Method for extracting protein multi-level structure by periodate oxidation

Abstract

The present invention discloses a method for extracting a multi-level structure of a protein by periodate oxidation, and relates to the field of protein separation and extraction. The steps of the method include: mixing a bio-based raw material and periodic acid; adjusting the pH of the reaction solution with an alkali solution; performing an oxidation reaction at room temperature in the dark; and dialyzing, filtering, settling, and centrifuging the reaction solution to obtain a multi-level structure protein product. The method provided by the present invention does not require complex pretreatment and defatting treatment of the bio-based raw material, has mild reaction conditions, and is simple to operate. By regulating the oxidation reaction time, different levels of protein biological structures can be obtained, and the method is highly efficient, controllable, and environmentally friendly.

Description

Technical Field

[0001] The present invention relates to the field of protein separation and extraction, and in particular to a method for extracting protein multi-level structures by oxidation with periodate. Background Art

[0002] Proteins are among the most abundant organic molecules in natural organisms, possessing a wide variety of types and functions. Due to their excellent bioactivity, biocompatibility, degradability, and absorbability, protein-based biomaterials are widely used in fields such as tissue engineering, bioelectronics, drug delivery, wound repair, and pharmaceutical manufacturing. The first step in utilizing natural proteins to create biofunctional materials is to fully extract the proteins from the bio-based raw materials. The integrity of the proteins during the extraction process directly determines the quality of the data obtained from downstream protein sample analysis and influences the performance of subsequent biomaterials.

[0003] Common methods for protein extraction, such as physical, chemical, and enzymatic degradation, inevitably disrupt cellular homeostasis, leading to protein degradation or instability, resulting in a relatively small size for most extracted proteins. Biological tissues or structures containing complex and stable hierarchical protein structures are particularly challenging for protein extraction and the preparation of functional materials. For example, biological structures containing keratin (such as hair, feathers, nails, horns, and hooves) possess strong mechanical strength and are difficult to dissolve and decompose, making the extraction of intact keratin extremely challenging. Physical methods often employ methods such as high-pressure hydrolysis, microwave radiation, and superheated hydrolysis to break and hydrolyze the disulfide bonds between or even within keratin molecules in hair fibers, thereby rendering the keratin soluble. Chemical methods typically utilize chemical reagents such as acids, bases, oxidants, or reducing agents to react with the disulfide bonds in hair to increase the solubility of keratin molecules. Biological methods primarily utilize keratinase to degrade and hydrolyze keratin into peptides. However, most of the above methods will destroy the disulfide bonds in the biological structure of keratin while also destroying the peptide bonds, leading to the destruction of the primary protein structure, resulting in the acquisition of some peptide mixtures. Some methods even use high temperatures to treat the biological structure, which will lead to the loss of some amino acids.

[0004] Therefore, the present invention aims to provide a method for stripping and extracting protein structures at different levels. The method uses mild, selectively oxidizable periodic acid as an oxidant, and carries out an oxidation reaction at room temperature in the dark to extract proteins. By controlling the reaction time, proteins of different levels of structure can be obtained, providing a new idea and approach for protein extraction methods. Summary of the Invention

[0005] In response to the deficiencies of the above-mentioned prior art, the present invention provides a method for extracting the multi-level structure of proteins through periodate oxidation. Mild and selectively oxidizable periodic acid is used as an oxidant, and an oxidation reaction is carried out at room temperature in the dark to extract proteins. By controlling the reaction time, proteins of different levels of structure are obtained. The method provided by the present invention is expected to achieve precise separation and extraction of multi-level protein structures, further promoting the application of proteins in the field of functional material preparation.

[0006] In order to achieve the above object, the specific technical solutions of the present invention are as follows:

[0007] The method for extracting protein multi-level structure by periodate oxidation comprises the following steps:

[0008] Step 1: Oxidation reaction of periodate

[0009] (1) Mix the crushed bio-based raw materials, pure water and periodic acid, stir to dissolve the periodic acid and keep stirring, add alkali solution to adjust the pH of the reaction solution. Under neutral conditions, the periodate ion form in the reaction is H2I2O 10 4- , the ion has a more negative charge and a larger ionic radius.

