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Method for preparing high-activity and low-molecular-weight heparin by enzymic method

A low-molecular-weight heparin and heparin technology are applied in the field of enzymatic preparation of high-activity low-molecular-weight heparin, which can solve the problems of severe reaction of heparin and destruction of biological activity function, and achieve the effect of reducing cost and production cost.

Inactive Publication Date: 2012-09-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the chemical degradation method of heparin reacts violently, so that some functional groups in the heparin molecule are more or less destroyed during the reaction process, so some biologically active functions are destroyed to varying degrees.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, PAPS regeneration system

[0023] The concentration of components in the PAPS regeneration system is: the concentration of PNPS is 5mM, the concentration of PAP is 20uM; the dosage of AST-IV in the PAPS regeneration system is 10mg / mM PAPS. The solution prepared according to the above composition is added to the solution for preparing high-activity low-molecular-weight heparin, as a sulfate group donor for the modification reaction of heparin biosynthetic enzyme 3-O-sulfatyltransferase.

Embodiment 2

[0024] Embodiment 2, the preparation of tool enzyme HepI, AST-IV and 3-OST-1

[0025] 1. Shake flask expression of recombinant E. coli

[0026] Pick a single colony of HepI, AST-IV and 3-OST-1 recombinant strains in 10 mL LB medium, add 10 μL ampicillin (50 mg / mL, final concentration 50 μg / mL) to each 10 mL medium, 37 ° C, Incubate overnight at 200 rpm. Then, according to the inoculum size of 1:100, 4 mL of the bacterial solution was inoculated into 400 mL of LB, and 400 μL of kanamycin was added. 37°C, 200rpm. After about 4 hours, its OD 600 reached between 0.6 and 0.8, adding 2 mL of inducer IPTG (0.2 M, so that the final concentration was 1 mM), at 22°C, 200 rpm, and induced for 12 hours. Centrifuge the above culture at 8000rpm, 4°C for 10min, collect the bacteria, resuspend to 20mL with BufferA (25mM Tris, 0.5M NaCl, 10mM imidazole, pH 7.4) pre-cooled at 4°C, and sonicate in an ice bath, the conditions are: 400W, working for 5s, intermittent for 10s, 200 cycles. After...

Embodiment 3

[0029] Embodiment 3, the preparation of low molecular weight heparin oligosaccharide

[0030] Using unfractionated heparin from Shanghai Sangong Technology Service Co., Ltd. as the raw material, the reaction conditions were as follows: 10 mg of unfractionated heparin was dissolved in 1 mL of the reaction solution, and the reaction was shaken (300) at 30°C. The reaction solution contains 50 mg / mL heparin, 25 mM ammonium acetate, 25 μM CaCl 2 , 0.25 μg / mL BSA, 3 IU / g heparin, pH 7.4. A232 to about 80, heat the reaction solution at 100°C for 5 minutes to terminate the reaction, separate and purify on a P10 column to obtain a heparin oligosaccharide solution with a molecular weight of 3000-8000, dialyze and desalt, and freeze-dry.

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Abstract

The invention discloses a method for preparing high-activity and low-molecular-weight heparin by an enzymic method. The method includes using heparin as substrate; degrading the heparin by HepI (histidine-tagged heparinase I) in a controlled manner; then utilizing a PAPS (3'-phosphoadenosine-5'-phosphosulfate) regeneration system; selectively decorating the heparin by heparin biological synthetase 3-O-sulfotransferase; and increasing the quantity of anticoagulation activity centers to obtain the low-molecular-weight heparin with high anticoagulation activity. The HepI, the 3-O-sulfotransferase and AST-IV which are adopted in the method can be prepared by means of high-density fermentation in high yield; the PAPS regeneration system uses PNPS (P-nitrophenol sulfonic acid potassium salt) as sulfate radical for enzyme modification reaction, and production cost is greatly reduced. The method provides a new way for enzymic method industrialization of the high-activity and low-molecular-weight heparin.

Description

technical field [0001] The invention relates to a method for preparing high activity low molecular weight heparin by enzymatic method. It 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 15000Da; heparin has been used as an anticoagulant and antithrombotic reagent for more than 60 years. The anticoagulant activity of heparin mainly binds antithrombin (AT-Ⅲ) through the pentose sugar active center containing 3-glucosamine sulfate, and induces a conformational change in it, so that the anticoagulant activity is first. The anticoagulant activity of heparin includes antithrombotic activity (anti-factor Xa) and anticoagulant activity (anti-factor IIa). Anticoagulant activity is also related to the molecular weight of Ga...

Claims

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Application Information

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IPC IPC(8): C12P19/18C12P19/04
Inventor 陈敬华刘卫超王敏王蕴聪张頔
Owner JIANGNAN UNIV
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