Amino acid-assisted papain imprinted polymer and its preparation method and application

Abstract

The present invention discloses an amino acid-assisted papain imprinted polymer, a preparation method, and applications, belonging to the technical field of papain adsorption materials. After chitosan modification and glutamic acid modification of orange peel charcoal material, papain and a bifunctional monomer are sequentially added for polymerization to obtain an amino acid-assisted papain imprinted polymer. Thanks to the high affinity brought about by the amino acid modification and the synergistic effect of the bifunctional monomer, the imprinted polymer has a large adsorption capacity for papain, high specificity, good separation efficiency, and stable performance during multiple cycles of use, meeting the needs of daily separation and purification, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of papain adsorption materials, and particularly relates to an amino acid-assisted papain imprinted polymer and a preparation method and application thereof. Background Art

[0002] Papain is an enzyme derived from papaya ( Carica papaya Papain is a thiol-containing endoenzyme found in the latex of unripe papaya fruits. This enzyme exhibits optimal catalytic activity at a pH of 6.0–7.0 and a temperature of 50–70°C. It specifically hydrolyzes peptide and ester bonds in proteins, and is highly effective at degrading macromolecular substrates such as myofibrillar protein and collagen. Papain has a wide range of applications in food processing (e.g., meat tenderizing and beer production), biomedicine (e.g., gastrointestinal diseases and wound repair), and daily chemical products (e.g., detergents and skincare products).

[0003] Traditional methods for extracting papain include ultrafiltration, salting-out, organic solvents, and affinity chromatography. Ultrafiltration is simple to operate, but it struggles to remove contaminants of similar molecular weight. Salting-out yields low-purity proteins and struggles to remove impurities. While organic solvents can improve purity, they can lead to residual solvents and low enzyme activity recovery. Affinity chromatography is complex and expensive, presenting limitations. Therefore, a more efficient and convenient papain extraction method is needed.

[0004] Molecular imprinting technology, a biomimetic separation technique that mimics antigen-antibody specific recognition, constructs three-dimensional imprinted pores through the interaction of template molecules with carriers and functional monomers, forming molecularly imprinted polymers (MIPs) with a "lock-and-key" structure. Molecularly imprinted polymers exhibit specific recognition and selective adsorption, high separation efficiency, strong stability, ease of operation, and good reusability, making them suitable for protein separation and purification. Currently, methods for preparing papain-imprinted polymers include: using polystyrene (PS) microspheres as carriers, poly-3-aminophenylboronic acid (pAPBA) as the functional monomer, and papain as the template, to form nanofilms on microbead surfaces in aqueous media. Yan Lu a,* , Chang-Ling Yan a, Xue-Jing Wang b , Gong-KeWang a . Applied Surface Science 256 (2009) 1341–1346.); A facile approach for imprinting protein on the surface of multi-walled carbon nanotubes. Ren Liu a , Mo Sha a , Sisi Jiang a , JingLuo a b , Xiaoya Liu a .Talanta.Volume 120, March 2014, Pages 76-83.), etc. However, the papain-imprinted polymers prepared by these methods all suffer from low adsorption capacity and imprinting factor. Therefore, it is urgent to prepare papain-imprinted polymers with better adsorption efficiency for the extraction and separation of papain. Summary of the Invention

[0005] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide an amino acid-assisted papain imprinted polymer and its preparation method and application, so as to solve the technical problem that the existing papain imprinted polymer has poor adsorption effect.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention discloses a method for preparing an amino acid-assisted papain imprinted polymer. The tangerine peel charcoal material is sequentially modified with chitosan and glutamic acid, and then papain and a bifunctional monomer are sequentially added for polymerization to obtain an amino acid-assisted papain imprinted polymer.

[0008] Preferably, the preparation method of the orange peel charcoal material is: adding ammonium ferric oxalate solution to orange peel powder, soaking, drying, powdering, and carbonizing to obtain the orange peel charcoal material.

[0009] Preferably, the steps of modifying the orange peel charcoal material with chitosan are: mixing the orange peel charcoal material with a chitosan solution and performing prepolymerization, adding a glutaraldehyde aqueous solution for polymerization, washing, and drying to obtain the obtained product.

[0010] Preferably, the ratio of orange peel charcoal material to chitosan solution is 4 mg:1 mL, and the concentration of chitosan solution is 1-3 mg / mL.

[0011] More preferably, the concentration of the chitosan solution is 2 mg / mL.

[0012] Preferably, the step of modifying the chitosan-modified product with glutamic acid is as follows: mixing the chitosan-modified product with a grafting agent, then adding a glutamic acid solution to react, washing the product, and drying it.

[0013] More preferably, the grafting reagent is phosphate buffer, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

[0014] Preferably, the ratio of the chitosan-modified product to the glutamic acid solution is 12 mg:1 mL, and the concentration of the glutamic acid solution is 4-16 mg / mL.

[0015] More preferably, the concentration of the glutamic acid solution is 12 mg / mL.

[0016] Preferably, the polymerization time is 9 to 18 h.

[0017] More preferably, the polymerization time is 15 h.

[0018] The second aspect of the present invention discloses a papain imprinted polymer obtained by the above-mentioned preparation method of the amino acid-assisted papain imprinted polymer.

