Silk fibroin modified silica gel chromatographic packing, preparation method and application thereof

Abstract

The application discloses a silk fibroin modified silica gel chromatographic packing and a preparation method and application thereof, and the preparation method comprises the following steps: adding protease into a regenerated silk fibroin solution, incubating, centrifuging, dissolving collected silk fibroin precipitate in a hydrogen bond inhibitor solution to obtain a hydrophobic silk fibroin solution; dispersing inorganic silica gel powder into the hydrophobic silk fibroin solution, performing dialysis treatment, cleaning the solid product after the dialysis treatment, and performing vacuum freeze-drying treatment to obtain the silk fibroin modified silica gel chromatographic packing. The preparation method provided by the application is green and environment-friendly, no toxic reagent and chemical crosslinking agent are left, and the prepared silk fibroin modified silica gel chromatographic packing has the advantages of regular surface structure, relatively uniform particle size and adjustable polarity, and can be applied to the field of chromatographic separation of biological macromolecules.

Description

Technical Field

[0001] This invention relates to the field of silica gel chromatography packing technology, specifically to a silk fibroin-modified silica gel chromatography packing, its preparation method, and its application. Background Technology

[0002] Chromatographic separation technology is widely used in biomedical fields such as protein purification. Traditional silica gel chromatography packing materials are often used for the separation of small molecule compounds due to their good mechanical stability and porous structure. However, they have significant drawbacks in the separation of biomacromolecules (especially proteins): insufficient specific binding capacity, low separation efficiency, poor biocompatibility, and easy denaturation of target proteins, making it difficult to meet the requirements for efficient separation of biomacromolecules.

[0003] Silk fibroin, a natural polymer extracted from silk, possesses excellent mechanical strength, tunable hydrophilicity / hydrophobicity, and biodegradability, and has been widely used in tissue engineering, medical devices, and cosmetics. Silk fibroin is composed of 18 amino acid residues, and its molecular structure alternates between highly ordered crystalline regions and loosely ordered amorphous regions. The crystalline regions are mainly formed by the repeating arrangement of amino acids with small side groups, such as glycine (G), alanine (A), and serine (S), exhibiting regular structure, hydrophobicity, and weak polarity. The amorphous regions, on the other hand, are rich in hydrophilic amino acids with larger side groups, are easily degraded, and have a disordered structure. Therefore, due to these structural characteristics, and problems such as easy hydrolysis, poor mechanical properties, and unstable adsorption performance, intact silk fibroin cannot be directly used as a chromatographic packing material or coating.

[0004] In the prior art, some patents involve the preparation of silk fibroin coatings or silica gel chromatography packing materials, but all have obvious limitations: Chinese patent CN110982429A discloses a method for preparing a silk fibroin coating for medical catheters, which is only used for optimizing medical catheter coatings and does not involve the modification of inorganic silica gel by silk fibroin or its application in the chromatographic separation of biomolecules, nor does it use enzymatic digestion technology to modify the structure of silk fibroin; Chinese patent CN113274993A discloses a method for preparing a silica gel matrix chromatography packing material for the separation of highly polar drugs and Chinese patent CN118663236A discloses a method for preparing a core-shell cholesterol-based silica gel chromatography packing material, both of which are modified by chemical bonding, have complex reaction processes, introduce a large number of toxic or non-environmentally friendly organic reagents, have poor biocompatibility, and do not involve silk fibroin, and are only suitable for the separation of highly polar drugs or tea saponin compounds, and cannot meet the separation requirements of non-polar / weakly polar biomolecules. Summary of the Invention

[0005] The purpose of this invention is to solve the technical problems of traditional silica gel chromatography packing materials in the separation of biomacromolecules, such as insufficient specific binding capacity, low separation efficiency, poor biocompatibility, and easy denaturation of target proteins. The invention provides a silk fibroin-modified silica gel chromatography packing material, its preparation method, and its application to achieve efficient and mild separation of biomacromolecules.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] The first aspect of this invention provides a method for preparing silk fibroin-modified silica gel chromatography packing material, comprising the following steps:

[0008] S1. Add protease to the regenerated silk fibroin solution and incubate. Collect the silk fibroin precipitate by centrifugation. Dissolve the silk fibroin precipitate in a hydrogen bond inhibitor solution to obtain a hydrophobic silk fibroin solution. The concentration of hydrophobic silk fibroin in the hydrophobic silk fibroin solution is 2-20 wt%.

[0009] S2. Under stirring conditions, inorganic silica gel powder is dispersed into the hydrophobic silk fibroin solution obtained in S1, followed by dialysis; the stirring rate is 20-100 rpm.

[0010] S3. Using methanol and / or ethanol as cleaning solvents, the solid product after dialysis is soaked and cleaned, and then subjected to vacuum freeze-drying to obtain the silk fibroin modified silica gel chromatography packing.

