Orange peel carbon surface trypsin imprinted polymer as well as preparation method and application thereof

Abstract

The invention discloses an orange peel carbon surface trypsin imprinted polymer as well as a preparation method and application thereof, and belongs to the technical field of trypsin extraction and separation materials. The preparation method comprises the following steps: by taking waste orange peel as a carbon source, preparing magnetic orange peel carbon as a carrier through hydrothermal carbonization and high-temperature calcination; further, trypsin is used as a template, caffeic acid and levodopa are used as bifunctional monomers, and the trypsin imprinted polymer is successfully prepared through a mild and green one-step synthesis method. Wherein the rigid molecular structure and the flexible molecular structure of the bifunctional monomer are synergistically complementary, so that the imprinted polymer is endowed with excellent trypsin adsorption performance. Meanwhile, the imprinted polymer can be quickly separated by an external magnetic field due to the good magnetic responsiveness of the carrier, so that the operation process is greatly simplified. The finally obtained trypsin imprinted polymer is high in recognition specificity, high in separation efficiency, simple and convenient to operate and good in stability, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the technical field of trypsin extraction and separation materials, specifically relating to trypsin-imprinted polymers on the carbon surface of orange peel, their preparation methods, and applications. Background Technology

[0002] Trypsin (EC 3.4.21.4) is a serine protease primarily found in the pancreas of vertebrates. Trypsin exhibits strong substrate specificity, selectively cleaving the peptide bonds at the carboxyl terminus of arginine (Arg) and lysine (Lys) residues. As an important industrial enzyme, trypsin has wide applications in various fields, including food processing (such as improving egg properties, tenderizing meat, and preparing hydrolyzed protein), medicine (such as anti-inflammatory and swelling-reducing agents, treating indigestion, and activating insulin precursors), textiles (silk degumming), leather tanning (deliming and softening, improving leather softness), and modern biotechnology (such as enzymatic digestion in proteomics research and tissue dissociation in cell culture).

[0003] Currently, trypsin is mainly derived from the pancreas of animals such as pigs, cattle, or sheep. Because pancreatic tissue contains multiple zymogens with similar properties, such as chymotrypsinogen and elastaseogen, the extraction and separation of trypsin presents a challenge. Traditional separation and purification methods mainly include salting-out precipitation, isoelectric point precipitation, organic solvent precipitation, ion exchange chromatography, and gel filtration chromatography. These methods generally have certain limitations: salting-out and isoelectric point precipitation are simple to operate and suitable for preliminary extraction, but they result in low purity, poor resolution, and are prone to co-precipitation of other enzymes; organic solvent precipitation can improve purity, but it easily leads to enzyme denaturation and inactivation, limiting activity recovery; ion exchange chromatography and gel filtration chromatography improve purification specificity, but the former requires frequent medium regeneration, and the latter has a slow separation speed and limited resolution, usually requiring a combination of methods. Therefore, developing a highly selective, easy-to-operate, and scalable method for the separation and purification of trypsin has significant practical significance and application demand.

[0004] Molecularly imprinted polymers (MIPs) open new pathways for capturing target molecules in complex systems by constructing specific recognition sites that match the spatial topology of the target molecule. The synthesized imprinted polymers possess advantages such as predictable structure, stable physicochemical properties, and strong recognition specificity, showing promising applications in extraction and separation. The reusability and stability of imprinted polymers further enhance their value in large-scale industrial production. Although this technology has made significant progress in the separation and purification of small molecules, its application to biomacromolecules is still in the exploratory stage. For biomacromolecules such as proteins, molecular imprinting technology can improve the separation efficiency and purity of proteins through strategies such as selecting carrier types and optimizing the coordination between template molecules and functional monomers. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a method for preparing and applying trypsin-imprinted polymers on the carbon surface of orange peel. The synthesized trypsin-imprinted polymers have the advantages of large adsorption capacity, strong specificity, high separation efficiency, and good stability, and can directly extract trypsin from complex biological matrices.

[0006] The first objective of this invention is to provide a method for preparing a trypsin-imprinted polymer on the surface of orange peel carbon. Waste orange peel is added to FeCl3 solution, and after hydrothermal carbonization and high-temperature calcination, magnetic orange peel carbon is obtained. Using the magnetic orange peel carbon as a carrier, trypsin and bifunctional monomers are added sequentially for imprinting polymerization to obtain a trypsin-imprinted polymer on the surface of orange peel carbon.

[0007] Preferably, the preparation method of magnetic orange peel carbon is as follows: orange peel is added to FeCl3 solution, and the reaction is carried out in a reactor at 180°C for 12 hours. The product is then washed, dried, and transferred to a tube furnace, where it is reacted at 650°C for 2 hours under a nitrogen atmosphere. After washing and drying, magnetic orange peel carbon is obtained.

