CN117777266A - Preparation method of natural spider silk protein/polypeptide with controllable molecular weight, spider silk protein/polypeptide and application thereof - Google Patents
Preparation method of natural spider silk protein/polypeptide with controllable molecular weight, spider silk protein/polypeptide and application thereof Download PDFInfo
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Abstract
The invention provides a preparation method of natural spider silk protein/polypeptide with controllable molecular weight, spider silk protein/polypeptide and application thereof, and belongs to the technical field of animal protein preparation. The preparation method provided by the invention comprises the following steps: 1) Freezing, ball milling and crushing natural spider silk to obtain spider silk powder; 2) Carrying out protein dissolution on spider silk powder to obtain spider silk protein hydrolysate; 3) Centrifuging the spider silk protein hydrolysate to remove impurities and obtain supernatant; 4) Purifying the supernatant to obtain purified spider silk protein hydrolysate; 5) Filtering and removing impurities from the purified spider silk protein hydrolysate to obtain purified protein/polypeptide hydrolysate. The invention adopts spider silk as raw material, and utilizes high-pressure forced hydrolysis process to make hydrolysis, extraction and purification of natural spider silk protein, and innovatively invents a quick, high-efficiency and controllable method for hydrolyzing natural spider silk, and the prepared spider silk protein/polypeptide has high purity, high concentration and controllable molecular weight.
Description
Technical Field
The invention belongs to the technical field of animal protein preparation, and particularly relates to a preparation method of natural spider silk protein/polypeptide with controllable molecular weight, spider silk protein/polypeptide and application thereof.
Background
Spider silk is a natural protein fiber of animal origin, is composed of proteins with multiple levels of different amino acids, and has unique mechanical, thermal, optical and biological properties. Natural spider silks have excellent strength, ultra-low temperature elasticity and flexibility, and excellent impact resistance. In addition, natural spider silks also exhibit unique moisture response, water collection, light transmission, heat conduction, and shape memory properties. Based on the many excellent properties described above, natural spider silk materials are considered as potential natural specialty fiber materials. In the aspect of biology, researches show that the natural spider silk has excellent biocompatibility and shows a plurality of biological characteristics of unique biological pollution resistance, hemostasis, healing promotion, scar resistance, whitening and the like; the natural spider silk and regenerated spider silk protein material is also considered as an ideal source material in the field of high-end biomedical materials by combining the excellent physical and chemical characteristics of the natural spider silk and regenerated spider silk protein material, and has great application potential in the field of high-end implantation biological materials (such as artificial joints, artificial tendons/ligaments, artificial blood vessels, nerve ducts and the like).
However, the acquisition of natural spider silk and spider silk proteins (spidroins) is a bottleneck problem in the current research field of spider silk new materials. Currently, although researchers have found that spider silk proteins can be manufactured in heterologous hosts (such as prokaryotic microorganisms, eukaryotes, plants, transgenic animals, etc.) with the development of genetic engineering techniques, the obtained spider silk protein/polypeptide fragments have problems of low yield, high cost, poor water solubility and genetic instability of the gene fragments, thereby causing the problems of difficult molding, poor mechanical properties and low biological activity of the subsequent regenerated spider silk protein materials, which severely limit the practical application thereof. Therefore, it is accepted that the preparation of regenerated spider silk protein or polypeptide aqueous solution by advanced hydrolysis process using natural spider silk as raw material is key to break through practical application of regenerated spider silk protein material; however, how to obtain an aqueous solution of spider silk proteins or polypeptides and their powder materials that are highly pure, of controllable molecular weight, of high solubility and that can be produced in large scale presents a number of challenges.
Disclosure of Invention
Accordingly, the present invention is directed to a method for preparing a natural spider silk protein/polypeptide having a controllable molecular weight. The invention adopts spider silk as raw material, and utilizes high-pressure forced hydrolysis process to hydrolyze, extract and purify natural spider silk protein, creatively invents a method for hydrolyzing natural spider silk rapidly, efficiently and controllably, and the prepared spider silk protein/polypeptide has high purity, high concentration and controllable molecular weight, greatly improves the preparation efficiency of spider silk protein solution and regenerated spider silk protein powder, and can be produced in large scale.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of natural spider silk protein/polypeptide with controllable molecular weight, which comprises the following steps:
1) Freezing, ball milling and crushing natural spider silk to obtain spider silk powder;
2) Carrying out protein dissolution on the spider silk powder in the step 1) to obtain spider silk protein hydrolysate;
3) Centrifuging the spider silk protein hydrolysate in the step 2) to remove impurities to obtain supernatant;
4) Purifying the supernatant in the step 3) to obtain purified spider silk protein hydrolysate;
5) Filtering and removing impurities from the purified spider silk protein hydrolysate in the step 4) to obtain purified protein/polypeptide hydrolysate.