[0011] This invention modifies the crystalline fibroin precipitate obtained after enzymatic hydrolysis with specific proteases by hydrogen-bonding crosslinking with inorganic silica gel, solving the technical problems of traditional silk fibroin's inability to be used as chromatographic packing material and the poor biocompatibility of traditional silica gel chromatographic packing material. The technical principle is as follows: Silk fibroin is regenerated through enzymatic hydrolysis with specific proteases, collecting only the crystalline precipitate rich in highly repetitive GAGAGS fragments and with a high β-sheet content. This precipitate possesses characteristics such as regular structure, hydrophobicity, non-polar / weakly polarity, excellent mechanical properties, and good biomolecular compatibility. The precipitate is dissolved in a hydrogen-bonding inhibitor solution to prepare a hydrophobic silk fibroin solution. Under appropriate concentration and stirring rate conditions, inorganic silica gel powder is added to stabilize and suspend it. The solution viscosity can be used to achieve stable suspension and uniform encapsulation of the silica gel. During dialysis, the hydrogen-bonding inhibitor is gradually removed, promoting hydrogen-bonding crosslinking between the peptides of the silk fibroin precipitate and the polar Si-OH groups on the silica gel surface, thereby achieving precise adjustment of the silica gel surface polarity. Subsequent soaking in methanol and / or ethanol can rapidly induce the formation of β-sheets of surface silk fibroin, achieving rapid sealing of the particle surface. This avoids excessive cross-linking between particles and accelerates the self-cross-linking of unbound pure silk fibroin to form small-diameter particles, facilitating subsequent sieving and removal. Finally, vacuum freeze-drying removes the cleaning solvent, yielding silk fibroin-modified silica gel chromatography packing material. This packing material possesses the advantages of regular surface structure, relatively uniform particle size, and adjustable polarity. It overcomes the defects of strong water solubility and easy hydrolysis of intact silk fibroin sequences, and solves the problems of insufficient specific adsorption of biomacromolecules and low separation efficiency of traditional silica gel.

[0012] Further, in S1, the concentration of silk fibroin in the regenerated silk fibroin solution is 0.5-10 wt%.

[0013] Further, in S1, the preparation method of the regenerated silk fibroin solution includes the following steps: adding silk to sodium carbonate solution and boiling, removing degummed silk and washing and drying it, then dissolving it in lithium bromide solution and dialysis purification to obtain the regenerated silk fibroin solution.

[0014] Further, in S1, the mass ratio of the protease to the silk fibroin in the regenerated silk fibroin solution is 1:(50-500).

[0015] Furthermore, in S1, the protease is selected from one or more of trypsin, α-chymotrypsin, pepsin, alkaline protease, papain, and proteinase K. All of these proteases are specific endopeptidases, capable of precisely identifying and cleaving the connecting peptides between the crystalline and amorphous regions, thus avoiding damage to the structure of the crystalline region by non-specific proteases.

[0016] Furthermore, in S1, the step of inactivating enzymes at 90-95 °C for 1-3 h is included before centrifugation.

[0017] Furthermore, in S1, the centrifugation speed is 7500-8500 rpm and the time is 5-20 min.

[0018] Further, in S1, the hydrogen bond inhibitor solution is selected from one or more of lithium bromide solution, sodium thiocyanate solution, zinc chloride solution, and calcium chloride-ethanol-water ternary solution. The function of the hydrogen bond inhibitor is to disrupt the hydrogen bond network inside the crystalline region of silk fibroin, allowing it to dissolve uniformly and form a solution.

[0019] Furthermore, the molar ratio of calcium chloride, ethanol and water is 1:2:8.

[0020] Furthermore, in S2, the particle size of the inorganic silica gel powder corresponds to 100-1000 mesh.

[0021] Further, in S2, the mass ratio of the inorganic silica gel powder to the hydrophobic silk fibroin in the hydrophobic silk fibroin solution is (2-20):1.

[0022] Furthermore, the stirring time is 20-60 min.

[0023] Furthermore, in S2, the dialysis treatment time is 8-48 h, and the temperature is 2-40 ℃.

[0024] Preferably, in step S3, the cleaning solvent is ethanol.

[0025] Furthermore, in S3, the soaking time is 10-60 min.

[0026] Furthermore, in S3, the vacuum degree of the vacuum freeze-drying process is 0-40 Pa, the temperature is -60 ℃ ~ -20 ℃, and the time is 6-24 h.

[0027] Furthermore, in step S3, the vacuum freeze-drying process further includes a step of sieving the product using a 1000-mesh sieve to remove pure silk fibroin particles. This step removes small self-crosslinked silk fibroin particles that are not bound to silica gel, preventing them from clogging the sieve plate in the chromatographic column and ensuring the smoothness of the separation process.

[0028] The second aspect of the present invention provides a silk fibroin-modified silica gel chromatography packing material prepared by the preparation method described in the first aspect.

[0029] The third aspect of this invention provides the application of the silk fibroin-modified silica gel chromatographic packing material described in the second aspect in the chromatographic separation of biomolecules.

[0030] The silk fibroin-modified silica gel chromatography packing material provided by this invention can be widely used for the separation and purification of biological macromolecules such as proteins and peptides, and is compatible with various chromatographic separation systems such as high performance liquid chromatography and column chromatography.

[0031] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0032] 1. This invention effectively overcomes the problem that traditional regenerated silk fibroin is difficult to use as a chromatographic packing material due to its strong water solubility, irregular chain structure, and easy hydrolysis. By using specific enzymatic digestion, silk fibroin precipitates rich in GAGAGS repeating fragments are collected. These precipitates have regular structure, hydrophobicity, and weak polarity, and can form hydrogen bonds with inorganic silica gel, thereby adjusting the polarity of the silica gel surface. This allows the modified product to be used as a high-performance chromatographic packing material in chromatographic separation processes.

[0033] 2. The silk fibroin-modified silica gel chromatography packing material prepared by this invention has excellent stability and durability. After elution with an acidic solution at pH=2 for 50 times, the silk fibroin shedding rate is less than 5%. Even after elution with organic solvents for 50 times, the silk fibroin shedding rate remains below 5%, which can meet the requirements for long-term repeated use.

[0034] 3. The silk fibroin-modified silica gel chromatography packing provided by this invention has significant advantages in the separation of biological macromolecules, especially protein components. The purity of the target components after separation is high, which fully demonstrates its excellent separation performance.