[0008] Preferably, the ratio of orange peel to FeCl3 solution is 1 mg: 3 mL, and the concentration of FeCl3 solution is 0.3 mol / L.

[0009] Preferably, the preparation method of the trypsin-imprinted polymer is as follows: trypsin and magnetic orange peel carbon are added to 20 mL of Tris buffer at pH 8.5 and pre-assembled for 1 h. Then, a bifunctional monomer is added, and the mixture is stirred at 25 °C for 15 h. An external magnetic field is applied to separate the imprinted polymer, and trypsin is removed by washing with elution buffer. After washing and drying, the trypsin-imprinted polymer is obtained.

[0010] Preferably, the ratio of magnetic orange peel carbon to bifunctional monomer is 1 mg:1.5 mg, and the bifunctional monomer includes caffeic acid and levodopa in a molar ratio of 1:3.

[0011] Preferably, the imprint aggregation time is 6–21 hours.

[0012] More preferably, the imprint aggregation time is 15 hours.

[0013] A second objective of the present invention is to provide an orange peel carbon surface trypsin-imprinted polymer prepared by the above method.

[0014] Preferably, when the molar ratio of caffeic acid to levodopa is 1:3 and the imprinting time is 15 h, the adsorption capacity of the orange peel carbon surface trypsin imprinted polymer for trypsin is 261 mg / g and the imprinting factor is 4.93.

[0015] A third objective of this invention is to provide the application of the above-mentioned orange peel carbon surface trypsin-imprinted polymer in trypsin extraction and separation.

[0016] Preferably, the concentration of the trypsin solution to be extracted is 100–700 μg / mL.

[0017] More preferably, the concentration of the trypsin solution to be extracted is 400 μg / mL.

[0018] Preferably, the adsorption time is 30-60 min.

[0019] More preferably, the adsorption time is 30 min.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention utilizes magnetic carbon material derived from waste orange peel as a carrier to prepare trypsin-imprinted polymers via a mild one-step method. Caffeic acid and levodopa are selected as bifunctional monomers. The rigid planar structure of caffeic acid and the flexible side chains of levodopa form spatial complementarity, constructing high-fidelity imprinted sites with multi-point synergistic recognition capabilities. The synthesis employs an environmentally friendly aqueous free radical polymerization process, requiring no organic solvents or vigorous reaction conditions at room temperature, aligning with green chemistry principles. The integrated magnetic nanounits (magnetic orange peel carbon) within the carrier can be rapidly separated using an external magnetic field, significantly improving separation efficiency. Data shows that this imprinted polymer achieves strong specificity, high adsorption capacity, and cycling stability for trypsin recognition through precise construction of biomimetic recognition sites, demonstrating significant technological advantages in the field of trypsin separation. Attached Figure Description

[0022] Figure 1 This is the isothermal adsorption curve of the trypsin-imprinted polymer on the carbon surface of orange peel in Example 1.

[0023] Figure 2 This is a dynamic adsorption curve of the trypsin-imprinted polymer on the carbon surface of orange peel in Example 1.

[0024] Figure 3 This is a selective adsorption curve of the trypsin-imprinted polymer on the carbon surface of orange peel in Example 1.

[0025] Figure 4 This is a graph showing the reusability of the trypsin-imprinted polymer on the carbon surface of orange peel in Example 1.

[0026] Figure 5 The graph shows the effect of different addition ratios of caffeic acid (CA) and levodopa (L-DOPA) in Examples 1-7 on the adsorption effect of trypsin-imprinted polymers on the carbon surface of orange peel.

[0027] Figure 6 The graph shows the effect of different self-polymerization times on the adsorption effect of trypsin-imprinted polymers on the carbon surface of orange peel in Examples 1, 8-12. Detailed Implementation

[0028] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0029] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0030] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0031] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0032] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0033] This invention provides a method for preparing trypsin-imprinted polymers on the carbon surface of orange peel, comprising the following steps:

[0034] Step 1: Orange peel was added to a 0.3 mol / L FeCl3 solution and reacted in a reactor at 180°C for 12 h. The product was then washed sequentially with ethanol and triple-distilled water. The resulting solid powder was transferred to a tube furnace and reacted at 650°C for 2 h under a nitrogen atmosphere. Afterward, it was washed sequentially with 1 mol / L hydrochloric acid solution, ethanol, and triple-distilled water, and dried to obtain magnetic orange peel carbon.

[0035] Step 2: Weigh 20.0 mg of trypsin and 60.0 mg of magnetic orange peel carbon and add them to 20 mL of Tris buffer (pH 8.5). Stir at 25°C for 1 h for pre-assembly. Then, add caffeic acid and levodopa, and stir at 25°C for 15 h. Apply an external magnetic field to separate the imprinted polymer, and wash with elution buffer to remove trypsin. Then wash with distilled water to remove the elution buffer, and dry to obtain the trypsin-imprinted polymer on the orange peel carbon surface.