Preferably, the step further comprises sterilizing and drying the purified protein/polypeptide hydrolysate to obtain the spider silk protein/polypeptide powder.
Preferably, the preparation method of the frozen ball mill grinding comprises the following steps: mixing natural spider silk and grinding balls, and ball milling under a freezing condition; the mass ratio of the natural spider silk to the grinding balls is 1 (50-100); the freezing temperature is-45 ℃ to-20 ℃; the ball milling conditions include: the rotating speed is 150-600rpm, and the time is 1-3h.
Preferably, the solvent used for protein dissolution of the spider silk powder in the step 2) includes: acids, bases or organic alcohols; the mass volume ratio of the spider silk powder to the solvent is 1: (100-200).
Preferably, the conditions for protein solubilization in step 2) include: the hydrolysis pressure is 3-12Mpa, the reaction time is 2-8h, and the treatment temperature is 30-100 ℃.
Preferably, the centrifugation conditions in step 3) include: the centrifugal speed is 6000-12000rpm, the centrifugal temperature is 20-25 ℃, and the centrifugal time is 5-20min.
Preferably, the purifying method in the step 4) includes: dialyzing and purifying; the conditions for dialysis purification include: the molecular weight cut-off of dialysis is more than 500Da, the dialysis time is 1-3 days, water is changed every 2-6 hours, and the water is changed for more than 8 times.
Preferably, the filtering and impurity removing in the step 5) includes: qualitative filter paper impurity removal and medical gauze impurity removal; the aperture of the qualitative filter paper is 5-15 mu m; the number of layers of the medical gauze is 3-5.
The invention also provides the natural spider silk protein/polypeptide prepared by the preparation method.
The invention also provides application of the natural spider silk protein/polypeptide in preparing functional regenerated spider silk protein materials.
According to the invention, natural spider silk is crushed by freezing ball milling to obtain spider silk raw material powder, so that the subsequent hydrolysis efficiency is improved; meanwhile, different hydrolysis systems (acid, alkali, organic alcohol and the like) are designed, and high pressure is applied to force the natural spider silk to hydrolyze, so that the hydrolysis efficiency is effectively improved; the spidroin protein/polypeptide water solutions with different molecular weights can be controllably obtained by means of dialysis and purification processes. Meanwhile, the regenerated spider silk protein/polypeptide powder material can be obtained by means of a freeze-drying or spray-drying process. The preparation method provided by the invention has the advantages of simplicity in operation, low cost and high feasibility of continuous preparation, and can be improved based on the existing relevant industrial equipment to realize large-scale production and preparation.
The spider silk protein/polypeptide aqueous solution or powder obtained by the invention has the advantages of controllable molecular weight, adjustable concentration and high dissolution efficiency, and provides an important raw material basis for designability, functional specificity and molding feasibility of high-end regenerated spider silk protein materials. The dissolution efficiency of the spider silk protein prepared by the preparation method is more than 75%, the concentration of the aqueous solution is 0.5-8wt% and the controllable range of molecular weight is 1000-150000g/mol.
Drawings
FIG. 1 is an SEM photograph of spider silk before and after freeze ball milling;
fig. 2 is a photograph of a solution of spider silk dissolved in different solvents (acid/base, organic alcohol, etc.);
FIG. 3 is a graph of weight average molecular weight of spider silk after dissolution in different solvents (acids/bases, organic alcohols, etc.), as measured by GPC;
FIG. 4 is a schematic diagram of a high temperature, high pressure reactor.
