Recombinant biomimetic affinity ligand for purifying bromelain, preparation method and application thereof

Abstract

The application relates to the technical field of biological separation engineering, and discloses a recombinant biomimetic affinity ligand for purifying bromelain as well as a preparation method and application of the recombinant biomimetic affinity ligand, which is prepared from the following raw materials in parts by weight: double-bonded silica-coated magnetic microspheres: 100 parts; monomer methyl acrylate: 150-250 parts; epoxy-containing monomer glycidyl methacrylate: 50-100 parts; crosslinking agent ethylene glycol dimethacrylate: 3-6 parts; and biomimetic affinity heptapeptide: 10-20 parts. The biomimetic affinity heptapeptide with a specific amino acid sequence is used as the affinity ligand, the hydrophobic residues and charged side chains in the sequence can specifically bind to the S2 sub-site and surrounding residues of the active center of bromelain to realize cavity complementation, high-specificity recognition of the target enzyme is realized, and the affinity medium prepared by using the ligand can effectively eliminate the interference of homologous impure proteins which are highly similar in structure and physicochemical properties.

Description

Technical Field

[0001] This invention relates to the field of bioseparation engineering technology, specifically to recombinant biomimetic affinity ligands for purifying bromelain, their preparation methods, and applications. Background Technology

[0002] Bromelain is a type of plant thiol protease extracted from pineapple stems or juice, and it has practical applications in the pharmaceutical, food processing, and daily chemical industries. With its expanding applications in high-value-added fields such as biopharmaceuticals, the requirements for the purity and specific activity of bromelain are correspondingly increasing.

[0003] Currently, industrial crude extraction of bromelain often employs salting out, organic solvent precipitation, or ultrafiltration. The resulting products typically contain a large number of contaminating proteins and have low enzyme specific activity. To obtain high-purity bromelain, further separation and purification usually require chromatographic techniques. Pineapple extracts are complex in composition, containing numerous homologous proteins highly similar to bromelain in molecular weight, isoelectric point, and spatial conformation, such as papain and other cysteine ​​proteases. Traditional gel filtration chromatography or ion exchange chromatography relies primarily on differences in the macroscopic physicochemical properties of protein molecules for separation, making it difficult to effectively distinguish and remove these homologous proteins with similar physicochemical properties. This limits the purity of the final purified product.

[0004] Affinity chromatography, based on specific intermolecular interactions, is an effective method for obtaining high-purity proteins. However, existing affinity chromatography techniques targeting bromelain often suffer from problems such as high ligand preparation costs, difficulty in directional coupling on the carrier surface, and poor physicochemical stability when using antibodies or natural protease inhibitors as affinity ligands. If conventional dye ligands or randomly synthesized short peptides are used, they lack recognition sites specific to the three-dimensional structure of the bromelain active site, resulting in insufficient spatial and electrostatic differential binding when encountering structurally similar homologous proteins. This non-specific or weakly specific adsorption makes the chromatography process susceptible to competitive interference from other proteins, leading to a high residual amount of homologous proteins in the product. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a recombinant biomimetic affinity ligand for purifying bromelain, its preparation method, and its application, solving the problem of difficulty in removing homologous contaminants during processing in existing technologies.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A recombinant biomimetic affinity medium for purifying bromelain is prepared from the following raw materials in parts by weight: Double-bonded silica-coated magnetic microspheres: 100 parts; Monomer methyl acrylate: 150-250 parts; Glycidyl methacrylate containing epoxy monomer: 50-100 parts; Crosslinking agent: ethylene glycol dimethacrylate: 3-6 parts; Bionic affinity heptapeptide: 10-20 parts; The amino acid sequence of the biomimetic affinity heptapeptide is H-Leu-Cys-Trp-Glu-Ile-Ala-Val-OH.

[0007] Preferably, the double-bonded silica-coated magnetic microspheres are obtained by the following preparation method: magnetic nanoparticles of iron oxide are suspended in a system containing ammonia water and reacted with tetraethyl orthosilicate in a sol-gel reaction to obtain silica-coated magnetic microspheres; then they are dispersed in anhydrous toluene and reacted with the silane coupling agent - methacryloxypropyltrimethoxysilane under heating reflux and nitrogen protection conditions to achieve surface double bonding.

[0008] Preferably, the epoxy group density on the surface of the affinity medium is 150-170 mol / g, the covalent coupling amount of the biomimetic affinity heptapeptide on the surface of the affinity medium is 41.6-46.8 mol / g, and the biomimetic affinity heptapeptide is bonded to the carrier through a secondary amine bond or thioether bond formed by the ring-opening addition of the free thiol group of its N-terminal amino or cysteine ​​side chain to the epoxy group.

