CN101259405B

CN101259405B - Preparation and application of nylon affinity membrane with reactive blue 4 as ligand

Info

Publication number: CN101259405B
Application number: CN2007101722397A
Authority: CN
Inventors: 朱利民; 苏赛男; 聂华丽; 陈天翔
Original assignee: Donghua University
Current assignee: Donghua University
Priority date: 2007-12-13
Filing date: 2007-12-13
Publication date: 2010-06-02
Anticipated expiration: 2027-12-13
Also published as: CN101259405A

Abstract

The invention relates to a preparing method and the application of a nylon affinity membrane which takes Reactive Blue 4 as a petunidin. The preparation includes: the nylon membrane after being activated is modified by chitosan; the modified nylon membrane is dipped into a Reactive Blue 4 dye and added with NaCL solution after heat preservation to react, added with Na2CO3 solution for reacting continuously after temperature rising, respectively washed by hot de-ionized water, methanol solution, NaCl solution, urea solution and de-ionized water to obtain the nylon affinity membrane which takesReactive Blue 4 as a petunidin. The application includes that pawpaw powders are sampled on the nylon affinity membrane which takes Reactive Blue 4 as a petunidin, washed by Tris-HCL buffer solution and eluted by NaSCN solution to obtain pawpaw prolease; the activity of the prolease and the protein content are tested; the purification times are calculated. The method is fast, simple and convenientand has large separation capacity; the prolease extracted has the advantages of good activity, high purity as well as being suitable for scale production.

Description

With the reactive blue 4 is the preparation method and the application of the nylon affinity membrane of aglucon

Technical field

The invention belongs to the nylon affinity membrane field, particularly relating to a kind of is the preparation method and the application of the nylon affinity membrane of aglucon with Reactive Blue 4.

Background technology

Papain (EC3.4.22.2) is a class thiol protease, extensively is present in root, stem, leaf and the fruit of papaya (Carica papaya), and wherein content is the abundantest in immature milk.Be widely used in food service industry, as the clarification of beer and tenderization and leather, weaving, daily use chemicals and the pharmaceuticals industry of meat.The purifying major part of papain is to adopt the precipitation method, but this method still is mixed with other protease, can not reach the demand of pharmaceuticals industry.

At present, the method of separation and purification papain comprises that crude separation separates with refining, wherein crude separation comprises salting out method, isoelectric point precipitation, organic solvent classification partition method etc., and refining separation comprises gel filtration, ion-exchange chromatography, adsorption chromatography, hydrophobic chromatography and covalent chromatography etc.Although these methods all have certain advantage, all there are shortcomings such as complex operation, the sample activity recovery is low, purification effect is undesirable, when especially from the weak solution of large volume, extracting a spot of bioactivator.

The affinity film chromatography The Application of Technology can make the protein purification multiple improve; and have select that specificity is good, pressure drop is little, weak point consuming time, large biological molecule sex change probability in separation process are little, allow faster charging rate, can reuse characteristics such as effectively reduce cost; compare easier realization scale purifies and separates with the post affinity chromatography.

Summary of the invention

The purpose of this invention is to provide a kind of method of utilizing the advanced quick purifying velardon of affinity film chromatography technology, the enzymatic activity that this method is quick, easy, fractional dose big, extract is good, and the purity height is applicable to large-scale production.

Of the present invention a kind of be the preparation method of the nylon affinity membrane of aglucon with Reactive Blue 4, comprise the following steps:

(1) nylon membrane after the activation immerses in the chitosan solution, and behind the room temperature reaction, 80-90 ℃ of oven dry dashed with 1vol.% acetic acid and deionized water, carries out the modification of nylon membrane;

(2) nylon membrane after chitin modified immerses in Reactive Blue 4 dye solutions, and behind the insulation 30-50min, adding mass fraction is 15%～20%NaCl solution, 55 ℃～60 ℃ constant temperature oscillating reactions 30-40min, and the bath temperature that raises then adds Na ₂CO ₃Solution, 65 ℃～80 ℃ are continued constant temperature oscillating reactions 4-5h, use the deionized water of heat respectively, methanol solution, 2mol/LNaCl solution, urea liquid and deionized water fully wash, and are able to Reactive Blue 4 and are the nylon affinity membrane of aglucon.

Described nylon membrane activation is meant that nylon membrane after the hydrolysis, activates with formaldehyde in 25-30 ℃ of 1mol/LHCl;

Described chitosan solution is meant that mass fraction is 2% chitosan solution (with the 1vol.% acetate dissolution);

Described Reactive Blue 4 dyes concentrations are 5mg/mL～15mg/mL, preferred 8mg/mL～12mg/mL;

Described Na ₂CO ₃Solution is meant that mass fraction is 15%～20%Na ₂CO ₃Solution;

Described methanol solution is meant that volume fraction is 10% methanol solution;

Described urea liquid is meant the 6mol/L urea liquid.

Of the present invention is the application of the nylon affinity membrane of aglucon with Reactive Blue 4, comprises step:

(1) papaya powder in the Tris-HCl buffer solution, being splined on Reactive Blue 4 is the nylon affinity membrane of aglucon, after the flushing of Tris-HCl buffer solution, uses the NaSCN eluant solution, obtains papain;

(2) tested enzyme activity and protein content calculate the purifying multiple.

