Preparation method of flavobacterium heparinum heparinases I, II and III
A technology of Flavobacterium heparin and heparinase, which is applied in the field of preparation of heparinase I, II, and III, can solve the problems of large activity loss, easy inactivation of heparinase, and low yield, and achieve easy repeatable results, The effect of simple process
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[0031] a. Inoculate Flavobacterium heparinus into 50ml seed medium, culture at 23°C, 150rpm for 1 day;
[0032] b. Insert the fermentation medium at 5% inoculum size, culture at 23°C, 150rpm for 2-3 days, centrifuge at 4°C, 10,000rpm for 15-30 minutes, and collect the precipitate;
[0033] c. Suspend the precipitate in 100ml, 25mM Tris-HCl ① In the buffer solution, sonicate in an ice bath for 1 hour, 4°C, 10000rpm, centrifuge for 30min, add dropwise 8ml of 0.5g / ml protamine, stir, 4°C, 10000rpm, centrifuge for 30min, take the supernatant;
[0034] d. Use Tris-HCl on the last one ① Buffer-equilibrated SP column, equilibrate 3 column volumes with the same buffer, and use Tris-HCl ① 0-0.5M linear gradient elution containing sodium chloride in the buffer, collecting several tubes with heparanase activity and heparanase activity in the eluate, which are distributed in two activity peaks I and II;
[0035] e. Use 50mM Tris-HCl on the last one after dialysis of the active peak II ...
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