CN111335030A - Preparation method of carboxylated fibroin nanofiber adsorption film and method for recovering lysozyme - Google Patents
Preparation method of carboxylated fibroin nanofiber adsorption film and method for recovering lysozyme Download PDFInfo
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Abstract
A preparation method of a carboxylated fibroin nanofiber adsorption film and a method for recovering lysozyme by using the same comprise the following steps: 1) spinning into a nano-scale silk fibroin film; 2) performing modified grafting treatment to prepare a carboxylated fibroin nanofiber adsorption film; 3) adsorbing lysozyme in the solution to be recovered; 4) and recovering the lysozyme adsorbed by the carboxylated fibroin nanofibers. According to the invention, a green biomass silkworm cocoon material is used as a raw material, a nano-scale silk fibroin membrane material is prepared by using an electrostatic spinning technology, then, 3-sodium carboxyl benzene sulfonate and the silk fibroin membrane material are subjected to modified grafting by adopting a DEC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) reaction, and finally, a carboxylated silk fibroin nanofiber adsorption membrane material is prepared, has a good adsorption effect on lysozyme, can desorb the lysozyme by changing experimental conditions, and can be repeatedly used.
Description
Technical Field
The invention relates to the technical field of lysozyme recovery, in particular to a preparation method of a carboxylated fibroin nanofiber adsorption film and a lysozyme recovery method thereof.
Background
The lysozyme is a hydrolase which acts on the cell wall of microorganisms specifically, and in the nature, the content of the lysozyme in the white of birds and poultry eggs is more, about 0.2-0.4%. Lysozyme, usually extracted from egg white, consists of 129 amino acid residues, is a basic protein, has four pairs of disulfide bonds, and is rich in basic amino acids. With the improvement of living standards and the progress of science and technology, the addition of chemicals to medicines and foods is beginning to be restricted by many countries, and therefore lysozyme having natural activity is receiving great attention because of its high efficiency and safety. As a safe and nontoxic protein, lysozyme can selectively decompose microbial cell walls without affecting other tissues, so that the lysozyme has good bacteriolysis effect, is a safe and reliable bactericide with good performance, and is widely applied to the industries of food preservation, pharmaceutical preparations, chemical engineering and the like. Compared with the prior art, the scale of the lysozyme manufacturing industry in China still has a large difference, and the production technology is still relatively lagged behind. Therefore, the production supply of domestic high-activity egg white lysozyme cannot meet the increasing demand of China, and the output of lysozyme directly influences the application of lysozyme. Therefore, the selection of an economical and efficient lysozyme separation and purification method is still the current research focus. The prior extraction method of egg white lysozyme mainly comprises a direct extraction method, an ultrafiltration method, a crystallization method, an ion exchange method, a chromatography method, an affinity separation method and the like. Although the above method has a certain extraction effect, there are some problems associated with the method. For example, when lysozyme is produced by a crystallization method, eutectic crystals are easy to occur, and the lysozyme cannot be repeatedly used.
Disclosure of Invention
One of the purposes of the invention is to provide a preparation method of a carboxylated fibroin nanofiber adsorption film.
The invention is realized by the technical scheme, and the method comprises the following specific steps:
1) cocoon silk is taken as a raw material, and a nano-scale silk fibroin membrane material is spun by adopting an electrostatic spinning technology;
2) and 3-sodium carboxyl benzene sulfonate is used for modifying and grafting the silk fibroin membrane to prepare the carboxylated silk fibroin nanofiber adsorption membrane.
Further, the specific steps of spinning the nano-scale silk fibroin cellulose membrane material in the step 1) are as follows:
1-1) degumming cocoon silk by using a sodium carbonate solution at a high temperature to obtain silk fibroin; the concentration range of the sodium carbonate solution is 0.3 wt% -0.8 wt%, and the temperature range is 90-100 ℃;
1-2) dissolving the silk fibroin fibers in a lithium bromide solution, dialyzing and filtering, taking out the silk fibroin fibers, and freeze-drying to obtain regenerated silk fibroin; the concentration range of the lithium bromide solution is 8mol L-1～10mol L-1The silk fibroin fiber and the lithium bromide solution respectively comprise the following components in parts by weight: 1-3 parts of silk fibroin fiber and 700-900 parts of lithium bromide solution;
1-3) dissolving the regenerated silk fibroin in a formic acid solution to form a spinning solution; the concentration range of the formic acid solution is 90 wt% -98 wt%, and the regenerated silk fibroin and the formic acid solution respectively comprise the following components in parts by weight: 1-3 parts of regenerated silk fibroin and 500-800 parts of formic acid solution;
1-4) spinning the spinning solution as a raw material into a nano-scale silk fibroin film material by adopting an electrostatic spinning technology, wherein the electrostatic spinning technology parameters are voltage of 20 kV-30 kV, receiving range of 5 cm-10 cm and filling speed range of 0.7mL h-1～ 1.1mLh-1The temperature is 20-30 ℃, the humidity is 35-55 percent, and the aperture range of the nozzle is 0.3-0.5 mm.
