CN101564656A - Preparation of nanometer composite affinity membrane used for rapidly and efficiently separating and purifying thiol protease - Google Patents

Abstract

The invention relates to the preparation of a nano-composite affinity membrane for fast and efficient separation and purification of thiol protease, comprising: (1) mixing hexafluoroisopropanol HFIP and formic acid mixture into a reaction vessel as a solvent system; (2) Nylon 6 and chitosan powder reaction container; (3) placed in a water bath oscillator to obtain nylon 6/chitosan spinning solution; (4) electrospinning with spinning solution to obtain nanofiber membrane, Drying; (5) Cut into circular membranes and activate; (6) After washing several times, put them into the dye solution for reaction; (7) Add NaCl solution for treatment; (8) Add Na ₂ CO ₃ for color fixation reaction (9) cooling, washing. The preparation method of the invention is simple in operation, less time-consuming and large in adsorption capacity; the prepared nano-affinity membrane is fast, convenient, cheap and efficient, can efficiently purify thiol protease, and is suitable for large-scale production.

Description

Preparation of Nanocomposite Affinity Membrane for Rapid and Efficient Separation and Purification of Thiol Protease

technical field

The invention belongs to the field of preparation of nanocomposite affinity membranes, in particular to the preparation of nanocomposite affinity membranes for fast and efficient separation and purification of thiol proteases.

Background technique

In recent years, with the increasing development of life science, biotechnology, pharmacy and medical technology, people have higher and higher requirements for the separation, extraction and purification of biological macromolecules. Affinity chromatography is a technique commonly used in laboratories and industries for the separation and purification of biomacromolecules. It separates biomacromolecules based on the affinity between biomacromolecules and specific immobilized ligands, that is, specific reversible binding and dissociation. It has the characteristics of mild operating conditions, no pollution, no phase change, etc. The membrane separation process has simple equipment, easy to scale up, low cost, fast separation speed, and continuous operation. It has been applied in many aspects.

Chitosan (CS) is a natural polymer, the molecule contains extremely strong reactive groups amino and hydroxyl, and has the advantages of excellent solvent resistance and alkali resistance. Combining it on porous membranes of various materials can make composite ultrafiltration membranes with different pore sizes. At present, chitosan polymers are mostly used to prepare pervaporation (or pervaporation) composite membranes, but blending chitosan and synthetic polymer nylon to make ultrafiltration composite membranes has not been reported.

Contents of the invention

The technical problem to be solved by the present invention is to provide the preparation of a nano-composite affinity membrane for fast and efficient separation and purification of thiol protease. The method is simple to operate, less time-consuming, and has a large adsorption capacity; the prepared nano-affinity membrane is fast and simple , cheap, efficient, can efficiently purify thiol protease, and is suitable for large-scale production.

The preparation of the nanocomposite affinity membrane for fast and efficient separation and purification of thiol protease of the present invention includes:

(1) The mixture of hexafluoroisopropanol (1,1,1,3,3,3,3-hexafluoroisopropanol) and formic acid with a volume ratio of 9 to 12:1 is used as a solvent system and mixed into the reaction vessel at a temperature of 40～60℃, water bath shaking reaction for 20～45min;

(2) Nylon 6 and chitosan powder are slowly added in the above-mentioned reaction vessel under stirring conditions, and continue stirring for 2～3h to fully swell, wherein the mass concentration of nylon 6 particles is 5～8wt%, and the mass concentration of chitosan is 0～2wt%;

(3) Place the above-mentioned reaction vessel in a water-bath oscillator, under reflux condensation, slowly heat to 40-60°C, shake for 15h-30h until completely dissolved, and obtain nylon 6/chitosan spinning solution;

(4) Extract the nylon 6/chitosan spinning solution with a syringe, fix it on the electrospinning device, adjust the spinning parameters for electrospinning, the static voltage is 10-18kv; the receiving screen is received by aluminum foil grounding, the needle and the receiving screen The distance is 10～20cm, get nylon 6/chitosan ultrafine nanofiber film;

(5) The collected film is dried in an oven and a vacuum drier successively;

(6) Cutting the dried nylon 6/chitosan nanofiber membrane into circular membranes with a diameter of 47 mm, putting it into 50-60° C., and activating it in a NaOH solution of 10-15 wt % for 20-30 min;

(7) Nylon 6/chitosan nanofiber film after activation, after washing several times with hot water, put into the dye solution and react;

(8) add NaCl solution to process, dyes are adsorbed on the nylon 6/chitosan nanofiber membrane;

(9) Adjust the temperature of the water bath, add Na ₂ CO ₃ to carry out the color fixation reaction;

(10) cooling, after fully washing with hot water, methanol, NaCl, urea, double distilled water successively, or preserve in Tris-HCl buffer solution, obtain the nylon 6/chitosan nanofiber affinity film with dye as ligand .

