A kind of enzymolysis preparation method of anticoagulant leech peptide
The technology of hirudin and anticoagulation is applied in the field of enzymatic hydrolysis and preparation of biologically active peptides, which can solve the problems of purity of natural hirudin, uneven activity level, long cycle, waste of raw materials, etc., and achieves simple and controllable reaction process, Mild reaction conditions and environmentally friendly effects
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Embodiment 1
[0022] Take fresh Hirudo phenanthae that has been refrigerated for no more than 24 hours, add 4 times of water and mix it in a beater for homogenization, centrifuge the homogenate at 10000r / min for 10min, and refrigerate the supernatant for later use, weigh 9g of the precipitate and 100ml of water and mix it in a triangle In the bottle, adjust the pH to 8.0, add Alcalase 2.4L enzyme (alkaline protease, produced by Novozymes), shake in a water bath constant temperature oscillator at 55°C and 150 rpm, and react for 1.5h. Inactivate the enzyme for 10 minutes, adjust the pH to 7.0 after cooling, and freeze-dry to obtain anticoagulant leech peptide powder with anticoagulant activity greater than 500AT-U / g.
Embodiment 2
[0024] Take fresh Hirudo phenanthae that has been refrigerated for no more than 24 hours, add 3 times of water and mix it in a beater for homogenization, centrifuge the homogenate at 10,000r / min for 10min, and refrigerate the supernatant for later use, weigh 12g of the precipitate and mix it with 100ml of water in a triangular In the bottle, adjust the pH to 8.5, add Alcalase 2.4L enzyme, place in a water bath constant temperature oscillator at 55°C and 150 rpm, shake, react for 2 hours, inactivate the enzyme in a 90°C water bath for 10 minutes, and adjust the pH to 7.0 after cooling. After freeze-drying, the anticoagulant leech peptide powder with anticoagulant activity greater than 600AT-U / g can be obtained.
Embodiment 3
[0026] Take fresh Hirudo phenanthae that has been refrigerated for no more than 24 hours, add 2 times the water and mix it in a beater for homogenization, centrifuge the homogenate at 10,000r / min for 10min, and refrigerate the supernatant for later use, weigh 15g of the precipitate and 100ml of water and mix it in a triangle In the bottle, adjust the pH to 9.0, add Alcalase 2.4L enzyme, place in a water bath constant temperature oscillator at 55°C and 150 rpm, shake, react for 2.5 hours, inactivate the enzyme in a 90°C water bath for 10 minutes, and adjust the pH to 7.0 after cooling After freeze-drying, the anticoagulant hirudopeptide powder with anticoagulant activity greater than 600AT-U / g can be obtained.
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