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Application of heparinase II to preparation of LMWH (low-molecular-weight heparin) by depolymerization of heparosan

A technology for low molecular weight heparin and heparin precursors, which is applied in the field of preparing low molecular weight heparin by depolymerizing heparin precursors with recombinant heparinase II, and achieves the effects of cost saving, mild reaction conditions and easy separation

Active Publication Date: 2013-03-27
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Due to the specificity of the enzyme, heparinase is only used to depolymerize heparin at present, and there is no report on the depolymerization of heparin precursor by heparinase

Method used

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  • Application of heparinase II to preparation of LMWH (low-molecular-weight heparin) by depolymerization of heparosan
  • Application of heparinase II to preparation of LMWH (low-molecular-weight heparin) by depolymerization of heparosan
  • Application of heparinase II to preparation of LMWH (low-molecular-weight heparin) by depolymerization of heparosan

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: the acquisition of recombinant heparanase II

[0038] 1. E. coli BL21(DE3) / pET15b-hepB expresses heparanase II

[0039] According to the reported Flavobacterium heparinum heparanase II gene sequence (Genbank access number U27585.1) and amino acid sequence (Genbank access number AAB18277.1)

[0040] Design primers (HepII-F: 5'-CATGCCATGGATGAAAAGACAATTAT-3'

[0041] HepII-R: 5'-GGAATTCCATATGTTATCTCAAAAAACGGT-3' is a restriction site, the upstream primer is added with NcoI restriction site, and the downstream primer is with NdeI restriction site), amplified from Flavobacterium heparinum genomic DNA to heparin Enzyme II (Heparinase II) gene fragment (SEQ ID No.1), cloned into pET-15b expression vector (Novagen, WI) to obtain recombinant vector pET15b-hepB, which contains an N-terminal His6-tag tag for easy recombination protein follow-up Ni 2+ Column affinity chromatography. Transform E. coli BL21(DE3) with pET15b-hepB to obtain recombinant strain E. coli ...

Embodiment 2

[0050] Example 2: Effect of reaction conditions on the depolymerization effect of heparanase II on heparosan:

[0051] (1) Heparanase Ⅱ depolymerization method of heparosan

[0052] A certain amount of heparosan was weighed and dissolved in different enzymatic hydrolysis buffers to dissolve completely, preheated in a water bath at 25°C for 10 minutes, added different amounts of heparanase II, and carried out the degradation reaction at 25°C, at different reaction times sampling. Boiling water bath for 5 min to inactivate the enzyme and cool to room temperature.

[0053] After being treated with Heparinase II, it was found that the crude product of heparosan depolymerized, from Figure 5 It can be seen from the figure that as the action time of Heparinase II increases, the large molecular weight bands of heparosan become less and less, and the small molecular weight bands gradually increase. By 4 h, almost all of them are small molecules. It shows that heparinase II has a go...

Embodiment 3

[0067] Embodiment 3: Preparation of low molecular weight heparin

[0068] (1) Depolymerization of heparosan:

[0069] Dissolve 0.05 g heparosan in 1 mL of enzymatic digestion buffer B (100 mM sodium acetate, 0.01% BSA and 10 mM calcium acetate, the solvent is water, pH7.0) to make it completely dissolved, and preheat it in a water bath at 25 °C 10 min, add 100 μL of heparinase II with a total activity of 199 U / mL (Hep II, obtained according to the method in Example 1), carry out the degradation reaction at 25°C for 1.5 h, and place in a boiling water bath for 5 min after the degradation reaction to deactivate the enzyme live, cooled to room temperature. See embodiment 2 for specific results.

[0070] (2) Deacetylation and sulfation

[0071] Dissolve 5 mg of depolymerized Heparosan in 1 mL of 2M sodium hydroxide solution, react at 60°C for 3 h, dilute the reaction solution to 5 mL after the reaction, cool and adjust the pH to 7 with 1M hydrochloric acid, and then add carboxy...

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Abstract

The invention provides an application of heparinase II to the preparation of LMWH (low-molecular-weight heparin) by the depolymerization of a heparosan and a method for preparing the LMWH by depolymerizing the heparosan by the recombinant heparinase II. The invention provides a new application of the heparinase II to the depolymerization of the heparosan and a new method for preparing the LMWH by the heparinase. The method has the advantages of mild reaction condition, easy separation of the product and the enzyme and convenience of production, thereby having a significant application prospect.

Description

(1) Technical field [0001] The invention relates to the application of heparanase II in depolymerizing heparin precursor to prepare low molecular weight heparin, and a method for preparing low molecular weight heparin by depolymerizing heparin precursor with recombinant heparanase II. (2) Background technology [0002] Heparin is a member of the glycosaminoglycan family consisting of repeating disaccharide units linked by uronic acid and D-glucosamine through α (1→4) glycosidic bonds. It is a mixture of various large and small molecules, and its molecular weight is polydisperse, generally between 3000 and 30000D, with an average molecular weight of 15000D (about 45 monosaccharides). As one of the most widely used and most effective anticoagulant and antithrombotic reagents in the world, it has been used in clinical diagnosis for more than 60 years. But its side effects are also becoming more and more obvious, such as a large amount of use of heparin can cause bleeding, oste...

Claims

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Application Information

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IPC IPC(8): C12P19/26C12N15/56C12N15/70C12N9/26
Inventor 钟卫鸿吴石金黄海婵邱乐泉钟莉吕沈聪赵雷严子琴王畅
Owner ZHEJIANG UNIV OF TECH
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