[0035] 4. This invention enables flexible adjustment of silica gel polarity. By controlling the concentration of the hydrophobic silk fibroin solution or the mass of the inorganic silica gel, the amount of silk fibroin coating on the surface of the inorganic silica gel can be precisely controlled. Thus, the polarity of the silica gel surface can be flexibly adjusted according to actual separation needs, adapting to separation scenarios of different biomacromolecules.

[0036] 5. The preparation method provided by this invention is environmentally friendly. It uses only a small amount of conventional chemical reagents, does not introduce toxic or unknown toxic organic reagents, and does not use chemical cross-linking agents such as glutaraldehyde. It achieves physical cross-linking only through hydrogen bonding, which reduces the impact on the environment and avoids the contamination of the separated products by chemical reagent residues. Attached Figure Description

[0037] Figure 1 The images show scanning electron microscope (SEM) images of the chromatographic packing materials prepared in Examples 1, 2, and 6; from left to right, they are Example 1 (scale bar 50 µm), Comparative Example 2 (scale bar 50 µm), and Comparative Example 6 (scale bar 5 µm).

[0038] Figure 2The image shows the high-performance liquid chromatography (HPLC) spectrum of the separation eluent of the mixed sample of lysozyme and bovine serum albumin prepared in Example 1 using the chromatographic packing material.

[0039] Figure 3 The image shows the HPLC detection spectrum of the eluent from the mixed sample of lysozyme and bovine serum albumin prepared by the chromatographic packing material in Comparative Example 1. Detailed Implementation

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0041] This invention provides a method for preparing silk fibroin-modified silica gel chromatography packing material, comprising the following steps:

[0042] S1. Add protease to the regenerated silk fibroin solution, incubate, centrifuge, collect the silk fibroin precipitate, dissolve the silk fibroin precipitate in hydrogen bond inhibitor solution to obtain hydrophobic silk fibroin solution.

[0043] S2. Disperse the inorganic silica gel powder into the hydrophobic silk fibroin solution obtained in S1, and then perform dialysis.

[0044] S3. The solid product after washing and dialysis is then freeze-dried under vacuum to obtain silk fibroin modified silica gel chromatography packing.

[0045] In this invention, after silk fibroin is enzymatically hydrolyzed by a specific protease, the connection sites between the crystalline and amorphous regions are cleaved, and the enzymatic hydrolysate is separated into a supernatant and a precipitate layer. The supernatant mainly consists of water-soluble amorphous fragments, while the precipitate layer is a crystalline region centered on a highly repetitive and regular GAGAGS sequence. Since the GAGAGS fragments do not contain large side chain groups, they can form the lowest-energy antiparallel β-sheet conformation through close packing via hydrogen bonds. This gives the precipitate a regular structure, hydrophobicity, nonpolar / weakly polarity, and good biomolecular compatibility, effectively overcoming the inherent defects of undigested silk fibroin and making it applicable to the field of biomolecular chromatographic separation. Dissolving this silk fibroin precipitate in a hydrogen bond inhibitor solution and then using it to coat inorganic silica gel with a surface rich in polar Si-OH groups allows for precise adjustment of the silica gel surface polarity, achieving the purpose of silica gel modification.

[0046] In one embodiment of the present invention, in S1, the hydrogen bond inhibitor solution is selected from one or more of lithium bromide solution, sodium thiocyanate solution, zinc chloride solution, and calcium chloride-ethanol-water ternary solution.

[0047] In one embodiment of the present invention, in S1, the concentration of hydrophobic silk fibroin in the hydrophobic silk fibroin solution is 2-20 wt%.

[0048] In one embodiment of the present invention, in S2, the mass ratio of the inorganic silica gel powder to the hydrophobic silk fibroin in the hydrophobic silk fibroin solution is (2-20):1.

[0049] In one embodiment of the present invention, in S2, inorganic silica powder is dispersed into the hydrophobic silk fibroin solution under stirring conditions; the stirring rate is 20-100 rpm and the time is 20-60 min.

[0050] As a highly shear-sensitive biomacromolecule, silk fibroin, in a hydrophobic solution of a specific concentration, will cause the solution to reach a certain viscosity and undergo slight cross-linking under appropriate stirring rates. This allows the inorganic silica gel powder to remain stably suspended in the solution, ensuring uniform encapsulation of the silica gel by the silk fibroin. In the hydrogen bond inhibitor system, the Si-OH groups of the inorganic silica gel and the GAGAGS peptides in the silk fibroin precipitate are both in an activated state (uncross-linked or slightly cross-linked). During dialysis, the hydrogen bond inhibitor is gradually removed, and the GAGAGS peptides undergo hydrogen bond cross-linking with the Si-OH groups, tightly encapsulating the inorganic silica gel and further regulating the surface polarity of the silica gel.

[0051] In one embodiment of the present invention, in step S3, the specific cleaning operation is as follows: first soak in a cleaning solvent for 10-60 minutes, and then clean; the cleaning solvent is methanol and / or ethanol.

[0052] In one embodiment of the present invention, in S3, the vacuum degree of the vacuum freeze-drying process is 0-40 Pa, the temperature is -60 ℃ ~ -20 ℃, and the time is 6-24 h.

[0053] When silk fibroin-coated silica particles are immersed in methanol and / or ethanol for cleaning, the methanol and / or ethanol can rapidly induce the silk fibroin on the particle surface to form a β-sheet conformation, achieving rapid sealing of the silica particle surface. This avoids excessive physical cross-linking between particles and accelerates the self-cross-linking of unbound silk fibroin to form small-diameter particles, which are then easily removed by sieving. After removing the cleaning solvent using a vacuum freeze-drying process, modified silica particles with silk fibroin coating, regular surface structure, relatively uniform particle size, and adjustable polarity are finally obtained.

