Method for preparing superoxide dismutase from cattle cardiac muscle tissue, prepared superoxide dismutase and application thereof
A technology of superoxide and dismutase, applied in the field of bioengineering technology and protein biochemistry, to achieve the effect of simple process operation, simple operation and increased flux
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Embodiment 1
[0129]Take 100 g of frozen beef heart, slice it, mince it with a meat grinder, add 500 ml of 10% acetic acid solution, soak it for 3 days, fully expand the myocardial tissue, grind it with a homogenizer, and centrifuge at 5000 rpm for 20 minutes (Hitachi high-speed refrigerated centrifuge) machine CR21GIII), about 500 milliliters of clear liquid was obtained, 88 grams of solid ammonium sulfate (about 30% saturation) was added, stirred at room temperature for 6 hours, filtered, and 198 grams of ammonium sulfate (about 60% saturation) was added to the supernatant, stirred at room temperature After 6 hours, the precipitate was filtered. Add 100 ml of 20 millimolar Tris-Cl (pH 8.0) to the precipitate to dissolve the precipitate, filter to obtain a transparent liquid, and then use a centrifugal ultrafiltration tube with a molecular weight cut-off of 30,000 (Millipore, USA) to remove ammonium sulfate. Method: Use a centrifugal ultrafiltration tube to concentrate to 1 ml volume, then...
Embodiment 2
[0130] Embodiment 2 (scale-up test)
[0131] Get 1000 g of frozen beef heart, chop it up, add 5000 ml of 10% acetic acid solution, soak for 3 days, grind with a homogenizer, and centrifuge at 5000 rpm for 20 minutes (Hitachi high-speed refrigerated centrifuge CR21GIII), to obtain about 5000 ml of clear liquid, add 880 grams of solid ammonium sulfate (about 30% saturation), stirred at room temperature for 6 hours, filtered, and 990 grams of ammonium sulfate (about 60% saturation) was added to the supernatant, stirred at room temperature for 6 hours, filtered to obtain a precipitate. Add 1000 ml of 20 millimolar Tris-Cl (pH 8.0) to dissolve the precipitate, filter to obtain a transparent liquid, and then use a hollow fiber ultrafiltration machine (TM-130, Beijing Xubang Membrane Equipment Co., Ltd.) with a molecular weight cut-off of 30,000 for ultrafiltration To remove ammonium sulfate, method: Use an ultrafiltration column to concentrate to 100 ml, then add deionized water to ...
Embodiment 3
[0132] Embodiment 3 (pilot test)
[0133] Take 10 kg of frozen beef heart, chop it up, add 50 liters of 10% acetic acid solution, soak for 3 days, grind with a homogenizer, and centrifuge at 5000 rpm for 20 minutes (super-capacity refrigerated centrifuge CL8R, Changsha Xiangyi Centrifuge Instrument Co., Ltd.) , the clarified liquid was filtered to obtain about 50 liters, added 8800 grams of solid ammonium sulfate (about 30% saturation), stirred at room temperature for 6 hours, filtered, added 9900 grams of ammonium sulfate (about 60% saturation) to the supernatant, and filtered to obtain a precipitate . Add 10 liters of 20 millimolar Tris-Cl (pH 8.0) to dissolve the precipitate, filter to obtain a transparent liquid, and then use a hollow fiber ultrafiltration machine (PM-300, Beijing Xubang Membrane Equipment Co., Ltd.) with a molecular weight cut-off of 30,000 for ultrafiltration Remove ammonium sulfate, method: use an ultrafiltration column to concentrate to 1 liter volume...
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