Further, the concentration of the sodium carbonate solution in the step 1-1) is 0.5 wt%;
the concentration range of the lithium bromide solution in the step 1-2) is 9mol L -12 parts of silk fibroin fiber and 800 parts of lithium bromide solution;
in the step 1-3), the concentration of the formic acid solution is 98%, the weight part of the regenerated fibroin is 2, and the weight part of the formic acid solution is 650.
Further, 2-1) preparing a 3-sodium carboxyl benzene sulfonate solution with the concentration range of 1g L-1～5g L-1；
2-2) concentration range of 3g L-1～7g L-1Adding 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) into a 3-sodium carboxybenzenesulfonate solution, and fully stirring at room temperature for 50-70 minutes to prepare a mixed solution; the weight portions of the 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and the 3-sodium carboxybenzenesulfonate solution are respectively as follows: 1-5 parts of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 2-6 parts of 3-sodium carboxybenzenesulfonate solution;
2-3) putting the fibroin membrane into the mixed solution and fully stirring, taking out the carboxylated fibroin nanofiber adsorption membrane after the reaction is finished, fully washing the carboxylated fibroin nanofiber adsorption membrane with an acetone solution, and drying the membrane in a vacuum oven to obtain a carboxylated fibroin nanofiber adsorption membrane material; the silk fibroin film and the mixed solution respectively comprise the following components in parts by weight: 1-5 parts of silk fibroin cellulose membrane and 400-800 parts of mixed solution.
Further, the concentration of the sodium 3-carboxybenzenesulfonate solution in step 2-1) is 3g L-1；
The concentration of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) in step 2-2) was 5g L-15 parts of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 1000 parts of 3-sodium carboxybenzenesulfonate solution;
in the step 2-3), the weight parts of the silk cellulose membrane is 1-3, and the weight parts of the mixed solution is 1000.
The invention also aims to provide a method for recovering lysozyme by using the carboxylated fibroin nanofiber adsorption film.
The invention is realized by the technical scheme, and the method comprises the following specific steps:
3) placing the carboxylated fibroin nanofiber adsorption film in the solution to be recovered, and adsorbing lysozyme in the solution to be recovered;
4) and taking out the carboxylated fibroin nanofiber adsorption film from the solution to be recovered, and recovering the lysozyme adsorbed by the carboxylated fibroin nanofiber.
Further, the specific method for adsorbing lysozyme in the solution to be recovered in the step 3) is as follows:
3-1) adjusting the pH value of the solution to be recovered to 3-5, and fully stirring the solution;
3-2) placing the carboxylated fibroin nanofiber adsorption membrane material in a solution to be recovered for 8-14 hours, fully stirring, and gradually adsorbing lysozyme onto the fiber membrane; when the temperature of the recovered solution is 20-30 ℃, the concentration of the lysozyme is 3g L-1～7gL-1。
Further, the pH value in the step 3-1) is 4;
the temperature in the step 3-2) is 25 ℃, and the lysozyme concentration is 5g L-1And time 12 hours.
Further, the specific method for recovering lysozyme adsorbed by the carboxylated fibroin nanofibers in step 4) is as follows:
4-1) taking out the carboxylated fibroin nanofiber adsorption film adsorbing the lysozyme from the solution to be recovered, and placing the film in a new water phase; the new water phase and the carboxylated fibroin nanofiber adsorption film respectively comprise the following components in parts by weight: 200-600 parts of new water phase and 1-3 parts of carboxylated fibroin nanofiber adsorption film; the temperature of the new water phase is 20-30 ℃, and the humidity is 35-45%;
4-2) adding 0.5mol L of sodium hydroxide-1～1.5mol L-1The sodium chloride solution is used for 3 to 5 hours, the temperature is 20 to 30 ℃, and the lysozyme on the carboxylated fibroin nanofiber adsorption film is gradually desorbed into a new water phase.
