture medium becomes turbid (otherwise, the cultivation time should be extended), transplant the strain on the agar plate and cultivate for 16-18h, which is the strain for production. It can be stored in a 40C refrigerator and can be used for 1 month. Strain beef broth medium; 370;17h→Strain culture plate colony one to three-level seed culture Put the plate colony inoculum in 5ml seed medium, and culture it at 370C for 5-8h. When receiving tertiary seeds, cultivate in a 10L seed pot with a seed amount of 2% to 3%. After culturing at 370C for 10 to 13 hours, the tertiary seed culture solution can be obtained. Plate colony culture medium; 370;6h^→{first-level seed culture medium; first-level seed culture medium; 370;6h^→secondary seed culture; second-level seed culture medium; 370;11h^→third-level seed culture Fermentation culture of tertiary seed culture liquid takes 150L of fermentation medium, the inoculation amount is 5%-10%, the culture temperature drops to about 330 under the tank pressure of 19.62kPa (under 0.2kg), and sodium carbonate (3mol /L) Adjust the pH to maintain the acidity between pH 7.3 and 7.4, and ferment for about 12 hours. Take samples and measure the activity unit of streptokinase. The fermentation will be terminated when the activity of streptokinase reaches the peak. The cycle is about 14-16h, and the yield It is streptokinase 519U/ml, deoxyribonuclease 1677U/ml. Three-level seed culture medium → fermentation broth adsorption and elution fermentation broth is adjusted to pH 7.5～8 with sodium hydroxide solution (100g/L or 10%) Then slowly add 1ml of cholesterol ethanol solution (5%) to every 100ml of fermentation broth, and stir well for 5min. Then add 724 resin that has passed 80 meshes or more to every 100ml of fermentation broth. After the foam disappears, adjust the pH to 4.8～5 with acetic acid solution (10%). Stir continuously to make it adsorb for 30min, and let it stand for 15min to discard the supernatant. , The resin is washed twice with tap water with pH 4.8-5, and once with distilled water with pH 4.8-5. After washing, it is filtered and drained, transferred to a container, and a small amount of ice distilled water is added, which is suitable for stirring. Below 15^0C, add sodium hydroxide (100g/L or 10%) dropwise while stirring until the pH reaches 7.2. Filter through a Buchner funnel, and rinse the resin once with a small amount of distilled water. Combine the filtrate and washing solution to obtain the eluent. Cholesterol ethanol solution of fermentation broth; 724 resin → adsorption adsorbent H2OnaOH; pH 7 → elution eluent salting out Add 243g of ammonium sulfate per kilogram of eluent to make the saturation of ammonium sulfate reach 40%, and use sodium hydroxide at any time (100g/L) Adjust pH 7～7.2, dissolve all ammonium sulfate at about 100C, centrifuge to obtain [Ⅰ] precipitate, for preparing streptokinase; [Ⅱ] mother liquor, for preparing deoxyribonuclease, and then process separately . Eluent (NH4)2SO4; 100C → salting out → [Ⅰ] precipitation (for the production of streptokinase) → mother liquor [Ⅱ] (for the production of deoxyribonuclease) sediment-preparation of streptokinase calcium phosphate treatment, precipitation Crush the precipitate, add a certain amount of distilled water and adjust the pH to 7.2 to dissolve it. After dissolving, adjust pH 8-9 with sodium hydroxide (100g/L), add 10-17ml of sodium phosphate (152g/L or 15.2%) per 100ml solution (volume is 1/10～1/6 of the solution) under thorough stirring ), then add 5-8ml of calcium acetate (226g/L or 22.6%), centrifuge, collect the clear liquid, adjust the pH to 7.2 with hydrochloric acid (10%) to obtain the enzyme solution. Place the enzyme solution in a salt ice bath at 00C, slowly add 30ml of ice methanol to every 100ml enzyme solution, at this time the temperature should be lower than 80C, then adjust the pH to 