Extraction method of spider silk fibroin

Abstract

Compared with the prior art, the method for extracting the spider silk fibroin has the advantage that the content of the spider silk fibroin can be remarkably increased.

Description

Background Art

[0001] The current chemical extraction technology of natural spider silk fibroin mainly relies on the solvent dissolution method, among which the lithium bromide (LiBr) method and the calcium chloride / ethanol / water ternary system are the cores of the existing processes. The LiBr method dissolves spider silk fibers with a high-concentration LiBr solution at high temperature, breaking the hydrogen bond network of fibroin to achieve separation. However, this process easily causes irreversible breakage of the fibroin molecular chain, resulting in a significant reduction in the molecular weight of the final product (usually lower than 200 kDa), and the residual LiBr is difficult to completely remove, affecting the protein purity. Although the calcium chloride / ethanol / water ternary system can weaken the crystalline region structure of fibroin through Ca 2 + chelation, it needs to react for a long time under high temperature (>80 °C), which not only triggers the hydrolysis reaction of fibroin, causing uncontrollable degradation of the β-sheet conformation, but also greatly reduces the content of high-molecular-weight fibroin in the extraction solution.

[0002] The limitations of the existing technology are mainly reflected in the following aspects: First, the strong corrosiveness of the solvent system and the high-temperature reaction conditions lead to the destruction of the multi-level structure of fibroin, an increase in the dispersion degree of the molecular weight distribution, and it is difficult for the product to retain the high strength and toughness characteristics of natural spider silk; Second, the degumming and purification processes are cumbersome. The repeated infiltration and cleaning of chemical reagents easily cause loss of protein activity, and the residual solvent may introduce the risk of biological toxicity; Third, the dissolution efficiency and selectivity of the existing methods for fibroin are insufficient, and it is difficult to achieve the directional enrichment of high-purity and high-molecular-weight fibroin. Therefore, there is an urgent need to develop an efficient and low-damage extraction technology in the market to improve the yield and functionality of fibroin while avoiding molecular weight attenuation, so as to break through the key bottleneck in the industrial application of natural spider silk materials. Summary of the Invention

[0003] The present invention relates to a method for extracting spider silk fibroin with a hexafluoroisopropanol / papain system, which has the following advantages compared with the existing technology: 1. High protein content. Most of them on the market are 12 wt%, and the present invention can be increased to about 24 wt%; 2. The molecular weight can be guaranteed (controllable). The molecular weight less than 10 kDa can be effectively intercepted by an ultrafiltration membrane with a molecular weight of 10 kDa, and it can be used for preparing hydrogel materials, etc.; 3. Compared with the commonly used hexafluoroisopropanol for extracting fibroin on the market, the dosage of hexafluoroisopropanol is greatly reduced, significantly reducing the extraction cost; 4. Reducing the dosage of hexafluoroisopropanol and increasing papain can reduce the toxicity of the extracted fibroin. Detailed Description of the Invention

[0004] The present invention relates to a method for extracting spider silk fibroin, characterized in that:

[0005] Step 1: Weigh an appropriate amount of the silk of *Macrothele jingdongensis*, wash away the excess impurities with water, dry the washed silk in an oven at 40 - 50 °C, and place it in a drying oven to cool to a constant weight. Weigh the dried silk, cut it into pieces, and place it in a flask. Prepare a degumming solution with silk: sodium carbonate: water in a mass ratio of 1:1 - 2:1 - 2. Boil it under standard atmospheric pressure for 1 - 2 hours, then wash it with deionized water, and repeat this process 2 - 5 times. Dry it overnight at a temperature of 50 - 70 °C to obtain degummed silk fibers;

[0006] Step 2: Add the degummed silk fibers and HFIP to the hexafluoroisopropanol solvent (HFIP) in a mass - to - volume ratio of 1:1 - 2 (mg / ml). Place the round - bottom flask containing the silk fiber - HFIP blend in a thermostatic magnetic stirrer, and carry out condensation reflux at 50 - 60 °C and 500 - 700 rpm for 4 - 8 h. Filter to obtain a natural silk protein solution, and rotary evaporate to remove HFIP;

[0007] Step 3: Prepare an aqueous solution of papain by mixing water and papain, where the mass - to - volume ratio of papain to water is 1:1 (mg / mL), and the mass - to - volume ratio of the amount of papain to HFIP is 1:0.625 - 2.5 (mg / mL). Add the previously prepared natural silk protein solution for further enzymatic digestion. Carry out water - bath at 65 - 70 °C, with a pH of 7, and heat for enzymatic digestion for 120 - 150 min. Then, dilute the digested solution with a buffer first, put it into an ultrafiltration membrane with a molecular weight cut - off of 10 kDa to effectively retain the silk fibroin protein and let the papain pass through. Centrifuge at 7000 - 9000 rpm for 10 - 20 minutes to prepare a silk fibroin solution, and dry it to obtain silk fibroin protein.

