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Natto kinase purifying process and its freeze dried powder for injection

A nattokinase and process technology, which is applied in the direction of freeze-drying transportation, enzyme, powder transportation, etc., can solve the problems of complicated nattokinase purification process and many steps, and achieve high yield, simplified process and high yield.

Inactive Publication Date: 2003-11-05
沈阳抗生素厂
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention solves the problem of complex nattokinase purification process and many steps at present, and the problem of no nattokinase freeze-dried powder injection, and provides a nattokinase purification process and injection-based nattokinase purification process with simple steps. Freeze-dried powder products

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Get 191 mg of nattokinase crude product, dissolve in 20 ml of 1.5 mol / L ammonium sulfate phosphate buffer (PH7.0, 0.05 mol / L), leave it overnight at room temperature, centrifuge at 2000 rpm for 15 minutes, draw the supernatant , plus an appropriate amount of ammonium sulfate, the final concentration is 4.5 mol / L, and after standing overnight at room temperature, centrifuge at 2000 rpm for 15 minutes, remove the supernatant, take the precipitate, and dissolve it completely in 20 ml of 1.4-1.8 Mole / L Ammonium Sulfate Phosphate Buffer (PH7.0, 0.05 Mole / L). Upper hydrophobic chromatography column [filler: OctylSepharose 4 Fast Flow, 2.5×60 cm, pre-balanced with 1.4-1.8 mol / L ammonium sulfate phosphate buffer solution (PH6.0-12.0, 0.01-0.5 mol / L)] , Gradient elution, that is, the concentration of ammonium sulfate is gradually reduced from 1.4 to 1.8 mol / liter to zero, and the time is about 6 hours. There is a large passing peak at the beginning, no activity. When the ammon...

Embodiment 2

[0020] Take 150 mg of nattokinase crude product, dissolve it in 20 ml of phosphate buffer (pH7.4, 0.02 mol / liter), and bathe in water at 43°C for 20 hours. Centrifuge at 2000 rpm for 15 minutes, take the supernatant and add an appropriate amount of ammonium sulfate to make the final concentration 1.4-1.8 mol / L. Put the entire supernatant on a hydrophobic chromatography column [filler: Octyl Sepharose 4 Fast Flow, pre-balanced with 1.4-1.8 mol / L ammonium sulfate phosphate buffer (PH6.0-12.0, 0.01-0.5 mol / L)], gradient Elution, that is, the concentration of ammonium sulfate is gradually reduced from 1.4 to 1.8 mol / L to zero, and there is a large passing peak at the beginning, which is inactive. When the ammonium sulfate concentration was close to zero, a peak of activity appeared, and the peak was collected, and ultrafiltration desalted to obtain 40 milliliters of nattokinase essence, and the yield was 2.0% (mg protein mg÷mg crude product×100%).

[0021] The nattokinase product...

Embodiment 3

[0023] Get 240 mg of nattokinase crude product and dissolve it in 10 ml of phosphate buffered saline (pH7.4, 0.02 mol / liter), bathe in water at 43°C for 20 hours, centrifuge at 2000 rpm for 15 minutes, remove the precipitate, take the supernatant, add Appropriate amount of ammonium sulfate to make the final concentration of 4.5 mol / L, centrifuge at 3000 rpm for 20 minutes, remove the supernatant, add 3 ml of phosphate buffer (PH7.4, 0.02 mol / L) to the precipitate to dissolve. Gel filtration column [filler: Sephadex G-75, imported from Sweden, distributed by China Pharmaceutical Company, 2.6 × 100 cm, balanced with phosphate buffer solution [PH7.4, 0.02 mol / L] in advance, with phosphoric acid Salt buffer (pH7.4, 0.02 mol / liter) elution, the peak of the highest activity appears first, and finally there is a large peak, inactive, and the elution time is about 16 hours. The highest activity peak was collected, desalted and concentrated by ultrafiltration to obtain 50 milliliters o...

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Abstract

The present invention relates to biochemical medicine. The natto kinase purifying process includes the steps of: dissolving coarse natto kinase in water in concentration of 0.1-10 wt%; setting at 0-55 deg.c for 0.5 hr to 6 months and / or salt separation, centrifugation or filtering to eliminate insoluble matter, slat separating supernatant to collect the precipitate; and chromatography for further purification to obtain purified natto kinase product with single band or quasi-single band. The freeze dried powder of natto kinase is prepared with the purified natto kinase product and through adding additives for injection, mixing and freeze drying.

Description

Technical field: [0001] The invention belongs to biochemical medicine, and mainly relates to the improvement of nattokinase purification process and the preparation of freeze-dried powder injection. Background technique: [0002] Natto (Natto) is a traditional soybean fermented food in Japan, similar to the water bean drum in China's light bean drum, which was introduced to Japan since the Tang Dynasty. The unique taste of natto is produced by the fermentation of a type of Bacillus subtilis, Bacillus subtilis / natto. In 1987, Sami et al. [1] reported for the first time that there was a fibrinolytic enzyme in natto—nattokinase (Nattokinase, NK). Subsequently, it was found that nattokinase was derived from Bacillus natto in natto, which was a subtilisin-like serine protease, and the initial synthesis product was pre-pro-nattokinase (pre-pro- NK), which excises 106 amino acid residues at the N-terminus during secretion to form a single-chain polypeptide with 275 amino acid res...

Claims

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Application Information

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IPC IPC(8): A61K9/19A61K38/48C12N9/56
Inventor 刘跃金李静姜洋张云湖
Owner 沈阳抗生素厂
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