[0019] Preferably, when the concentration of the chitosan solution is 2 mg / mL and the concentration of the glutamic acid solution is 12 mg / mL, the adsorption capacity of papain by the papain-imprinted polymer is 197 mg / g, and the imprinting factor is 6.12.

[0020] The third aspect of the present invention discloses the use of the amino acid-assisted papain imprinted polymer in the extraction and separation of papain.

[0021] The fourth aspect of the present invention discloses a method for extracting papain, comprising mixing the amino acid-assisted papain imprinted polymer with a papain solution to be extracted, adsorbing, eluting, dialyzing, concentrating, and drying to obtain papain.

[0022] Preferably, the concentration of the papain solution to be extracted is 50-350 μg / mL.

[0023] Further preferably, the concentration of the papain solution to be extracted is 250 μg / mL.

[0024] Preferably, the adsorption time is not less than 20 min.

[0025] More preferably, the adsorption time is 20 to 90 min.

[0026] More preferably, the adsorption time is 40 min.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides an amino acid-assisted method for preparing papain-imprinted polymers. Using a biomass-derived carbon material (orange peel carbon material) as a carrier, amino acid (glutamic acid) groups are grafted onto the biomass-derived carbon material surface in combination with a bifunctional monomer to produce the papain-imprinted polymer. This method is simple and easy to operate. In this method, 1) the orange peel carbon material is amino-modified with chitosan to facilitate subsequent grafting of glutamic acid groups. 2) Glutamic acid, which has both amino and carboxyl groups, exhibits enhanced affinity for papain adsorption, improving the adsorption efficiency of the synthesized imprinted polymer. The glutamic acid modification significantly enhances the imprinted polymer's affinity for the template protein. 3) Papain and the bifunctional monomer are added for polymerization. The interaction between the bifunctional monomer and the template protein further enhances the selectivity of the imprinted polymer. This method not only improves the separation efficiency of papain, but also leverages the environmentally friendly nature of biomass-derived carbon materials, reduces costs, and enhances the polymer's affinity through amino acid modification, providing a new approach for the extraction and separation of papain. The papain molecularly imprinted polymer prepared by this method has an adsorption capacity of 197 mg / g for papain and an imprinting factor of 6.12. It exhibits high specific adsorption capacity and high separation efficiency, can effectively recognize and bind to target proteins, is reusable, and exhibits stable performance over multiple cycles, meeting large-scale separation requirements. In summary, the present invention provides an efficient, economical, and practical papain-imprinted polymer material with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a static adsorption curve of the papain-imprinted polymer in Example 1;

[0030] Figure 2 This is the adsorption kinetics curve of the papain imprinted polymer in Example 1;

[0031] Figure 3 This is a selective adsorption curve of the papain imprinted polymer in Example 1;

[0032] Figure 4 This is a graph showing the reusability of the papain-imprinted polymer of Example 1;

[0033] Figure 5The graphs show the effects of different chitosan addition amounts on the adsorption of papain imprinted polymers and non-imprinted polymers in Examples 1 to 3;

[0034] Figure 6 The graph shows the effect of different addition amounts of glutamate on the adsorption of papain imprinted polymer and non-imprinted polymer in Examples 1, 4-6;

[0035] Figure 7 This is a graph showing the effect of different polymerization times on the adsorption effect of papain imprinted polymer and non-imprinted polymer in Examples 1, 7 to 9. DETAILED DESCRIPTION

[0036] To facilitate understanding of the features and effects of the present invention by those skilled in the art, the following provides a general description and definition of the terms and expressions used in the specification and claims. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary meanings as understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions in this specification shall prevail.

[0037] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0038] All features, such as values, amounts, contents, and concentrations, described herein as numerical ranges or percentage ranges are provided for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to include and specifically disclose all possible subranges and individual values ​​within the range (including integers and fractions).

[0039] In this document, unless otherwise specified, “include,” “including,” “contains,” “has” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0040] In this document, for the sake of brevity, not all possible combinations of the various technical features in each embodiment or example are described. Therefore, as long as there are no contradictions in the combination of these technical features, the various technical features in each embodiment or example can be combined in any way, and all possible combinations should be considered to be within the scope of this specification.

[0041] The present invention provides a method for preparing an amino acid-assisted papain imprinted polymer, comprising the following steps:

[0042] Step 1: Amino modification of orange peel carbon material

[0043] 1) Adding ammonium ferric oxalate solution to orange peel powder, soaking, drying, pulverizing, and carbonizing to obtain orange peel charcoal material;

[0044] 2) Add the tangerine peel carbon material to a chitosan solution with a concentration of (1-3) mg / mL at a dosage ratio of 4 mg:1 mL, stir and prepolymerize for 1 h, then add 50 wt% glutaraldehyde aqueous solution for polymerization. The reaction product is washed with distilled water and dried to obtain chitosan-modified tangerine peel carbon material;

[0045] Step 2: Amino acid modification

[0046] The chitosan-modified tangerine peel carbon material obtained in step 1 was mixed with a grafting reagent, and a glutamic acid solution with a concentration of (4-16) mg / mL was added dropwise to react. The product was washed with distilled water and dried to obtain a glutamic acid-modified tangerine peel carbon material;

[0047] Among them, the grafting reagents include but are not limited to phosphate buffer, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and can also be other commonly used reagents for grafting amino and carboxyl groups; the dosage ratio of chitosan-modified orange peel carbon material to glutamic acid solution is 12 mg:1 mL.