Further, in the step 4-1), 400 parts of new water phase and 2 parts of carboxylated fibroin nanofiber adsorption film are adopted, the temperature is 25 ℃, and the humidity is 40%;
adding 1.0mol L of sodium hydroxide in the step 4-2)-1At a temperature of 25 ℃ for 4 hours.
Due to the adoption of the technical scheme, the invention has the following advantages:
the invention makes the carboxylated fibroin nano-fiber adsorption membrane material applied to the adsorption and recovery of lysozyme, the method is different from the traditional lysozyme recovery method, and the silk fiber has better biocompatibility as a green and environment-friendly biomass fiber, the side chain of the silk fibroin nano-fiber membrane material has the amino acid reaction groups of arginine, serine, threonine and the like, so that the possibility of fixing other functional molecules through modification is provided, more importantly, the preparation method is simple, the adopted EDC reaction ensures that the surface of the silk fibroin nano-fiber membrane material is rich in carboxylated negative ions, has stronger adsorption effect on lysozyme, can desorb the lysozyme from a membrane material by methods of adding sodium chloride, changing the pH value and the like to achieve the aim of recycling, and the membrane material has small fiber diameter and large specific surface area, and can be repeatedly used, thereby having good application prospect.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof.
Drawings
The drawings of the present invention are described below.
FIG. 1 is a schematic view of the preparation of a silk fibroin membrane material and the adsorption and recovery process of lysozyme;
FIG. 2 is a graph showing the results of the experiment in example 1;
FIG. 3 is a graph showing the results of the experiment in example 2;
FIG. 4 is a graph showing the results of the experiment in example 3;
FIG. 5 is a graph showing the desorption of lysozyme by sodium chloride in example 4;
fig. 6 is a surface morphology diagram of the carboxylated silk fibroin nanofiber adsorbent membrane of example 4 after repeated use.
Detailed Description
The invention is further illustrated by the following figures and examples.
Example 1:
a preparation method of a carboxylated fibroin nanofiber adsorption film and a method for recovering lysozyme by using the same are shown in figure 1, and specifically comprise the following steps:
1) cocoon silk is used as a raw material, and a nano-scale silk fibroin film material is spun by adopting an electrostatic spinning technology.
1-1) degumming cocoon silk by using a sodium carbonate solution at a high temperature to obtain silk fibroin; the concentration of the sodium carbonate solution is 0.3 wt%, and the temperature is 90 ℃;
1-2) dissolving the silk fibroin fibers in a lithium bromide solution, dialyzing and filtering, taking out the silk fibroin fibers, and freeze-drying to obtain regenerated silk fibroin; the concentration of the lithium bromide solution is 8mol L-1The silk fibroin fiber and the lithium bromide solution respectively comprise the following components in parts by weight: 1 part of silk fibroin fiber and 700 parts of lithium bromide solution;
1-3) dissolving the regenerated silk fibroin in a formic acid solution to form a spinning solution; the concentration of the formic acid solution is 90 wt%, and the regenerated fibroin and the formic acid solution respectively comprise the following components in parts by weight: 1 part of regenerated silk fibroin and 500 parts of formic acid solution;
1-4) spinning the spinning solution into a nano-scale silk fibroin film material by adopting an electrostatic spinning technology. Voltage 25kV, receiving range 7cm, perfusion speed 1.0mL h-1The temperature is 25 ℃, the humidity is 40 percent, and the aperture range of the nozzle is 0.4 mm.
2) And 3-sodium carboxyl benzene sulfonate is used for modifying and grafting the silk fibroin membrane to prepare the carboxylated silk fibroin nanofiber adsorption membrane.