Detailed Description
The invention provides a preparation method of natural spider silk protein/polypeptide with controllable molecular weight, which comprises the following steps:
1) Freezing, ball milling and crushing natural spider silk to obtain spider silk powder;
2) Carrying out protein dissolution on the spider silk powder in the step 1) to obtain spider silk protein hydrolysate;
3) Centrifuging the spider silk protein hydrolysate in the step 2) to remove impurities to obtain supernatant;
4) Purifying the supernatant in the step 3) to obtain purified spider silk protein hydrolysate;
5) Filtering and removing impurities from the purified spider silk protein hydrolysate in the step 4) to obtain purified protein/polypeptide hydrolysate.
In the present invention, the natural spider silk is preferably a primary spider silk of a Jingzhaosan spider, more preferably a primary spider silk of a clean Jingzhaosan spider.
In the present invention, the size of the spider silk powder particles is preferably less than 10 μm.
The invention preferably adopts manual picking and ethanol ultrasonic treatment to remove impurities before freezing, ball milling and crushing the natural spider silk.
The invention adopts liquid nitrogen to treat for 5-20min before freezing and ball milling, and keeps the temperature of the ball milling system lower than 40 ℃ below zero.
In the present invention, the preparation method of the freeze ball mill pulverizing preferably comprises: mixing natural spider silk and grinding balls, and ball milling under freezing condition.
In the present invention, the mass ratio of the natural spider silk to the grinding balls is preferably 1 (50-100).
In the present invention, the temperature of the freezing is preferably-45℃to-20 ℃.
In the present invention, the conditions for the ball milling preferably include: the rotating speed is 150-600rpm, and the time is 1-3h.
In the present invention, the grinding ball preferably includes: at least one of zirconia ball milling and corundum ball milling.
In the present invention, the solvent used for the protein solubilization of the spider silk powder in step 2) preferably includes: an acid, a base or an organic alcohol.
In the present invention, the acidic solution preferably includes hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, oxalic acid, acetic acid.
In the present invention, the alkaline solution preferably includes sodium hydroxide, sodium bicarbonate, ammonia water, calcium hydroxide.
In the present invention, the organic alcohol solvent is preferably hexafluoroisopropanol, methanol, isopropanol.
In the present invention, the acid-alkali soluble system is preferably low in concentration, and the solution concentration is preferably 0.1 to 3mol/L.
In the invention, the mass-volume ratio of the spider silk powder to the solvent is preferably 1:
(100-200)。
in the present invention, the conditions for protein solubilization in step 2) preferably include: the hydrolysis pressure is 3-12Mpa, the reaction time is 2-8h, and the treatment temperature is 30-100 ℃.
In the present invention, the centrifugation conditions in the step 3) preferably include: the centrifugal speed is 6000-12000rpm, the centrifugal temperature is 20-25 ℃, and the centrifugal time is 5-20min.
In the present invention, the purification method in the step 4) preferably comprises: and (5) dialyzing and purifying.
In the present invention, the conditions for dialysis purification preferably include: the molecular weight cut-off of dialysis is more than 500Da, the dialysis time is 1-3 days, water is changed every 2-6 hours, and the water is changed for more than 8 times.
In the present invention, the dialysis purification is preferably purification using a dialysis bag.
In the present invention, the filtering and impurity removing in the step 5) preferably includes: qualitative filter paper impurity removal and medical gauze impurity removal.
In the present invention, the pore size of the qualitative filter paper is preferably 5-15 μm; the number of layers of the medical gauze is preferably 3-5.
In the present invention, the step further preferably includes sterilizing and drying the purified protein/polypeptide hydrolysate to obtain the spider silk protein/polypeptide powder.
In the present invention, the sterilization is preferably high-pressure steam sterilization, and the temperature of the sterilization is preferably 110-130 ℃, and the pressure is preferably 0.2-1Mpa.
In the invention, the sterilization is preferably ultraviolet irradiation sterilization, the power of the ultraviolet lamp is preferably 40-120W, and the irradiation time is preferably 0.5-4h.
In the present invention, the drying preferably includes freeze-drying and spray-drying.
In the invention, the freeze drying process is preferably to pour the spider silk protein/polypeptide solution into a PS culture dish, seal the dish with a preservative film, and then place the dish in a freeze dryer, and set the freeze drying temperature to be < -40 ℃ and the freeze drying time to be 1-3d.