[0048] Step 3: Aggregation

[0049] The glutamic acid-modified orange peel charcoal material obtained in step 2 is added to a Tris buffer, and then a template molecule (i.e., papain) is added for prepolymerization. Then, dopamine hydrochloride and 3,4-dihydroxyphenylacetic acid are added at a molar ratio of 2:1 and polymerized for 9-18 hours. The obtained reaction product is separated and washed with a mixed aqueous solution of sodium dodecyl sulfate and glacial acetic acid to remove the template molecule, thereby obtaining an amino acid-assisted papain-imprinted polymer.

[0050] Among them, the mass ratio of glutamic acid-modified orange peel carbon material to template molecule is 8:3.

[0051] In this article, the orange peel charcoal material used can also be replaced by biomass-derived charcoal materials such as wood charcoal material, rice husk charcoal material, wheat straw charcoal material, corn cob charcoal material, banana peel charcoal material or zucchini charcoal material.

[0052] The glutamic acid used herein can also be replaced by any other amino acid.

[0053] Below in conjunction with Table 1 and specific examples, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.In addition, should be understood that after reading the content of the present invention description, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0054] Conventional instruments and equipment in the art are used in the following examples. Experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. In the following examples, glacial acetic acid (Article No.: 73562B), ammonia water (Article No.: 81922G), phosphate buffer solution (Article No.: 046103662) and sodium lauryl sulfate (Article No.: 25701I) used were all purchased from Shanghai Titan Technology Co., Ltd.; ammonium ferric oxalate (Article No.: C33510), chitosan (Article No.: 922587), glutaraldehyde (Article No.: 902042), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (Product No.: 939374), N-hydroxysuccinimide (Product No.: 903973), glutamic acid (Product No.: 288636), Tris buffer (Product No.: 992232), papain (Product No.: 916928), dopamine hydrochloride (Product No.: 907334), and 3,4-dihydroxyphenylacetic acid (Product No.: 436848) were purchased from Beijing Bailingwei Technology Co., Ltd.; all other raw materials were conventional commercial products with specifications conventional in the field.

[0055] 1. Preparation of amino acid-assisted papain-imprinted polymers

[0056] Table 1 Different reaction conditions optimized during the synthesis of molecularly imprinted polymers in Examples 1 to 9

[0057]

[0058] Example 1

[0059] The preparation method of amino acid-assisted papain imprinted polymer comprises the following steps:

[0060] 1) 5 g of orange peel powder was added to 15 mL of 0.6 M ammonium ferric oxalate aqueous solution, soaked at 60°C for 2 days, oven-dried at 90°C, and pulverized to obtain treated orange peel powder. The treated orange peel powder was then carbonized in a tube furnace at 600°C for 2 hours under a nitrogen atmosphere. The product was then rinsed with anhydrous ethanol and then distilled water to obtain orange peel carbon.

[0061] 2) Weigh 400 mg of tangerine peel carbon material and add it to 100 mL of 0.1% (v / v) glacial acetic acid aqueous solution containing 2 mg / mL chitosan. Stir and prepolymerize for 1 hour. Adjust the pH of the solution to 8.0 with dilute ammonia. Add 10 μL of 50 wt% glutaraldehyde aqueous solution and stir at room temperature for 3 hours. Wash the reaction product with distilled water and dry it in an oven at 50°C to obtain chitosan-modified tangerine peel carbon material.

[0062] 3) Weigh 360 mg of chitosan-modified tangerine peel carbon material and add it to 30 mL of phosphate buffer (pH 7.0). Add 287.52 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 172.62 mg of N-hydroxysuccinimide and stir at room temperature for 2 hours. Add 30 mL of a 12 mg / mL glutamic acid solution dropwise and stir at room temperature for 6 hours. Dry in an oven to obtain the glutamic acid-modified tangerine peel carbon material.

[0063] 4) Weigh 80 mg of glutamic acid-modified orange peel charcoal material into 30 mL of Tris buffer (pH 8.3), then add 30 mg of papain and stir at room temperature for 2 h for prepolymerization. Then, add 91.42 mg of dopamine hydrochloride and 40.58 mg of 3,4-dihydroxyphenylacetic acid, and stir at room temperature for 15 h for polymerization. The resulting polymer was separated using a magnet and washed with a 2% (w / v) sodium dodecyl sulfate-2% (v / v) glacial acetic acid aqueous solution to remove the template molecules. The polymer was then dried at 50°C to obtain amino acid-assisted papain-imprinted polymers (MIPs).

[0064] Example 2

[0065] The preparation method of amino acid-assisted papain imprinted polymer comprises the following steps:

[0066] 1) 5 g of orange peel powder was added to 15 mL of 0.6 M ammonium ferric oxalate aqueous solution, soaked at 60°C for 2 days, oven-dried at 90°C, and pulverized to obtain treated orange peel powder. The treated orange peel powder was then carbonized in a tube furnace at 600°C for 2 hours under a nitrogen atmosphere. The product was then rinsed with anhydrous ethanol and then distilled water to obtain orange peel carbon.