[0036] The present invention will be further illustrated below with reference to Table 1 and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims. In the following examples, the lysozyme (catalog number: 302969), human serum albumin (catalog number: A1653), bovine serum albumin (catalog number: 900523), trypsin (catalog number: 954773), papain (catalog number: 916928), bromelain (catalog number: 810010), ferric chloride (catalog number: 916296), levodopa (catalog number: 115511), caffeic acid (catalog number: C804976), Tris buffer (catalog number: 992232), glacial acetic acid (catalog number: 73562B), and sodium dodecyl sulfate (catalog number: 25701I) used were all purchased from Shanghai Titan Technology Co., Ltd.; all other raw materials were conventional commercially available products with specifications that are standard in the art.

[0037] Example: Preparation of trypsin-imprinted polymer on carbon surface of orange peel

[0038] Table 1 lists the optimized reaction conditions during the synthesis of trypsin-imprinted polymers in Examples 1 to 12.

[0039] Serial Number CA:L-DOPA ratio CA addition amount (mg) L-DOPA dosage (mg) Time (h) Example 1 1:3 21.01 68.99 15 Example 2 1:0 90.00 0.00 15 Example 3 2:1 58.16 31.84 15 Example 4 1:1 42.97 47.03 15 Example 5 1:2 28.22 61.78 15 Example 6 1:4 16.73 73.27 15 Example 7 0:1 0.00 90.00 15 Example 8 1:3 21.01 68.99 6 Example 9 1:3 21.01 68.99 9 Example 10 1:3 21.01 68.99 12 Example 11 1:3 21.01 68.99 18 Example 12 1:3 21.01 68.99 21

[0040] Example 1

[0041] A method for preparing trypsin-imprinted polymers on the carbon surface of orange peel includes the following steps:

[0042] Step 1: Add orange peel to a 0.3 mol / L FeCl3 solution, transfer to a reaction vessel, and react at 180°C for 12 h. Wash the reaction product sequentially with ethanol and triple-distilled water, and dry in an oven at 60°C for 12 h. Transfer the dried solid powder to a tube furnace and react at 650°C for 2 h under a nitrogen atmosphere. Afterward, wash sequentially with 1 mol / L hydrochloric acid solution, ethanol, and triple-distilled water.

[0043] After drying, magnetic orange peel carbon is obtained;

[0044] Step 2: Weigh 20.0 mg of trypsin and add it to 20 mL of Tris buffer (pH 8.5), then sonicate to dissolve. Add 60.0 mg of magnetic orange peel carbon and stir at 25°C for 1 h for pre-assembly. Then, add 21.01 mg of caffeic acid and 68.99 mg of levodopa, and stir at 25°C for 15 h. Apply an external magnetic field to separate the imprinted polymer, and wash with a mixed aqueous solution of 2% (w / v) sodium dodecyl sulfate and 2% (v / v) glacial acetic acid to remove trypsin. Dry at 60°C for 12 h to obtain orange peel carbon surface trypsin-imprinted polymers (T-MIPs).

[0045] Example 2

[0046] A method for preparing trypsin-imprinted polymers on the carbon surface of orange peel includes the following steps:

[0047] Step 1: Add orange peel to a 0.3 mol / L FeCl3 solution, transfer to a reaction vessel, and react at 180°C for 12 h. Wash the reaction product sequentially with ethanol and triple-distilled water, and dry in an oven at 60°C for 12 h. Transfer the dried solid powder to a tube furnace and react at 650°C for 2 h under a nitrogen atmosphere. Afterward, wash sequentially with 1 mol / L hydrochloric acid solution, ethanol, and triple-distilled water.

[0048] After drying, magnetic orange peel carbon is obtained;

[0049] Step 2: Weigh 20.0 mg of trypsin and add it to 20 mL of Tris buffer (pH 8.5), then sonicate to dissolve. Add 60.0 mg of magnetic orange peel carbon and stir at 25°C for 1 h for pre-assembly. Then, add 90.00 mg of caffeic acid and stir at 25°C for 15 h. Apply an external magnetic field to separate the imprinted polymer, and wash with a mixed aqueous solution of 2% (w / v) sodium dodecyl sulfate and 2% (v / v) glacial acetic acid to remove trypsin. Dry at 60°C for 12 h to obtain orange peel carbon surface trypsin-imprinted polymers (T-MIPs).

