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High-efficiency preparation method of heparinase I from Flavobacterium heparinum

A technology of Flavobacterium heparin and heparinase, applied in the field of producing crude enzymes using osmotic stress, can solve problems such as complex chromatography processes

Inactive Publication Date: 2019-01-11
深圳市长征生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of CS column chromatography in the prior art requires a complicated chromatographic process

Method used

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  • High-efficiency preparation method of heparinase I from Flavobacterium heparinum
  • High-efficiency preparation method of heparinase I from Flavobacterium heparinum
  • High-efficiency preparation method of heparinase I from Flavobacterium heparinum

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Fermentation preparation of heparinase: scrape two-ring bacteria from Flavobacterium heparin plate or slant and receive in 50ml seed culture medium (0.5% of beef extract, 1% of peptone, 0.5% of yeast powder, 0.5% of NaCl, pH 7.0), 23°C, 150rpm, cultured for 1 day. The 5% inoculum was inserted into the secondary seed medium and cultivated under the same conditions for 1 day. Then insert 4L fermentation medium according to 5% inoculum amount and culture for 2 days. Fermentation medium composition: heparin 8g / L, K 2 HPO 4 2.5g / L, NaH 2 PO 4 2.5g / L, NH 4 Cl 2.0g / L, MgCl 2 0.5g / L, histidine 0.5g / L, methionine 0.5g / L, trace element NaMoO 3 , CuCl 2 , FeCl 2 、CoCl 2 , MnCl 2 , CaCl 2 1×10 each -4 M, pH 7.0. Cultured for 2 days at 23°C, 150 rpm. The bacterial solution was centrifuged at 10,000 rpm at 4°C for 30 minutes, and the precipitate was collected.

[0045] Suspend the enzyme-containing bacterial cells in 40ml sucrose solution at 4°C for 2 hours, centrifu...

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Abstract

The invention relates to a high-efficiency preparation method of heparinase I of Flavobacterium heparinum, including the following steps: Flavobacterium heparinum is culture in a seed culture medium,subject to culture in fermentation medium, centrifugal sedimentation, a crude enzyme solution is obtained under osmotic stress, the crude enzyme is purified by CS (Cellufine Sulfate) column chromatography, and the specific activity of the prepared heparanase I is 135IU / mg, the pure enzyme activity yield is as high as 57%, and the purification yield is nearly doubled compared with the existing method. The invention has the advantages of simple process, easy amplification, less equipment requirement, low reagent cost, etc.

Description

technical field [0001] The invention relates to a preparation method of heparanase I, in particular to a method for producing crude enzyme by using osmotic stress and purifying heparanase I by one-step CS chromatography. Background technique [0002] Heparanase refers to a class of enzymes that can specifically cleave the glycosidic bonds of heparin and heparin-like main chains. It was first discovered and isolated from Flavobacterium heparinus, and later, hepatic enzymes were found in some microorganisms and animal tissues. prime enzyme. The application of heparinase is very extensive, such as: removing residual heparin in blood, preventing coagulation, producing low molecular weight heparin, and studying the structure of heparin. [0003] There are three kinds of heparanases that have been isolated and purified from Flavobacterium heparinus, namely heparanase I, II and III, and the enzymatic characteristics of these three enzymes are different. Among the three heparinase...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12R1/20
CPCC12N9/88C12Y402/02007
Inventor 马小来郭进京陈冀中
Owner 深圳市长征生物科技有限公司
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