[0067] 2) Weigh 400 mg of tangerine peel carbon material and add it to 100 mL of 0.1% (v / v) glacial acetic acid aqueous solution containing 1 mg / mL chitosan. Stir and prepolymerize for 1 hour. Adjust the pH of the solution to 8.0 with dilute ammonia. Add 10 μL of 50 wt% glutaraldehyde aqueous solution and stir at room temperature for 3 hours. Wash the reaction product with distilled water and dry it in an oven at 50°C to obtain chitosan-modified tangerine peel carbon material.

[0068] 3) Weigh 360 mg of chitosan-modified tangerine peel carbon material and add it to 30 mL of phosphate buffer (pH 7.0). Add 287.52 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 172.62 mg of N-hydroxysuccinimide and stir at room temperature for 2 hours. Add 30 mL of a 12 mg / mL glutamic acid solution dropwise and stir at room temperature for 6 hours. Dry in an oven to obtain the glutamic acid-modified tangerine peel carbon material.

[0069] 4) Weigh 80 mg of glutamic acid-modified orange peel charcoal material into 30 mL of Tris buffer (pH 8.3), then add 30 mg of papain and stir at room temperature for 2 h for prepolymerization. Then, add 91.42 mg of dopamine hydrochloride and 40.58 mg of 3,4-dihydroxyphenylacetic acid, and stir at room temperature for 15 h for polymerization. The resulting polymer was separated using a magnet and washed with a 2% (w / v) sodium dodecyl sulfate-2% (v / v) glacial acetic acid aqueous solution to remove the template molecules. The polymer was then dried at 50°C to obtain amino acid-assisted papain-imprinted polymers (MIPs).

[0070] Example 3

[0071] The preparation method of amino acid-assisted papain imprinted polymer comprises the following steps:

[0072] 1) 5 g of orange peel powder was added to 15 mL of 0.6 M ammonium ferric oxalate aqueous solution, soaked at 60°C for 2 days, oven-dried at 90°C, and pulverized to obtain treated orange peel powder. The treated orange peel powder was then carbonized in a tube furnace at 600°C for 2 hours under a nitrogen atmosphere. The product was then rinsed with anhydrous ethanol and then distilled water to obtain orange peel carbon.

[0073] 2) Weigh 400 mg of tangerine peel carbon material and add it to 100 mL of 0.1% (v / v) glacial acetic acid aqueous solution containing 3 mg / mL chitosan. Stir and prepolymerize for 1 hour. Adjust the pH of the solution to 8.0 with dilute ammonia. Add 10 μL of 50 wt% glutaraldehyde aqueous solution and stir at room temperature for 3 hours. Wash the reaction product with distilled water and dry it in an oven at 50°C to obtain chitosan-modified tangerine peel carbon material.

[0074] 3) Weigh 360 mg of chitosan-modified tangerine peel carbon material and add it to 30 mL of phosphate buffer (pH 7.0). Add 287.52 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 172.62 mg of N-hydroxysuccinimide and stir at room temperature for 2 hours. Add 30 mL of a 12 mg / mL glutamic acid solution dropwise and stir at room temperature for 6 hours. Dry in an oven to obtain the glutamic acid-modified tangerine peel carbon material.

[0075] 4) Weigh 80 mg of glutamic acid-modified orange peel charcoal material into 30 mL of Tris buffer (pH 8.3), then add 30 mg of papain and stir at room temperature for 2 h for prepolymerization. Then, add 91.42 mg of dopamine hydrochloride and 40.58 mg of 3,4-dihydroxyphenylacetic acid, and stir at room temperature for 15 h for polymerization. The resulting polymer was separated using a magnet and washed with a 2% (w / v) sodium dodecyl sulfate-2% (v / v) glacial acetic acid aqueous solution to remove the template molecules. The polymer was then dried at 50°C to obtain amino acid-assisted papain-imprinted polymers (MIPs).

[0076] Example 4

[0077] The preparation method of amino acid-assisted papain imprinted polymer comprises the following steps:

[0078] 1) 5 g of orange peel powder was added to 15 mL of 0.6 M ammonium ferric oxalate aqueous solution, soaked at 60°C for 2 days, oven-dried at 90°C, and pulverized to obtain treated orange peel powder. The treated orange peel powder was then carbonized in a tube furnace at 600°C for 2 hours under a nitrogen atmosphere. The product was then rinsed with anhydrous ethanol and then distilled water to obtain orange peel carbon.

[0079] 2) Weigh 400 mg of tangerine peel carbon material and add it to 100 mL of 0.1% (v / v) glacial acetic acid aqueous solution containing 2 mg / mL chitosan. Stir and prepolymerize for 1 hour. Adjust the pH of the solution to 8.0 with dilute ammonia. Add 10 μL of 50 wt% glutaraldehyde aqueous solution and stir at room temperature for 3 hours. Wash the reaction product with distilled water and dry it in an oven at 50°C to obtain chitosan-modified tangerine peel carbon material.

[0080] 3) Weigh 360 mg of chitosan-modified tangerine peel carbon material and add it to 30 mL of phosphate buffer (pH 7.0). Add 287.52 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 172.62 mg of N-hydroxysuccinimide, and stir at room temperature for 2 hours. Add 30 mL of a 4 mg / mL glutamic acid solution dropwise, stir at room temperature for 6 hours, and oven dry to obtain the glutamic acid-modified tangerine peel carbon material.