[0050] Example 3

[0051] A method for preparing trypsin-imprinted polymers on the carbon surface of orange peel includes the following steps:

[0052] Step 1: Add orange peel to a 0.3 mol / L FeCl3 solution, transfer to a reaction vessel, and react at 180°C for 12 h. Wash the reaction product sequentially with ethanol and triple-distilled water, and dry in an oven at 60°C for 12 h. Transfer the dried solid powder to a tube furnace and react at 650°C for 2 h under a nitrogen atmosphere. Afterward, wash sequentially with 1 mol / L hydrochloric acid solution, ethanol, and triple-distilled water.

[0053] After drying, magnetic orange peel carbon is obtained;

[0054] Step 2: Weigh 20.0 mg of trypsin and add it to 20 mL of Tris buffer (pH 8.5), then sonicate to dissolve. Add 60.0 mg of magnetic orange peel carbon and stir at 25°C for 1 h for pre-assembly. Then, add 58.16 mg of caffeic acid and 31.84 mg of levodopa, and stir at 25°C for 15 h. Apply an external magnetic field to separate the imprinted polymer, and wash with a 2% (w / v) sodium dodecyl sulfate-2% (v / v) glacial acetic acid mixed aqueous solution to remove trypsin. Dry at 60°C for 12 h to obtain orange peel carbon surface trypsin-imprinted polymers (T-MIPs).

[0055] Example 4

[0056] A method for preparing trypsin-imprinted polymers on the carbon surface of orange peel includes the following steps:

[0057] Step 1: Add orange peel to a 0.3 mol / L FeCl3 solution, transfer to a reaction vessel, and react at 180°C for 12 h. Wash the reaction product sequentially with ethanol and triple-distilled water, and dry in an oven at 60°C for 12 h. Transfer the dried solid powder to a tube furnace and react at 650°C for 2 h under a nitrogen atmosphere. Afterward, wash sequentially with 1 mol / L hydrochloric acid solution, ethanol, and triple-distilled water.

[0058] After drying, magnetic orange peel carbon is obtained;

[0059] Step 2: Weigh 20.0 mg of trypsin and add it to 20 mL of Tris buffer (pH 8.5), then sonicate to dissolve. Add 60.0 mg of magnetic orange peel carbon and stir at 25°C for 1 h for pre-assembly. Then, add 42.97 mg of caffeic acid and 47.03 mg of levodopa, and stir at 25°C for 15 h. Apply an external magnetic field to separate the imprinted polymer, and wash with a 2% (w / v) sodium dodecyl sulfate-2% (v / v) glacial acetic acid mixed aqueous solution to remove trypsin. Dry at 60°C for 12 h to obtain orange peel carbon surface trypsin imprinted polymers (T-MIPs).

[0060] Example 5

[0061] A method for preparing trypsin-imprinted polymers on the carbon surface of orange peel includes the following steps:

[0062] Step 1: Add orange peel to a 0.3 mol / L FeCl3 solution, transfer to a reaction vessel, and react at 180°C for 12 h. Wash the reaction product sequentially with ethanol and triple-distilled water, and dry in an oven at 60°C for 12 h. Transfer the dried solid powder to a tube furnace and react at 650°C for 2 h under a nitrogen atmosphere. Afterward, wash sequentially with 1 mol / L hydrochloric acid solution, ethanol, and triple-distilled water.

[0063] After drying, magnetic orange peel carbon is obtained;

[0064] Step 2: Weigh 20.0 mg of trypsin and add it to 20 mL of Tris buffer (pH 8.5), then sonicate to dissolve. Add 60.0 mg of magnetic orange peel carbon and stir at 25°C for 1 h for pre-assembly. Then, add 28.22 mg of caffeic acid and 61.78 mg of levodopa, and stir at 25°C for 15 h. Apply an external magnetic field to separate the imprinted polymer, and wash with a mixed aqueous solution of 2% (w / v) sodium dodecyl sulfate and 2% (v / v) glacial acetic acid to remove trypsin. Dry at 60°C for 12 h to obtain orange peel carbon surface trypsin-imprinted polymers (T-MIPs).

[0065] Example 6

[0066] A method for preparing trypsin-imprinted polymers on the carbon surface of orange peel includes the following steps:

[0067] Step 1: Add orange peel to a 0.3 mol / L FeCl3 solution, transfer to a reaction vessel, and react at 180°C for 12 h. Wash the reaction product sequentially with ethanol and triple-distilled water, and dry in an oven at 60°C for 12 h. Transfer the dried solid powder to a tube furnace and react at 650°C for 2 h under a nitrogen atmosphere. Afterward, wash sequentially with 1 mol / L hydrochloric acid solution, ethanol, and triple-distilled water.

