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A kind of preparation method of low molecular weight heparin

A low-molecular-weight heparin and heparin technology, applied in the direction of fermentation, can solve the problems of poor stability and high cost, and achieve the effects of high enzyme activity, improved efficiency, and fast reaction speed

Active Publication Date: 2016-04-06
CHANGSHAN BIOCHEM PHARM JIANGSU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these studies have improved the yield, purification method and preparation system of heparanase I, the heparanase I used in the enzymatic hydrolysis of heparin still has a certain problem of poor stability, resulting in the cost of the enzyme being still expensive, which limits the Development of Enzymatic Preparation of Low Molecular Weight Heparin

Method used

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  • A kind of preparation method of low molecular weight heparin
  • A kind of preparation method of low molecular weight heparin
  • A kind of preparation method of low molecular weight heparin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1）

[0022] (Example 1) Reaction solution ΔA in the process of histidine-tagged heparanase I degrading heparin to prepare low molecular weight heparin 232 The change of the number average molecular weight Mn and ΔA 232 The relationship between the value and the relationship between the liquid number average molecular weight Mn and the weight average molecular weight Mw

[0023] (1). Histidine-tagged heparanase I degrades heparin to prepare low molecular weight heparin, the reaction solution ΔA 232 The change

[0024] Using unfractionated heparin (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.) as raw material, add 2000 mg of unfractionated heparin to 100 mL containing 5 mM CaCl 2 and 100mM NaCl in water, and adjust the pH to 7.0 with HCl to obtain a heparin solution.

[0025] Put 100mL of heparin solution at a constant temperature of 30°C, add 4000mIU of histidine-labeled heparinase I for reaction, take out part of the reaction solution at regular intervals, measure i...

Embodiment 2）

[0035] (Example 2) Histidine-tagged heparanase I controlled degradation of heparin to prepare low molecular weight heparin 1

[0036] Prepare 20mg / mL heparin, 5mMCaCl 2 , 100mMNaCl, pH 7.0 reaction solution, put it in a constant temperature water bath at 15°C to preheat for 12min, add 2000mIU of histidine-labeled heparinase I for reaction, after a period of reaction, measure its ΔA 232 The value is 34.8, add hydrochloric acid to adjust the pH to 2.0 to terminate the reaction, filter the reaction solution with a 0.22 micron fiber membrane to remove protein, collect the initial filtrate and filter it with an ultrafiltration membrane with a molecular weight cut-off of 3000Da, add 3-4 times the volume of the filtrate to the filtrate The ethanol was mixed and centrifuged, the precipitate was collected and washed with acetone, and the number average molecular weight Mn and weight average molecular weight Mw of the precipitate were measured after being evaporated to dryness under red...

Embodiment 3）

[0037] (Example 3) Histidine-tagged heparanase I controlled degradation of heparin to prepare low molecular weight heparin 2

[0038] Prepare 20mg / mL heparin, 5mMCaCl 2 , 100mMNaCl, pH 7.0 reaction solution, put it in a constant temperature water bath at 20°C and preheat it for 10 minutes, then add 2000mIU of histidine-labeled heparinase I for reaction, after a period of reaction, measure its ΔA 232 The value is 34.8, add hydrochloric acid to adjust the pH to 2.0 to terminate the reaction, filter the reaction solution with a 0.22 micron fiber membrane to remove protein, collect the initial filtrate and filter it with an ultrafiltration membrane with a molecular weight cut-off of 3000Da, add 3-4 times the volume of the filtrate to the filtrate The ethanol was mixed and centrifuged, the precipitate was collected and washed with acetone, and the number average molecular weight Mn and weight average molecular weight Mw of the precipitate were measured after being evaporated to dry...

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Abstract

The invention relates to a preparation method of low molecular weight heparin. The preparation method is characterized in that histidine-tag heparin I is used for degrading heparin at a certain reaction temperature, a reaction liquid is purified so as to obtain the low molecular weight heparin, in the method, the certain reaction temperature is 15-40 DEG C, and the reaction process is ended when the deltaA 232 of the reaction liquid reaches 15-50. The preparation method has the advantages that the appropriate reaction temperature is adopted so as to ensure the relatively high enzymatic activity and the relatively fast reaction speed; meanwhile, the reaction endpoint can be accurately judged, and the control on the degree of the number-average molecular weight degradation reaction of the low molecular weight heparin is realized by detecting the absorbance at 232nm, so that the molecular weight of the low molecular weight heparin is in a target range, and the efficiency of degrading the heparin is improved. The histidine heparin I prepared by the method can be used for preparing the high quality low molecular weight heparin at relatively low cost and has enormous industrial application value.

Description

technical field [0001] The invention relates to a preparation method of low molecular weight heparin, which belongs to the field of biomedicine. Background technique [0002] Heparin is a mucopolysaccharide formed by hexuronic acid (L-iduronic acid, D-glucuronic acid) and D-glucosamine sulfate alternately with 1→4 glycosidic bonds. Its molecular weight is between 3000-40000Da, and the average molecular weight It is 15,000 Da. Heparin has been used as an anticoagulant and antithrombotic reagent for many years. In addition, heparin also has anti-inflammatory, anti-allergic, anti-viral, anti-cancer, and blood lipid-regulating functions. However, because heparin has anticoagulant activity, a large amount of use will cause side effects such as bleeding, induced thrombocytopenia, and osteoporosis, which greatly limits the clinical application of heparin. [0003] Low-molecular-weight heparin (LMWH) (. Linhardt, R.J. Gunay, N.S. Production and chemical processing of flow molecular...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/26C12P19/14
Inventor 丁建文陈敬华程咏梅邓超宋志新管惠娟张华
Owner CHANGSHAN BIOCHEM PHARM JIANGSU CO LTD