[0081] 4) Weigh 80 mg of glutamic acid-modified orange peel charcoal material into 30 mL of Tris buffer (pH 8.3), then add 30 mg of papain and stir at room temperature for 2 h for prepolymerization. Then, add 91.42 mg of dopamine hydrochloride and 40.58 mg of 3,4-dihydroxyphenylacetic acid, and stir at room temperature for 15 h for polymerization. The resulting polymer was separated using a magnet and washed with a 2% (w / v) sodium dodecyl sulfate-2% (v / v) glacial acetic acid aqueous solution to remove the template molecules. The polymer was then dried at 50°C to obtain amino acid-assisted papain-imprinted polymers (MIPs).

[0082] Example 5

[0083] The preparation method of amino acid-assisted papain imprinted polymer comprises the following steps:

[0084] 1) 5 g of orange peel powder was added to 15 mL of 0.6 M ammonium ferric oxalate aqueous solution, soaked at 60°C for 2 days, oven-dried at 90°C, and pulverized to obtain treated orange peel powder. The treated orange peel powder was then carbonized in a tube furnace at 600°C for 2 hours under a nitrogen atmosphere. The product was then rinsed with anhydrous ethanol and then distilled water to obtain orange peel carbon.

[0085] 2) Weigh 400 mg of tangerine peel carbon material and add it to 100 mL of 0.1% (v / v) glacial acetic acid aqueous solution containing 2 mg / mL chitosan. Stir and prepolymerize for 1 hour. Adjust the pH of the solution to 8.0 with dilute ammonia. Add 10 μL of 50 wt% glutaraldehyde aqueous solution and stir at room temperature for 3 hours. Wash the reaction product with distilled water and dry it in an oven at 50°C to obtain chitosan-modified tangerine peel carbon material.

[0086] 3) Weigh 360 mg of chitosan-modified tangerine peel carbon material and add it to 30 mL of phosphate buffer solution (pH 7.0). Add 287.52 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 172.62 mg of N-hydroxysuccinimide, and stir at room temperature for 2 hours. Add 30 mL of 8 mg / mL glutamic acid solution dropwise, stir at room temperature for 6 hours, and oven dry to obtain glutamic acid-modified tangerine peel carbon material.

[0087] 4) Weigh 80 mg of glutamic acid-modified orange peel charcoal material into 30 mL of Tris buffer (pH 8.3), then add 30 mg of papain and stir at room temperature for 2 h for prepolymerization. Then, add 91.42 mg of dopamine hydrochloride and 40.58 mg of 3,4-dihydroxyphenylacetic acid, and stir at room temperature for 15 h for polymerization. The resulting polymer was separated using a magnet and washed with a 2% (w / v) sodium dodecyl sulfate-2% (v / v) glacial acetic acid aqueous solution to remove the template molecules. The polymer was then dried at 50°C to obtain amino acid-assisted papain-imprinted polymers (MIPs).

[0088] Example 6

[0089] The preparation method of amino acid-assisted papain imprinted polymer comprises the following steps:

[0090] 1) 5 g of orange peel powder was added to 15 mL of 0.6 M ammonium ferric oxalate aqueous solution, soaked at 60°C for 2 days, oven-dried at 90°C, and pulverized to obtain treated orange peel powder. The treated orange peel powder was then carbonized in a tube furnace at 600°C for 2 hours under a nitrogen atmosphere. The product was then rinsed with anhydrous ethanol and then distilled water to obtain orange peel carbon.

[0091] 2) Weigh 400 mg of tangerine peel carbon material and add it to 100 mL of 0.1% (v / v) glacial acetic acid aqueous solution containing 2 mg / mL chitosan. Stir and prepolymerize for 1 hour. Adjust the pH of the solution to 8.0 with dilute ammonia. Add 10 μL of 50 wt% glutaraldehyde aqueous solution and stir at room temperature for 3 hours. Wash the reaction product with distilled water and dry it in an oven at 50°C to obtain chitosan-modified tangerine peel carbon material.

[0092] 3) Weigh 360 mg of chitosan-modified tangerine peel carbon material and add it to 30 mL of phosphate buffer (pH 7.0). Add 287.52 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 172.62 mg of N-hydroxysuccinimide, and stir at room temperature for 2 hours. Add 30 mL of a 16 mg / mL glutamic acid solution dropwise, stir at room temperature for 6 hours, and oven dry to obtain the glutamic acid-modified tangerine peel carbon material.

[0093] 4) Weigh 80 mg of glutamic acid-modified orange peel charcoal material into 30 mL of Tris buffer (pH 8.3), then add 30 mg of papain and stir at room temperature for 2 h for prepolymerization. Then, add 91.42 mg of dopamine hydrochloride and 40.58 mg of 3,4-dihydroxyphenylacetic acid, and stir at room temperature for 15 h for polymerization. The resulting polymer was separated using a magnet and washed with a 2% (w / v) sodium dodecyl sulfate-2% (v / v) glacial acetic acid aqueous solution to remove the template molecules. The polymer was then dried at 50°C to obtain amino acid-assisted papain-imprinted polymers (MIPs).