[0008] Preferably, the extraction method of silk fibroin protein from spider silk is as follows:

[0009] Step 1: Weigh an appropriate amount of the silk of *Macrothele jingdongensis*, wash away the excess impurities with water, dry the washed silk in an oven at 50 °C, and place it in a drying oven to cool to a constant weight. Weigh the dried silk, cut it into pieces, and place it in a 1.0 L round - bottom flask. Prepare a degumming solution with silk: sodium carbonate: water in a mass ratio of 1:2:2. Boil it under standard atmospheric pressure for 1 hour, and then wash it with deionized water several times. Repeat this process three times, and dry it overnight at a temperature of 65 °C to obtain degummed silk fibers;

[0010] Step 2: Add the degummed silk fibers and HFIP to the hexafluoroisopropanol solvent (HFIP) in a mass - to - volume ratio of 1:1 - 2 (mg / mL). Place the round - bottom flask containing the silk fiber - HFIP blend in a thermostatic magnetic stirrer, and carry out condensation reflux at 58 °C and 500 rpm for 6 h. Filter it with a 400 - mesh stainless - steel sieve to obtain a natural silk protein solution, and rotary evaporate to remove HFIP;

[0011] Step 3: Prepare an aqueous solution of papain, where the mass-volume ratio of papain to water is 1:1 (mg / mL), and the mass-volume ratio of papain to HFIP is 1:0.625 - 2.5 (mg / mL). Add the natural silk fibroin solution prepared with HFIP beforehand for further enzymatic digestion. Incubate in a water bath at 65 °C, with a pH of 7. After heating and enzymatic digestion for 120 min, prepare a phosphate buffer solution (PBS) with a pH of 7.3 for sample dilution and membrane cleaning during ultrafiltration. Dilute the enzymatically digested solution with the buffer first, and then place it into an ultrafiltration membrane with a molecular weight cut-off of 10 kDa to effectively retain silk fibroin and allow papain to pass through. Centrifuge at 8000 rpm for 10 minutes to prepare a silk fibroin solution, and obtain silk fibroin after drying.

[0012] In some embodiments, the mass-volume ratio of degummed silk fibers to HFIP in step 2 is 1:1 (mg / ml).

[0013] In some embodiments, the mass-volume ratio of papain to HFIP is 1:0.625 - 2.5 (mg / mL).

[0014] In some embodiments, the mass-volume ratio of degummed silk fibers to HFIP in step 2 is 1:1 (mg / ml),

[0015] The mass-volume ratio of papain to HFIP is 1:0.625 (mg / mL).

[0016] In some embodiments, the mass-volume ratio of degummed silk fibers to HFIP in step 2 is 1:2 (mg / ml),

[0017] The mass-volume ratio of papain to HFIP is 1:2.5 (mg / mL).

[0018] The present invention also protects the silk fibroin prepared by the preparation method described in any one of the above.

[0019] Specific implementation cases

[0020] The following examples can enable those skilled in the art of this specialty to understand the present invention more comprehensively, but do not limit the present invention to the scope of the described examples.

[0021] Example 1: Extract silk fibroin using hexafluoroisopropanol (HFIP) (Control Group 1)

[0022] Weigh an appropriate amount of the silk of *Macrothele jingdongensis*, wash away the excess impurities with water, dry the washed silk in an oven at 50 °C, and place it in a drying oven to cool to a constant weight. Weigh 500 mg of the dried silk, cut it into pieces, and place it in a 1.0 L round-bottom flask. Prepare a degumming solution with silk: sodium carbonate: water in a mass ratio of 1:2:2. Boil the silk in the solution under 1 standard atmosphere for 1 hour, and then wash it several times with deionized water. Repeat this process three times, and dry it overnight at 65 °C to obtain degummed silk fibers (250 mg). Add the degummed silk fibers and HFIP to 500 mL of hexafluoroisopropanol solvent (HFIP) in a mass-to-volume ratio of 1:2 (mg / mL). Place the round-bottom flask containing the silk-HFIP blend in a constant-temperature magnetic stirrer, and carry out condensation reflux at 58 °C and 500 rpm for 6 h. Filter it through a 400-mesh stainless-steel sieve to obtain a natural silk fibroin solution, rotary evaporate to remove HFIP, and dry. Dry it in an oven at 40 °C to prepare silk fibroin at 16 wt% (wt% = mass of dried silk fibroin / mass of silk).

