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Method for separating and purifying natto kinase by ion exchange

A technology of ion exchange chromatography and nattokinase, which is applied in the field of separation and purification of nattokinase, can solve the problems of unsuitability to nattokinase, limited operating capacity, high production cost, etc., and achieves high level of production automation, convenient operation, and short production cycle short effect

Inactive Publication Date: 2005-03-16
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has a high recovery rate, but the operating capacity is limited, the production cost is high, and it is not suitable for the preparation of large-scale nattokinase

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] 1) Engineering bacteria fermentation

[0049] Pick a single bacterium colony expressing nattokinase from the solid medium and insert it into 10ml of seed medium, cultivate it on a shaker at 150rpm for 8 hours, insert it into the fermentation medium according to 1% inoculum size, and then incubate at 28°C and 150rpm Cultivate 100 hours in the lower fermenter;

[0050] 2) Fractional precipitation of ammonium sulfate

[0051] Use a refrigerated centrifuge to centrifuge the fermented liquid at a speed of 2000 rpm for 8 min at 0° C., collect the supernatant, and add 10% saturated (NH 4 ) 2 SO 4 Precipitation, 2000rpm centrifugal collection supernatant, continue to add (NH 4 ) 2 SO 4 To 60% saturation, centrifuge at 2000rpm to collect the precipitate, resuspend the precipitate in 10mM, pH6 phosphate buffer, centrifuge at 2000rpm for 10min to collect the supernatant, and store it at 0°C for later use;

[0052] 3) Gel chromatography separation

[0053] Use the chromatogra...

Embodiment 2

[0059] 1) Engineering bacteria fermentation

[0060] Pick a single bacterium colony expressing nattokinase from the solid medium and insert it into 200ml seed medium, cultivate it on a shaking table at 300rpm for 25 hours, insert it into the fermentation medium according to 5% inoculum size, and then incubate at 37°C and 300rpm Cultivate 150 hours in the lower fermenter;

[0061] 2) Fractional precipitation of ammonium sulfate

[0062] Use a refrigerated centrifuge to centrifuge the fermented liquid at a speed of 10,000 rpm for 15 min at 20° C., collect the supernatant, and add 40% saturated (NH 4 ) 2 SO 4 Precipitation, 10000rpm centrifugal collection supernatant, continue to add (NH 4 ) 2 SO 4 To 85% saturation, centrifuge at 10000rpm to collect the precipitate, resuspend the precipitate in 100mM, pH8 phosphate buffer, centrifuge at 10000rpm for 30min to collect the supernatant, and store it at 20°C for later use;

[0063] 3) Gel chromatography separation

[0064] Us...

Embodiment 3

[0070] 1) Engineering bacteria fermentation

[0071] Pick a single bacterium colony expressing nattokinase from the solid medium and insert it into 50ml seed medium, culture it on a shaking table at 240rpm for 10 hours, insert it into the fermentation medium according to 4% inoculum size, and then incubate at 30°C and 220rpm 120 hours of culture in the fermenter;

[0072] 2) Fractional precipitation of ammonium sulfate

[0073] Use a refrigerated centrifuge to centrifuge the fermented liquid at a speed of 8,000 rpm for 20 min at 4° C., collect the supernatant, and add 20% saturated (NH 4 ) 2 SO 4 Precipitation, 8000rpm centrifugal collection supernatant, continue to add (NH 4 ) 2 SO 4 To 60% saturation, centrifuge at 8000rpm to collect the precipitate, resuspend the precipitate in 10mM pH6.4 phosphate buffer, centrifuge at 8000rpm for 20min to collect the supernatant, and store it at 4°C for later use;

[0074] 3) Gel chromatography separation

[0075] Use a chromatogr...

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PUM

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Abstract

The invention discloses a method for separating and purifying Natto kinase mainly using ion exchange chromatography. The steps are composed of crude enzyme liquid preparation, gel chromatography and ion exchange chromatography, activating bacillus subtilis through slant medium, medium culture and fermenting in ferment medium, natto kinase being secreted to fermented solution by bacillus subtilis, centrifuging to collecting upper clear solution, then proceeding (NH#-[4])#-[2]SO#-[4] graded precipitation, redissolving using phosphorus acid buffer, adding redissolved solution to gel chromatography column to proceeding gel filtration, collecting elution peak containg natto kinase, adding fraction of having natto kinase activity from gel filtration to inon exchange chromatography column, proceeding adsorbing, collecting elution peak containg natto kinase, freeze drying to get purified natto kinase. The invention has merits of condition, low cost, simple process , easy operation ,high purity and short production period, etc.

Description

technical field [0001] The invention relates to a method for separating and purifying nattokinase by using ion exchange as the main means. Background technique [0002] Thrombotic disease is a serious threat to human life and health, and its morbidity and mortality rank first among various diseases. Thrombolysis is the treatment of choice for this type of disease. However, the current thrombolytic agents, such as urokinase, streptokinase, recombinant tissue plasminogen activator, etc., have various disadvantages such as short half-life, large side effects, and high price, making it difficult to become an applicable mass drug. In 1987, Sumi found an enzyme with strong fibrinolytic activity in natto, a traditional fermented food in Japan, and named it nattokinase (NK). Studies have shown that nattokinase is a serine protease that can significantly dissolve thrombus in vitro and in vivo, significantly shorten the dissolution time (ELT) of euglobulin, and stimulate venous endo...

Claims

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Application Information

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IPC IPC(8): C07K1/14C12N9/56
Inventor 梅乐和高大海林东强姚善泾
Owner ZHEJIANG UNIV
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