CN102432681A

CN102432681A - Method for separating and purifying trypsin inhibitor from mung bean

Info

Publication number: CN102432681A
Application number: CN2011103958023A
Authority: CN
Inventors: 蔡少娟
Original assignee: SHANTOU ZHONGQIAN NETWORK TECHNOLOGY Co Ltd
Current assignee: SHANTOU ZHONGQIAN NETWORK TECHNOLOGY Co Ltd
Priority date: 2011-12-04
Filing date: 2011-12-04
Publication date: 2012-05-02

Abstract

The invention provides a method for separating and purifying trypsin inhibitor from mung bean. A crude extract of a mung bean trypsin inhibitor through mung bean smashing, filtering, salting out centrifugation, sediment dissolving, ultrafiltration, dialysis and centrifugation; and the trypsin inhibitor with higher extraction ratio through affinity chromatography and purification by taking chitosan particles as carriers. The method is lower in cost and simple and easy in operation.

Description

A kind of from mung bean the method for separation and purification trypsin inhibitor

Technical field

The present invention relates to biological technical field, particularly a kind of from mung bean the method for separation and purification trypsin inhibitor.

Technical background

Trypsin inhibitor is one type can suppress the active micromolecule polypeptide of trypsin hydrolyzing, is prevalent in the storage organ of plant, in seed, piece root and stem tuber, and particularly pulse family, woody section and plant of Solanaceae.The existence of trypsin inhibitor is also arranged in the blood of animal, seminal fluid, pancreas, first fresh milk, serum, egg albumen, placenta, the urine and in the mikrobe such as yeast, streptomyces in addition.

The proteolytic enzyme type that suppresses according to them can be divided into Serine, halfcystine, aspartic acid and inhibitors of metalloproteinase.Because they can suppress in the insect gut and the intravital proteolytic enzyme of some pathogenic micro-organisms, so proteinase inhibitor has important effect plant to infecting of insect and pathogenic agent in the system of defense.In addition, it also is used to study the interaction of protein-protein, or is used to cancer, AIDS patient's treatment.Inhibitor gene is changed in the purpose plant, can also improve pest-resistant, the resistance against diseases of plant.

Mung bean trypsin is the multifunctional dual-head proteinase inhibitor that separation and purification obtains from soybean, except possessing other proteinase inhibitor pest-resistant characteristics aspect disease-resistant, also has the advantage of many uniquenesses.As: higher and stable than vigor, broad spectrum is strong, and multiple protein enzymes such as trypsinase, Quimotrase, kallikrein are had stronger restraining effect.And these proteolytic enzyme are main digestive ferments of some Agricultural pests enteron aisles, and therefore, the application of mung bean trypsin aspect plant anti-insect has wide prospect.

In recent years, mung bean trypsin is studied hotter at home and abroad, mainly concentrates on the active structure center of studying, and suppresses mechanism and aspects such as gene clone and conversion.Research is extracted from mung bean, separates, and purifying then seldom, and is more economical as for considering to attempt other, and effective process for extracting does not domesticly see that as yet relevant report is arranged.

Summary of the invention

The present invention provide a kind of from mung bean the method for separation and purification trypsin inhibitor, trypsin inhibitor that can enough lower cost separation and purification higher degrees.

The present invention realizes through following steps:

(1) mung bean is smashed to pieces, added the 0.1mol/L PBS damping fluid of pH7.6, fully smash into pasty state to pieces;

(2) above-mentioned pasty state liquid is left standstill 20min, use filtered through gauze, collect extract,, discard deposition in 75 ℃ of heating in water bath sex change 30min;

(3) get supernatant and transfer pH to 6.5, add solid ammonium sulfate to 70% saturation ratio in the time of stirring, 4 ℃ are spent the night, and with the centrifugal 15min of liquid, discard deposition;

(4) the filter membrane ultrafiltration of 10kDa and 30kDa is successively used in precipitate with deionized water dissolving, collects the component of 10kDa～30kDa;

The component of (5) collecting is used 0.05mol/L, the Tris-HC dialysed overnight of pH8.0, and it is centrifugal to concentrate the back, collects supernatant;

(6) get chitosan in addition and use acetate dissolution, the dissolving back adds Tween80 fully;

(7) add ETHYLE ACETATE, after stirring, drip span80 again, be heated to 50 ℃ of reaction 30min;

(8) connect above-mentioned step, be warming up to 60 ℃, add formolite reaction 30min;

(9) add LUTARALDEHYDE after reaction is accomplished, regulate pH to 9.0, be warming up to 80 ℃ of reaction 1h;