[0068] After drying, magnetic orange peel carbon is obtained;

[0069] Step 2: Weigh 20.0 mg of trypsin and add it to 20 mL of Tris buffer (pH 8.5), then sonicate to dissolve. Add 60.0 mg of magnetic orange peel carbon and stir at 25°C for 1 h for pre-assembly. Then, add 16.73 mg of caffeic acid and 73.27 mg of levodopa, and stir at 25°C for 15 h. Apply an external magnetic field to separate the imprinted polymer, and wash with a mixed aqueous solution of 2% (w / v) sodium dodecyl sulfate and 2% (v / v) glacial acetic acid to remove trypsin. Dry at 60°C for 12 h to obtain orange peel carbon surface trypsin-imprinted polymers (T-MIPs).

[0070] Example 7

[0071] A method for preparing trypsin-imprinted polymers on the carbon surface of orange peel includes the following steps:

[0072] Step 1: Add orange peel to a 0.3 mol / L FeCl3 solution, transfer to a reaction vessel, and react at 180°C for 12 h. Wash the reaction product sequentially with ethanol and triple-distilled water, and dry in an oven at 60°C for 12 h. Transfer the dried solid powder to a tube furnace and react at 650°C for 2 h under a nitrogen atmosphere. Afterward, wash sequentially with 1 mol / L hydrochloric acid solution, ethanol, and triple-distilled water.

[0073] After drying, magnetic orange peel carbon is obtained;

[0074] Step 2: Weigh 20.0 mg of trypsin and add it to 20 mL of Tris buffer (pH 8.5), then sonicate to dissolve. Add 60.0 mg of magnetic orange peel carbon and stir at 25°C for 1 h for pre-assembly. Then, add 90.00 mg of levodopa and stir at 25°C for 15 h. Apply an external magnetic field to separate the imprinted polymer, and wash with a mixed aqueous solution of 2% (w / v) sodium dodecyl sulfate and 2% (v / v) glacial acetic acid to remove trypsin. Dry at 60°C for 12 h to obtain orange peel carbon surface trypsin-imprinted polymers (T-MIPs).

[0075] Example 8

[0076] A method for preparing trypsin-imprinted polymers on the carbon surface of orange peel includes the following steps:

[0077] Step 1: Add orange peel to a 0.3 mol / L FeCl3 solution, transfer to a reaction vessel, and react at 180°C for 12 h. Wash the reaction product sequentially with ethanol and triple-distilled water, and dry in an oven at 60°C for 12 h. Transfer the dried solid powder to a tube furnace and react at 650°C for 2 h under a nitrogen atmosphere. Afterward, wash sequentially with 1 mol / L hydrochloric acid solution, ethanol, and triple-distilled water.

[0078] After drying, magnetic orange peel carbon is obtained;

[0079] Step 2: Weigh 20.0 mg of trypsin and add it to 20 mL of Tris buffer (pH 8.5), then sonicate to dissolve. Add 60.0 mg of magnetic orange peel carbon and stir at 25°C for 1 h for pre-assembly. Then, add 21.01 mg of caffeic acid and 68.99 mg of levodopa, and stir at 25°C for 6 h. Apply an external magnetic field to separate the imprinted polymer, and wash with a mixed aqueous solution of 2% (w / v) sodium dodecyl sulfate and 2% (v / v) glacial acetic acid to remove trypsin. Dry at 60°C for 12 h to obtain orange peel carbon surface trypsin-imprinted polymers (T-MIPs).

[0080] Example 9

[0081] A method for preparing trypsin-imprinted polymers on the carbon surface of orange peel includes the following steps:

[0082] Step 1: Add orange peel to a 0.3 mol / L FeCl3 solution, transfer to a reaction vessel, and react at 180°C for 12 h. Wash the reaction product sequentially with ethanol and triple-distilled water, and dry in an oven at 60°C for 12 h. Transfer the dried solid powder to a tube furnace and react at 650°C for 2 h under a nitrogen atmosphere. Afterward, wash sequentially with 1 mol / L hydrochloric acid solution, ethanol, and triple-distilled water.

[0083] After drying, magnetic orange peel carbon is obtained;

[0084] Step 2: Weigh 20.0 mg of trypsin and add it to 20 mL of Tris buffer (pH 8.5), then sonicate to dissolve. Add 60.0 mg of magnetic orange peel carbon and stir at 25°C for 1 h for pre-assembly. Then, add 21.01 mg of caffeic acid and 68.99 mg of levodopa, and stir at 25°C for 9 h. Apply an external magnetic field to separate the imprinted polymer, and wash with a mixed aqueous solution of 2% (w / v) sodium dodecyl sulfate and 2% (v / v) glacial acetic acid to remove trypsin. Dry at 60°C for 12 h to obtain orange peel carbon surface trypsin-imprinted polymers (T-MIPs).