[0094] Example 7

[0095] The preparation method of amino acid-assisted papain imprinted polymer comprises the following steps:

[0096] 1) 5 g of orange peel powder was added to 15 mL of 0.6 M ammonium ferric oxalate aqueous solution, soaked at 60°C for 2 days, oven-dried at 90°C, and pulverized to obtain treated orange peel powder. The treated orange peel powder was then carbonized in a tube furnace at 600°C for 2 hours under a nitrogen atmosphere. The product was then rinsed with anhydrous ethanol and then distilled water to obtain orange peel carbon.

[0097] 2) Weigh 400 mg of tangerine peel carbon material and add it to 100 mL of 0.1% (v / v) glacial acetic acid aqueous solution containing 2 mg / mL chitosan. Stir and prepolymerize for 1 hour. Adjust the pH of the solution to 8.0 with dilute ammonia. Add 10 μL of 50 wt% glutaraldehyde aqueous solution and stir at room temperature for 3 hours. Wash the reaction product with distilled water and dry it in an oven at 50°C to obtain chitosan-modified tangerine peel carbon material.

[0098] 3) Weigh 360 mg of chitosan-modified tangerine peel carbon material and add it to 30 mL of phosphate buffer (pH 7.0). Add 287.52 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 172.62 mg of N-hydroxysuccinimide and stir at room temperature for 2 hours. Add 30 mL of a 12 mg / mL glutamic acid solution dropwise and stir at room temperature for 6 hours. Dry in an oven to obtain the glutamic acid-modified tangerine peel carbon material.

[0099] 4) Weigh 80 mg of glutamic acid-modified orange peel charcoal material into 30 mL of Tris buffer (pH 8.3), then add 30 mg of papain and stir at room temperature for 2 h for prepolymerization. Then, add 91.42 mg of dopamine hydrochloride and 40.58 mg of 3,4-dihydroxyphenylacetic acid, and stir at room temperature for 9 h for polymerization. The resulting polymer was separated using a magnet and washed with a 2% (w / v) sodium dodecyl sulfate-2% (v / v) glacial acetic acid aqueous solution to remove the template molecules. The polymer was then dried at 50°C to obtain amino acid-assisted papain-imprinted polymers (MIPs).

[0100] Example 8

[0101] The preparation method of amino acid-assisted papain imprinted polymer comprises the following steps:

[0102] 1) 5 g of orange peel powder was added to 15 mL of 0.6 M ammonium ferric oxalate aqueous solution, soaked at 60°C for 2 days, oven-dried at 90°C, and pulverized to obtain treated orange peel powder. The treated orange peel powder was then carbonized in a tube furnace at 600°C for 2 hours under a nitrogen atmosphere. The product was then rinsed with anhydrous ethanol and then distilled water to obtain orange peel carbon.

[0103] 2) Weigh 400 mg of tangerine peel carbon material and add it to 100 mL of 0.1% (v / v) glacial acetic acid aqueous solution containing 2 mg / mL chitosan. Stir and prepolymerize for 1 hour. Adjust the pH of the solution to 8.0 with dilute ammonia. Add 10 μL of 50 wt% glutaraldehyde aqueous solution and stir at room temperature for 3 hours. Wash the reaction product with distilled water and dry it in an oven at 50°C to obtain chitosan-modified tangerine peel carbon material.

[0104] 3) Weigh 360 mg of chitosan-modified tangerine peel carbon material and add it to 30 mL of phosphate buffer (pH 7.0). Add 287.52 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 172.62 mg of N-hydroxysuccinimide and stir at room temperature for 2 hours. Add 30 mL of a 12 mg / mL glutamic acid solution dropwise and stir at room temperature for 6 hours. Dry in an oven to obtain the glutamic acid-modified tangerine peel carbon material.

[0105] 4) Weigh 80 mg of glutamic acid-modified orange peel charcoal material into 30 mL of Tris buffer (pH 8.3), then add 30 mg of papain and stir at room temperature for 2 h for prepolymerization. Then, add 91.42 mg of dopamine hydrochloride and 40.58 mg of 3,4-dihydroxyphenylacetic acid, and stir at room temperature for 12 h for polymerization. The resulting polymer was separated using a magnet and washed with a 2% (w / v) sodium dodecyl sulfate-2% (v / v) glacial acetic acid aqueous solution to remove the template molecules. The polymer was then dried at 50°C to obtain amino acid-assisted papain-imprinted polymers (MIPs).

[0106] Example 9

[0107] The preparation method of amino acid-assisted papain imprinted polymer comprises the following steps:

[0108] 1) 5 g of orange peel powder was added to 15 mL of 0.6 M ammonium ferric oxalate aqueous solution, soaked at 60°C for 2 days, oven-dried at 90°C, and pulverized to obtain treated orange peel powder. The treated orange peel powder was then carbonized in a tube furnace at 600°C for 2 hours under a nitrogen atmosphere. The product was then rinsed with anhydrous ethanol and then distilled water to obtain orange peel carbon.

