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Preparation and purification method of different domain oligosaccharides of heparan sulfate/heparin

A technology of heparan sulfate and heparin oligosaccharides, which is applied in the direction of fermentation and can solve problems such as unseen

Pending Publication Date: 2019-06-21
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the preparation of oligosaccharides in different regions of HS / Hp

Method used

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  • Preparation and purification method of different domain oligosaccharides of heparan sulfate/heparin
  • Preparation and purification method of different domain oligosaccharides of heparan sulfate/heparin
  • Preparation and purification method of different domain oligosaccharides of heparan sulfate/heparin

Examples

Experimental program
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Embodiment 1

[0036] Take the preparation of five crude samples of HS / Hp oligosaccharides containing different domain structures (disaccharide, tetrasaccharide, hexasaccharide, octasaccharide and decasaccharide) as an example:

[0037] 1. Preparation of Heparan Sulfate / Heparin (HS / Hp) Oligosaccharides

[0038]Weigh 200 mg commercially available heparin and dissolve it in 10 mL Tris-HCl buffer (containing 20 mmol / L Tris and 5 mmol / L calcium chloride, adjust the pH to 7.40 with HCl), add 20mIU heparinase I, and heat the enzyme in an oven at 40°C After 24 hours of solution, the reaction was terminated by inactivation at 100°C for 5 minutes, centrifuged at 12,000 r / min for 10 minutes, and the supernatant was collected and freeze-dried at -80°C for 5 hours.

[0039] Separation and collection of disaccharides, tetrasaccharides, hexasaccharides, octasaccharides and decasaccharides

[0040] Take 140mg of the above freeze-dried sample, and use 3mL 0.2M NH 4 HCO 3 Dissolved and separated by polyac...

Embodiment 2

[0049] Take the preparation of fifteen low-sulfated HS / Hp oligosaccharide pure products as an example:

[0050] 1. Enzymatic hydrolysis of heparan sulfate / heparin (HS / Hp)

[0051] Weigh 100 mg of commercially available heparin and dissolve it in 5 mL of Tris-HCl buffer (containing 20 mmol / L Tris and 5 mmol / L calcium chloride, adjust the pH to 7.40 with HCl), add 10 mIU of heparinase I and perform enzymatic hydrolysis in an oven at 40 °C After 24 h, the reaction was terminated by inactivation at 100 °C for 5 min, centrifuged at 12000 r / min for 10 min, and the supernatant was collected and freeze-dried.

[0052] Preparation of oligosaccharides

[0053] Take 140mg of the above freeze-dried sample, and use 3mL 0.2M NH 4 HCO 3 Dissolved, separated by polyacrylamide gel chromatography column (Bio-GelP-10), and collected the corresponding chromatographic peaks of each heparin oligosaccharide. Volatilize NH in an oven at 55 °C for 72 h 4 HCO 3 After vacuum freeze-drying at -80°C...

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Abstract

The invention relates to a preparation and purification method of different domain oligosaccharides of heparan sulfate / heparin (HS / Hp). According to the method herein, commercially available heparin is used as a raw material, a high-sulfur disaccharide with three sulfite radicals is specifically enzymatically hydrolyzed with heparinase I, enzymatic hydrolysis conditions are controlled precisely toarrive at partial enzymatic hydrolysis so as to obtain a highly sulfated domain oligosaccharide containing the high-sulfur disaccharide; lowly sulfated oligosaccharide fragments with NS and NAc structures in HS / Hp are enriched so as to prepare a series of lowly sulfated domain oligosaccharides with different structures; a series of lowly sulfated HS / Hp oligosaccharides, from disaccharide to decasaccharide, are separated and purified through gel chromatography and ion exchange high-performance liquid chromatography. The method established herein to prepare different domain oligosaccharides ofHS / Hp is fast and simple and has low cost; important carbohydrate library samples are provided for the study on structure and functionality of HS / Hp; important precursors are also provided for the development of novel heparin drugs.

Description

technical field [0001] The invention provides a preparation and purification method of oligosaccharides in different regions of heparan sulfate / heparin, and belongs to the field of preparation and purification of natural products. Background technique [0002] Heparan sulfate (HS) and heparin (Hp) are the most complex carbohydrates in the glycosaminoglycan (GAG) family, mainly composed of different disaccharide units linked by alternating β(1-4) glycosidic bonds to form linear sulfation of polysaccharides. The HS long chain contains three domains with different degrees of sulfation, high sulfated domain (S-domain), low sulfated domain (NA / NS domain) and sulfur-free domain (NA domain), which are arranged alternately to form a complex chain structure. However, 70-80% of the long chain of Hp is high-sulfur disaccharide, which is similar to the high-sulfur region of HS. Studies have found that the highly sulfated and lowly sulfated regions of HS / Hp use their own specific stru...

Claims

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Application Information

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IPC IPC(8): C12P19/14C12P19/12C12P19/00C12P19/04
Inventor 魏峥苏晓明梁群焘
Owner FUZHOU UNIV
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