[0054] Based on the "like dissolves like" principle, silk fibroin, which has good biocompatibility, is combined with inorganic silica gel, which has a supporting and porous structure, so that the resulting silk fibroin-modified silica gel particles can be used as high-performance chromatographic packing materials for the chromatographic separation of biomacromolecules.

[0055] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0057] The sodium carbonate (Na2CO3) used in the following examples was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number 10019260; lithium bromide (LiBr) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number L108934; sodium thiocyanate (NaSCN) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number S140873; zinc chloride (ZnCl2) was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number 10023818; calcium chloride (CaCl2) was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number JJ2974; ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number 100092683; methanol was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number 10014128; and trypsin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The following products were purchased: α-chymotrypsin (C804761) from Shanghai Maclean Biochemical Technology Co., Ltd.; pepsin (P110928) from Shanghai Aladdin Biochemical Technology Co., Ltd.; proteinase K (JK-038) from Shanghai Jingke Chemical Technology Co., Ltd.; papain (P3250) from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; alkaline protease (QN0397) from Beijing Bio-Lab Technology Co., Ltd.; phosphate buffer (PBS) solution (bzw2106f) from Nanjing Bianzhen Biotechnology Co., Ltd.; inorganic silica gel (PA00955) from Guangdong Wengjiang Chemical Reagent Co., Ltd.; and petroleum ether (10015218) from Sinopharm Chemical Reagent Co., Ltd.

[0058] Example 1

[0059] A method for preparing a silk fibroin-modified silica gel chromatography packing material includes the following steps:

[0060] S1. After boiling silk in 0.5 g / L Na2CO3 solution for 1 h, the degummed silk was removed, washed and dried, dissolved in 9.3 M lithium bromide solution, and purified by dialysis to obtain a 0.5 wt% regenerated silk fibroin solution. An equal volume of the 0.5 wt% regenerated silk fibroin solution and a 0.01 wt% trypsin solution were mixed and reacted at 37 ℃ for 24 h. After enzyme inactivation at 95 ℃ for 2 h, the mixture was centrifuged at 8000 rpm and washed for 10 min to obtain silk fibroin precipitate. The silk fibroin precipitate was dissolved in 9.3 M lithium bromide solution to obtain a 2 wt% hydrophobic silk fibroin solution.

[0061] S2. Disperse 200 g of 100-1000 mesh inorganic silica powder into 500 mL of 2 wt% hydrophobic silk fibroin solution at a stirring frequency of 20 rpm, stir for 20 min, and dialyze at 2 °C for 48 h using water as the medium.

[0062] S3. The solid product after dialysis was soaked in ethanol for 10 min and washed repeatedly. Then it was dried under vacuum of 10 Pa and temperature of -60 ℃ for 6 h. The product was sieved using a 1000-mesh sieve to remove pure silk fibroin particles, and silk fibroin modified silica gel chromatography packing was obtained.

[0063] Example 2

[0064] A method for preparing a silk fibroin-modified silica gel chromatography packing material includes the following steps:

[0065] S1. After boiling silk in 0.5 g / L Na2CO3 solution for 1 h, the degummed silk was removed, washed and dried, dissolved in 9.3 M lithium bromide solution, and purified by dialysis to obtain a 10 wt% regenerated silk fibroin solution. An equal volume of the 10 wt% regenerated silk fibroin solution and a 0.02 wt% α-chymotrypsin solution were mixed and reacted at 37 ℃ for 24 h. After enzyme inactivation at 95 ℃ for 2 h, the mixture was centrifuged at 8000 rpm for 10 min to obtain silk fibroin precipitate. The silk fibroin precipitate was dissolved in 4.0 M sodium thiocyanate solution to obtain a 20 wt% hydrophobic silk fibroin solution.

[0066] S2. Disperse 200 g of 100-1000 mesh inorganic silica powder into 500 mL of 20 wt% hydrophobic silk fibroin solution at a stirring frequency of 100 rpm, stir for 60 min, and dialyze at 25 ℃ for 20 h with water as the medium.

[0067] S3. The dialysis solid product was soaked in ethanol for 60 min and washed repeatedly. Then it was dried for 24 h under vacuum of 20 Pa and temperature of -20 ℃. The product was then sieved through a 1000-mesh sieve to remove pure silk fibroin particles, thus obtaining silk fibroin-modified silica gel chromatography packing.

[0068] Example 3

[0069] A method for preparing a silk fibroin-modified silica gel chromatography packing material includes the following steps:

[0070] S1. After boiling silk in 0.5 g / L Na2CO3 solution for 1 h, the degummed silk was removed, washed and dried, dissolved in 9.3 M lithium bromide solution, and purified by dialysis to obtain a 5 wt% regenerated silk fibroin solution. The 5 wt% regenerated silk fibroin solution was mixed with an equal volume of 0.05 wt% pepsin solution, reacted at 37 ℃ for 24 h, and then the enzyme was inactivated at 95 ℃ for 2 h. After centrifugation at 8000 rpm for 10 min, the silk fibroin precipitate was obtained. The silk fibroin precipitate was dissolved in 6.0 M zinc chloride solution to obtain a 10 wt% hydrophobic silk fibroin solution.

[0071] S2. Disperse 250 g of 100-1000 mesh inorganic silica powder into 500 mL of 10 wt% hydrophobic silk fibroin solution at a stirring frequency of 50 rpm, stir for 30 min, and dialyze at 40 ℃ for 12 h with water as the medium.