[0109] 2) Weigh 400 mg of tangerine peel carbon material and add it to 100 mL of 0.1% (v / v) glacial acetic acid aqueous solution containing 2 mg / mL chitosan. Stir and prepolymerize for 1 hour. Adjust the pH of the solution to 8.0 with dilute ammonia. Add 10 μL of 50 wt% glutaraldehyde aqueous solution and stir at room temperature for 3 hours. Wash the reaction product with distilled water and dry it in an oven at 50°C to obtain chitosan-modified tangerine peel carbon material.

[0110] 3) Weigh 360 mg of chitosan-modified tangerine peel carbon material and add it to 30 mL of phosphate buffer (pH 7.0). Add 287.52 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 172.62 mg of N-hydroxysuccinimide and stir at room temperature for 2 hours. Add 30 mL of a 12 mg / mL glutamic acid solution dropwise and stir at room temperature for 6 hours. Dry in an oven to obtain the glutamic acid-modified tangerine peel carbon material.

[0111] 4) Weigh 80 mg of glutamic acid-modified orange peel charcoal material into 30 mL of Tris buffer (pH 8.3), then add 30 mg of papain and stir at room temperature for 2 h for prepolymerization. Then, add 91.42 mg of dopamine hydrochloride and 40.58 mg of 3,4-dihydroxyphenylacetic acid, and stir at room temperature for 18 h for polymerization. The resulting polymer was separated using a magnet and washed with a 2% (w / v) sodium dodecyl sulfate-2% (v / v) glacial acetic acid aqueous solution to remove the template molecules. The polymer was then dried at 50°C to obtain amino acid-assisted papain-imprinted polymers (MIPs).

[0112] 2. Performance Study of Amino Acid-Assisted Papain Imprinted Polymers

[0113] Non-imprinted polymers (NIPs) were prepared as a control group. The synthesis and elution methods of NIPs were the same as those of MIPs in the corresponding example, except that the template molecule papain was not added during the synthesis.

[0114] 1. Investigate the effect of papain concentration on the adsorption capacity of the synthesized imprinted polymer

[0115] Papain solutions of varying concentrations (50, 100, 150, 200, 250, 300, and 350 μg / mL) were prepared and subjected to shaker adsorption experiments using the MIPs and NIPs prepared in Example 1 for 1 hour. The supernatant after adsorption was analyzed using a fluorescence spectrophotometer at an excitation wavelength of 282 nm. The amount of papain adsorbed by the MIPs and NIPs was calculated based on the change in papain concentration before and after adsorption.

[0116] The results are as follows Figure 1 As shown in the figure, within the concentration range of 50–250 μg / mL, the adsorption of papain by both MIPs and NIPs increased to varying degrees with increasing papain concentration. At 250 μg / mL, the adsorption curves for MIPs and NIPs began to flatten and no longer increased with increasing papain concentration, reaching adsorption equilibrium. These results indicate that the optimal initial concentration for the adsorption experiment is 250 μg / mL.

[0117] 2. Investigate the effect of adsorption time on the adsorption capacity of the synthesized imprinted polymer

[0118] A papain solution with a concentration of 250 μg / mL was prepared, and shaker adsorption experiments were performed using the MIPs and NIPs prepared in Example 1. The supernatants at different adsorption times (10, 20, 30, 40, 50, 60, 70, and 90 min) were collected and detected using a fluorescence spectrophotometer at an excitation wavelength of 282 nm. The amount of papain adsorbed by the MIPs and NIPs was calculated based on the change in papain concentration in the solution before and after adsorption.

[0119] The results are as follows Figure 2 As shown in the figure, at the beginning of adsorption, the adsorption of template protein by both MIPs and NIPs increased rapidly within a short period of time. At this point, the growth rate of MIPs' adsorption was much greater than that of NIPs. When the adsorption time exceeded 40 minutes, the adsorption of MIPs and NIPs stopped increasing over time, and the adsorption of papain gradually reached equilibrium. These results indicate that the optimal adsorption time for the adsorption experiment is 40 minutes.

[0120] 3. Verification of the selectivity of the synthesized imprinted polymer for the template molecule papain

[0121] Papain solution, HSA (human serum albumin) solution, BSA (bovine serum albumin) solution, Lyz (lysozyme) solution, BR (bromelain) solution, and pepsin (pepsin) solution were prepared at a concentration of 250 μg / mL, respectively. The MIPs and NIPs prepared in Example 1 were used to perform shaker adsorption experiments for 40 min. The supernatant after adsorption was detected by a fluorescence spectrophotometer. The adsorption amount of each protein by the MIPs and NIPs was calculated based on the change in the corresponding protein concentration in each solution before and after adsorption.

[0122] The results are as follows Figure 3 As shown in the figure, compared to NIPs, MIPs exhibited the highest adsorption capacity for papain, reaching 197 mg / g, with an imprinting factor (IF) of 6.12, demonstrating very high selectivity. In comparison, the IFs for the reference proteins HSA, BSA, Lysine, BR, and pepsin ranged from 0.73 to 1.30, indicating nonspecific adsorption of these proteins with no selectivity. These experimental results demonstrate that the synthesized MIPs possess highly specific recognition capabilities for papain.