[0010] (2) The oxidation reaction was carried out by stirring at 1000 rpm in the dark at room temperature.

[0011] Step 2: Purification and separation of protein hierarchical structures

[0012] (1) After the oxidation reaction is completed, add an appropriate amount of ethylene glycol to quench the unreacted periodic acid, then dialyze and filter the reaction solution, and collect the filtrate.

[0013] (2) Allow the filtrate to settle for 24 hours, then slowly remove the sample and collect the sediment P1 and the upper turbid liquid P2.

[0014] (3) The precipitate P1 is washed with water several times and centrifuged, and the upper turbid liquid P2 is centrifuged and washed with water, and then dried to obtain the multi-level structure of the protein.

[0015] Preferably, the protein comprises keratin.

[0016] Preferably, the length of the crushed bio-based feedstock is less than 1 cm.

[0017] Preferably, the ratio of the bio-based raw material, pure water, and periodic acid is 1:(20-100):(1-7); further preferably, the ratio of the bio-based raw material, pure water, and periodic acid is 1:(20-100):4.

[0018] Preferably, the alkali solution is LiOH solution or ammonia water, and the pH is adjusted to 6.5-7.

[0019] Preferably, the alkali solution is a KOH solution, and the pH is adjusted to 8.5-11.

[0020] Preferably, the reaction time of the oxidation reaction is 1 to 15 days.

[0021] Preferably, the molecular weight cut-off of the dialysis bag used for dialysis is 7000-14000 Da.

[0022] Preferably, the dialysis treatment further comprises changing the water multiple times until the conductivity is less than 1 μS / cm.

[0023] Preferably, the filter cloth used for the filtration is 200-800 mesh.

[0024] The method of the present invention uses periodic acid as a reaction reagent. By adjusting the ratio of bio-based raw materials to periodic acid and the concentration of the reaction solution, adjusting the pH of the reaction solution with an appropriate alkali solution, and controlling the reaction time, a protein hierarchical structure product is isolated. Using the method of the present invention to extract the hierarchical keratin structure, the precipitate P1 is washed with water multiple times and centrifuged to form clean cortical cells at the bottom. The upper turbid liquid P2 is centrifuged at 10,000 rpm to precipitate filaments, and the supernatant is keratin nanoparticles. The filamentous precipitate is washed with water and centrifuged multiple times at 10,000 rpm to obtain relatively pure filaments. The cortical cells, filaments, and keratin nanoparticles are frozen at low temperature for 24 hours and then freeze-dried in a vacuum for 24-48 hours to obtain the dried keratin hierarchical structure. During the oxidation reaction, the reaction system with LiOH adjusted to a pH of 6.5-7 was optimal. The highest yield of cortical cell products was achieved within 1-3 days, with a maximum yield of 20%. The best yield was achieved within 3-6 days, with a maximum yield of 16%. Within 6-10 days, the product was primarily keratin nanoparticles, with a maximum yield of 76%. The isolated cortical cells exhibited a spindle-shaped morphology, ranging from 150-200 microns in length and 5-10 microns in diameter. The isolated filaments, derived from the exfoliation of cortical cells, were filamentous, with a diameter of 0.1-0.4 microns and a length of 2-10 microns. The isolated protein nanoparticles were spherical with a core-shell structure and a diameter of 50-200 nm.

[0025] Compared with the prior art, the present invention is beneficial in that:

[0026] (1) The method provided by the present invention for extracting the multi-level structure of protein by periodate oxidation avoids the severe damage to the keratin structure caused by traditional methods and greatly preserves the natural micro-nanostructure of the protein, which provides an ideal raw material for the preparation of high-performance protein-based advanced functional materials.