[0072] S3. The dialysis solid product was soaked in ethanol for 30 min and washed repeatedly. Then it was dried for 12 h under vacuum of 30 Pa and temperature of -30 ℃. The product was then sieved through a 1000-mesh sieve to remove pure silk fibroin particles, thus obtaining silk fibroin modified silica gel chromatography packing.

[0073] Example 4

[0074] A method for preparing a silk fibroin-modified silica gel chromatography packing material includes the following steps:

[0075] S1. After boiling silk in 0.5 g / L Na2CO3 solution for 1 h, the degummed silk was removed, washed and dried, dissolved in 9.3 M lithium bromide solution, and purified by dialysis to obtain a 4 wt% regenerated silk fibroin solution. The 4 wt% regenerated silk fibroin solution was mixed with an equal volume of 0.03 wt% alkaline protease solution, reacted at 37 ℃ for 24 h, and the enzyme was inactivated at 95 ℃ for 2 h. After centrifugation at 8000 rpm for 10 min, silk fibroin precipitate was obtained. The silk fibroin precipitate was dissolved in a ternary solution of calcium chloride-ethanol-water (molar ratio 1:2:8) to obtain a 5 wt% hydrophobic silk fibroin solution.

[0076] S2. Disperse 100 g of 100-1000 mesh inorganic silica powder into 500 mL of 5 wt% hydrophobic silk fibroin solution at a stirring frequency of 60 rpm, stir for 40 min, and dialyze at 37 ℃ for 12 h with water as the medium.

[0077] S3. The solid product after dialysis was soaked in ethanol for 40 min and washed repeatedly. Then it was dried under vacuum of 40 Pa and temperature of -50 ℃ for 8 h. The product was sieved using a 1000 mesh sieve to remove pure silk fibroin particles, and silk fibroin modified silica gel chromatography packing was obtained.

[0078] Example 5

[0079] A method for preparing a silk fibroin-modified silica gel chromatography packing material includes the following steps:

[0080] S1. After boiling silk in 0.5 g / L Na2CO3 solution for 1 h, the degummed silk was removed, washed and dried, dissolved in 9.3 M lithium bromide solution, and purified by dialysis to obtain a 3 wt% regenerated silk fibroin solution. The 3 wt% regenerated silk fibroin solution was mixed with an equal volume of 0.02 wt% papain solution, reacted at 37 ℃ for 24 h, and then the enzyme was inactivated at 95 ℃ for 2 h. After centrifugation at 8000 rpm for 10 min, silk fibroin precipitate was obtained. The silk fibroin precipitate was dissolved in 9.3 M lithium bromide solution to obtain a 15 wt% hydrophobic silk fibroin solution.

[0081] S2. Disperse 200 g of 100-1000 mesh inorganic silica powder into 500 mL of 15 wt% hydrophobic silk fibroin solution at a stirring frequency of 80 rpm, stir for 50 min, and dialyze at 25 ℃ for 16 h with water as the medium.

[0082] S3. The solid product after dialysis was soaked in ethanol for 50 min and washed repeatedly. Then it was dried under vacuum of 25 Pa and temperature of -55 ℃ for 6 h. The product was sieved through a 1000-mesh sieve to remove pure silk fibroin particles, and silk fibroin modified silica gel chromatography packing was obtained.

[0083] Example 6

[0084] A method for preparing a silk fibroin-modified silica gel chromatography packing material includes the following steps:

[0085] S1. After boiling silk in 0.5 g / L Na2CO3 solution for 1 h, the degummed silk was removed, washed and dried, dissolved in 9.3 M lithium bromide solution, and purified by dialysis to obtain a 6 wt% regenerated silk fibroin solution. The 6 wt% regenerated silk fibroin solution was mixed with an equal volume of 0.02 wt% proteinase K solution, reacted at 37 ℃ for 24 h, and the enzyme was inactivated at 95 ℃ for 2 h. After centrifugation at 8000 rpm for 10 min, silk fibroin precipitate was obtained. The silk fibroin precipitate was dissolved in 9.3 M lithium bromide solution to obtain a 12 wt% hydrophobic silk fibroin solution.

[0086] S2. Disperse 300 g of 100-1000 mesh inorganic silica powder into 500 mL of 12 wt% hydrophobic silk fibroin solution at a stirring frequency of 40 rpm, stir for 25 min, and dialyze at 25 ℃ for 8 h with water as the medium.

[0087] S3. The solid product after dialysis was soaked in ethanol for 45 min and washed repeatedly. Then it was dried under vacuum of 15 Pa and temperature of -30 ℃ for 6 h. The product was sieved using a 1000-mesh sieve to remove pure silk fibroin particles, and silk fibroin modified silica gel chromatography packing was obtained.

[0088] Comparative Example 1

[0089] A method for preparing a silk fibroin-modified silica gel chromatography packing is basically the same as in Example 1, except that: in S1, silk is added to a 0.5 g / L Na2CO3 solution and boiled for 1 h, then the degummed silk is taken out, washed and dried, dissolved in a 9.3 M lithium bromide solution, and purified by dialysis to obtain a 2 wt% regenerated silk fibroin solution; no subsequent enzymatic hydrolysis treatment is performed.

[0090] Comparative Example 2

[0091] A method for preparing a silk fibroin-modified silica gel chromatography packing is basically the same as that in Example 1, except that in S1, the concentration of the hydrophobic silk fibroin solution is 0.5 wt%.

[0092] Comparative Example 3

[0093] A method for preparing a silk fibroin-modified silica gel chromatography packing is basically the same as that in Example 1, except that in S2, the stirring frequency is 500 rpm.