In the invention, the spray drying process is preferably to pump the spider silk protein/polypeptide solution into a spray dryer, atomize the spider silk protein/polypeptide solution through a high-pressure nozzle and then deliver the atomized spider silk protein/polypeptide solution to hot air, wherein the temperature of the hot air is preferably 60-80 ℃; evaporating and drying at high speed, and separating gas and solid to obtain spider silk protein powder.
The invention also provides the natural spider silk protein/polypeptide prepared by the preparation method.
The invention also provides application of the natural spider silk protein/polypeptide in preparing functional regenerated spider silk protein materials.
In the present invention, the regenerated spider silk protein material preferably includes fibers, films, coatings and scaffolds.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A preparation method of a high-pressure auxiliary controllable hydrolyzed natural spider silk polypeptide aqueous solution comprises the following steps:
(1) Freezing ball milling pretreatment: taking 1g of spider silk, manually picking, ultrasonically treating with ethanol to remove impurities, grinding by using a freezing ball mill, setting a ball milling process (a zirconia ball milling tank, the mass ratio of the spider silk to the grinding balls is 1:100, the temperature is-50 ℃, the rotating speed is 600rpm, the time is 2 hours), and grinding the natural spider silk balls into powder, wherein the particle size is less than 5 mu m;
(2) High-pressure forced hydrolysis of acid solution system: loading the spider silk powder obtained by ball milling into a high-pressure hydrolysis reactor according to the mass ratio of 1:100, adding 30mL of 0.5M hydrochloric acid solution, applying 3.5Mpa, controlling the hydrolysis temperature to 60 ℃, and performing a hydrolysis reaction for 5h at a magnetic stirring rotating speed of 500 rpm;
(3) And (3) centrifugal impurity removal: centrifuging the obtained spider silk protein hydrolysate to remove impurities, obtaining supernatant, and centrifuging at 12000rpm for 10min;
(4) And (3) dialysis and purification: placing the centrifugal impurity-removed spider silk protein solution in a dialysis bag with molecular retention more than 1000Da, dialyzing in a large amount of deionized water system, changing deionized water every 2-3h, dialyzing for 10 times, and thoroughly removing small molecular impurities such as hydrochloric acid and the like in the solution;
(5) Filtering and removing impurities: filtering the dialyzed hydrolysate by qualitative filter paper with the aperture of 10 mu m to obtain pale yellow spider silk protein hydrolysate;
(6) Sterilizing and preserving: sealing the spider silk hydrolysate in a clean glass container, sterilizing with high pressure steam (120deg.C, 0.2 Mpa), and refrigerating at 4deg.C;
(7) Protein concentration and molecular weight characterization: the solid content concentration in the obtained protein aqueous solution is 1.8wt% by using a differential weight method; the weight average molecular weight of the obtained protein polypeptide is 1800g/mol by aqueous phase GPC molecular weight measurement;
(8) Spider silk protein powder: pouring spider silk hydrolysate into a culture dish, sealing with a preservative film, placing into a freeze dryer, setting the freeze drying temperature to-60 ℃, freeze-drying for 2 days, taking out, and grinding to obtain white regenerated spider silk protein powder;
wherein the spider silk adopts primary spider silk of Wuzhishan Jingzhaosan spider in Hainan.
