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Method for preparing flavobacterium heparinum heparinase I

A technology of Flavobacterium heparin and heparinase, applied in the direction of microorganism-based methods, biochemical equipment and methods, lyase, etc., can solve the problems of low heparinase and the inability of heparinase I to be effectively protected by calcium chloride , to achieve the effect of large preparation, increased yield and stable

Active Publication Date: 2012-03-07
SHENZHEN HEPALINK PHARMA GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This results in heparanase I not being effectively protected by calcium chloride
It can also be seen from the reported purification table that the yield of heparanase in the octyl-sepharose column chromatography step is seriously lower than that of other steps

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 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. Then press to insert 1L of fermentation medium. 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. 23°C, 150rpm, cultured for 2 days. Centrifuge the bacterial solution at 10,000rpm at 4°C for 20 minutes, collect the precipitate, and suspend it in 100ml, 50mM Tris-HCl ① (pH7.5, containing CaCl 2 10mM) buffer solution, sonicate in an ice bath for 1 hour (150W, 5s, 5s). 4°C, 10000rpm, centrifuge for 30min, add 8ml of protamine (0.5g / ml) dropwise, stir, 4°C, 10000rpm, centrifuge for 30min, the supernatant is the c...

Embodiment 2

[0029] Repeat the steps of fermentative preparation of heparanase in Example 1.

[0030] Add a tube of 10mM Tris-HCl to the sonication solution of Flavobacterium heparinus ① (with 10mM CaCl 2 , pH7.0) equilibrated SP column (2.5×30cm), equilibrate 200ml with the same buffer, then elute 200ml with the linear gradient of sodium chloride in the same buffer 0-0.5M, collect 0.25M-0.3M chloride Sodium concentration of the heparanase elution peak in several tubes. Combined with 10mM Tris-HCl after dialysis ① (with 10mM CaCl 2 , pH7.0) equilibrated SP column (1.5×40cm), equilibrate 100ml with the same buffer solution, and then use the same concentration of Tris-HCl containing 25mM sodium chloride and 0.1% heparin ② 300ml of buffer solution was used to elute, and each tube of the elution peak with higher activity was collected. After dialysis, a strip of 10mM Tris-HCl ① (with 10mM CaCl 2 , pH7.0) equilibrated SP column (1.5×40cm), equilibrated 100ml with the same buffer, then el...

Embodiment 3

[0033] Repeat the steps of fermentative preparation of heparanase in Example 1.

[0034] Add a tube of 30mM Tris-HCl to the sonication liquid of Flavobacterium heparinus ① (with 10mM CaCl 2 , pH6.5) equilibrated SP column (2.5×30cm), equilibrate 200ml with the same buffer, then elute 200ml with the linear gradient of 0-0.5M sodium chloride in the same buffer, collect 0.25M-0.3M chloride Sodium concentration of the heparanase elution peak in several tubes. Combined with 30mM Tris-HCl after dialysis ① (with 10mM CaCl 2 , pH 6.5) equilibrated SP column (1.5×40cm), equilibrated 100ml with the same buffer solution, and then used the same concentration of Tris-HCl containing 35mM sodium chloride and 0.15% heparin ② 300ml of buffer solution was used to elute, and each tube of the elution peak with higher activity was collected. After dialysis, a 20mM Tris-HCl ① (with 10mM CaCl 2 , pH 6.5) equilibrated SP column (1.5×40cm), equilibrate 100ml with the same buffer solution, then ...

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Abstract

The invention provides a method for preparing heparinase I. The method for preparing the heparinase I comprises the following steps of: inoculating flavobacterium heparinum serving as a raw material to a seed culture medium for culture; then inoculating the flavobacterium heparinum to a fermentation culture medium; centrifugally collecting precipitate; performing ultrasonication on the precipitate; performing centrifugation again to obtain crude enzyme liquid of the flavobacterium heparinum heparinase I; and performing SP-sepharose FF chromatographic purification on the crude enzyme liquid for three times to obtain the high-purity flavobacterium heparinum heparinase I, wherein the SP-sepharose FF chromatographic purification for three times is protected by calcium chloride in the whole course, so that the yield of pure enzymic activity is greatly increased. The method for preparing the heparinase I has the characteristics of simple process, easy amplification, large preparation amount of products at a time, low cost of reagents and the like. The specific activity of the prepared heparinase I reaches 223 IU / mg and the yield of the pure enzymic activity reaches 30 percent. Compared with the conventional newest method, the method for preparing the flavobacterium heparinum heparinase I has the advantages of increasing the specific activity by more than two times, and the purification yield by about one time.

Description

technical field [0001] The invention relates to a preparation method of heparanase I, in particular to a method for preparing heparanase I through SP-Sepharose FF chromatographic separation under the full protection of calcium chloride. 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. At present, there are about 10 kinds of heparinases reported in academic papers, and only three enzymes from Flavobacterium heparinus have been studied in detail, namely heparinase I, heparinase II and heparinase III. Heparanase ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12R1/20
Inventor 马小来李锂李坦
Owner SHENZHEN HEPALINK PHARMA GRP CO LTD
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