[0094] Comparative Example 4

[0095] A method for preparing a silk fibroin-modified silica gel chromatography packing is basically the same as that in Example 1, except that in S3, the dialyzed solid product is soaked in pure water for 10 min and repeatedly washed.

[0096] Comparative Example 5

[0097] A method for preparing a silk fibroin-modified silica gel chromatography packing is basically the same as that in Example 1, except that in S3, the dialyzed solid product is soaked in ethanol for 10 min, repeatedly washed, and then dried in a conventional 80 ℃ oven for 6 h. The product is then sieved using a 1000-mesh sieve to remove pure silk fibroin particles, thus obtaining the silk fibroin-modified silica gel chromatography packing.

[0098] Comparative Example 6

[0099] A method for preparing a silk fibroin precipitation chromatography packing material includes the following steps:

[0100] S1. After boiling silk in 0.5 g / L Na2CO3 solution for 1 h, the degummed silk was removed, washed and dried, dissolved in 9.3 M lithium bromide solution, and purified by dialysis to obtain a 0.5 wt% regenerated silk fibroin solution. An equal volume of the 0.5 wt% regenerated silk fibroin solution and a 0.01 wt% trypsin solution were mixed and reacted at 37 ℃ for 24 h. After enzyme inactivation at 95 ℃ for 2 h, the mixture was centrifuged at 8000 rpm and washed for 10 min to obtain silk fibroin precipitate. The silk fibroin precipitate was dissolved in 9.3 M lithium bromide solution to obtain a 2 wt% hydrophobic silk fibroin solution.

[0101] S2. Stir a 2 wt% hydrophobic silk fibroin solution at a stirring frequency of 20 rpm for 20 min, and dialyze it with water at 2 ℃ for 48 h.

[0102] S3. The solid product after dialysis is soaked in ethanol for 10 min, washed repeatedly, and then dried under vacuum of 10 Pa and temperature of -60 ℃ for 6 h to obtain silk fibroin precipitation chromatography packing.

[0103] Figure 1 SEM images of the chromatographic packing materials prepared in Example 1, Comparative Example 2, and Comparative Example 6 are shown below. Figure 1 As can be seen, the chromatographic packing material of Example 1 exhibits spherical particles with uniform particle size and smooth, regular surface, laying a microscopic foundation for stable separation performance. The chromatographic packing material of Comparative Example 2 has rough particle surface and irregular morphology. This is because the concentration of its hydrophobic silk fibroin solution is only 0.5 wt%, and the insufficient viscosity leads to silica gel sedimentation and the inability of silk fibroin to be uniformly coated. Such defects directly lead to a decrease in the specific surface area of ​​the packing material and uneven adsorption sites. The chromatographic packing material of Comparative Example 6 only exhibits a large number of amorphous silk fibroin particles with small particle size and severe aggregation. It completely lacks silica gel as a core support. Since the packing material is prepared by only using pure enzymatically digested silk fibroin, it has neither a rigid support structure nor particle aggregation and rough surface due to protein self-crosslinking. It is not only easy to lose, but also cannot control polarity, and does not have the application value of chromatographic packing material.

[0104] Comparative Example 7

[0105] A method for preparing an inorganic silica gel chromatographic packing material includes the following steps:

[0106] S1. Disperse 200 g of 100-1000 mesh inorganic silica powder into 500 mL of pure water at a stirring frequency of 20 rpm, stir for 20 min, and dialyze at 2 °C for 48 h using water as the medium.

[0107] S2. The dialysis solid product was soaked in ethanol for 10 min, washed repeatedly, and then dried under vacuum of 10 Pa and temperature of -60 ℃ for 6 h. The product was then sieved using a 1000-mesh sieve to obtain inorganic silica gel chromatographic packing material.

[0108] Test Example 1

[0109] The chromatographic packing materials prepared in Examples 1-6 and Comparative Examples 1-7 were characterized and tested. The specific test items and methods are as follows:

[0110] (1) Amino acid content test: According to GB / T 32016-2015 "Determination of Amino Acids in Silk", an amino acid analyzer was used for detection. The chromatographic packing was diluted with concentrated hydrochloric acid and placed in a sealed tube, hydrolyzed at 110 ℃ for 22 h, and then redissolved in 0.01 M hydrochloric acid solution after nitrogen blowing treatment. After filtration, the test was performed, and the mass ratio of glycine (G), alanine (A) and serine (S) to the total amino acids was calculated.

[0111] (2) Observation of properties: The chromatographic packing was placed in a colorless cold white light environment without background interference, and the morphological changes and appearance characteristics of the finished product were observed by the naked eye. The microstructure was observed and analyzed by scanning electron microscopy (SEM).

[0112] (3) Particle size test: The particle size of the chromatographic packing material of Comparative Example 6 was tested according to the particle size and particle size distribution determination method in Chinese Pharmacopoeia 0982; the particle size range of the chromatographic packing materials of the other examples and comparative examples was calculated according to the mesh number of the sieve used for sieving.

[0113] (4) Acid elution stability test: The chromatographic packing material was packed into the chromatographic column and eluted repeatedly 50 times with an acidic solution of pH=2 at approximately 5 column volumes. The protein content on the surface of the packed particles before and after elution was determined according to the protein content determination method in Chinese Pharmacopoeia 0731, and the protein loss rate was calculated.

[0114] (5) Organic solvent elution stability test: The chromatographic packing material was packed into the chromatographic column and eluted repeatedly with petroleum ether at a volume of about 5 times the column volume for 50 times. The protein content on the surface of the packed particles before and after elution was determined according to the protein content determination method in Chinese Pharmacopoeia 0731, and the protein loss rate was calculated.