Example 2
A preparation method of a high-pressure auxiliary controllable hydrolyzed natural spider silk protein polypeptide aqueous solution comprises the following steps:
(1) Freezing ball milling pretreatment: taking 1g of spider silk, manually picking, removing impurities by ethanol ultrasonic treatment, grinding by using a freezing ball mill, setting a ball milling process (corundum ball milling tank, the mass ratio of spider silk to grinding balls is 1:100, the temperature is-60 ℃, the rotating speed is 400rpm, the time is 1 h), and grinding natural spider silk balls into powder, wherein the particle size is less than 3 mu m;
(2) High-pressure forced hydrolysis of alkali solution system: loading the spider silk powder obtained by ball milling into a polytetrafluoroethylene-lined high-pressure reaction container, wherein the mass ratio is 2:100, adding 30mL of 0.25M sodium hydroxide solution, applying pressure of 6Mpa, controlling the hydrolysis temperature to 50 ℃, and performing hydrolysis reaction for 6h at a magnetic stirring rotating speed of 500 rpm;
(3) And (3) centrifugal impurity removal: centrifuging the obtained spider silk protein hydrolysate to remove impurities, obtaining supernatant, and centrifuging at 10000rpm for 15min;
(4) And (3) dialysis and purification: placing the centrifugal impurity-removed spider silk protein solution in a dialysis bag with molecular retention of more than 500Da, dialyzing in a large amount of deionized water system, changing deionized water every 2-3h, dialyzing for 10 times, and thoroughly removing small molecular impurities such as inorganic salts and the like in the solution; filtering and removing impurities: filtering solid impurities in the dialysis process of the dialyzed hydrolysate by medical gauze with the aperture of 5 layers to obtain yellowish spider silk protein hydrolysate;
(5) Sterilizing and preserving: sealing the spider silk hydrolysate in a clean glass container, sterilizing with high pressure steam (120deg.C, 0.2 Mpa), and refrigerating at 4deg.C;
(6) Protein concentration and molecular weight characterization: the solid content concentration in the obtained protein aqueous solution is 4wt% by using a differential weight method; the weight average molecular weight of the resulting protein polypeptide was 11500g/mol as determined by aqueous GPC molecular weight;
(7) Spider silk protein powder preparation: pumping spider silk hydrolysate into a spray dryer, atomizing the spider silk hydrolysate by a high-pressure spray nozzle, then connecting the spider silk hydrolysate with 80 ℃ hot air in a spray drying chamber, evaporating and drying the solution at a high speed to obtain powder, and separating the powder from air flow by a cyclone separator to obtain white spider silk protein powder;
wherein the spider silk adopts primary spider silk of Wuzhishan Jingzhaosan spider in Hainan.
Example 3
A preparation method of a high-pressure auxiliary controllable hydrolyzed natural spider silk protein polypeptide aqueous solution comprises the following steps:
(1) Freezing ball milling pretreatment: taking 1g of spider silk, manually picking, ultrasonically treating with ethanol to remove impurities, grinding by using a freezing ball mill, setting a ball milling process (a zirconia ball milling tank, the mass ratio of the spider silk to the grinding balls is 1:100, the temperature is-40 ℃, the rotating speed is 500rpm, the time is 1 h), and grinding the natural spider silk balls into powder, wherein the particle size is less than 10 mu m;
(2) Swelling of the inorganic salt system: loading the spider silk powder obtained by ball milling into a polytetrafluoroethylene lining high-pressure reaction container, wherein the mass ratio is 2:100, adding 30mL of 9.3M lithium bromide solution, applying pressure of 6Mpa, controlling the temperature to 60 ℃, and performing a swelling reaction for 1h at a magnetic stirring rotating speed of 500 rpm;
(3) And (3) centrifugal separation: centrifuging the swelled solution, removing supernatant, centrifuging at 8000rpm for 10min, taking out swelled spider silk, cleaning with deionized water for 3 times, and oven drying at 60deg.C;
(4) Acid solution system hydrolysis: loading the swelled spider silk into a high-pressure hydrolysis reactor according to the mass ratio of 1:10, adding 20mL of formic acid solution, applying 2Mpa of pressure, controlling the hydrolysis temperature to 60 ℃, and performing hydrolysis reaction for 4 hours at a magnetic stirring rotating speed of 500 rpm;
(5) And (3) centrifugal impurity removal: centrifuging the obtained spider silk protein hydrolysate to remove impurities, obtaining supernatant, and centrifuging at 10000rpm for 10min;
(6) Filtering and removing impurities: filtering the supernatant obtained by centrifugation by using a water-based filter membrane with the pore diameter of 0.45 mu m to obtain orange-red spider silk protein hydrolysate;
(7) Sterilizing and preserving: placing the spider silk hydrolysate under a 40W ultraviolet lamp, irradiating for 4h for physical sterilization, and preserving at 4deg.C;
(8) Protein concentration characterization: the solid content concentration in the obtained protein aqueous solution is 6wt% by using a differential weight method;
wherein the spider silk adopts primary spider silk of Wuzhishan Jingzhaosan spider in Hainan.