In the step (1), the mass concentration of hexafluoroisopropanol (HIFP) is 99%-100%, and the water content is less than 0.1%.

The specification of the syringe in the step (4) is 5ml, the inner diameter of the needle is about 0.4-0.7mm; the ejection flow rate is 0.5-2ml/h.

The series drying method in the step (5) is to first dry the spun film at room temperature for 12 hours, then put it in an oven for 4-6 hours at 90°C, and finally put it in a vacuum drier for 24 hours at 60-70°C.

The dye in the step (6) is the dye Cibacron Blue F3GA with a concentration of 5-15 mg/ml.

The step (7) NaCl solution treatment is to use 20-30wt% NaCl solution in a water bath at 40-80° C. for 20-45 minutes.

In the step (8) Na ₂ CO ₃ color fixing reaction, 25-30wt% Na ₂ CO ₃ is used to shake and react in a water bath at 60-90° C. for 3-6 hours.

The washing in the step (9) is to wash with 40°C-50°C hot water, methanol with a content of 99.5-100%, 2M NaCl, and 6M urea, and the washing degree is that the washing liquid after washing is colorless.

The prepared affinity membrane is applied to the separation and purification of thiol protease.

In this method, nylon 6 is used as the main spinning material, and chitosan rich in hydrophilic functional groups -OH and -NH ₂ is added, and the blending is successfully realized by adjusting the solvent and related spinning condition parameters; The obtained nano-composite material is used as a raw material, and the dye is used as a ligand, and the prepared nano-affinity membrane is fast, simple, cheap and efficient, can efficiently purify thiol protease, and is suitable for large-scale production.

The electrospinning method is an important method for preparing ultrafine fibers. It is significantly different from the traditional method. The electrostatic force is used as the traction force, and the polymer solution or melt is charged with thousands to tens of thousands of volts of high-voltage static electricity. The polymer droplets are stretched under the action of electric field force. When the electric field force is strong enough, the polymer droplets can overcome the surface tension to form a jet stream, and the solvent evaporates or solidifies during the jetting process, and finally falls on the receiving device, forming a fiber mat similar to a non-woven fabric. Fibers prepared by electrospinning are much thinner than traditional spinning methods, with diameters generally ranging from tens of nanometers to hundreds of nanometers, and the smallest diameter can reach 1nm.

The electrospinning method can spin more than 50 different polymers, such as polyester, polyurethane, polyethylene, polypropylene, polyvinyl alcohol, polyaniline, polyacrylonitrile, etc., into diameters ranging from less than a few nanometers to more than 1 μm The resulting electrospun fibers were collected on negative plates and deposited as nonwovens. On the one hand, nylon 6 nanofibers prepared by electrospinning can prepare high-performance nylon nanofibers and improve the wearability of nylon fibers; The resulting fiber can combine the advantages of both, not only has the toughness and wear resistance of nylon, but also endows the composite membrane with new biological characteristics, and has been widely used in many aspects.

The membrane matrix material plays an important role in the preparation of the affinity membrane, because it not only determines the bonded spacer arm and ligand type, but also directly determines the separation effect. Cellulose is the most abundant natural polymer in nature, and it is also one of the earliest media used for affinity separation. Because the cellulose molecule contains a large amount of -OH, it endows cellulose with good hydrophilicity, biocompatibility and flexibility. Reactivity, and the cellulose membrane is cheap, low cost when used for affinity separation, suitable for industrial applications. In recent years, the application of cellulose in biochemical separation has attracted more and more attention.

Beneficial effect

(1) The method of the present invention is simple to operate, takes less time, and has a large adsorption capacity;

(2) The raw materials used in the present invention are cheap and easy to obtain, the source is convenient, and they themselves contain abundant reactive functional groups, without modification treatment, saving time and effort, and reducing the preparation cost; it is convenient for large-scale extraction and purification;

(3) The nano-affinity membrane prepared by the invention is fast, convenient, cheap and efficient, can efficiently purify thiol protease, and is suitable for large-scale production.

