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Freeze-drying preservation method for heparin flavobacterium heparinum heparinase I, II and III

A preservation method and technology of heparinase, applied in the direction of enzyme stabilization, etc., can solve the problems of incomplete information and different results, etc.

Inactive Publication Date: 2012-07-18
SHENZHEN HEPALINK PHARMA GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There are very few research reports on lyoprotection of heparanase that can be retrieved, mainly in the research of Daniel et al. The information is not comprehensive and some results differ from ours

Method used

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Experimental program
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Embodiment

[0061] The heparanase used is isolated and purified from Flavobacterium heparinus after fermentation and cultivation. For the enzyme preparation process, see the invention patent "A method for preparing heparinase I, II, and III" submitted by the inventor earlier, application number 201110241260.4.

[0062] a. Take the prepared heparinase I, II and III and dialyze overnight in 100 times the volume of 10mM Tris-HCl buffer solution (pH7.0). ml EP tube, 50ul per tube;

[0063] b. Add an appropriate amount of pre-prepared 10mM Tris-HCl buffer solution (pH7.0) containing 100mM calcium chloride and 10mM Tris-HCl buffer solution (pH7.0) containing 50% (w / v) trehalose, respectively Add it into the heparinase solution, dilute to 100ul, shake gently, so that the heparanase I solution contains 20mM calcium chloride and 5% trehalose in addition to the buffer, and make the heparanase II solution The solution contains calcium chloride 10mM and trehalose 10%, so that the heparanase III solu...

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Abstract

The invention relates to a freeze-drying preservation method for heparin flavobacterium heparinum heparinase I, II and III, in particular to a protective agent optionally comprising calcium chloride and trehalose, and the protective agent is sued for protecting activity of the heparinase I, II and III during freeze-drying. The enzyme freeze-drying powder prepared according to the method provided by the invention is convenient to store, transport and use, and the application value is improved.

Description

technical field [0001] The present invention relates to a method for freeze-drying preservation of heparinase I, II, III, in particular to a protective agent composed of calcium chloride and trehalose, which is used for the preservation of heparinase I, II, III in the process of freeze-drying active protection. 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 studied i...

Claims

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Application Information

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IPC IPC(8): C12N9/96
Inventor 马小来史绍鹏李锂
Owner SHENZHEN HEPALINK PHARMA GRP CO LTD
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