Described Tris-HCl buffer solution is the Tris-HCl buffer solution of 0.05mol/L～1mol/L pH 8.0～8.5, the Tris-HCl buffer solution of preferred 0.05mol/L pH 8.5;

The papaya powder solution that described papaya powder solution is 10.0～15.0mg/mL;

Described NaSCN solution is meant the NaSCN solution of 1mol/L pH 6.0.

Beneficial effect of the present invention:

(1) method of the present invention is simple to operate, and is consuming time less, and purifying multiple and enzymatic activity are higher;

(2) sample source is convenient, is convenient to extract on a large scale purifying.

Description of drawings

Fig. 1 is the absorb-elute curve that utilizes nylon affinity membrane separation and purification papain from papaya powder.

The specific embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Embodiment 1

The activation of nylon membrane and bonding shitosan carry out modification, and concrete steps are as follows:

(1) the hydrolysis 24h in 25 ℃ of 1mol/LHCl of nylon membrane elder generation activates with formaldehyde then.The nylon membrane immersion 20mL formalin of 10 hydrolysis (＞36.5wt.%), add 0.2mL phosphoric acid (85wt.%), in 60 ℃ of reaction 7h, 40-50 ℃ of hot water washing is repeatedly.

(2) nylon membrane that above-mentioned formaldehyde is activated, immersion 10mL mass fraction is 2% chitosan solution (with the 1vol.% acetate dissolution), and room temperature reaction 1h changes 80 ℃ of baking ovens then over to, take out behind the 1h, use 1vol.% acetic acid and the unreacted shitosan of deionized water flush away respectively.Shitosan content on the nylon membrane can be tested by the method for ninhydrin.

Embodiment 2

With Reactive Blue 4 is the preparation of the nylon affinity membrane of aglucon, and concrete steps are as follows:

It is in Reactive Blue 4 dye solutions of 10mg/mL that above-mentioned nylon membrane after chitin modified immerses concentration, behind 60 ℃ of insulation 40min, adding mass fraction is 20%NaCl solution, constant temperature oscillating reactions 30min, raise then bath temperature to 80 ℃, the adding mass fraction is 25%Na ₂CO ₃Solution, continue constant temperature oscillating reactions 4h, use the deionized water of heat respectively, volume fraction is 10% methanol solution, 2mol/L NaCl solution, 6mol/L urea liquid and deionized water fully wash, up to cleaning solution be colourless till, obtaining with Reactive Blue 4 is the nylon affinity membrane of aglucon, is stored in the distilled water.

Embodiment 3

With the casein is the ultraviolet spectrophotometry enzyme sign alive of substrate, and concrete steps are as follows:

Get the enzyme liquid of 0.1mL, add 0.7mL 0.05mol/L Tris-HCl buffer solution (pH 8.0), (above-mentioned buffer solution includes the L-cysteine of 0.5mol/L and the EDTA of 0.02mol/L to add the papain activator of 0.2mL again, pH8.0) 35 ℃ of insulations are constant, 1% (W/V) casein solution (the above-mentioned buffer solution preparation) 1mL that adds same preheating, 35 ℃ of reaction 15mim add 3mL 5% trichloroacetic acid (TCA) solution cessation reaction (the control group experiment adds substrate after adding TCA earlier).Leave standstill, 8000 rev/mins of centrifugal 20min get filtrate colorimetric under the 280nm wavelength.

Embodiment 4

Utilize nylon affinity membrane separation and purification papain from papaya powder solution, concrete steps are as follows:

Build up membrane stack on 15 nylon affinity membranes, put into the film bridge, send into buffer solution and eluent with peristaltic pump, at first be 8.5 Tris-HCl buffer solution balance 15min with 0.05mol/L pH, flow velocity is 1.0mL/min, the papaya powder solution of 10mg/mL of sending into 35mL with peristaltic pump is by the film bridge subsequently, then the protein that does not adsorb with the Tris-HCl buffer solution flush away of 0.05mol/L pH 8.5; At last the eluent (pH6.0) with the NaSCN of 1mol/L carries out wash-out, obtains the target protein papain.

Claims

1. one kind is the preparation method of the nylon affinity membrane of aglucon with Reactive Blue 4, comprises the following steps:

(2) nylon membrane after chitin modified immerses in Reactive Blue 4 dye solutions, and behind the insulation 30-50min, adding mass fraction is 15%～20%NaCl solution, 55 ℃～60 ℃ constant temperature oscillating reactions 30-40min, and the bath temperature that raises then adds Na ₂CO ₃Solution, 65 ℃～80 ℃ are continued constant temperature oscillating reactions 4-5h, and deionized water, methanol solution, NaCl solution, urea liquid and the deionized water with heat fully washs respectively, is able to Reactive Blue 4 and is the nylon affinity membrane of aglucon;

The nylon membrane activation is meant that nylon membrane after the hydrolysis, activates with formaldehyde in 25-30 ℃ of 1mol/L HCl;

Described Reactive Blue 4 dyes concentrations are 5mg/mL～15mg/mL;

Described washing is to be that 10% methanol solution, 2mol/LNaCl solution, 6mol/L urea liquid and deionized water fully wash with hot deionized water, volume fraction respectively;

Described chitosan solution is that the mass fraction with the 1vol.% acetate dissolution is 2% chitosan solution.

2. according to claim 1 is the preparation method of the nylon affinity membrane of aglucon with Reactive Blue 4, it is characterized in that: described Reactive Blue 4 dyes concentrations are 8mg/mL～12mg/mL.