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Process of producing heparan sulfate by using chondrosulphatase

A technology of heparan sulfate and chondroitin sulfate enzyme, which is applied in the direction of fermentation, can solve the problems of difficult removal, high cost and low yield, and achieve the effects of short production cycle, strong catalytic ability and high yield

Inactive Publication Date: 2015-01-21
DONGYING TIANDONG PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has low yield, long period, high cost and introduces impurity copper ions that are difficult to remove and is not selected.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Under the condition of stirring, 100kg of crude heparin sodium was dissolved in 1000L of 2% sodium chloride solution by mass percentage, and the concentration below is all by mass percentage unless otherwise specified. Adjust the temperature of the feed liquid to 37°C, then adjust the pH value of the feed liquid to 8.5 with sodium hydroxide with a concentration of 6 mol / L, and add 1.5Kg of trypsin to enzymatically digest it for 3 hours. After the enzymolysis is completed, the feed liquid is adjusted to a pH value of 7.0, and the feed liquid is completely absorbed by a strong base anion exchange resin until there is no feed liquid. Prepare a sodium chloride solution with twice the resin volume and a concentration of 5.3% to elute the resin column. The obtained eluate was precipitated with 1.6 times the volume of medicinal ethanol for 4 hours, the supernatant was discarded and dried at 60° C. for 5 hours to obtain 17.7 Kg of the eluate. Dissolve this precipitate in 177L ...

Embodiment 2

[0021] Under stirring conditions, dissolve 100kg of crude heparin sodium in 1000L of 2% sodium chloride solution. Adjust the temperature of the feed liquid to 35° C., then adjust the pH value of the feed liquid to 8.3 with sodium hydroxide with a concentration of 6 mol / L, and add 1.3 Kg of crude heparin sodium to enzymatically digest with trypsin for 4 h. After the enzymolysis is completed, the feed liquid is adjusted to a pH value of 7.2, and the feed liquid is completely absorbed by a strong base anion exchange resin until there is no feed liquid. Prepare a sodium chloride solution with twice the resin volume and a concentration of 4.6% to elute the resin column. The obtained eluate was precipitated with 2.0 times the volume of medicinal ethanol for 4 hours, the supernatant was discarded and dried at 60° C. for 5 hours to obtain 17.6 Kg of the eluate. Dissolve the precipitate in 176L of purified water, adjust the pH value of the feed solution to 8.3, and add 0.52Kg of CaCl ...

Embodiment 3

[0023] Under stirring conditions, dissolve 100kg of crude heparin sodium in 1000L of 2% sodium chloride solution. Adjust the temperature of the feed liquid to 38°C, then adjust the pH value of the feed liquid to 8.2 with sodium hydroxide with a concentration of 6mol / L, and add 1Kg of crude heparin sodium to enzymatically digest with trypsin for 4h. After the enzymolysis is completed, the feed liquid is adjusted to a pH value of 6.9, and the feed liquid is completely absorbed by a strong base anion exchange resin until there is no feed liquid. Prepare a sodium chloride solution with twice the resin volume and a concentration of 3.7% to elute the resin column. The obtained eluate was precipitated with 2.0 times the volume of medicinal ethanol for 4 hours, the supernatant was discarded and dried at 60° C. for 5 hours to obtain 16.2 Kg of the eluate. Dissolve the precipitate in 176L purified water, adjust the pH value of the feed solution to 8.0, add 0.6Kg of CaCl 2 , after addi...

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Abstract

The invention provides a process of producing heparan sulfate by using chondrosulphatase. The process comprises the following steps: firstly, performing enzymolysis, resin adsorption and sodium chloride solution elution on a heparin sodium crude product to obtain eluate; performing enzymolysis on the eluate by using chondrosulphatase ABC or chondrosulphatase B; exclusively cutting off specificity of 1-4 glucosidic bonds between disaccharide units in a dermatan sulfate structure by virtue of the chondrosulphatase ABC and the chondrosulphatase B to obtain a degradation reaction product unsaturated disaccharide; and then, performing refining steps such as ultrafiltration, heavy metal removal and oxidization to obtain a heparan sulfate fine product. The process not only can be used for overcoming the characteristic that negative charges on the surfaces of the heparan sulfate and the dermatan sulfate are similar in density and difficult to separate, but also can be used for avoiding heavy metal ion impurities brought in heparan sulfate produced according to the conventional route, and thus, the product yield is increased, and the economic benefits are remarkable.

Description

technical field [0001] The invention relates to a process for producing heparan sulfate, in particular to a process for producing heparan sulfate by using chondroitin sulfate enzyme. Background technique [0002] Heparan sulfate is a glycosaminoglycan widely present on the surface of animal cells and is the main component of the drug sulodexide. It has important biological functions in cell signal transduction, proliferation and differentiation, immune regulation, cell adhesion, and information transmission of nerve cells. Heparan sulfate is similar to heparin in the composition of glycosyl groups and glycosidic linkages, but it has the advantages of stronger antithrombotic activity and weaker anticoagulant activity than heparin. Because its negative charge density and molecular weight are similar to those of dermatan sulfate, the separation and purification of heparan sulfate has become a difficult point restricting drug development. It has been shown in the literatur...

Claims

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Application Information

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IPC IPC(8): C12P19/26
Inventor 杨晓宏郭林李荣崔慧斐李福川陈少鹏张中志
Owner DONGYING TIANDONG PHARM CO LTD
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