[0123] 4. Verification of the reusability of papain polymer

[0124] A 250 μg / mL papain solution was prepared and subjected to a shaker adsorption experiment using the MIPs prepared in Example 1 for 40 minutes. The supernatant after adsorption was analyzed using a fluorescence spectrophotometer. The amount of papain adsorbed by the MIPs was calculated based on the change in papain concentration before and after adsorption. The MIPs were eluted with a 2% (w / v) sodium dodecyl sulfate-2% (v / v) glacial acetic acid aqueous solution to remove the template papain. The eluate was washed with distilled water and dried to obtain regenerated MIPs. The regenerated MIPs were then used as adsorbents for five subsequent adsorption-desorption cycles to evaluate their reusability.

[0125] The results are as follows Figure 4 As shown in the figure, after six adsorption-desorption cycles, the adsorption of papain by the MIPs decreased by only 9.0%, indicating that the imprinted polymer still retained 91.0% of its adsorption capacity. These experimental results demonstrate that the prepared MIPs have good reusability and can meet the requirements of daily experiments.

[0126] 5. Investigate the effect of chitosan addition on the adsorption effect of papain imprinted polymer

[0127] Shaking table adsorption experiments were performed using the MIPs and NIPs prepared in Example 1, Example 2, and Example 3, respectively. The supernatant after adsorption was detected using a fluorescence spectrophotometer at an excitation wavelength of 282 nm. The adsorption amount of the template protein by the MIPs and NIPs was calculated based on the change in papain concentration in the solution before and after adsorption.

[0128] The results are as follows Figure 5 As shown, it can be seen that when the addition amount is 2 mg / mL, the prepared MIPs has the best adsorption effect on papain, so this ratio is selected as the optimal preparation condition to synthesize MIPs.

[0129] 6. Investigate the effect of glutamic acid addition on the adsorption of papain imprinted polymer

[0130] Shaking table adsorption experiments were performed using the MIPs and NIPs prepared in Examples 1, 4, 5, and 6, respectively. The supernatant after adsorption was detected using a fluorescence spectrophotometer at an excitation wavelength of 282 nm. The adsorption amount of the template protein by the MIPs and NIPs was calculated based on the change in papain concentration in the solution before and after adsorption.

[0131] The results are as follows Figure 6As shown, it can be seen that when the addition amount is 12 mg / mL, the prepared MIPs has the best adsorption effect on papain, so this ratio is selected as the optimal preparation condition to synthesize MIPs.

[0132] 7. Investigate the effect of polymerization reaction time on the adsorption effect of papain imprinted polymer

[0133] Shaking table adsorption experiments were performed using the MIPs and NIPs prepared in Examples 1, 7, 8, and 9, respectively. The supernatant after adsorption was detected using a fluorescence spectrophotometer at an excitation wavelength of 282 nm. The adsorption amount of the template protein by the MIPs and NIPs was calculated based on the change in papain concentration in the solution before and after adsorption.

[0134] The results are as follows Figure 7 As shown in the figure, as the polymerization reaction time increases from 9 to 15 h, the adsorption capacity of both MIPs and NIPs gradually increases, with the adsorption capacity of MIPs increasing at a faster rate than that of NIPs, leading to a corresponding increase in the imprinting factor of the synthesized MIPs. When the reaction time exceeds 15 h, the adsorption capacity and imprinting factor of the synthesized MIPs for papain decrease. Therefore, the MIPs synthesized at a polymerization reaction time of 15 h have the highest adsorption capacity and the best imprinting factor for papain.

[0135] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing an amino acid-assisted papain imprinted polymer, characterized in that: The orange peel charcoal material was modified with chitosan and glutamic acid in sequence, and then papain, dopamine hydrochloride and 3,4-dihydroxyphenylacetic acid were added in sequence for polymerization to obtain amino acid-assisted papain-imprinted polymer.

2. The method for preparing an amino acid-assisted papain imprinted polymer according to claim 1, wherein: The dosage ratio of orange peel charcoal material to chitosan solution was 4 mg:1 mL, and the concentration of chitosan solution was 1~3 mg / mL.

3. The method for preparing an amino acid-assisted papain imprinted polymer according to claim 2, wherein: The concentration of chitosan solution was 2 mg / mL.

4. The method for preparing an amino acid-assisted papain imprinted polymer according to claim 1, wherein: The dosage ratio of the chitosan-modified product to the glutamic acid solution was 12 mg:1 mL, and the concentration of the glutamic acid solution was 4-16 mg / mL.

5. The method for preparing an amino acid-assisted papain imprinted polymer according to claim 4, wherein: The concentration of the glutamate solution was 12 mg / mL.

6. The method for preparing an amino acid-assisted papain imprinted polymer according to claim 1, wherein: The polymerization time is 9~18 h. 7 . The papain imprinted polymer obtained by the preparation method of the amino acid-assisted papain imprinted polymer according to any one of claims 1 to 6 .

8. The papain imprinted polymer according to claim 7, wherein When the concentration of chitosan solution was 2 mg / mL and the concentration of glutamic acid solution was 12 mg / mL, the adsorption capacity of papain imprinted polymer for papain was 197 mg / g, and the imprinting factor was 6.

12.

9. Use of the amino acid-assisted papain imprinted polymer according to claim 7 or 8 in the extraction and separation of papain.

10. A method for extracting papain, characterized in that: The amino acid-assisted papain imprinted polymer according to claim 7 or 8 is mixed with a papain solution to be extracted, subjected to adsorption, elution, dialysis, concentration, and drying to obtain papain.

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