5.2～5.4 with hydrochloric acid (10%), and let it stand for 15 ~30min. Centrifuge and collect the precipitate to obtain the refined product of the enzyme. [Ⅰ] Precipitation H2O; Na3PO4; Ca(Ac)2→calcium phosphate treatment enzyme solution methanol; pH5.3→precipitation crude product precipitation isoelectric point treatment, precipitation Add appropriate amount of distilled water to the crude product and adjust the pH to 7.2. , Make it completely dissolve. Adjust the pH to 5 with hydrochloric acid (1mol/L) in the ice bath, perform precipitation, collect the precipitate, then add the same volume of the original solution to distilled water with pH 7.2, and place in an ice bath to cool to 00C, under the condition of less than 50C In each 100ml ice solution, gradually add 30ml ice methanol, adjust the pH to 5.4～5.5 with hydrochloric acid (1mol/L), let it stand for 15～30min, and centrifuge to obtain the precipitate. Crude product precipitation distilled water; pH7.2→enzyme crude liquid methanol; pH5.4→precipitation sediment pre-adsorption, isoelectric point treatment, dissolve the precipitate in distilled water with pH 6.6 to make the concentration of the enzyme solution 500, 000 to 1 million units /ml, add the same amount of pH6.6 phosphate buffer solution (0.2mol/L), and then add 200g of DEAE-cellulose that has been balanced with pH6.6 phosphoric acid rinse for every 300 million units/ml enzyme solution concentration, Stir thoroughly for 15 minutes, filter, and wash DEAE-fiber with pH 6.6 phosphate buffer (0.1 mol/L) for 2 times, combine the filtrate and washing liquid, adjust pH 5 with hydrochloric acid (1 mol/L) to precipitate, and collect the streptokinase precipitate by centrifugation , Prepared into a distilled water solution with a concentration of 50-100 U/ml and a pH of 7.2 to obtain a refined enzyme solution. Precipitate DEAE-cellulose column; pH 6.6 → washing filtrate HCl; distilled water pH 5.5 → 7.2 → enzyme solution adsorption and elution: 800g DEAE-cellulose (for refining) is used per 100 million units to calculate the feed, add an appropriate amount PH6.6 phosphate buffer (0.01mol/L), stir evenly, pass the enzyme solution through the chromatographic column for adsorption, after completion, wash with a small amount of ph6.6 phosphate buffer (0.01mol/L), with pH6.6 phosphate Buffer (0.08mol/L for elution, collect streptokinase eluate above 20000U/ml.

Double-chain enzymes can be used as adjuvant drugs for the treatment of empyema, hemothorax, hematoma, chronic suppuration of drainage sinus, osteomyelitis, ulcers and infectious wounds. Also used for anterior chamber hemorrhage and vitreous hemorrhage. However, intracavity injections can cause fever and bleeding. Fever can be at least treated with glucocorticoid shock. 6-aminocaproic acid for bleeding is effective. This product office is an adjuvant therapy drug. It can only be applied on the basis of antibacterial treatment, surgical debridement and drainage. This product is banned in patients with acute suppurative peak fossa tissue inflammation, active pulmonary tuberculosis and bronchial pleural fistula to avoid the spread of the lesion. Low fibrinogen, low fibrinogen, liver function and abnormal blood are also prohibited. This product can be injected into the cavity of the affected part, or applied externally. Intraluminal injection, for hemothorax and empyema, 100,000 units of SK and 50,000 units of SD can be injected into 10ml of normal saline. For maxillary sinus empyema, 10,000 to 15,000 units of SK and 5000 to 7,500 units of SD (dissolved in 2 to 3 ml of saline) can be injected. Oral, 4 times a day, 1 tablet each time, continuous use for 4 to 6 days, drip, with 1000 units/ml solution for local instillation, `1 to 2h 1 time, local injection, intraocular ball or subconjunctival injection, Injection around the lesion, the concentration is 1000～2000 units/ml.