[0027] (2) The method provided by the present invention does not require complex pretreatment of biological raw materials, nor does it require defatting of biological materials. The method is simple to operate and can obtain different levels of protein biological structures by simply regulating simple reaction conditions. It is highly efficient, controllable, and environmentally friendly, and is more conducive to industrial development.

[0028] (3) The method of the present invention is also applicable to the separation and extraction of various animal and plant proteins, such as collagen, lysozyme, soy protein isolate, serum protein, etc., and has wide applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Shown is a flow chart of a method for separating hair cortex cells, filaments and keratin nanoparticles according to the present invention;

[0030] Figure 2 Shown is a schematic diagram of the hair structure;

[0031] Figure 3 Shown is the SEM image of hair fiber;

[0032] Figure 4 The following are the morphologies of samples obtained after periodate oxidation of hair: (a) shows the morphology of cortical cells under an optical microscope; (b) shows the morphology of filaments under an optical microscope; and (c) shows a transmission electron microscope image of protein nanoparticles.

[0033] Figure 5 The figure shows the sample morphology obtained after the periodate oxidation reaction of lysozyme, where (a) is the transmission electron microscopy morphology of the original sample of lysozyme without oxidation, and (b) is the transmission electron microscopy image of the lysozyme protein nanoparticles obtained after the oxidation reaction. DETAILED DESCRIPTION

[0034] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0035] The present invention provides a method for extracting protein multi-level structure by periodate oxidation, comprising the following steps:

[0036] Step 1: Oxidation reaction of periodate

[0037] (1) Mix the crushed bio-based raw materials, pure water and periodic acid, stir to dissolve the periodic acid and keep stirring, add alkali solution to adjust the pH of the reaction solution. Under neutral conditions, the periodate ion form in the reaction is H2I2O 104- , the ion has a more negative charge and a larger ionic radius.

[0038] (2) The oxidation reaction was carried out by stirring at 1000 rpm in the dark at room temperature.

[0039] Step 2: Purification and separation of protein hierarchical structures

[0040] (1) After the oxidation reaction is completed, add an appropriate amount of ethylene glycol to quench the unreacted periodic acid, then dialyze and filter the reaction solution, and collect the filtrate.

[0041] (2) Allow the filtrate to settle for 24 hours, then slowly remove the sample and collect the sediment P1 and the upper turbid liquid P2.

[0042] (4) The precipitate P1 is washed with water several times and centrifuged, and the upper turbid liquid P2 is centrifuged and washed with water, and then dried to obtain the multi-level structure of the protein. The separation process is as follows Figure 1 shown.

[0043] In some examples, the shredded bio-based feedstock is less than 1 cm in length.

[0044] In some examples, the ratio of the bio-based raw material, pure water, and periodic acid is 1:(20-100):(1-7).

[0045] In some examples, the alkaline solution is a LiOH solution or ammonia water, and the pH is adjusted to 6.5-7.

[0046] In some examples, the alkaline solution is a KOH solution, and the pH is adjusted to 8.5-11.

[0047] In some examples, the dialysis bag used for dialysis has a molecular weight cut-off range of 7000-14000 Da.

[0048] In some examples, the dialysis treatment further includes changing the water multiple times until the conductivity is less than 1 μS / cm.

[0049] In some examples, the filter cloth used for the filtration has a mesh size of 200-800.

[0050] In the following examples, periodic acid, potassium hydroxide, lithium hydroxide, and ammonia were all purchased from MacLean's Reagent Company. The bio-based raw materials used were: hair from a barber shop with an average diameter of 100 μm; feathers from a slaughterhouse; collagen and lysozyme were also purchased from MacLean's Reagent Company.

[0051] Example 1

[0052] The method of extracting the multi-level structure of proteins by periodate oxidation in this embodiment includes the following steps:

[0053] Step 1: Periodate oxidation reaction

[0054] (1) Take 10 reaction bottles, cut hair and feathers into small pieces less than 1 cm, weigh 0.5 g of hair and feathers respectively and place them in 5 reaction bottles. Add 50 mL of room temperature pure water and 2.0 g of periodic acid to each reaction bottle, stir on a stirrer to dissolve the periodic acid and keep stirring, add LiOH and adjust the pH to 7.