Description of drawings

Figure 1 is a scanning electron microscope photo of nylon 6/chitosan nanofibers prepared by electrospinning and the affinity membrane obtained after treatment: A is nylon 6/chitosan nanofiber without any treatment; B is affinity membrane;

Fig. 2 is the impact curve of the change of reaction concentration on papain adsorption;

Fig. 3 is the dynamic adsorption test curve to papain.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

Preparation of nanocomposite affinity membranes for fast and efficient separation and purification of thiol proteases, including:

(1) The mixture of hexafluoroisopropanol (1,1,1,3,3,3,3-hexafluoroisopropanol) and formic acid with a volume ratio of 9 to 12:1 is used as a solvent system and mixed into the reaction vessel at a temperature of 40°C, shaking reaction in a water bath for 20-45 minutes, wherein the mass concentration of hexafluoroisopropanol is >99%;

(2) Nylon 6 and chitosan powder are slowly added in the above-mentioned reaction vessel under stirring conditions, and continue to stir for 2 to 3 hours to completely swell, wherein the mass concentration of nylon 6 is 5wt%, and the mass concentration of chitosan is 2wt%;

(3) Place the above-mentioned reaction vessel in a water-bath oscillator, under reflux condensation, slowly heat to 40°C, shake for 15h-30h until completely dissolved, and obtain nylon 6/chitosan spinning solution;

(4) Extract the nylon 6/chitosan spinning solution with a syringe, fix it on the electrospinning device, adjust the spinning parameters for electrospinning, the static voltage is 10-18kv; the receiving screen is received by aluminum foil grounding, the needle and the receiving screen The distance is 10～20cm, get nylon 6/chitosan ultrafine nanofiber film;

(5) Dry the spun film at room temperature for 12 hours, then dry it in an oven at 90°C for 4-6 hours, and finally put it in a vacuum dryer at 60-70°C for 24 hours;

(6) cutting the dried nylon 6/chitosan nanofiber membrane into circular membranes with a diameter of 47 mm, putting it into 50-60° C., and activating it in a 10-15% NaOH solution for 20-30 min;

(7) Nylon 6/chitosan nanofiber film after activation, after washing several times with hot water, put into the dye Cibacron Blue F3GA solution that concentration is 5～15mg/ml and react;

(8) process 20-45min with the NaCl solution of 20～30wt% in 40 ℃ of water-bath oscillations, dyestuff is adsorbed on the nylon 6/chitosan nanofiber film;

(9) Adjust the temperature of the water bath, and react with 25wt% Na ₂ CO ₃ in a 90°C water bath for 3 to 6 hours;

(10) Cool, wash with 50°C hot water, methanol, 2M NaCl, 6M urea with a content of 99.5% to 100% successively, and after fully washing with double distilled water, the washing degree is that the washing liquid after washing is colorless or preserved In Tris-HCl buffer solution, the nylon 6/chitosan nanofiber affinity membrane with dye Cibacron Blue F3GA as ligand was obtained.

Example 2

Weigh 6 groups of 0.1g cellulose affinity membranes, add buffer and enzyme solutions with concentrations of 0, 0.5, 1.0, 1.5, and 2.0mg/ml respectively, shake in a water bath at 37°C for 3 hours, and measure the adsorption at 280nm before and after adsorption. The absorbance; Calculate the concentration of the papain with the largest adsorption amount, and use it as the following experiment. Under the same conditions, the cellulose membrane without any treatment was used as a parallel comparison experiment to measure the non-specific adsorption amount and compare it with the affinity membrane adsorption amount. It was found that the optimal enzyme dosage was 2.0mg/ml. Figure 2 is the adsorption curves of affinity membrane and untreated nylon 6/chitosan nanofibers to different concentrations of papain.

Example 3

Stack 20 affinity membranes into a membrane stack and load them into the membrane bridge. At room temperature, first equilibrate the membrane stack with buffer solution, then load the papaya powder solution prepared with buffer solution at an appropriate flow rate, and then wash the membrane stack with buffer solution to remove unadsorbed protein on the membrane until it flows out The absorbance A ₂₈₀ value of liquid is close to 0, at last, elute the adsorbed papain with appropriate eluent, collect components in separate tubes, the component volume of each tube is 4ml, measure the absorbance A ₂₈₀ value of each component, with A ₂₈₀ is the ordinate, the volume is the abscissa, and the adsorption-elution curve of papain is obtained. Fig. 3 is the dynamic adsorption test curve to papain.

