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Method for immobilizing heparanase I

A technology of heparinase and chitosan microspheres, which is applied in the direction of immobilization on/in the organic carrier, can solve the problems of low immobilization efficiency and high cost, and achieve high stability, high immobilization efficiency, high The effect of reusability

Inactive Publication Date: 2012-11-28
SHENZHEN HEPALINK PHARMA GRP CO LTD
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  • Abstract
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Problems solved by technology

At present, there are few studies on the immobilization of heparanase I, Howard Bernstein et al [Bernstein, H., (1987), Appl. Biochem. Biotech. 16, 129-143] Using CNBr-activated Sepharose 4B as the immobilization material, heparanase I was immobilized by the covalent cross-linking method, which realized the immobilization of heparanase, but the immobilization efficiency of this method was low. The results obtained by using the same materials and methods can only achieve the immobilization of 1IU heparanase I per ml of material, and the cost of immobilizing CNBr-activated Sepharose 4B is relatively high

Method used

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Embodiment

[0038] The heparinase used is isolated and purified from the fermented and cultured cells of Flavobacterium heparinus. For the enzyme preparation process, refer to the example in "A Preparation Method for Flavobacterium Heparinus Heparinase I" of Invention Patent Application No. 200910039360.1.

[0039] a, getting 1g of commercially available chitosan powder with a purity of 99% is dissolved in 50ml of 2% acetic acid to obtain an acidic chitosan sol, and 0.6g of commercially available chemically pure Span 80 (Sinopharm Chemical Reagent Co., Ltd. , chemically pure) was added into 100ml of liquid paraffin, stirred and mixed at a speed of 800rpm, the acidic chitosan sol was added dropwise into the liquid paraffin, after emulsification for 1h, 5ml of glutaraldehyde with a mass fraction of 25% was added as a crosslinking agent, The crosslinking temperature is 40°C, add 100ml equal volume of 2.5M NaOH and absolute ethanol mixture after 1h, let it stand for 10min after stirring, disca...

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Abstract

The invention relates to a method for immobilizing heparanase I, and in particular relates to the method for immobilizing heparanase I, which takes chitosan microspheres as immobilized materials. The immobilized heparanase I can be recovered and reutilized, is easily separated from a substrate and products, and has strong stability and other advantages.

Description

technical field [0001] The invention relates to a method for immobilizing heparinase I, in particular to a method for immobilizing heparinase I with chitosan as an immobilization material, so as to realize high immobilization efficiency and high stability of heparanase I and reusability. Background technique [0002] Heparinase 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. Later, heparin was also found in some microorganisms and animal tissues. Enzymes are present. Heparinase has the functions of removing residual heparin in the blood and preventing blood coagulation. It also plays an important role in the production of low-molecular-weight heparin and the study of the structure of heparin. However, free heparanase is easily inactivated, especially the activity of heparanase I solution is only 23% after storage for 96 hours. In addit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/10C12N11/04
Inventor 白佳珂史绍鹏马小来李锂
Owner SHENZHEN HEPALINK PHARMA GRP CO LTD
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