[0023] Example 2: Extract silk fibroin using papain (Control Group 2)

[0024] Weigh an appropriate amount of the silk of *Macrothele jingdongensis*, wash away the excess impurities with water, dry the washed silk in an oven at 50 °C, and place it in a drying oven to cool to a constant weight. Weigh 500 mg of the dried silk, cut it into pieces, and place it in a 1.0 L round-bottom flask. Prepare a degumming solution with silk: sodium carbonate: water in a mass ratio of 1:2:2. Boil the silk in the solution under 1 standard atmosphere for 1 hour, and then wash it several times with deionized water. Repeat this process three times, and dry it overnight at 65 °C to obtain degummed silk fibers (250 mg). Prepare an aqueous papain solution using 200 ml of water and 200 mg of papain, add the degummed silk fibers for enzymatic cleavage, with a pH of 7 and a temperature of 65 °C, and heat in a water bath for two hours to prepare a silk fibroin solution. Prepare phosphate buffer solution (PBS) for sample dilution and membrane cleaning during the ultrafiltration process. First, dilute the enzymatically cleaved silk fibroin solution with the buffer, place it in an ultrafiltration membrane with a molecular weight of 10 kDa to effectively retain the silk fibroin of the spider silk and allow the papain to pass through. Centrifuge at 8000 rpm for 10 minutes to prepare a silk solution, and dry it in an oven at 40 °C. After drying, obtain silk fibroin at 10 wt% (wt% = mass of dried silk fibroin / mass of silk).

[0025] Example 3: Extract silk fibroin using a combination of hexafluoroisopropanol and papain and parameter screening

[0026] Step 1: Preparation of degummed spider silk

[0027] Weigh an appropriate amount of the silk of *Macrothele jingdongensis*, wash away the excess impurities with water, dry the washed silk in an oven at 50 °C, and place it in a drying oven to cool to a constant weight. Weigh 500 mg of the dried silk, cut it into pieces, and place it in a 1.0 L round-bottom flask. Prepare a degumming solution with silk: sodium carbonate: water in a mass ratio of 1:2:2. Boil the silk in the solution under 1 standard atmosphere for 1 hour, and then wash it several times with deionized water. Repeat this process three times, and dry it overnight at 65 °C to obtain degummed silk fibers (250 mg). Step 2: Extraction with hexafluoroisopropanol (HFIP)

[0028] Weigh the mass of the degummed silk fibers as 250 mg, and add them to 500 mL of hexafluoroisopropanol solvent (HFIP) at a mass-to-volume ratio of 1:2 (mg / mL). Place the round-bottom flask containing the silk fiber-HFIP blend in a thermostatic magnetic stirrer, and carry out condensation reflux at 58 °C and 500 rpm for 6 h. Filter it through a 400-mesh stainless steel sieve to obtain a natural silk fibroin solution, and rotary evaporate to remove HFIP. Dry the above silk fibroin in an oven at 40 °C, and weigh it as 81.5 mg, accounting for 16.30 wt%. The calculation formula is m (mass of the final silk fibroin) ÷ m (initial 500 mg of silk).

[0029] Step 3: Extraction with hexafluoroisopropanol (HFIP) combined with papain

[0030] Prepare an aqueous solution of papain, and fully dissolve and stir 200 ml of water and 200 mg of papain. Add the natural silk fibroin solution extracted with HFIP in Step 2 in advance, and heat the solution in a water bath for further enzymatic hydrolysis to extract silk fibroin. Prepare a phosphate buffer solution (PBS) with a pH of 7.3 for sample dilution and membrane cleaning during the ultrafiltration process. Dilute the enzymatically digested solution with the buffer first, put it into an ultrafiltration membrane with a molecular weight of 10 kDa to effectively retain the silk fibroin of the spider silk, allow the papain to pass through, and centrifuge at 8000 rpm for 10 minutes to prepare a silk fibroin solution. Dry it in an oven at 40 °C. The inventor screened the extraction conditions of papain in Step 3. As shown in Table 1-3, it was found that the optimal conditions for papain enzymatic hydrolysis were a hydrolysis temperature of 65 °C, a hydrolysis pH of 7, and a hydrolysis time of 120 min.