Claims (8)

1. be used for the preparation of the nanometer composite affinity membrane of rapidly and efficiently separating and purifying thiol protease, comprise:

(1) with volume ratio be 9～12: 1 hexafluoroisopropanol HFIP and formic acid mixed liquor as dicyandiamide solution, blending is gone in the reaction vessel, temperature is 40～60 ℃, water-bath oscillating reactions 20～45min;

(2) nylon 6 and shitosan powder are slowly added in the above-mentioned reaction vessel under stirring condition, continue to stir 2～3h to complete swelling, wherein nylon 6 granular mass concentration are 5～8wt%, and the mass concentration of shitosan is 0～2wt%;

(3) above-mentioned reaction vessel is placed in the water bath chader, under reflux condensation mode, slowly be heated to 40～60 ℃, vibration 15h～30h gets nylon 6/ shitosan spinning solution to dissolving fully;

(4) extract nylon 6/ shitosan spinning solution with syringe, be fixed on the electrostatic spinning apparatus, regulate spinning parameter and carry out electrospinning, electrostatic pressure is 10～18kv; Receiving screen adopts the reception of aluminium foil ground connection, and the distance of syringe needle and receiving screen is 10～20cm, gets nylon 6/ shitosan superfine nano tunica fibrosa, and is dry then;

(5) dried nylon 6/ chitosan nano fiber membrane is cut into circular film, puts into 50～60 ℃, concentration is to activate 20-30min in the NaOH solution of 10～15wt%;

(6), behind the hot water wash several, put in the dye solution and react with above-mentioned tunica fibrosa;

(7) with the NaCl solution of 20～30wt% at 40 ℃～80 ℃ water-bath oscillation treatment 20～45min, dyestuff is adsorbed onto on nylon 6/ chitosan nano fiber membrane;

(8) regulate bath temperature, with 25～30wt%Na ₂CO ₃At 60 ℃～90 ℃ water-bath oscillating reactions 3～6h;

(9) cooling, washing, or be stored in the Tris-HCl buffer solution, obtaining with the dyestuff is the nylon 6/ chitosan nano fiber affinity membrane of aglucon.

2. the preparation that is used for the nanometer composite affinity membrane of rapidly and efficiently separating and purifying thiol protease according to claim 1 is characterized in that: the mass concentration of described step (1) hexafluoroisopropanol HIFP is 99%～100%, and water content is less than 0.1%.

3. the preparation that is used for the nanometer composite affinity membrane of rapidly and efficiently separating and purifying thiol protease according to claim 1 is characterized in that: the syringe specification in the described step (4) is 5ml, and the syringe needle internal diameter is about 0.4～0.7mm; The ejection flow velocity is 0.5～2ml/h.

4. the preparation that is used for the nanometer composite affinity membrane of rapidly and efficiently separating and purifying thiol protease according to claim 1, it is characterized in that: the drying means in the described step (4) is earlier with dry 12h under the spun film room temperature, put into 90 ℃ of drying 4～6h of baking oven then, put into 60～70 ℃ of dry 24h of vacuum desiccator at last.

5. the preparation that is used for the nanometer composite affinity membrane of rapidly and efficiently separating and purifying thiol protease according to claim 1 is characterized in that: the diameter of described step (5) circular film is 47mm.

6. the preparation that is used for the nanometer composite affinity membrane of rapidly and efficiently separating and purifying thiol protease according to claim 1 is characterized in that: described step (6) dyestuff is dyestuff Cibacron Blue F3GA, and concentration is 5～15mg/ml.

7. the preparation that is used for the nanometer composite affinity membrane of rapidly and efficiently separating and purifying thiol protease according to claim 1, it is characterized in that: the washing in the described step (9) is to be 99.5～100% methyl alcohol, 2～3M NaCl, the washing of 3～6M urea with 40～50 ℃ of hot water, content, the washing degree be the cleaning solution after washing be colourless.

8. the preparation that is used for the nanometer composite affinity membrane of rapidly and efficiently separating and purifying thiol protease according to claim 1 is characterized in that: described nanometer composite affinity membrane is applied to the separation and purification of thiol protease.

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