[0115] (6) Resolution Test: The chromatographic column was packed with chromatographic packing material. Lysozyme and bovine serum albumin, two biomolecules, were dissolved and mixed in pure water to prepare a mixed sample. This sample was then added to the packed chromatographic column. After optimizing the separation method, elution was performed using 5 column volumes of PBS buffer. The eluent was collected in fractions according to the elution sequence. All eluent was divided into 5 equal volumes and transferred to 5 test tubes, which were labeled tube 1, tube 2, tube 3, tube 4, and tube 5, respectively, to collect the eluent that flowed out in sequence during the elution process. The eluent in each tube was detected by HPLC. The detection conditions were set as follows: the chromatographic column was BioBasic. TM PREP SEC 120 (30 cm × 7.8 mm), column temperature maintained at 40 ℃, mobile phase is pure water, flow rate is controlled at 0.8 mL / min, and detection wavelength is set at 280 nm.

[0116] The test results are shown in Tables 1 and 2. Figure 2 and Figure 3 As shown:

[0117] Table 1

[0118]

[0119] Note: Comparative Example 7 prepared pure inorganic silica gel without introducing silk fibroin for modification. Since the three indicators of amino acid content, acid elution protein loss rate and organic solvent elution protein loss rate are all based on silk fibroin, Comparative Example 7 does not need to perform the above tests. The relevant data are represented by " / ".

[0120] Table 2

[0121]

[0122] Figure 2 The image shows the HPLC detection chromatogram of the eluent from the mixed sample of lysozyme and bovine serum albumin prepared in Example 1, demonstrating the separation effect of the chromatographic packing material of Example 1: lysozyme showed a characteristic peak only in the eluent of tube 2, and bovine serum albumin showed a characteristic peak only in the eluent of tube 3, with no other interfering peaks in the eluents of either tube. This result directly proves that the silk fibroin-modified silica gel chromatographic packing material prepared in Example 1 can achieve effective baseline-level separation by specifically interacting with the two proteins, resulting in a significant difference in their retention times in the chromatographic column. This further demonstrates the excellent performance of the silk fibroin-modified silica gel chromatographic packing material prepared in this invention in the chromatographic separation of biomacromolecules.

[0123] Comparative Example 1 directly used undigested regenerated silk fibroin solution to modify inorganic silica gel to prepare chromatographic packing material. Because the regenerated silk fibroin had not undergone specific protease hydrolysis, the proportion of glycine (G), alanine (A), and serine (S) was only 76%, far lower than the 94%-96% in Examples 1-6; moreover, polar and non-polar segments coexisted in the molecular chain, resulting in an irregular structure, high water solubility, and easy chain breakage and hydrolysis. Although silica gel could achieve uniform suspension during the preparation process, and the final product exhibited a relatively uniform particle size and regular surface morphology, after repeated elution with acidic solutions and organic solvents, a large amount of silk fibroin was hydrolyzed and lost, with a loss rate far exceeding the standard of less than 5% in the examples, indicating significantly insufficient durability and stability of the packing material. Simultaneously, the loss of silk fibroin led to increased surface polarity of the packing material and a decrease in column efficiency. The HPLC detection chromatogram of the separation eluent of the mixed sample of lysozyme and bovine serum albumin is shown below. Figure 3 As shown, lysozyme and bovine serum albumin had extremely short retention times, and both were detected in the first elution tube, indicating that effective separation was not achieved.

[0124] In Comparative Example 2, the concentration of the hydrophobic silk fibroin solution was only 0.5 wt%. At this concentration, the solution viscosity was insufficient to stably suspend the 100-1000 mesh inorganic silica gel powder. The silica gel rapidly settled to the bottom of the solution, resulting in the silk fibroin failing to uniformly coat the silica gel particles. Consequently, the resulting filler had incomplete surface coating defects. This type of filler exhibits irregular surface morphology and a low specific surface area, leading to decreased column efficiency. Furthermore, due to structural defects, after repeated elution with acidic solutions and organic solvents, the silk fibroin loss rate exceeded 5%, indicating inferior durability and stability compared to the example. During the separation of lysozyme and bovine serum albumin, both proteins eluted simultaneously in the first eluent tube, failing to achieve effective separation.

[0125] In Comparative Example 3, the stirring rate was increased to 500 rpm. Silk fibroin, being a highly shear-sensitive biomacromolecule, was subjected to excessively high stirring rates, leading to the formation of self-crosslinking flocs. The hydrogen bond network of these flocs was closed, making it difficult for them to crosslink with the Si-OH groups on the silica gel surface. The resulting particles had irregular surface morphology, insufficient silk fibroin coating, and physical crosslinking. The adhesion between silica gel and silk fibroin relied solely on van der Waals forces, resulting in weak bonding. After repeated elution with acidic solutions and organic solvents, the silk fibroin loss rate exceeded 5%, indicating poor packing durability and stability. Furthermore, the silk fibroin failed to form effective hydrogen bond crosslinks with the silica gel. Protein loss during elution increased the polarity of the silica gel surface, decreased column efficiency, and resulted in extremely short retention times for lysozyme and bovine serum albumin, which also eluted in the first eluent tube, failing to achieve effective separation.