[0031] Table 1 Screening of the hydrolysis temperature of papain in Step 3 (hydrolysis pH is 7, hydrolysis time is 120 min)

[0032] 50℃ 55℃ 60℃ 65℃ 70℃ 75℃ 18.64 wt% 20.10 wt% 22.70 wt% 23.77 wt% 23.30 wt% Inactivated

[0033] Table 2 Screening of the hydrolysis pH of papain in Step 3 (hydrolysis temperature is 65 °C, hydrolysis time is 120 min)

[0034] 5 6 7 8 9 16.60 wt% 19.50 wt% 24.30 wt% 22.14 wt% 18.20 wt%

[0035] Table 3 Screening of papain hydrolysis time in Step 3 (hydrolysis temperature 65°C, hydrolysis pH 7)

[0036] 60 min 90 min 120 min 150 min 180 min 14.63 wt% 21.00 wt% 24.52 wt% 24.31 wt% 24.20 wt%

[0037] Example 4 Extraction of silk fibroin using hexafluoroisopropanol combined with papain (HFIP weight reduction)

[0038] Weigh an appropriate amount of the silk of Macrothele gigas from Jingdong, wash away the excess impurities with water, dry the washed silk in an oven at 50°C, and place it in a drying oven until it cools to a constant weight. Weigh 500 mg of the dried silk, cut it into pieces, and place it in a 1.0 L round-bottom flask. Prepare a degumming solution with silk: sodium carbonate: water in a mass ratio of 1:2:2. Boil the silk in the solution under 1 standard atmosphere for 1 hour, and then wash it several times with deionized water. Repeat this process three times, and dry it overnight at 65°C to obtain degummed silk fibers (250 mg). Add the degummed silk fibers and HFIP to 250 mL of hexafluoroisopropanol solvent (HFIP) at a mass-to-volume ratio of 1:1 (mg / mL). Place the round-bottom flask containing the silk fiber-HFIP blend in a thermostatic magnetic stirrer, and carry out condensation reflux at 58°C and 500 rpm for 6 h. Filter through a 400-mesh stainless steel sieve to obtain a natural silk protein solution, and rotary evaporate to remove HFIP. Prepare an aqueous papain solution (400 ml of water and 400 mg of papain), add it to the silk protein solution prepared with HFIP beforehand for further enzymatic digestion, heat in a water bath at 65°C, pH 7, for 120 minutes. Prepare a phosphate buffer solution (PBS) with a pH of 7.3 for sample dilution and membrane cleaning during the ultrafiltration process. Dilute the enzymatically digested solution with the buffer first, and place it in an ultrafiltration membrane with a molecular weight cut-off of 10 kDa to effectively retain the silk fibroin of the silk, allowing the papain to pass through. Centrifuge at 8000 rpm for 10 minutes to prepare a silk fibroin solution, and dry it in an oven at 40°C. After drying, obtain silk fibroin at 23.82 wt% (wt% = mass of dried silk fibroin / mass of silk)

[0039] Under the same other conditions, compared with Example 3, the dosage of HFIP in Example 4 decreased significantly, which can reduce the residue of HFIP in silk fibroin and avoid the toxicity brought when implanted into the human body. At the same time, the price of HFIP is higher than that of papain, and reducing the dosage of HFIP can greatly reduce the cost.

