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Process for separating and purifying natto kinase by reverse micelle method

A nattokinase, separation and purification technology, applied in the field of separation and purification of biologically active substances, can solve the problems of complex nattokinase purification process, numerous steps, etc., and achieve the effects of low cost, high-efficiency extraction, and high recovery rate of enzyme activity

Inactive Publication Date: 2005-11-02
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problem of complex nattokinase purification process and numerous steps, and to provide a method for separating and purifying nattokinase by reverse micelles method, which is simple in operation, easy to enlarge, and convenient for continuous operation

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  • Process for separating and purifying natto kinase by reverse micelle method
  • Process for separating and purifying natto kinase by reverse micelle method

Examples

Experimental program
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Effect test

Embodiment 1

[0036] 1. Separation and purification of nattokinase from the fermentation broth produced by liquid fermentation

[0037] With Bacilliis natto NLSSe (Chinese patent, application number 02121352.6) as the strain, first carry out seed culture, take a glycerol spore strain and inoculate it in a 100ml Erlenmeyer flask with 20ml seed medium, and culture it on a shaker at 37°C and 170rpm for 12 Hours later (OD660nm is about 7-8), refrigerate at 0-4°C for later use. Liquid seeds will keep for a week.

[0038] Then carry out fermentation culture: 100ml Erlenmeyer flask, liquid volume is 20ml, inoculum size is 5%, 37°C, 170rpm shaker culture for 48 hours, 5000rpm, 10min centrifugation, after the supernatant is tested for enzyme activity, cryopreservation, storage time Not longer than 9 days.

[0039] In this embodiment, the reverse micelles solution is composed of AOT / octane, and its weight ratio is AOT:octane=20:878; AOT and octane are weighed according to the above ratio, mixed eve...

Embodiment 2

[0044] Soybeans are soaked with three times the amount of water for one day and night (room temperature 15°C), then high-pressure cooking, 121°C, 15 minutes, inoculated with 3% inoculum after cooling, and fermented for 24 hours at 40°C to obtain natto. Natto is soaked with twice the amount of normal saline to extract nattokinase to obtain a nattokinase crude extract.

[0045] In this embodiment, the reverse micelles solution is composed of AOT and isooctane, and its weight ratio is AOT:isoctane=100:800. Weigh the AOT and isooctane respectively according to the above proportions, mix them evenly, and let them stand until they are completely dissolved to obtain the reverse micelles solution. Adjust the pH of the fermentation broth to 6; adjust the ionic strength of the fermentation broth to 0.2M with 2M sodium chloride. The fermentation broth and the reverse micelles solution were added into the Erlenmeyer flask at a ratio of 1:1, and the two phases were shaken on a shaker at a...

Embodiment 3

[0049] The preparation method of nattokinase crude extract is as in Example 1. In this embodiment, the reverse micelles solution is composed of cetyltrimethylammonium bromide, n-hexanol, n-hexane and water, and the ratio of parts by weight is 100:100:800:0.5. Mix evenly and let it stand until it is completely dissolved to obtain a reverse micellar solution. Adjust the pH of the fermentation broth to 4.5; adjust the ionic strength of the fermentation broth to 0.2M with 2M sodium chloride. The reverse micelle solution and the fermentation broth were added to the Erlenmeyer flask at a ratio of 1:25, and the two phases were shaken on a shaker at a speed of 240rpm for 5min, and the temperature was controlled at 20°C; the mixed solution was centrifuged at a speed of 4000rpm for 5min to obtain a clear fraction. phase, to obtain the reverse micelles solution loaded with nattokinase;

[0050] The aqueous stripping phase consisted of isopropanol, potassium bromide, glycine-sodium hydr...

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Abstract

The invention relates to a method for separating and purifying bean-kinase in opposition-micelle process, in particular to a new method for extraction and purification of bean-kinase of bean-kinase coarse extraction liquid prepared from fermenting the solid or liquid directly in mother liquid, including the steps of extraction and back extraction; and the related extraction steps contains: proportionally mixing the bean-kinase coarse extraction as aqueous phase with opposition-micelle solution, extracting in 10 to 35 Deg. C, and letting bean-kinase into opposition-micelle solution; back extraction in back extraction liquid, centrifuging in 20 to 45 Deg. C, separating and the purified bean-kinase water solution prepared. It can solve the present problem that craft of purifying bean-kinase is complicated and cycle is long. The recovery ratio of enzyme is more than 80 percent, and the purifying factor more than 3, and at the same time has an effect of concentration decolouration. The cycle is short and facilitating to continuous operation and enlarging by separating and purifying bean-kinase with opposition-micelle process.

Description

field of invention [0001] The invention belongs to the technical field of separation and purification of biologically active substances, and in particular relates to a method for directly separating and purifying nattokinase from fermentation broth by using reverse micellar extraction technology. technical background [0002] Natto is a traditional food in Japan with a history of 2,000 years. In 1987, Japanese scholar SumiH (Experimentia, 1987, 43, 1110-1111) discovered an enzyme with efficient thrombolytic activity from the traditional Japanese soybean fermented food natto (natto), and named it nattokinase ( Nattokinase, referred to as NK). Relevant scientific research shows that nattokinase has efficient thrombolytic ability, good safety, no allergic reaction, and low production cost. As a new generation of thrombolytic drug, nattokinase has an extremely attractive market prospect. [0003] At present, the separation and purification process of nattokinase often adopts t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A23L11/00C12N9/00
Inventor 刘俊果邢建民沈睿阳承利刘会洲
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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