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Method of producing heparin oligosaccharide using heparinase

A technology of heparin oligosaccharide and heparinase, applied in the field of enzyme engineering, can solve the problems of high price, low anticoagulation, high antiproliferative activity and the like

Inactive Publication Date: 2003-07-16
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, currently there are very few heparinases with practical value. Flavobacterium heparinase is the only commercial heparinase, but it is expensive ($696 / 11 units, Sigma), and the low-molecular-weight heparin produced by degrading heparin still remains High anticoagulant activity, not suitable for the preparation of heparin oligosaccharides with low anticoagulant and high antiproliferative activity

Method used

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  • Method of producing heparin oligosaccharide using heparinase
  • Method of producing heparin oligosaccharide using heparinase
  • Method of producing heparin oligosaccharide using heparinase

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Embodiment

[0019] 1. Strain cultivation and identification: Inoculate the heparanase-producing strain HC-6155 on a common beef juice slant medium, cultivate it at 30°C for 48 hours, and then insert it into a 500ml Erlenmeyer flask containing 50ml of fermentation medium , at 30 DEG C, cultivate 16 hours on a shaker at 200 rpm, then transfer to a 500ml Erlenmeyer flask filled with 50ml of the same medium at a 2% inoculum size, and cultivate for 36 hours under the same conditions. It is identified by external morphology, physiological and biochemical reaction characteristics and molecular biological methods. The external shape of the bacteria is rod-shaped, non-motile Gram-negative bacteria, which produce light purple pigments, and do not use citric acid, KNO 3 , Tween, malonic acid, etc., do not produce H 2 S gas can utilize glucose, sucrose, arabinose, and rhamnose. And according to the phylogenetic analysis results based on the 16s rRNA gene sequence, it was confirmed that the strain w...

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Abstract

A process for preparing heparin oligose from heparinase includes such steps as culturing sphingobacterium (CGMCC No.0660), preparing non-cell coarse enzyme liquid, extracting and purifying heparinase, degradating heparin by the heparinase at 20-30 deg.C to obtain heparin oligose mixture, ultrafilter, gel filter for fractionation and evaporation concentration. The product has the activity of resisting smooth muscle hyperplasia.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering. Sphingobacterium [0002] (Sphingobacterium sp.HC-6155) Heparanase produced by CGMCC0660 and the enzyme engineering technology for producing heparin oligosaccharides with anti-proliferative activity of smooth muscle cells by using the enzyme. technical background [0003] Heparanase is a lyase that can degrade heparin substances. The main feature of degrading heparin is the production of uronic acid with unsaturated double bonds and glucosamine with reducing ends. There are not many reports about the source of heparinase. At present, the heparinase of microbial origin has the heparin Flavobacterium heparinum (Flavobacterium heparinum ATCC 13125) reported by Calliher P.M. (1981), the heparin Prevot's heparinase reported by Watanabe M. (1998) Prevotella heparinolytica, Bacteroides stercoris HJ-15 isolated from rumen reported by Linhardt R.J. (1986) and Bacteroides stercoris HJ-15 isola...

Claims

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Application Information

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IPC IPC(8): C12P19/00
Inventor 程秀兰杨敬高宁国张树政
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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