[0040] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for extracting spider silk fibroin protein, characterized in that: Step 1: Weigh an appropriate amount of the silk of Macrothele gigas from Jingdong, wash away the excess impurities with water, dry the washed silk in an oven at 40-50 °C, and place it in a drying oven to cool to a constant weight; weigh the dried silk, cut it into pieces, and place it in a flask. The silk: sodium carbonate: water is made into a degumming solution according to the mass ratio of 1:1-2:1-2, boil it for 1-2 hours under standard atmospheric pressure, and then wash it with deionized water. This process is repeated 2-5 times, and dry it overnight at a temperature of 50-70 °C to obtain degummed silk fibers; Step 2: Add the degummed silk fibers and HFIP to the hexafluoroisopropanol solvent (HFIP) according to the mass-volume ratio of 1:1-2 (mg / ml). Place the round-bottom flask containing the silk fiber-HFIP blend in a thermostatic magnetic stirrer, and carry out condensation reflux at 50-60 °C and 500-700 rpm for 4-8 h, filter to obtain a natural silk fibroin protein solution, and rotary evaporate to remove HFIP; Step 3: Prepare an aqueous solution of papain by mixing water and papain, wherein the mass-volume ratio of papain to water is 1:1 (mg / mL), and the mass-volume ratio of the dosage of papain to HFIP is 1:0.625-2.5 (mg / mL). Add it to the previously prepared natural silk fibroin protein solution for further enzymatic digestion. Water bath at 65-70 °C, pH 7, heat and enzymatically digest for 120-150 min, then dilute the enzymatically digested solution with a buffer solution, put it into an ultrafiltration membrane with a molecular weight of 10 kDa to effectively retain the spider silk fibroin protein and allow the papain to pass through; centrifuge at 7000-9000 rpm for 10-20 minutes to prepare a fibroin solution, and obtain fibroin protein after drying.

2. The method for extracting spider silk fibroin protein according to claim 1, characterized in that: Step 1: Weigh an appropriate amount of the silk of Macrothele gigas from Jingdong, wash away the excess impurities with water, dry the washed silk in an oven at 50 °C, and place it in a drying oven to cool to a constant weight; weigh the dried silk, cut it into pieces, and place it in a 1.0 L round-bottom flask. The silk: sodium carbonate: water is made into a degumming solution according to the mass ratio of 1:2:2, boil it for 1 hour under standard atmospheric pressure, and then wash it with deionized water several times; this process is repeated three times, and dry it overnight at a temperature of 65 °C to obtain degummed silk fibers; Step 2: Add the degummed silk fibers and HFIP to the hexafluoroisopropanol solvent (HFIP) according to the mass-volume ratio of 1:1-2 (mg / mL). Place the round-bottom flask containing the silk fiber-HFIP blend in a thermostatic magnetic stirrer, and carry out condensation reflux at 58 °C and 500 rpm for 6 h, filter with a 400-mesh stainless steel sieve to obtain a natural silk fibroin protein solution, and rotary evaporate to remove HFIP; Step 3: Prepare an aqueous solution of papain, where the mass-volume ratio of papain to water is 1:1 (mg / mL), and the mass-volume ratio of papain to HFIP is 1:0.625 - 2.5 (mg / mL). Add the natural silk fibroin solution prepared with HFIP in advance for further enzymatic digestion. Incubate in a water bath at 65 °C, with a pH of 7. After heating and enzymatic digestion for 120 min, prepare phosphate buffer solution (PBS) with a pH of 7.3 for sample dilution and membrane cleaning during the ultrafiltration process. Dilute the enzymatically digested solution with buffer first, and place it in an ultrafiltration membrane with a molecular weight cut-off of 10 kDa to effectively retain silk fibroin and allow papain to pass through. Centrifuge at 8000 rpm for 10 minutes to prepare a silk fibroin solution, and obtain silk fibroin after drying.

3. The method for extracting spider silk fibroin according to any one of claims 1-2, characterized in that: The mass-volume ratio of the degummed silk fiber to HFIP in Step 2 is 1:1 (mg / ml).

4. The method for extracting spider silk fibroin according to any one of claims 1-3, characterized in that: The mass-volume ratio of papain to HFIP is 1:0.625 - 2.5 (mg / mL).

5. The method for extracting spider silk fibroin according to any one of claims 1-2, characterized in that: The mass-volume ratio of the degummed silk fiber to HFIP in Step 2 is 1:1 (mg / ml), and the mass-volume ratio of papain to HFIP is 1:0.625 (mg / mL).

6. The method for extracting spider silk fibroin according to any one of claims 1-2, characterized in that: The mass-volume ratio of the degummed silk fiber to HFIP in Step 2 is 1:2 (mg / ml), and the mass-volume ratio of papain to HFIP is 1:2.5 (mg / mL).

7. Silk fibroin prepared by the preparation method according to any one of claims 1 - 6.