[0126] Comparative Example 4 omitted the ethanol soaking and washing step. The role of ethanol is to induce rapid hydrogen bonding cross-linking of silk fibroin, achieving rapid sealing of the packing surface. Because this step was omitted, the surface morphology of the packing in Comparative Example 4 was irregular. After multiple elutions with acidic solution and organic solvents, the silk fibroin loss rate exceeded 5%, and its durability and stability were inferior to the examples. Simultaneously, the irregular surface of the packing reduced the specific surface area, and the loss of packing material from the column caused a decrease in column efficiency. The retention time of bovine serum albumin was shortened, and it eluted simultaneously with lysozyme in the second eluent tube, failing to achieve effective separation.

[0127] Comparative Example 5 used a conventional drying process to prepare the packing material. Conventional drying easily causes particle adhesion. After physical crushing, the surface morphology of the packing material is irregular, which not only easily leads to in-column runoff but also exposes a large number of Si-OH groups on the silica gel surface. After multiple elutions with acidic solution and organic solvent, the silk fibroin loss rate exceeded 5%. Affected by the combined effects of reduced specific surface area and in-column runoff, column efficiency decreased, bovine serum albumin retention time was shortened, and it eluted in the second eluent tube along with lysozyme, failing to achieve effective separation.

[0128] Comparative Example 6 used pure silk fibroin after enzymatic digestion to prepare the packing material. The pure silk fibroin particles were small (D99 < 13 μm), theoretically capable of complete removal through a 1000-mesh sieve (corresponding to a particle size of approximately 13 μm); however, these particles had extremely irregular surface morphology and lacked inorganic silica gel rich in Si-OH groups as a core and support, resulting in both easy protein loss and inability to control the packing material polarity. After multiple elutions with acidic solution and organic solvents, the silk fibroin loss rate exceeded 5%; during the separation of lysozyme and bovine serum albumin, both proteins eluted simultaneously in the second eluent tube, failing to achieve effective separation.

[0129] Comparative Example 7 used pure inorganic silica gel as the chromatographic packing material. The surface of pure inorganic silica gel is rich in polar Si-OH groups, lacking the coating and polarity regulation needed for enzymatically cleaved silk fibroin proteins, resulting in insufficient biocompatibility and specific adsorption capacity for protein-based biomolecules. When separating lysozyme and bovine serum albumin, both proteins had extremely short retention times and eluted simultaneously in the first eluent tube, failing to achieve effective separation.

[0130] In summary, this invention successfully prepared silk fibroin-modified silica gel chromatography packing material through a core process involving specific protease hydrolysis to regenerate silk fibroin, precise enrichment of GAGAGS crystalline regions, and hydrogen-bonded cross-linking coating of inorganic silica gel. Test results show that the chromatography packing materials prepared in Examples 1-6 all possess characteristics such as high G+A+S amino acid content (94%-96%), uniform particle size, and regular surface. After repeated elution with acidic solutions and organic solvents, the protein loss rate was less than 5%, demonstrating excellent stability and durability. In biomolecular separation tests, precise separation of lysozyme and bovine serum albumin was achieved, with the two target proteins eluting separately in the second and third eluent tubes, respectively, demonstrating significant separation efficiency. The preparation method of this invention significantly improves the biocompatibility and specific adsorption capacity of silica gel chromatography packing material, effectively solving the technical problems of low separation efficiency and easy protein denaturation caused by traditional silica gel packing materials. The prepared silk fibroin-modified silica gel chromatography packing material has outstanding application value in the field of biomolecular chromatographic separation.

[0131] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art should understand that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a silk fibroin-modified silica gel chromatography packing material, characterized in that, Includes the following steps: S1. Add protease to the regenerated silk fibroin solution and incubate. Collect the silk fibroin precipitate by centrifugation. Dissolve the silk fibroin precipitate in a hydrogen bond inhibitor solution to obtain a hydrophobic silk fibroin solution. The concentration of hydrophobic silk fibroin in the hydrophobic silk fibroin solution is 2-20 wt%. The protease is selected from one or more of trypsin, α-chymotrypsin, pepsin, papain, and proteinase K. The hydrogen bond inhibitor solution is selected from one or more of lithium bromide solution, sodium thiocyanate solution, zinc chloride solution, and calcium chloride-ethanol-water ternary solution. S2. Under stirring conditions, inorganic silica gel powder is dispersed into the hydrophobic silk fibroin solution obtained in S1, followed by dialysis; the stirring rate is 20-100 rpm. S3. Using methanol and / or ethanol as cleaning solvents, the solid product after dialysis is soaked and cleaned, and then subjected to vacuum freeze-drying to obtain the silk fibroin modified silica gel chromatography packing.

2. The preparation method according to claim 1, characterized in that, In S1, the concentration of silk fibroin in the regenerated silk fibroin solution is 0.5-10 wt%.

3. The preparation method according to claim 1, characterized in that, In S1, the mass ratio of the protease to the silk fibroin in the regenerated silk fibroin solution is 1:(50-500).

4. The preparation method according to claim 1, characterized in that, In S2, the particle size of the inorganic silica gel powder corresponds to 100-1000 mesh; And / or, the mass ratio of the inorganic silica gel powder to the hydrophobic silk fibroin in the hydrophobic silk fibroin solution is (2-20):

1.

5. The preparation method according to claim 1, characterized in that, In S2, the dialysis treatment time is 8-48 h and the temperature is 2-40 ℃.

6. The preparation method according to claim 1, characterized in that, In S3, the vacuum degree of the vacuum freeze-drying process is 0-40 Pa, the temperature is -60 ℃ ~ -20 ℃, and the time is 6-24 h.

7. A silk fibroin-modified silica gel chromatography packing material prepared by the preparation method according to any one of claims 1-6.

8. The application of the silk fibroin-modified silica gel chromatographic packing material as described in claim 7 in the chromatographic separation of biomacromolecules.

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