2-1) preparing a 3-carboxyl sodium benzenesulfonate solution with the concentration of 1g L-1；
2-2) concentration range 3g L-1Adding 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) into a 3-sodium carboxybenzenesulfonate solution, and fully stirring at room temperature to prepare a mixed solution; the weight portions of the 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and the 3-sodium carboxybenzenesulfonate solution are respectively as follows: 1 part of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 2 parts of 3-sodium carboxybenzenesulfonate solution;
2-3) putting the silk fibroin membrane into the mixed solution and fully stirring, taking out the silk fibroin membrane material after the reaction is finished, and fully washing and drying to obtain a carboxylated silk fibroin nanofiber adsorption membrane material; the silk fibroin film and the mixed solution respectively comprise the following components in parts by weight: 1 part of silk fibroin film and 400 parts of mixed solution; and after the reaction is finished, taking out the carboxylated fibroin nanofiber adsorption film, fully washing the adsorption film with an acetone solution, and drying the adsorption film in a vacuum oven for later use.
3) Placing 1 part of carboxylated fibroin nanofiber adsorption film in 200 parts of solution to be recovered, and adsorbing lysozyme in the solution to be recovered; containing lysozyme 3g L of the solution to be recovered-1。
3-1) adjusting the pH value of the solution to be recovered to 3, and fully stirring the solution;
3-2) placing the carboxylated fibroin nanofiber adsorption membrane material into a solution to be recovered, fully stirring, and gradually adsorbing lysozyme onto the fiber membrane; the temperature is 20 ℃ in the experimental process, and the concentration of the lysozyme in the solution to be recovered is 3g L-1The recovery time was 12 hours.
4) And taking out the carboxylated fibroin nanofiber adsorption film from the solution to be recovered, and recovering the lysozyme adsorbed by the carboxylated fibroin nanofiber.
4-1) taking 1 part of the carboxylated fibroin nanofiber adsorption film adsorbing lysozyme out of 200 parts of the solution to be recovered, and placing the film in a new water phase, wherein the temperature of the new water phase is 20 ℃, and the experimental humidity is 35%.
4-2) adding 0.5mol L of sodium hydroxide-1After 4 hours, the temperature is kept at 25 ℃, lysozyme on the carboxylated fibroin nanofiber adsorption film is gradually desorbed into a new water phase, and the experimental result is shown in figure 2.
Example 2:
1) cocoon silk is used as a raw material, and a nano-scale silk fibroin film material is spun by adopting an electrostatic spinning technology.
1-1) degumming cocoon silk by using a sodium carbonate solution at a high temperature to obtain silk fibroin; the concentration of the sodium carbonate solution is 0.8 wt%, and the temperature is 100 ℃;
1-2) dissolving the silk fibroin fiber in a lithium bromide solution,after dialysis and filtration, taking out the silk fibroin and freeze-drying the silk fibroin to obtain regenerated silk fibroin; the concentration of the lithium bromide solution is 10mol L-1The silk fibroin fiber and the lithium bromide solution respectively comprise the following components in parts by weight: 3 parts of silk fibroin fiber and 900 parts of lithium bromide solution;
1-3) dissolving the regenerated silk fibroin in a formic acid solution to form a spinning solution; the concentration of the formic acid solution is 98 wt%, and the regenerated fibroin and the formic acid solution respectively comprise the following components in parts by weight: 3 parts of regenerated silk fibroin and 800 parts of formic acid solution;
1-4) spinning the spinning solution into a nano-scale silk fibroin film material by adopting an electrostatic spinning technology. Voltage 25kV, receiving range 7cm, perfusion speed 1.0mL h-1The temperature is 25 ℃, the humidity is 40 percent, and the aperture range of the nozzle is 0.4 mm.
2) And 3-sodium carboxyl benzene sulfonate is used for modifying and grafting the silk fibroin membrane to prepare the carboxylated silk fibroin nanofiber adsorption membrane.
2-1) preparing a 3-carboxyl sodium benzenesulfonate solution with the concentration of 5g L-1；
2-2) concentration range 7g L-1Adding 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) into a 3-sodium carboxybenzenesulfonate solution, and fully stirring at room temperature to prepare a mixed solution; the weight portions of the 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and the 3-sodium carboxybenzenesulfonate solution are respectively as follows: 5 parts of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 6 parts of 3-sodium carboxybenzenesulfonate solution;
2-3) putting the silk fibroin membrane into the mixed solution and fully stirring, taking out the silk fibroin membrane material after the reaction is finished, and fully washing and drying to obtain a carboxylated silk fibroin nanofiber adsorption membrane material; the silk fibroin film and the mixed solution respectively comprise the following components in parts by weight: 3 parts of silk fibroin film and 800 parts of mixed solution; and after the reaction is finished, taking out the carboxylated fibroin nanofiber adsorption film, fully washing the adsorption film with an acetone solution, and drying the adsorption film in a vacuum oven for later use.
