CN101003795A

CN101003795A - Method for preparing peroxidase of radish

Info

Publication number: CN101003795A
Application number: CN 200610160056
Authority: CN
Inventors: 王林嵩; 王琳; 马剑敏; 徐存拴; 王丽; 车淑红; 王歌
Original assignee: Henan Normal University
Current assignee: Henan Normal University
Priority date: 2006-12-27
Filing date: 2006-12-27
Publication date: 2007-07-25

Abstract

This invention discloses a method for preparing radish peroxydase. The method comprises: (1) pulverizing fresh radish at 0-15 deg.C, and separating the juice; (2) adding ammonium sulfate 30-50% into the juice, standing for 10-18 h, removing the precipitate, collecting the supernatant, adding ammonium sulfate 75-90%, standing for 10-18 h, decanting the supernatant, collecting the precipitate, diluting with water, and filtering; (3) filtering with an ultrafiltration membrane, recovering the filtrate, and collecting the concentrated liquid; (4) freeze-drying the concentrated liquid to obtain crude radish peroxydase, and performing column chromatography to obtain purified radish peroxydase. The method has such advantages as low peroxydase activity loss, low energy consumption, and easy operation. The radish peroxydase is safe and effective when used in animals and plants.

Description

The preparation method of peroxidase of radish

Technical field:

The present invention relates to biotechnology, specifically a kind of is raw material with the method for saltouing, ultrafiltration and dialysis apparatus extract peroxidase with bright radish.

Background technology:

Peroxidase extensively exists in the organism; has the removing superoxide; the protection cell exempts to be damaged by superoxide, removes effects such as cutaneous pigmentation, as fields such as the additive of foods and cosmetics and biochemical reagents wide application prospect and bigger economic benefit is being arranged.But be difficult for screening owing to extract the appropriate materials of peroxidase, the leaching process complexity, reasons such as cost height have restricted its development and use, are the method that raw material is produced peroxidase with bright radish particularly, though report is at home and abroad arranged at present.But producing peroxidase of radish with direct salting-out process does not then appear in the newspapers.

Summary of the invention:

It is wide to the purpose of this invention is to provide a kind of raw material sources, the preparation method of the peroxidase of radish that extraction process is simple, cost is low.

Technical solution of the present invention is finished as follows, it is to be raw material with bright radish, its preparation method is: in envrionment temperature is under the 0-15 ℃ of situation, 1. get bright radish, isolate radish Normal juice after the fragmentation, 2. substep adds ammonium sulfate in radish Normal juice, and the saturation ratio that joins ammonium sulfate for the first time is 30-50%, leaves standstill 10-18 hour, abandon throw out, get supernatant liquor, continue to add ammonium sulfate in supernatant liquor, making its saturation ratio is 75-90%, left standstill 10-18 hour, abandon supernatant liquor then, the taking precipitate dilute with water refilters, and 3. above-mentioned filtrate is used ultrafiltration membrance filter, filtrate being by filter membrane sees through liquid, can further recycle, remaining is concentrated solution, 4. the concentrated solution lyophilize is the peroxidase of radish raw product; The pure product of peroxidase of radish will be obtained behind the concentrated solution column chromatography purification.Extracting peroxidase of radish with present method, to have an enzyme loss of living little, and energy consumption is low, simple operation and other advantages, and security and validity were good when its zymin was applied to animals and plants.

Embodiment:

Specific embodiments of the invention are as follows: get 1 ton of bright radish, be broken into pasty state, place and isolate radish juice in the high speed freezing centrifuge, after the radish slag is used the damping fluid lixiviate again, volume increases 3-5 doubly, but the peroxidase of gained only is 1/10 in the Normal juice, the gained radish juice is added the ammonium sulfate powder when making its saturation ratio reach 30-50% step by step, left standstill 10-18 hour, discard throw out after, get supernatant liquor, continuing to add the ammonium sulfate powder in supernatant liquor makes its saturation ratio reach 75-90%, left standstill 10-18 hour, and abandoned supernatant liquor then, with the throw out dilute with water, filter, with filtrate filtered with 20 * 17cm, 100kD PES ultra-filtration membrane ultrafiltration desalination by the seeing through liquid and can further recycle of filter membrane, is Powdered with the concentrated solution lyophilize by film not, obtain the peroxidase of radish raw product of about 0.37 kilopond, this enzyme raw product has 2-3 kind isozyme band through electrophoresis detection, and the RZ value approximates 1, and this enzyme raw product promptly can be used for the field lower to the enzyme purity requirement.Also can will obtain about pure product 22 grams of peroxidase of radish behind above-mentioned concentrated solution process PHE hydrophobic chromatography, ISO hydrophobic chromatography, negatively charged ion or cation exchange column chromatography, the sieve chromatography column purification as required, the RZ value approximates 3.It is widely used in the biological health field, removes superoxide, and the protection cell is avoided the superoxide infringement, removes cutaneous pigmentation, and as the additive and the biochemical reagents of foods and cosmetics.

Claims

1, the preparation method of peroxidase of radish, it is to be raw material with bright radish, its preparation method is: in envrionment temperature is under the 0-15 ℃ of situation, 1. get bright radish, isolate radish Normal juice after the fragmentation, 2. substep adds ammonium sulfate in radish Normal juice, and the saturation ratio that joins ammonium sulfate for the first time is 30-50%, leaves standstill 10-18 hour, abandon throw out, get supernatant liquor, continue to add ammonium sulfate in supernatant liquor, making its saturation ratio is 75-90%, left standstill 10-18 hour, abandon supernatant liquor then, the taking precipitate dilute with water refilters, and 3. above-mentioned filtrate is used ultrafiltration membrance filter, filtrate being by filter membrane sees through liquid, can further recycle, remaining is concentrated solution, 4. will obtain the pure product of peroxidase of radish behind the concentrated solution process column chromatography purification.

2, the preparation method of peroxidase of radish, it is to be raw material with bright radish, its preparation method is: in envrionment temperature is under the 0-15 ℃ of situation, 1. get bright radish, isolate radish Normal juice after the fragmentation, 2. substep adds ammonium sulfate in radish Normal juice, and the saturation ratio that joins ammonium sulfate for the first time is 30-50%, leaves standstill 10-18 hour, abandon throw out, get supernatant liquor, continue to add ammonium sulfate in supernatant liquor, making its saturation ratio is 75-90%, left standstill 10-18 hour, abandon supernatant liquor then, the taking precipitate dilute with water refilters, and 3. above-mentioned filtrate is used ultrafiltration membrance filter, filtrate being by filter membrane sees through liquid, can further recycle, remaining is concentrated solution, 4. the concentrated solution lyophilize is the peroxidase of radish raw product.