Example 4
A preparation method of a high-pressure auxiliary controllable dissolution natural spider silk protein polypeptide solution comprises the following steps:
(1) Freezing ball milling pretreatment: taking 1g of spider silk, manually picking, removing impurities by ethanol ultrasonic treatment, grinding by using a freezing ball mill, setting a ball milling process (corundum ball milling tank, the mass ratio of spider silk to grinding balls is 1:100, the temperature is-40 ℃, the rotating speed is 600rpm, the time is 2 hours), and grinding natural spider silk balls into powder, wherein the particle size is less than 2 mu m;
(2) Organic alcohol system dissolution: loading the spider silk powder obtained by ball milling into a polytetrafluoroethylene lining high-pressure reaction container, wherein the mass ratio is 1:100, 20mL of hexafluoroisopropanol is added, the pressure is applied to 6Mpa, the hydrolysis temperature is controlled to be room temperature, the magnetic stirring rotating speed is 1200rpm, and the hydrolysis reaction is carried out for 8 hours;
(3) And (3) centrifugal impurity removal: centrifuging the obtained spider silk protein solution to remove impurities, obtaining supernatant, and centrifuging at 12000rpm for 20min;
(4) Filtering and removing impurities: filtering the supernatant obtained by centrifugation by using an organic filter membrane with the pore diameter of 0.22 mu m to obtain a semitransparent white spider silk protein solution;
(5) Sterilizing and preserving: placing the spider silk solution under 40W ultraviolet lamp, irradiating for 4 hr for physical sterilization, and refrigerating at 4deg.C;
(6) Protein concentration and molecular weight characterization: determining the solid content concentration of the obtained protein solution to be 1wt% by using a differential weight method; the weight average molecular weight of the resulting protein polypeptide was 120000g/mol as determined by GPC molecular weight of the organic phase;
wherein the spider silk adopts primary spider silk of Wuzhishan Jingzhaosan spider in Hainan.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A method for preparing a natural spider silk protein/polypeptide with controllable molecular weight, comprising the steps of:
1) Freezing, ball milling and crushing natural spider silk to obtain spider silk powder;
2) Carrying out protein dissolution on the spider silk powder in the step 1) to obtain spider silk protein hydrolysate;
3) Centrifuging the spider silk protein hydrolysate in the step 2) to remove impurities to obtain supernatant;
4) Purifying the supernatant in the step 3) to obtain purified spider silk protein hydrolysate;
5) Filtering and removing impurities from the purified spider silk protein hydrolysate in the step 4) to obtain purified protein/polypeptide hydrolysate.
2. The method according to claim 1, wherein the step further comprises sterilizing and drying the purified protein/polypeptide hydrolysate to obtain the spidroin protein/polypeptide powder.
3. The method of claim 1, wherein the method of preparing the cryo-ball mill grind comprises: mixing natural spider silk and grinding balls, and ball milling under a freezing condition; the mass ratio of the natural spider silk to the grinding balls is 1 (50-100); the freezing temperature is-45 ℃ to-20 ℃; the ball milling conditions include: the rotating speed is 150-600rpm, and the time is 1-3h.
4. The method according to claim 1, wherein the solvent used for the proteolytic processing of the spider silk powder in step 2) comprises: acids, bases or organic alcohols; the mass volume ratio of the spider silk powder to the solvent is 1: (100-200).
5. The method according to claim 1, wherein the conditions for protein solubilization in step 2) include: the hydrolysis pressure is 3-12Mpa, the reaction time is 2-8h, and the treatment temperature is 30-100 ℃.
6. The method according to claim 1, wherein the centrifugation conditions in step 3) include: the centrifugal speed is 6000-12000rpm, the centrifugal temperature is 20-25 ℃, and the centrifugal time is 5-20min.
7. The method according to claim 1, wherein the purification in step 4) comprises: dialyzing and purifying; the conditions for dialysis purification include: the molecular weight cut-off of dialysis is more than 500Da, the dialysis time is 1-3 days, water is changed every 2-6 hours, and the water is changed for more than 8 times.
8. The method according to claim 1, wherein the filtering and removing impurities in the step 5) comprises: qualitative filter paper impurity removal and medical gauze impurity removal; the aperture of the qualitative filter paper is 5-15 mu m; the number of layers of the medical gauze is 3-5.
9. The natural spider silk protein/polypeptide produced by the production method according to any one of claims 1 to 8.
10. Use of a natural spider silk protein/polypeptide according to claim 9 for the preparation of a functional regenerated spider silk protein material.
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