[0085] Example 10

[0086] A method for preparing trypsin-imprinted polymers on the carbon surface of orange peel includes the following steps:

[0087] Step 1: Add orange peel to a 0.3 mol / L FeCl3 solution, transfer to a reaction vessel, and react at 180°C for 12 h. Wash the reaction product sequentially with ethanol and triple-distilled water, and dry in an oven at 60°C for 12 h. Transfer the dried solid powder to a tube furnace and react at 650°C for 2 h under a nitrogen atmosphere. Afterward, wash sequentially with 1 mol / L hydrochloric acid solution, ethanol, and triple-distilled water.

[0088] After drying, magnetic orange peel carbon is obtained;

[0089] Step 2: Weigh 20.0 mg of trypsin and add it to 20 mL of Tris buffer (pH 8.5), then sonicate to dissolve. Add 60.0 mg of magnetic orange peel carbon and stir at 25°C for 1 h for pre-assembly. Then, add 21.01 mg of caffeic acid and 68.99 mg of levodopa, and stir at 25°C for 12 h. Apply an external magnetic field to separate the imprinted polymer, and wash with a mixed aqueous solution of 2% (w / v) sodium dodecyl sulfate and 2% (v / v) glacial acetic acid to remove trypsin. Dry at 60°C for 12 h to obtain orange peel carbon surface trypsin-imprinted polymers (T-MIPs).

[0090] Example 11

[0091] A method for preparing trypsin-imprinted polymers on the carbon surface of orange peel includes the following steps:

[0092] Step 1: Add orange peel to a 0.3 mol / L FeCl3 solution, transfer to a reaction vessel, and react at 180°C for 12 h. Wash the reaction product sequentially with ethanol and triple-distilled water, and dry in an oven at 60°C for 12 h. Transfer the dried solid powder to a tube furnace and react at 650°C for 2 h under a nitrogen atmosphere. Afterward, wash sequentially with 1 mol / L hydrochloric acid solution, ethanol, and triple-distilled water.

[0093] After drying, magnetic orange peel carbon is obtained;

[0094] Step 2: Weigh 20.0 mg of trypsin and add it to 20 mL of Tris buffer (pH 8.5), then sonicate to dissolve. Add 60.0 mg of magnetic orange peel carbon and stir at 25°C for 1 h for pre-assembly. Then, add 21.01 mg of caffeic acid and 68.99 mg of levodopa, and stir at 25°C for 18 h. Apply an external magnetic field to separate the imprinted polymer, and wash with a 2% (w / v) sodium dodecyl sulfate-2% (v / v) glacial acetic acid mixed aqueous solution to remove trypsin. Dry at 60°C for 12 h to obtain orange peel carbon surface trypsin imprinted polymers (T-MIPs).

[0095] Example 12

[0096] A method for preparing trypsin-imprinted polymers on the carbon surface of orange peel includes the following steps:

[0097] Step 1: Add orange peel to a 0.3 mol / L FeCl3 solution, transfer to a reaction vessel, and react at 180°C for 12 h. Wash the reaction product sequentially with ethanol and triple-distilled water, and dry in an oven at 60°C for 12 h. Transfer the dried solid powder to a tube furnace and react at 650°C for 2 h under a nitrogen atmosphere. Afterward, wash sequentially with 1 mol / L hydrochloric acid solution, ethanol, and triple-distilled water.

[0098] After drying, magnetic orange peel carbon is obtained;

[0099] Step 2: Weigh 20.0 mg of trypsin and add it to 20 mL of Tris buffer (pH 8.5), then sonicate to dissolve. Add 60.0 mg of magnetic orange peel carbon and stir at 25°C for 1 h for pre-assembly. Then, add 21.01 mg of caffeic acid and 68.99 mg of levodopa, and stir at 25°C for 21 h. Apply an external magnetic field to separate the imprinted polymer, and wash with a mixed aqueous solution of 2% (w / v) sodium dodecyl sulfate and 2% (v / v) glacial acetic acid to remove trypsin. Dry at 60°C for 12 h to obtain orange peel carbon surface trypsin-imprinted polymers (T-MIPs).

[0100] Study on the adsorption properties of trypsin-imprinted polymer on the carbon surface of orange peel (experimental example)

[0101] Non-imprinted polymers (T-NIPs) were prepared as a control group. The synthesis and elution methods of T-NIPs were the same as those of T-MIPs in the corresponding examples, except that the template molecule trypsin was not added during the synthesis process.

[0102] 1. Isothermal adsorption experiment of trypsin-imprinted polymer on the carbon surface of orange peel

[0103] A series of trypsin solutions with concentration gradients (100, 200, 300, 400, 500, 600, 700 μg / mL) were prepared. T-MIPs and T-NIPs prepared in Example 1 were mixed with the above solutions and adsorbed by shaking at room temperature for 1 h. After adsorption was complete, the supernatant was collected, and the concentration was measured using a fluorescence spectrophotometer at an excitation wavelength of 282 nm. The adsorption capacity of T-MIPs and T-NIPs for the target protein was calculated by comparing the concentration difference of trypsin in the solution before and after adsorption.