3) Placing 1 part of carboxylated fibroin nanofiber adsorption film in 200 parts of solution to be recovered, and adsorbing lysozyme in the solution to be recovered; containing lysozyme 7g L of the solution to be recovered-1。
3-1) adjusting the pH value of the solution to be recovered to 5, and fully stirring the solution;
3-2) placing the carboxylated fibroin nanofiber adsorption membrane material into a solution to be recovered, fully stirring, and gradually adsorbing lysozyme onto the fiber membrane; the temperature is 30 ℃ in the experimental process, and the concentration of the lysozyme in the solution to be recovered is 7g L-1The recovery time was 12 hours.
4) And taking out the carboxylated fibroin nanofiber adsorption film from the solution to be recovered, and recovering the lysozyme adsorbed by the carboxylated fibroin nanofiber.
4-1) taking 3 parts of the carboxylated fibroin nanofiber adsorption film adsorbing lysozyme out of 600 parts of the solution to be recovered, and placing the film in a new water phase, wherein the temperature of the new water phase is 30 ℃, and the experimental humidity is 45%.
4-2) adding 1.5mol L of sodium hydroxide-1After 4 hours, the temperature is kept at 25 ℃, lysozyme on the carboxylated fibroin nanofiber adsorption film is gradually desorbed into a new water phase, and the experimental result is shown in fig. 3.
Example 3:
1) cocoon silk is used as a raw material, and a nano-scale silk fibroin film material is spun by adopting an electrostatic spinning technology.
1-1) degumming cocoon silk by using a sodium carbonate solution at a high temperature to obtain silk fibroin; the concentration of the sodium carbonate solution is 0.5 wt%, and the temperature is 100 ℃;
1-2) dissolving the silk fibroin fibers in a lithium bromide solution, dialyzing and filtering, taking out the silk fibroin fibers, and freeze-drying to obtain regenerated silk fibroin; the concentration of the lithium bromide solution was 9mol L-1The silk fibroin fiber and the lithium bromide solution respectively comprise the following components in parts by weight: 2 parts of silk fibroin fiber and 800 parts of lithium bromide solution;
1-3) dissolving the regenerated silk fibroin in a formic acid solution to form a spinning solution; the concentration of the formic acid solution is 98 wt%, and the regenerated fibroin and the formic acid solution respectively comprise the following components in parts by weight: 2 parts of regenerated silk fibroin and 700 parts of formic acid solution;
1-4) spinning the spinning solution into a nano-scale silk fibroin film material by adopting an electrostatic spinning technology. Voltage 25kV, receiving range 7cm, and perfusion speedDegree of 1.0mL h-1The temperature is 25 ℃, the humidity is 40 percent, and the aperture range of the nozzle is 0.4 mm.
2) And 3-sodium carboxyl benzene sulfonate is used for modifying and grafting the silk fibroin membrane to prepare the carboxylated silk fibroin nanofiber adsorption membrane.
2-1) preparing 3-carboxyl sodium benzenesulfonate solution with the concentration of 3g L-1；
2-2) concentration range 5g L-1Adding 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) into a 3-sodium carboxybenzenesulfonate solution, and fully stirring at room temperature to prepare a mixed solution; the weight portions of the 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and the 3-sodium carboxybenzenesulfonate solution are respectively as follows: 3 parts of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 4 parts of 3-sodium carboxybenzenesulfonate solution;
2-3) putting the silk fibroin membrane into the mixed solution and fully stirring, taking out the silk fibroin membrane material after the reaction is finished, and fully washing and drying to obtain a carboxylated silk fibroin nanofiber adsorption membrane material; the silk fibroin film and the mixed solution respectively comprise the following components in parts by weight: 2 parts of silk fibroin film and 400 parts of mixed solution; and after the reaction is finished, taking out the carboxylated fibroin nanofiber adsorption film, fully washing the adsorption film with an acetone solution, and drying the adsorption film in a vacuum oven for later use.