[0055] (2) Stir at 1000 rpm at room temperature in the dark for 1 day, 3 days, 6 days, 10 days, and 15 days.

[0056] Step 2: Purification and separation of protein hierarchical structures

[0057] (1) After the reaction is completed, add an appropriate amount of ethylene glycol to the reaction flask to quench the unreacted periodic acid. Then transfer the reaction solution into a 7000-14000 Da dialysis bag for dialysis, changing the water several times until the conductivity is less than 1 μS / cm.

[0058] (2) Filter the dialyzed sample using a 200-800 mesh filter cloth to remove the hair or feathers that have not reacted completely (collect, dry and weigh to calculate the weight of the unreacted material), and collect the filtrate.

[0059] (3) Allow the filtrate to settle for 24 hours, then slowly remove the sample and collect the precipitate P1 and the upper turbid liquid P2.

[0060] (4) The precipitate P1 was washed and centrifuged at 1000 rpm multiple times to obtain clean cortical cells at the bottom. The upper turbid liquid P2 was centrifuged at 10,000 rpm to obtain filaments, and the supernatant was keratin nanoparticles. The filaments were washed and centrifuged at 10,000 rpm multiple times to obtain relatively pure filaments. The cortical cells, filaments, and keratin nanoparticles were dried, and the yields were calculated. The separation results are shown in Table 1.

[0061] Table 1: Separation and extraction results of Example 1

[0062]

[0063] Example 2

[0064] The method for extracting protein multi-level structure by periodate oxidation in this example is basically the same as that in Example 1, except that the alkali solution added in this example is ammonia water and the pH is adjusted to 7. The separation results are shown in Table 2.

[0065] Table 2: Separation and extraction results of Example 2

[0066]

[0067] Example 3

[0068] The method for extracting protein multi-level structure by periodate oxidation in this example is basically the same as that in Example 1, except that the alkali solution added in this example is KOH solution and the pH is adjusted to 10. The separation results are shown in Table 3.

[0069] Table 3: Separation and extraction results of Example 3

[0070]

[0071] Example 4

[0072] The method of extracting the multi-level structure of proteins by periodate oxidation in this embodiment includes the following steps:

[0073] Step 1: Periodate oxidation reaction

[0074] (1) Weigh 0.5 g of lysozyme into 5 reaction bottles respectively. Add 50 mL of pure water at room temperature and 2.0 g of periodic acid to each reaction bottle. Stir on a stirrer to dissolve the periodic acid and keep stirring. Add LiOH and adjust the pH to 7.

[0075] (2) Stir at 1000 rpm at room temperature in the dark for 1 day, 2 days, 3 days, 5 days, and 7 days.

[0076] Step 2: Purification and separation of protein hierarchical structures

[0077] (1) After the reaction is completed, add an appropriate amount of ethylene glycol to the reaction flask to quench the unreacted periodic acid. Then transfer the reaction solution into a 7000-14000 Da dialysis bag for dialysis, changing the water several times until the conductivity is less than 1 μS / cm.

[0078] (2) The dialyzed sample was centrifuged at 10,000 rpm for 10 min to remove large particle impurities. The obtained clear supernatant was relatively pure protein nanoparticles, which were dried and the yield was calculated. The separation results are shown in Table 4.

[0079] Example 5

[0080] The method for extracting protein multi-level structure by periodate oxidation in this example is basically the same as that in Example 4, except that the alkali solution added in this example is KOH solution and the pH is adjusted to 10. The separation results are shown in Table 4.

