Method for separating and purifying nattokinase by magnetic microsphere

A technology of nattokinase and magnetic microspheres, which is applied in the direction of transferase, etc., can solve the problems of numerous steps and complicated separation process of nattokinase, and achieve the effect of simple operation steps, short cycle and realization of adsorption

Inactive Publication Date: 2006-12-13
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problems of complex nattokinase separation process and numerous steps, and provide a nattokinase separation and purification method with simple operation, low production cost, short cycle and easy large-scale production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] (1) Preparation of nattokinase fermentation broth

[0031] A single colony of nattokinase was picked from the solid medium and inserted into 20 mL of seed medium, cultivated in a shaking table for 12 hours, inserted into the fermentation liquid medium according to 2% inoculum size, and cultivated in a fermenter at 25°C for 36 hours. After the liquid fermentation is finished, the bacterium is removed by centrifugation to obtain a nattokinase fermentation liquid.

[0032] (2) Immobilization of affinity ligands on magnetic microspheres

[0033] The self-made magnetic polymethyl methacrylate microspheres were modified by surface functionalization to obtain magnetic microspheres with epoxy functional groups on the surface. Under the condition of 60°C, using sodium hydroxide as a catalyst and aminobenzamidine as an affinity ligand, the immobilization reaction is carried out on the surface of the magnetic microspheres to obtain the magnetic microspheres containing the benzami...

Embodiment 2

[0037] (1) Preparation of nattokinase fermentation broth

[0038] A single colony of nattokinase was picked from the solid medium and inserted into 100 mL of seed medium, cultivated in a shaker for 24 hours, inserted into the fermentation liquid medium according to 5% inoculum size, and cultivated in a fermenter at 30°C for 48 hours. After the liquid fermentation is finished, the bacterium is removed by centrifugation to obtain a nattokinase fermentation liquid.

[0039] (2) Immobilization of affinity ligands on magnetic microspheres

[0040] The self-made magnetic polyvinyl alcohol microspheres were modified by surface functionalization to obtain magnetic microspheres with epoxy functional groups on the surface. Under the condition of 70°C, using sodium hydroxide as a catalyst and aminophenylboronic acid as an affinity ligand, the immobilization reaction is carried out on the surface of the magnetic microspheres to obtain the magnetic microspheres containing the phenylboroni...

Embodiment 3

[0044] (1) Preparation of nattokinase fermentation broth

[0045] A single colony of nattokinase was picked from the solid medium and inserted into 40 mL of seed medium, cultivated in a shaker for 16 hours, inserted into the fermentation liquid medium according to 4% inoculum size, and cultivated in a fermenter at 37 ° C for 24 hours. After the liquid fermentation is finished, the bacterium is removed by centrifugation to obtain a nattokinase fermentation liquid.

[0046] (2) Immobilization of affinity ligands on magnetic microspheres

[0047] The self-made magnetic polyvinyl alcohol microspheres were modified by surface functionalization to obtain magnetic microspheres with epoxy functional groups on the surface. Under the condition of 60°C, using sodium hydroxide as a catalyst and aminobenzamidine as an affinity ligand, the immobilization reaction is carried out on the surface of the magnetic microspheres to obtain the magnetic microspheres containing the benzamidine on the...

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PUM

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Abstract

The invention provides a method for separating and purifying beankinase with magnetic microsphere. The method includes adding the magnetic microsphere coupled with friendly aglucon on the surface into fermentation liquor of beankinase, slowly mixing and sufficiently mixing them under an ambient temperature to make the beankinase specifically adsorb on the magnetic microsphere surface; separating the magnetic microsphere adsorbed by beankinase with magnet from the fermentation liquor, cleaning once with buffer solution to remove the foreign matter, then performing analysis with analytic liquor, and collecting the beankinase eluent; finally obtaining beankinase product by cryodesiccation. The separated beankinase has 85% vitality and 6.0 purified factor. The invention has advantages that it is of short cycle, simple operation, low cost of manufacture and is easy for mass production.

Description

technical field [0001] The invention relates to the technical field of separation of bioactive substances, in particular to a method for separating and purifying nattokinase from fermented liquid by using magnetic microspheres. Background technique [0002] Cardiovascular system diseases caused by thrombosis and arteriosclerosis seriously threaten human life and health, and its morbidity and mortality rank first among various diseases. Thrombolysis is the mainstay of treatment for these diseases. Nattokinase is a potential new oral thrombolytic drug because of its high-efficiency thrombolytic ability, no side effects, low cost, and being produced by bacterial fermentation. [0003] At present, the separation and purification process of nattokinase often adopts traditional protein purification methods, such as salting out, gel chromatography, hydrophobic chromatography, ion exchange chromatography and other purification methods. When Nakaniski K etc. (referring to U.S. Pat....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12
Inventor 阳承利邢建民官月平刘会洲
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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