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A kind of preparation method of Flavobacterium heparinase heparanase I, ii, iii

A technology of Flavobacterium heparin and heparinase, which is applied in the preparation of heparinase I, III, and II, can solve the problems of easy inactivation of heparinase, large loss of activity, and low yield, and achieve simple process and good results easily repeatable effect

Active Publication Date: 2011-12-21
SHENZHEN HEPALINK PHARMA GRP CO LTD
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AI Technical Summary

Problems solved by technology

[0003] There are many research reports on the purification of heparanase, but because heparanase is an enzyme that is easily inactivated, most of the reported purification processes have the disadvantages of great loss of activity and low yield.

Method used

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Examples

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Embodiment 1

[0031] a. Inoculate Flavobacterium heparinus into 50ml seed medium, culture at 23°C, 150rpm for 1 day;

[0032] b. Insert the fermentation medium according to the inoculum size of 5%, culture at 23°C, 150rpm for 2-3 days, centrifuge at 4°C, 10000rpm 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 0.5g / ml protamine 8ml, 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 ac...

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PUM

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Abstract

The invention provides a preparation method of flavobacterium heparinum heparinases I, II and III, which comprises the following steps: inoculating flavobacterium heparinum used as the raw material into a seed culture medium for culture, then inoculating into a fermentation culture medium, centrifuging, collecting the precipitate, carrying out ultrasonication on the precipitate, and centrifuging to obtain a crude enzyme liquid of flavobacterium heparinum heparinases I, II and III; carrying out SP-Sepharose FF on the crude enzyme liquid to obtain an enzyme II and enzymes I and III through chromatographic separation; carrying out SP-Sepharose FF on the enzymes I and III to obtain a heparinase I and a heparinase III through separation; respectively carrying out SP-Sepharose FF on the obtained enzyme I and enzyme III twice to obtain high-purity heparinases I and III through purification; and respectively carrying out HEP-Sepharose 4B, HA-Ultrogel and SP-Sepharose FF on the obtained enzymeII to obtain a high-purity heparinase II.

Description

technical field [0001] The present invention relates to a preparation method of heparinase I, II and III, in particular to a special chromatographic separation technique for preparing heparinase I and II under the protection of calcium chloride throughout the process and eluted with heparin buffer , the method of III. Background technique [0002] Heparanase refers to a class of enzymes that can specifically cleave the glycosidic bonds of heparin and heparin-like main chains. It was first discovered and isolated from Flavobacterium heparinus, and later, hepatic enzymes were found in some microorganisms and animal tissues. prime enzyme. The application of heparinase is very extensive, such as: removing residual heparin in blood, preventing coagulation, producing low molecular weight heparin, and studying the structure of heparin. At present, there are about 10 kinds of heparinases reported in academic papers, and only three enzymes from Flavobacterium heparinus have been st...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12R1/20
Inventor 马小来史绍鹏李锂
Owner SHENZHEN HEPALINK PHARMA GRP CO LTD
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