| 摘要 |
FIELD: biotechnology, medicine, biochemistry, food industry, scientific aims. SUBSTANCE: method for preparing the collagenase preparation involves homogenization of crab hepatopancreas, separation of extract by centrifugation, precipitation of collagenase with ammonium sulfate, repeated centrifugation to separate the collagenase deposit, dissolving the latter in acetate-ammonium buffer and purification of the preparation solution by chromatography method followed by elution of sorbed enzymes, concentrating, desalting eluate and lyophilic drying the preparation. The preparation is purified by method of affinity chromatography and sorption of collagenase is carried on macroporous silica as an affinity sorbent containing polymixin M as a covalently bound antibiotic. Elution is carried out with tris-buffer with calcium acetate and concentrating and desalting are carried out by ultrafiltration method. Homogenization of tissue is carried out in acetate- ammonium buffer containing calcium acetate at pH 6.2-6.4. Precipitation is carried out by addition of ammonium sulfate to 60-70% of saturation and sorption in the presence of acetate-ammonium buffer at pH 8.0-8.5 and ultrafiltration on device with membrane retaining proteins with molecular mass above 10 kDa. Invention provides the yield of the end product up to 75-100%, increase of specific activity of collagenase by 2-3 times and reduced time process. EFFECT: improved preparing method of collagenase. 2 ex |