[0104] Experimental results are as follows Figure 1 As shown, within the trypsin concentration range of 100–400 μg / mL, the adsorption capacity of both T-MIPs and T-NIPs increased with increasing substrate concentration. When the substrate concentration reached 400 μg / mL, the rate of increase in adsorption capacity slowed down, indicating that the system had reached adsorption saturation, and the adsorption capacity no longer increased with further increases in substrate concentration. Therefore, the optimal initial trypsin concentration for subsequent adsorption experiments was 400 μg / mL.

[0105] 2. Dynamic adsorption experiment of trypsin-imprinted polymer on the carbon surface of orange peel

[0106] A trypsin solution with a concentration of 400 μg / mL was prepared and subjected to isothermal oscillation adsorption experiments with T-MIPs and T-NIPs prepared in Example 1, respectively. Supernatants were collected at different adsorption time points (10, 20, 30, 40, 50, 60, 70, and 90 min). The concentration of trypsin in the supernatant at each time point was measured using a fluorescence spectrophotometer at an excitation wavelength of 282 nm. Based on the changes in the concentration of the target protein before and after adsorption, the adsorption amounts of T-MIPs and T-NIPs at different time points were calculated.

[0107] Experimental results are as follows Figure 2 As shown, in the initial adsorption stage (0-30 min), the adsorption capacity of both T-MIPs and T-NIPs for the target protein showed a rapid upward trend, with the adsorption rate of T-MIPs being significantly higher than that of T-NIPs during this stage. After adsorption time exceeded 30 min, the adsorption capacity of both stopped increasing with time, and the adsorption tended to reach equilibrium. Therefore, the optimal adsorption time for subsequent adsorption experiments was 30 min.

[0108] 3. Selective adsorption experiment of trypsin-imprinted polymer on the carbon surface of orange peel

[0109] Reference protein solutions with a concentration of 400 μg / mL were prepared: trypsin (Try), human serum albumin (HSA), bovine serum albumin (BSA), bromelain (BR), lysozyme (Lyz), and papain. T-MIPs and T-NIPs prepared in Example 1 were then used to adsorb the aforementioned reference protein solutions by isothermal shaking for 30 min. After adsorption, the supernatant was collected, and the residual protein concentration was determined by fluorescence spectrophotometry. Based on the difference in target analyte concentration before and after adsorption, the adsorption capacity of T-MIPs and T-NIPs for different proteins was calculated.

[0110] Experimental results are as follows Figure 3 As shown, compared to T-NIPs, T-MIPs exhibit superior adsorption performance for trypsin, with an adsorption capacity of 261 mg / g and an imprinting factor (IF) of 4.93. In contrast, the imprinting factors of T-MIPs for reference proteins (HSA, BSA, Lyz, BR, and Papain) ranged from 0.56 to 1.29, showing no specific adsorption. Therefore, the prepared T-MIPs possess high specificity for trypsin recognition.

[0111] 4. Repeatability experiment of trypsin-imprinted polymer on carbon surface of orange peel

[0112] A 400 μg / mL trypsin solution was prepared and the T-MIPs prepared in Example 1 were used for adsorption under isothermal shaking for 30 min. After adsorption, the supernatant was collected, and the residual trypsin concentration was determined by fluorescence spectrophotometry. The adsorption capacity of the T-MIPs for the target protein was calculated based on the concentration difference before and after adsorption. Subsequently, the adsorbed T-MIPs were eluted with a 2% (w / v) sodium dodecyl sulfate-2% (v / v) glacial acetic acid mixed aqueous solution. The eluent was washed away with distilled water and dried to obtain regenerated T-MIPs. The regenerated T-MIPs were used in five subsequent consecutive adsorption-desorption cycle experiments to evaluate their reusability.

[0113] Experimental results are as follows Figure 4 As shown, after six complete adsorption-desorption cycles, the adsorption capacity decay rate of T-MIPs for trypsin was only 8.3%, indicating that it still retained up to 91.7% of its initial adsorption capacity. Therefore, the prepared T-MIPs have good regeneration stability and recycling performance.

[0114] 5. Optimization of trypsin-imprinted polymers on the carbon surface of orange peel

[0115] (1) Effect of different addition ratios of caffeic acid (CA) and levodopa (L-DOPA) on the adsorption efficiency of T-MIPs and T-NIPs

[0116] The T-MIPs and T-NIPs prepared in Examples 1-7 were used to conduct shaker adsorption experiments. After shaking, the supernatant was collected, and the change in trypsin concentration in the solution before and after adsorption was determined by fluorescence spectrophotometry. The adsorption amount of template protein by T-MIPs and T-NIPs was calculated.

