Method of producing heparin oligosaccharide using heparinase

A technology of Sphingosinobacterium and heparin oligosaccharides, which is applied in the field of enzyme engineering and can solve the problems of high price, high anti-proliferation activity, and unsuitable preparation

Inactive Publication Date: 2006-03-08
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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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

[0020] 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 16 S The results of phylogenetic analysis based on the rRNA gene sequence confirmed that the strain was...

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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. It mainly relates to a sphingobacterium sp.HC-6155 CGMCC 0660 and its enzyme production conditions and the enzyme engineering technology of using the enzyme to produce heparin oligosaccharides with anti-smooth muscle cell hyperplasia and other activities. technical background [0002] 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 includes Flavobacterium heparinum ATCC 13125 reported by Calliher P.M. (1981) and Prevotella heparinum reported by Watanabe M. (1998). (Prevotella heparinolytica), Bacteroides sp. isolated from the rumen reported by LinhardtR.J. (1986) and Bacteroides sp. isolated from human intestine reported by KimB.T. (20...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/00C12R1/01
Inventor 程秀兰高宁国杨敬张树政
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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