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Method suitable for industrialized production for separating and purifying natto kinase

A nattokinase, separation and purification technology, applied in the field of separation and purification, can solve problems such as low yield and difficult industrial production, and achieve the effects of stable separation process, easy activation and regeneration, and optimization of ion exchange media and elution conditions.

Inactive Publication Date: 2011-02-23
HUBEI GOLE BIO TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In a word, the current separation and purification methods all have the disadvantages of difficult industrial production and low yield.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1. Pretreatment of fermentation broth

[0023] The about 10L fermented liquid of fermented gained carries out plate and frame filtration immediately after putting into tank. Transfer to settling tank. Grind finely (NH 4 ) 2 SO 4 According to 20% saturation, it was added into the precipitation tank, stored at 4°C overnight, the precipitate was discarded, and the supernatant was collected for later use. Move the supernatant to another precipitation tank, add ethanol to 75% saturation, store overnight at 4°C, discard the supernatant, collect the precipitate, and dissolve it with 10mmol / L pH 9.0 phosphate buffer. Set the volume to 1L, and measure various indicators.

[0024] 2. Anion exchange resin to remove impurities

[0025] Use Styrene-DVB macroporous strongly basic styrene-based anion exchange resin (Nankai University Chemical Plant) to remove impurities and pigments. The specifications of the anion exchange chromatography column are 7.5cm in inner diameter and ...

Embodiment 2

[0034] 1. Pretreatment of fermentation broth

[0035] About 10L of the fermented liquid obtained in the fermenter is put into the tank and immediately centrifuged at 8000r / min for 10min. Collect the supernatant for later use. Grind (NH4) 2 SO 4Add to the above supernatant according to 20% saturation, store overnight at 4°C, centrifuge at 10000r / min for 30min, and discard the precipitate. Take about 7L of the supernatant and add ethanol to 75% saturation, store at 4°C overnight and then centrifuge at 10,000r / min for 30min, discard the supernatant, and dissolve the precipitate with 10mmol / L pH 6.4 phosphate buffer. Dilute to 500mL. The enzyme activity was determined by spotting the supernatant and precipitate solution respectively.

[0036] 2. Anion exchange resin to remove impurities

[0037] Use Styrene-DVB macroporous strongly basic styrene-based anion exchange resin to remove impurities and pigments. The specifications of the anion exchange chromatography column are 5...

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Abstract

The invention discloses a method suitable for industrialized production for separating and purifying natto kinase, which relates to the technical field of separation and extraction of natto kinase for treating thrombus diseases and solves the problems that the conventional natto kinase purification process is complex and is not suitable for industrialized production. A pure natto kinase product is obtained in a separation and purification mode by adopting ethanol precipitation and crude extraction, cation and anion exchange resin chromatography and HPD-400 macroporous absorption resin refining. The common expensive material such as gel and the like is replaced by adopting a column chromatography filling material suitable for industrialized production, the yield can reach 59 percent, and the purity reaches over 95 percent; and the method has the characteristics of high yield, low cost and capability of scale production, basically solves the bottleneck problem of natto kinase separation and purification in the industrialized production, creates a condition for obtaining the natto kinase with high purity and high enzyme activity by efficient separation, and has good economic benefit and social benefit.

Description

technical field [0001] The invention mainly relates to a method for separating and purifying nattokinase suitable for industrial production. It belongs to the technical field of separation and purification. Background technique [0002] Thrombotic diseases include arterial thrombotic diseases and venous thrombotic diseases, which have become one of the main causes of death and disability in the world. Researchers from all over the world are actively carrying out various tasks on how to overcome the stubborn disease of thrombotic diseases. Therefore, thrombolytic drugs have huge market potential. [0003] Antithrombotic drugs commonly used at this stage are mainly divided into three categories: antiplatelet drugs, anticoagulant drugs and thrombolytic drugs. The currently used thrombolytic drugs bring certain curative effects, but also bring corresponding toxic and side effects. As a new generation of thrombolytic drugs, nattokinase has many advantages such as direct and di...

Claims

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Application Information

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IPC IPC(8): C12N9/54
Inventor 曹晓琴张云峰李军陈晓炜胡伶俐
Owner HUBEI GOLE BIO TECH DEV
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