[0081] Table 4: Separation and extraction results of Examples 4 and 5

[0082]

[0083] Comparative Example 1

[0084] The method for extracting the hierarchical structure of proteins in this comparative example has the same steps as those in Example 1, except that, in step 1, hair and feathers were cut into small segments less than one centimeter, 0.5 g of which was weighed and placed in a reaction flask. A 0.2 mol / L Na2HPO4·NaH2PO4 (pH=7) buffer solution and a 0.2 mol / L Na2CO3·NaHCO3 (pH=10) buffer solution at room temperature were added, and the mixture was stirred at 1000 rpm at room temperature for 15 days. Details and separation results are shown in Table 5.

[0085] Comparative Example 2

[0086] The method for extracting the multi-level structure of protein in this comparative example has the same steps as Example 1, except that no alkaline solution is used to adjust the pH value in this comparative example. The separation results are shown in Table 5.

[0087] Table 5: Separation and extraction results of Comparative Examples 1-2

[0088]

[0089] The present invention extracts the multi-level structure of keratin in hair by periodate oxidation ( Figure 2 Schematic diagram of hair structure. Figure 3 (SEM image of hair fiber), the morphology of the cortical cells and filaments obtained under optical microscope are as follows Figure 4 (a) and (b) are shown. The cortical cells are spindle-shaped under a microscope, with a length of 150-200 microns and a diameter of 5-10 microns. The filaments are the products obtained after the cortical cells are peeled off. Under a microscope, they are filamentous, with a diameter of 0.1-0.4 microns and a length of 2-10 microns. The keratin nanoparticles obtained were observed by transmission electron microscopy (TEM). Figure 4 (c) As can be seen from the figure, keratin nanoparticles have a spherical morphology with a shell-core structure and a diameter of 50~200 nm.

[0090] Comparing Comparative Examples 1-2 with Examples 1-5 shows that the protein stripping effect is very poor under alkaline conditions alone or under periodic acid conditions alone; periodic acid is only effective under appropriate alkaline conditions. Examples 1-5 show that the reaction efficiency of periodic acid varies under different alkaline conditions, and the degree of reaction can be controlled by adjusting the alkaline solution, pH, and reaction time. The use of periodate to strip and extract the multilayer structure of protein has advantages. First, the degree of stripping can be controlled by controlling the reaction time. In Examples 1-3, the cortical cell product obtained within 1-3 days of reaction is the largest, with the highest yield of about 20%; the filaments obtained within 3-6 days are the best, with the highest yield of about 16%; protein nanoparticles are mainly obtained within 6-10 days, with the highest yield reaching 76%; in addition, the oxidizing strength of periodic acid varies under different conditions. As can be seen from the comparison of Examples 1-3, the oxidizing strength of periodic acid in LiOH and ammonia water is relatively strong when the initial pH of the reaction solution is 7; the oxidizing strength of periodic acid in KOH is relatively weak when the pH of the reaction solution is 10; therefore, the degree of periodate oxidation stripping can also be controlled by adding different alkali solutions to adjust the pH to different values; second, the operation is simple, does not require complex pretreatment, does not introduce other chemical reagents, and has little harm to the human body and the environment. After the oxidation reaction is completed, it is only necessary to inactivate the unreacted periodic acid to ensure safety.

[0091] From Examples 4-5 and Figure 5 It can be seen that the protein nanoparticles extracted using periodate oxidation of lysozyme are also applicable. Figure 5 The following are transmission electron microscopic images of the lysozyme samples before and after periodate oxidation. (a) shows the original, unoxidized lysozyme sample, and (b) shows the lysozyme protein nanoparticles obtained after periodate oxidation. The figure shows that the unoxidized sample is disordered, but after periodate oxidation, it transforms into relatively uniform spherical particles with diameters ranging from 10 to 50 nanometers. Consistent with the behavior of collagen, the sample is initially disordered, but after oxidation, it transforms into relatively uniform spherical particles with diameters ranging from 10 to 30 nanometers. Table 4 shows that when periodate oxidation is used to extract the hierarchical protein structure from lysozyme, the yield reaches its highest level after 5 days of reaction, reaching approximately 85%, when LiOH solution is used as the base for pH adjustment. The reaction rate is relatively slow when KOH solution is used as the base for pH adjustment, which is consistent with the behavior of keratin extraction in Examples 1 and 3.