3) Placing 1 part of carboxylated fibroin nanofiber adsorption film in 200 parts of solution to be recovered, and adsorbing lysozyme in the solution to be recovered; containing lysozyme 5g L of the solution to be recovered-1。
3-1) adjusting the pH value of the solution to be recovered to 4, and fully stirring the solution;
3-2) placing the carboxylated fibroin nanofiber adsorption membrane material into a solution to be recovered, fully stirring, and gradually adsorbing lysozyme onto the fiber membrane; the temperature is 25 ℃ in the experimental process, and the concentration of the lysozyme in the solution to be recovered is 5g L-1The recovery time was 12 hours.
4) And taking out the carboxylated fibroin nanofiber adsorption film from the solution to be recovered, and recovering the lysozyme adsorbed by the carboxylated fibroin nanofiber.
4-1) taking 3 parts of the carboxylated fibroin nanofiber adsorption film adsorbing lysozyme out of 600 parts of the solution to be recovered, and placing the film in a new water phase, wherein the temperature of the new water phase is 25 ℃, and the experimental humidity is 40%.
4-2) adding 1.0mol L of sodium hydroxide-1After 4 hours, the temperature is kept at 25 ℃, lysozyme on the carboxylated fibroin nanofiber adsorption film is gradually desorbed into a new water phase, and the experimental result is shown in fig. 3.
Example 4:
preparing a carboxylated fibroin nanofiber adsorption film under the conditions of example 3, recovering lysozyme, and recycling for 10 times, wherein the experimental component temperature of the solution to be recovered, which is 2 parts of the carboxylated fibroin nanofiber adsorption film and 400 parts of the solution to be recovered, is 25 ℃, and the lysozyme concentration is 5g L-1The results of the experiment are shown in FIGS. 4 and 5.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the embodiments of the invention without departing from the spirit and scope of the invention, which is to be covered by the claims.
Claims (10)
1. A preparation method of a carboxylated fibroin nanofiber adsorption film is characterized by comprising the following specific steps:
1) cocoon silk is taken as a raw material, and a nano-scale silk fibroin membrane material is spun by adopting an electrostatic spinning technology;
2) and 3-sodium carboxyl benzene sulfonate is used for modifying and grafting the silk fibroin membrane to prepare the carboxylated silk fibroin nanofiber adsorption membrane.
2. The preparation method of the carboxylated silk fibroin nanofiber adsorbent membrane as claimed in claim 1, wherein the specific steps of spinning into the nanoscale silk fibroin membrane material in step 1) are as follows:
1-1) degumming cocoon silk by using a sodium carbonate solution at a high temperature to obtain silk fibroin; the concentration range of the sodium carbonate solution is 0.3 wt% -0.8 wt%, and the temperature range is 90-100 ℃;
1-2) dissolving the silk fibroin fibers in a lithium bromide solution, dialyzing and filtering, taking out the silk fibroin fibers, and freeze-drying to obtain regenerated silk fibroin; the concentration range of the lithium bromide solution is 8mol L-1～10mol L-1The silk fibroin fiber and the lithium bromide solution respectively comprise the following components in parts by weight: 1-3 parts of silk fibroin fiber and 700-900 parts of lithium bromide solution;
1-3) dissolving the regenerated silk fibroin in a formic acid solution to form a spinning solution; the concentration range of the formic acid solution is 90 wt% -98 wt%, and the regenerated silk fibroin and the formic acid solution respectively comprise the following components in parts by weight: 1-3 parts of regenerated silk fibroin and 500-800 parts of formic acid solution;
1-4) spinning the spinning solution as a raw material into a nano-scale silk fibroin film material by adopting an electrostatic spinning technology, wherein the electrostatic spinning technology parameters are voltage of 20 kV-30 kV, receiving range of 5 cm-10 cm and filling speed range of 0.7mL h-1～1.1mL h-1The temperature is 20-30 ℃, the humidity is 35-55 percent, and the aperture range of the nozzle is 0.3-0.5 mm.
3. The preparation method of the carboxylated silk fibroin nanofiber adsorption film as claimed in claim 2,
the concentration of the sodium carbonate solution in the step 1-1) is 0.5 wt%;
the concentration range of the lithium bromide solution in the step 1-2) is 9molL-12 parts of silk fibroin fiber and 800 parts of lithium bromide solution;
in the step 1-3), the concentration of the formic acid solution is 98%, the weight part of the regenerated fibroin is 2, and the weight part of the formic acid solution is 650.