(10) carry out suction filtration after reaction is accomplished, get filter residue and wash with acetone and absolute ethyl alcohol and be placed on 80 ℃ of dryings, obtain yellowish chitosan microballon;

(11) with the chitosan microballon in the above-mentioned steps as sorbent material, use 0.1mol/L, ph7.8 boric acid-borate buffer solution balance;

(12) get appearance on the arrowhead trypsin inhibitor crude extract;

(13) earlier with boric acid-borate buffer solution 4mL/min wash-out, use 0.25mol/L again instead, ph4.0 sodium-acetate-acetate buffer solution, the 2mL/min wash-out is used 0.5 mol/L at last, pH1.5 NaCl-HCl damping fluid, 2mL/min wash-out;

The method of this invention is easy, easy handling, and higher yields is arranged.

Embodiment

(1) gets mung bean 100g and smash to pieces, add the 0.1mol/L PBS damping fluid of 300ml pH7.6, fully smash into pasty state to pieces;

(4) deposition is used the 5ml deionized water dissolving, successively uses the filter membrane ultrafiltration of 10kDa and 30kDa, collects the component of 10kDa～30kDa;

The component of (5) collecting is used 0.05mol/L, the Tris-HCl dialysed overnight of pH8.0, and it is centrifugal to concentrate the back, collects supernatant;

(6) get chitosan 1.5g in addition with 300mL 0.5% acetate dissolution, the dissolving back adds the Tween80 of 1.5ml fully;

(7) add 0.8ml ETHYLE ACETATE, after stirring, drip the span80 of 1ml again, be heated to 50 ℃ of reaction 30min;

(8) connect above-mentioned step, be warming up to 60 ℃, add 2ml36% formolite reaction 30min;

(9) add the LUTARALDEHYDE of 10ml2% after reaction is accomplished, regulate pH to 9.0, be warming up to 80 ℃ of reaction 1h;

(12) get appearance on the arrowhead trypsin inhibitor crude extract;

The purification liquid that obtains behind the wash-out is measured its content with Xylene Brilliant Cyanine G G-250 staining, and the yield that obtains its trypsin inhibitor is 0.5%.

Claims

1. the method for a separation and purification trypsin inhibitor from mung bean is characterized in that following steps are arranged:

S1 smashs mung bean to pieces, filters, and saltouts, and is centrifugal, gets resolution of precipitate, ultrafiltration, and dialysis, centrifugal, make the mung bean trypsin crude extract;

S2 uses acetate dissolution with chitosan;

S3 adds Tween-80, ETHYLE ACETATE and span80 at the said lysate of S2, is heated to 50 ℃ of reaction 30min;

S4 is warming up to 60 ℃ with the said lysate of S3, adds formolite reaction 30min;

S5 adds LUTARALDEHYDE after the S4 reaction is accomplished, regulate pH to 9.0, is warming up to 80 ℃ of reaction 1h;

S6 carries out suction filtration after S5 reaction is accomplished, get filter residue and wash with acetone and absolute ethyl alcohol and be placed on 80 ℃ of dryings, obtains yellowish chitosan microballon;

S7 is that affiliation carrier carries out affinity chromatography with the chitosan microballon.

According to according to claim 1 said from mung bean the method for separation and purification trypsin inhibitor, it is characterized in that the said crude extract of S1, to add 0.1mol/L when smashing to pieces, the PBS damping fluid of pH7.6.

According to according to claim 1 said from mung bean the method for separation and purification trypsin inhibitor, it is characterized in that the said crude extract of S1, with the ammonium sulfate precipitation of 70% saturation ratio.

4. according to method, it is characterized in that the said crude extract of S1, use the filter membrane ultrafiltration of 10kDa and 30kDa respectively according to separation and purification trypsin inhibitor in the said mung bean of claim 1.

According to according to claim 1 said from mung bean the method for separation and purification trypsin inhibitor, it is characterized in that the said crude extract of S1, it is dialysis equilibrium liquid that dialysis needs use 0.05mol/L, the Tris-HCl of pH8.0.

According to according to claim 1 said from mung bean the method for separation and purification trypsin inhibitor, it is characterized in that the said affiliation carrier of S7, with 0.1mol/L, ph7.8 boric acid-borate buffer solution balance.

According to claim 6 said from mung bean the method for separation and purification trypsin inhibitor; It is characterized in that affinity chromatography elder generation uses 0.1mol/L, ph7.8 boric acid-borate buffer solution wash-out is used 0.25mol/L again instead; Ph4.0 sodium-acetate-acetate buffer solution; The 2mL/min wash-out is used 0.5 mol/L at last, the NaCl-HCl buffer solution elution of pH1.5.