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Culture medium for biologically synthesizing heparinase

A technology of biosynthesis and culture medium, applied in the field of culture medium for biosynthesis of heparanase, can solve the problem of low titer of heparanase

Inactive Publication Date: 2011-05-11
SHANGHAI DUOMIRUI BIOTECHNOLOGY LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the prior art, because the titer of heparinase produced by the fermentation of Flavobacterium heparinus is generally not high, it has become the main obstacle restricting the large-scale production of heparinase by fermentation, and it is an urgent problem to be solved.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Flavobacterium heparinum (Flavobacterium heparinum, ATCC 13125), 750ml shake flask, culture solution composition: (g / L) glucose 3; NH 4 Cl 4; soybean peptone 4.6; tryptone 2.3; L-Met 0.5; L-His 0.5; mixed phosphate 5; heparin sodium 2; trace elements 10 -4 M(NaMoO 3 , CuCl 2 , FeCl 2 、CoCl 2 , MnCl 2 , CaCl 2 ), pH 7.0, the seed age of the seed solution was 48 hours, inoculated with 10%, the liquid volume was 90ml / 750ml, and cultivated at 25°C for 32 hours. The cell culture medium was centrifuged (10,000r / min, 8min) to collect the cells, washed with a certain volume of 0.02mol / L, pH7.0 phosphate buffer, suspended cells, and ultrasonically disrupted in an ice bath. The broken liquid was centrifuged (14,000r / min, 30min) to collect the supernatant to obtain the cell-free crude enzyme solution. 15g / L (wet weight) of bacteria and 1,768U / L titer of heparanase can be obtained.

Embodiment 2

[0020] Flavobacterium heparinum (Flavobacterium heparinum, ATCC 13125), 750ml shake flask, culture solution composition: (g / L) glucose 3; NH 4 Cl 4; soybean peptone 4; tryptone 2; L-Met 0.25; L-His 0.25; mixed phosphate 5; heparin sodium 2; trace elements 10 -5 M(NaMoO 3 , CuCl 2 , FeCl 2 、CoCl 2 , MnCl 2 , CaCl 2 ), pH 7.0, the seed age of the seed solution was 48 hours, inoculated with 10%, the liquid volume was 90ml / 750ml, and cultivated at 25°C for 32 hours. The cell culture medium was centrifuged (10,000r / min, 8min) to collect the cells, washed with a certain volume of 0.02mol / L, pH7.0 phosphate buffer, suspended cells, and ultrasonically disrupted in an ice bath. The broken liquid was centrifuged (14,000r / min, 30min) to collect the supernatant to obtain the cell-free crude enzyme solution. 14g / L (wet weight) of bacteria and about 1,550U / L titer of heparanase can be obtained.

Embodiment 3

[0022] Flavobacterium heparinum (Flavobacterium heparinum, ATCC 13125), 750ml shake flask, culture solution composition: (g / L) glucose 3; NH 4 Cl 4; soybean peptone 4; tryptone 3; L-Met 0.25; L-His 0.25; mixed phosphate 5; heparin sodium 2; trace elements 10 -5 M(NaMoO 3 , CuCl 2 , FeCl 2 、CoCl 2 , MnCl 2 , CaCl 2 ), pH 7.0, the seed age of the seed solution was 48 hours, inoculated with 10%, the liquid volume was 90ml / 750ml, and cultivated at 25°C for 32 hours. The cell culture medium was centrifuged (10,000r / min, 8min) to collect the cells, washed with a certain volume of 0.02mol / L, pH7.0 phosphate buffer, suspended cells, and ultrasonically disrupted in an ice bath. The broken liquid was centrifuged (14,000r / min, 30min) to collect the supernatant to obtain the cell-free crude enzyme solution. 15g / L (wet weight) of bacteria and about 1,550U / L titer of heparanase can be obtained.

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PUM

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Abstract

The invention discloses a culture medium which utilizes Flavobacterium heparinum ATCC 13125 as bacterial to biologically synthesize heparinase, which comprises carbon source, nitrogen source, amino acid and heparin. The culture medium is characterized in that nitrogen source is composed by NH4Cl, soybean tryptone and tryptone. In the culture medium of the invention, the thallus yield of flavobacteriun heparinum can reach to 15g / L (wet weight). Enzyme production potency can reach 2,000U / L through adopting the culture medium of the invention to synthesize heparinase, and the production cost is low, the control is simple, and scale production is easy to achieve.

Description

technical field [0001] The invention relates to a culture medium, in particular to a culture medium for biosynthesizing heparinase with Flavobacterium heparinum ATCC 13125 as the strain. Background technique [0002] Heparanase can specifically cleave different sequences with specific modifications on heparin and heparan-like chains to produce different oligosaccharide fragments. Heparinase has many important uses, including the elimination of heparin in the body circulation, the determination of the precise structure of heparin, the study of the anticoagulant mechanism of heparin and the structure and function of heparin, the preparation of low molecular weight heparin and antitumor drugs, etc. It is reported abroad that the titer of 1,475U / L of heparanase produced by the fermentation of Flavobacterium heparinus can be achieved, while the domestic reports are also very low. Among them, the article "Process Optimization of Heparanase Production by Flavobacterium Heparin Fer...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/24
Inventor 田永辉陈雪齐艳艳赵文杰冯军程睛华林纲薛春佳朱裕辉魏晓东
Owner SHANGHAI DUOMIRUI BIOTECHNOLOGY LTD