4. The method for preparing the carboxylated fibroin nanofiber adsorption film according to claim 1, wherein the specific method for preparing the carboxylated fibroin nanofiber adsorption film in step 2) is as follows:
2-1) preparing 3-carboxyl sodium benzenesulfonate solution with the concentration range of 1g L-1～5g L-1；
2-2)The concentration range is 3g L-1～7g L-1Adding 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) into a 3-sodium carboxybenzenesulfonate solution, and fully stirring at room temperature for 50-70 minutes to prepare a mixed solution; the weight portions of the 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and the 3-sodium carboxybenzenesulfonate solution are respectively as follows: 1-5 parts of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 2-6 parts of 3-sodium carboxybenzenesulfonate solution;
2-3) putting the fibroin membrane into the mixed solution and fully stirring, taking out the carboxylated fibroin nanofiber adsorption membrane after the reaction is finished, fully washing the carboxylated fibroin nanofiber adsorption membrane with an acetone solution, and drying the membrane in a vacuum oven to obtain a carboxylated fibroin nanofiber adsorption membrane material; the silk fibroin film and the mixed solution respectively comprise the following components in parts by weight: 1-5 parts of silk fibroin cellulose membrane and 400-800 parts of mixed solution.
5. The preparation method of the carboxylated silk fibroin nanofiber adsorption film as claimed in claim 4,
the concentration of the sodium 3-carboxybenzenesulfonate solution in step 2-1) is 3g L-1；
The concentration of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) in step 2-2) was 5g L-15 parts of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 1000 parts of 3-sodium carboxybenzenesulfonate solution;
in the step 2-3), the weight parts of the silk cellulose membrane is 1-3, and the weight parts of the mixed solution is 1000.
6. A method for recovering lysozyme by using a carboxylated fibroin nanofiber adsorption film prepared by any one of claims 1 to 5, which is characterized by comprising the following specific steps:
3) placing the carboxylated fibroin nanofiber adsorption film in the solution to be recovered, and adsorbing lysozyme in the solution to be recovered;
4) and taking out the carboxylated fibroin nanofiber adsorption film from the solution to be recovered, and recovering the lysozyme adsorbed by the carboxylated fibroin nanofiber.
7. The method for recovering lysozyme through the carboxylated silk fibroin nanofiber adsorption film as claimed in claim 6, wherein the specific method for adsorbing lysozyme in the solution to be recovered in the step 3) is as follows:
3-1) adjusting the pH value of the solution to be recovered to 3-5, and fully stirring the solution;
3-2) placing the carboxylated fibroin nanofiber adsorption membrane material in a solution to be recovered for 8-14 hours, fully stirring, and gradually adsorbing lysozyme onto the fiber membrane; when the temperature of the recovered solution is 20-30 ℃, the concentration of the lysozyme is 3g L-1～7g L-1。
8. The method for recovering lysozyme through the carboxylated fibroin nanofiber adsorption film as claimed in claim 7, wherein:
the pH value in the step 3-1) is 4;
the temperature in the step 3-2) is 25 ℃, and the lysozyme concentration is 5g L-1And time 12 hours.
9. The method for recovering lysozyme through the carboxylated fibroin nanofiber adsorption film according to claim 6, wherein the specific method for recovering lysozyme adsorbed by the carboxylated fibroin nanofiber in the step 4) is as follows:
4-1) taking out the carboxylated fibroin nanofiber adsorption film adsorbing the lysozyme from the solution to be recovered, and placing the film in a new water phase; the new water phase and the carboxylated fibroin nanofiber adsorption film respectively comprise the following components in parts by weight: 200-600 parts of new water phase and 1-3 parts of carboxylated fibroin nanofiber adsorption film; the temperature of the new water phase is 20-30 ℃, and the humidity is 35-45%;
4-2) adding 0.5mol L of sodium hydroxide-1～1.5mol L-1The sodium chloride solution is used for 3 to 5 hours, the temperature is 20 to 30 ℃, and the lysozyme on the carboxylated fibroin nanofiber adsorption film is gradually desorbed into a new water phase.
10. The method for recovering lysozyme through the carboxylated fibroin nanofiber adsorption film as claimed in claim 9, wherein:
in the step 4-1), 400 parts of new water phase and 2 parts of carboxylated fibroin nanofiber adsorption film are carried out at the temperature of 25 ℃ and the humidity of 40 percent;
adding 1.0mol L of sodium hydroxide in the step 4-2)-1At a temperature of 25 ℃ for 4 hours.
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