Hyaluronidase

Method 1. Chopping and extracting the testicles of cattle and sheep. Wash the frozen or fresh sheep testicles, peel off the inner and outer layers and accessory testicles, mince them, mince them with a meat grinder, weigh 100kg, and pour into In a good acetic acid solution (mixed with 90L of water, 0.6L of glacial acetic acid, and 10L of 1mol/L hydrochloric acid), in a freezer below -50, vigorously stir for 3 to 5 minutes every 15 minutes to fully extract it for 4 hours, and then proceed Pulling the slurry, the slurry is about 125～140L. The pulp residue is used for the second extraction of hyaluronidase. Sheep testis Hac;HCl;-50→Extract the extraction solution for salting out, suspend filtration to take the first slurry, and add 210g of ammonium sulfate (industrial use) to each liter of slurry under constant stirring. Let it dissolve safely. After resting for 1h, transfer to double-layer polyester bag to suspend the filter and repeat twice. The next day the clear filtrate was obtained, and 290 g of ammonium sulfate was added per liter with constant stirring. Allow it to dissolve completely. After standing for 1h, suspend the filter with a polyester bag, overnight, and remove the bag the next day to obtain a crude product. Extraction solution (NH42SO4→salting out; salting out the crude product by hanging filtration, dialysis, dissolve the fine product in 5L of ice-cold distilled water, slowly add 125g of ammonium sulfate solid, and keep stirring to dissolve it completely, let stand overnight in the cold storage, times Remove the oil and fat on the surface of the liquid, siphon the middle supernatant with a rubber tube, filter with a Buchner funnel, and finally filter the upper layer and bottom layer with a Buchner funnel, and combine the filtrates. Slowly add 750g of ammonium sulfate to the filtrate and stir thoroughly until it is completely dissolved , Place in the cold storage overnight. The next day, discard the supernatant (if the upper solution is turbid, you can place it in the cold storage for 3 to 5 days until the upper solution becomes clear) to obtain a salted out precipitate, which is then dissolved in 0.5L of ice Distilled water, dialyzed, about 100ml per bag, placed in a phosphate-citrate buffer solution of pH 6.5 [citrate 7.5g, disodium hydrogen phosphate (12 crystal water) 62g, sodium chloride 70g prepared by adding water Into 2500ml, adjusted to pH6.5 with citrus] 2500ml, dialyzed in cold storage for 24h to obtain dialysate. Enzyme activity is about 250, 000U/ml. Crude product (NH4) 2so4; phosphate-citric acid → pH6.5; salting out; The dialysis dialysate is depyrogenated and dried. The dialysate is centrifuged. The centrifuge is placed in ice-cold water. Add 50ml of Na3PO4·12H2O (150g/L or 15%) solution and slowly add calcium acetate (200g/L or 20%). ) 30ml, and keep stirring. Then adjust the pH to 8.5 with sodium hydroxide (0.5mol/L), continue stirring for 10min, filter with No. 4 vertical molten glass funnel (filter and put the bottle in an ice water bath), and the filtrate with hydrochloric acid (0.5 mol/L) Neutralized pH is 7, freeze-dried to obtain enzyme refined products. Dialysis solution Na3PO4; Ca (Ac) 2; pH8.5 → depyrogenated refined product HCl; pH7 → dried dry refined product The amount of refined products is dissolved in 5% aqueous solution of gelatin for injection, adjusted to pH 5-7 with sodium hydroxide, sterilized, filtered, and divided into 0.5ml bottles, freeze-dried to obtain hyaluronidase preparation for injection, each bottle Containing 100% or 150U of hyaluronidase, the yield is about 20, 000U/kg based on the weight of testis. 5% hydrolyzed gelatin of dried refined products; pH 6→ preparation of the freeze-dried preparation product method two microbial fermentation culture, fermentation medium; starch 1 part; 1 part cotton cake; 1 part gluten powder; 1 part dry yeast; 0.1 part dipotassium phosphate; Streptomyces; 300C; 3d → fermentation broth defoamer 0.4% precipitation, separation fermentation broth → concentrated acetone → crude crude product Chromatography column → Separation of refined products, frozen and dried refined products 5% hydrolyzed gelatin → Freeze-dried freeze-dried preparations

Pharmaceutic aid (diffusing agent in s.c. injections).