[0092] In the above examples, protein nanoparticles can still be isolated and extracted even when the reaction time is extended to 15-30 days, demonstrating that the method provided by the present invention for extracting protein hierarchical structures through periodate oxidation is relatively mild. The inventors also conducted related experiments using alkali solutions LiOH or KOH to adjust the reaction solution to other pH values ​​(e.g., pH = 10 with LiOH solution and pH = 7 with KOH solution), or using a buffer solution Na2HPO4·NaH2PO4 to adjust the reaction solution to pH = 7. During the pH adjustment process, a small amount of solid precipitation occurred, resulting in slightly inferior final extraction results compared to Examples 1-4.

[0093] Table 6 compares the method provided by the present invention with other research methods. As can be seen from the table, the method provided by the present invention can extract protein structures of varying micro- and nanometer sizes from protein-rich bio-based feedstocks. In contrast, the protein products extracted using the other methods in the table are limited, mostly small molecules.

[0094] In summary, the method of the present invention uses periodic acid as a reaction reagent, adjusts the addition ratio of bio-based raw materials and periodic acid, and the concentration of the reaction solution, adjusts the pH of the reaction solution with an appropriate alkali solution, and controls the reaction time to separate and obtain a protein multi-level structure product. The method provided by the present invention has mild reaction conditions, which can avoid the destruction of the protein multi-level structure and greatly preserve the natural micro-nanostructure of the protein, providing an ideal raw material for the preparation of high-performance keratin advanced functional materials. In addition, the method provided by the present invention is simple to operate, and by regulating the oxidation reaction time, different levels of protein biological structures can be obtained. It is efficient, controllable, and environmentally friendly, and is more conducive to industrial development.

[0095] Table 6: Comparison of the present invention with other methods previously studied

[0096]

[0097] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A method for extracting protein multi-level structure by periodate oxidation, characterized in that: The following steps are involved: (1) mixing a bio-based raw material, pure water and periodic acid, stirring to dissolve the periodic acid, and adding an alkali solution to adjust the pH of the reaction solution; the bio-based raw material comprises one of hair, feathers, nails, horns and hooves; the alkali solution is a LiOH solution, ammonia water or KOH solution, and when the alkali solution is a LiOH solution or ammonia water, the pH is adjusted to 6.5-7; when the alkali solution is a KOH solution, the pH is adjusted to 8.5-11; (2) Carry out oxidation reaction under stirring at room temperature in the dark; (3) dialyzing and filtering the reaction solution and collecting the filtrate; (4) Allow the filtrate to settle and collect the precipitate P1 and the upper turbid liquid P2; (5) The precipitate P1 is washed with water for multiple times and centrifuged, and the upper turbid liquid P2 is centrifuged and washed with water, and then dried to obtain a biomaterial with a multi-level protein structure.

2. The method for extracting protein multi-level structure by periodate oxidation according to claim 1, characterized in that: The protein includes keratin.

3. The method for extracting protein multi-level structure by periodate oxidation according to claim 1, characterized in that: The ratio of the bio-based raw material, pure water and periodic acid is 1: (20-100): (1-7).

4. The method for extracting protein multi-level structure by periodate oxidation according to claim 1, characterized in that: The ratio of the bio-based raw material, pure water and periodic acid is 1: (20-100):

4.

5. The method for extracting protein multi-level structure by periodate oxidation according to claim 1, characterized in that: The reaction time of the oxidation reaction is 1 to 15 days.

6. The method for extracting protein multi-level structure by periodate oxidation according to claim 1, characterized in that: The molecular weight cut-off of the dialysis bag used for the dialysis is 7000-14000 Da.

7. The method for extracting protein multi-level structure by periodate oxidation according to claim 1, characterized in that: The filter cloth used for the filtration is 200-800 meshes.

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