[0117] The results are as follows Figure 5 As shown, within a molar ratio range of 1:0 to 1:3, the adsorption capacity of T-MIPs for trypsin slowly increased with the increase of L-DOPA added during synthesis, and the imprinting factor was enhanced. When the amount of L-DOPA added continued to increase, the imprinting factor of the prepared T-MIPs for the template protein gradually decreased. Based on the above results, the prepared T-MIPs exhibited the best adsorption effect for trypsin when the molar ratio of caffeic acid to levodopa was 1:3 (Example 1). Therefore, this ratio was selected as the optimal preparation condition for synthesizing T-MIPs.

[0118] (2) Effect of different imprinting polymerization times on the adsorption efficiency of T-MIPs and T-NIPs

[0119] T-MIPs and T-NIPs prepared in Examples 1 and 8-12 were used to conduct shaker adsorption experiments. After shaking, the supernatant was collected, and the change in trypsin concentration in the solution before and after adsorption was determined by fluorescence spectrophotometry. The adsorption amount of template protein by T-MIPs and T-NIPs was calculated.

[0120] The results are as follows Figure 6 As shown, with the extension of the imprinting polymerization time from 6 h to 15 h, the adsorption capacity of both T-MIPs and T-NIPs increased, and the imprinting factor (IF) of T-MIPs also increased with the extension of reaction time. When the reaction time exceeded 15 h, the imprinting factor of the prepared T-MIPs against trypsin began to decrease. Therefore, the T-MIPs prepared after 15 h of imprinting polymerization reaction have both the largest adsorption capacity and the best imprinting factor.

[0121] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for preparing trypsin-imprinted polymers on the carbon surface of orange peel, characterized in that, Waste orange peels were added to FeCl3 solution and subjected to hydrothermal carbonization and high-temperature calcination to obtain magnetic orange peel carbon. Using the magnetic orange peel carbon as a carrier, trypsin and bifunctional monomers were added sequentially for imprinting polymerization to obtain trypsin-imprinted polymer on the surface of orange peel carbon.

2. The method for preparing the trypsin-imprinted polymer on the carbon surface of orange peel according to claim 1, characterized in that, The method for preparing the magnetic orange peel carbon is as follows: orange peel is added to FeCl3 solution, and the reaction is carried out in a reactor at 180°C for 12 hours. The product is washed, dried, and transferred to a tube furnace, where it is reacted at 650°C for 2 hours under a nitrogen atmosphere. After washing and drying, the magnetic orange peel carbon is obtained.

3. The method for preparing the trypsin-imprinted polymer on the carbon surface of orange peel according to claim 2, characterized in that, The ratio of orange peel to FeCl3 solution was 1 mg: 3 mL, and the concentration of FeCl3 solution was 0.3 mol / L.

4. The method for preparing the trypsin-imprinted polymer on the carbon surface of orange peel according to claim 1, characterized in that, The method for preparing the trypsin-imprinted polymer on the surface of the orange peel carbon is as follows: trypsin and magnetic orange peel carbon are added to 20 mL of pH Pre-assembled in 8.5% Tris buffer for 1 hour; then, bifunctional monomers were added and stirred at 25°C for 15 hours; the imprinted polymer was separated by applying an external magnetic field, and trypsin was removed by washing with elution buffer. After washing and drying, trypsin-imprinted polymer on orange peel carbon surface was obtained.

5. The method for preparing the trypsin-imprinted polymer on the carbon surface of orange peel according to claim 4, characterized in that, The ratio of magnetic orange peel carbon to bifunctional monomers is 1 mg:1.5 mg, and the bifunctional monomers include caffeic acid and levodopa in a molar ratio of 1:

3.

6. The method for preparing the trypsin-imprinted polymer on the carbon surface of orange peel according to claim 1, characterized in that, The imprint aggregation time is 6–21 hours.

7. The orange peel carbon surface trypsin-imprinted polymer prepared by the method according to any one of claims 1 to 6.

8. The orange peel carbon surface trypsin-imprinted polymer according to claim 7, characterized in that, When the molar ratio of caffeic acid to levodopa is 1:3 and the self-polymerization time is 15 h, the adsorption capacity of trypsin on the trypsin-imprinted polymer on the orange peel carbon surface is 261 mg / g, and the imprinting factor is 4.

93.

9. The application of the orange peel carbon surface trypsin-imprinted polymer according to claim 8 in trypsin extraction and separation.

10. The application of the orange peel carbon surface trypsin-imprinted polymer according to claim 9 in trypsin extraction and separation, characterized in that, The orange peel carbon surface trypsin imprinted polymer was mixed with the trypsin solution to be extracted, adsorbed for 30-60 min, eluted, dialyzed, concentrated, and dried to obtain trypsin.

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