[0009] A method for preparing a recombinant biomimetic affinity medium for purifying bromelain includes the following steps: S1. After coating the surface of the magnetite nanoparticles with a silica layer, the surface is modified with a silane coupling agent to obtain double bonded silica-coated magnetic microspheres. S2. The magnetic microspheres coated with double-bonded silica are dispersed in a mixed solvent, and methyl acrylate, glycidyl methacrylate and ethylene glycol dimethacrylate are added in sequence. After nitrogen gas is introduced to remove oxygen, an initiator is added to carry out a surface-initiated polymerization reaction to obtain a highly cross-linked polymer hybrid epoxy magnetic carrier. S3. The highly cross-linked polymer hybrid epoxy magnetic carrier is suspended in a buffer solution containing a biomimetic affinity heptapeptide for coupling reaction. After the reaction is completed, the free polypeptide is washed away to obtain the recombinant biomimetic affinity medium.

[0010] Preferably, in step S2, the mixed solvent is an ethanol-water mixture with a volume ratio of 1:1, the initiator is azobisisobutyronitrile, and the process parameters for the surface-initiated polymerization reaction are: mechanical stirring reaction at a constant temperature of 70°C for 24 hours.

[0011] Preferably, in step S3, the reaction system is controlled in advance as follows: a 0.1 mol / L phosphate buffer solution with a pH of 7.8 is used, and ethylenediaminetetraacetic acid with a final concentration of 1 mmol / L is added to the coupling reaction system; the process parameters of the coupling reaction are: shake the reaction in a constant temperature shaker at 28°C for 14 h, and strictly maintain the pH of the system between 7.8 and 0.1 throughout the process using dilute alkali solution.

[0012] A method for purifying bromelain using a recombinant biomimetic affinity medium includes the following steps: (1) Centrifuge the pineapple pomace extract to collect the supernatant, adjust the pH to 6.9-7.1, and add ethylenediaminetetraacetic acid to a final concentration of 1 mmol / L to obtain crude enzyme solution; (2) Add the recombinant biomimetic affinity medium to the crude enzyme solution, and oscillate and adsorb at 4°C. Then apply an external magnetic field for magnetic separation to obtain the enzyme-carrying magnetic medium. (3) Add pre-elution buffer to the enzyme-carrying magnetic medium for pre-elution, and then perform gradient main elution using main elution buffer containing a competing agent, and collect the main elution buffer; (4) After dialysis and desalting, the collected main eluent is loaded into a pre-equilibrated cation exchange chromatography column for linear gradient elution. The main elution peak of the target bromelain is collected and freeze-dried to obtain high-purity bromelain.

[0013] Preferably, in step (3), the pre-elution buffer is a 0.1 mol / L citrate buffer with pH 5.8 and the elution volume is 2 times the column volume; the main elution buffer is a 0.1 mol / L phosphate buffer containing reduced glutathione as a competing agent and its pH is controlled at 7.2.

[0014] Preferably, the main elution in step (3) is implemented as follows: the flow rate is controlled at 1.0 mL / min, and the concentration of reduced glutathione is eluted in 8 to 12 gradient steps in the range of 0 to 6 mmol / L, with each step eluting 1.5 to 2 times the column volume.

[0015] Preferably, in step (4), the cation exchange chromatography column is a CMSepharoseFF resin column with a carboxymethyl group, the linear gradient elution is carried out using a 0.1 mol / L phosphate buffer containing 0 to 0.5 mol / L sodium chloride, and the 280 nm ultraviolet absorption spectrum is monitored online during the elution process.

[0016] This invention provides a recombinant biomimetic affinity ligand for purifying bromelain, its preparation method, and its application. It offers the following advantages: 1. This invention uses a biomimetic heptapeptide with a specific amino acid sequence as an affinity ligand. The hydrophobic residues and charged side chains in this sequence can form a specific binding cavity complementary with the S2 subsite and surrounding residues of the active center of bromelain, thereby achieving highly specific recognition of the target enzyme. The affinity medium prepared using this ligand can effectively eliminate interference from homologous proteins with highly similar structures and physicochemical properties.

[0017] 2. This invention controls the pH of the peptide coupling reaction to around 7.8, promoting the ring-opening addition of the free thiol groups of the N-terminal amino or cysteine ​​side chains in the ligands to the epoxy groups, forming secondary amine bonds or thioether bonds with less steric hindrance and higher chemical bond energy. The polymer three-dimensional network structure with a specific degree of crosslinking limits excessive swelling of the carrier during regeneration. Combined with the optimized ligand bonding mode, this improves the compressive strength and chemical stability of the affinity medium.

[0018] 3. In the affinity elution stage, this invention combines pre-elution with a pH 5.8 buffer solution with gradient main elution containing a reducing glutathione competitor to remove non-specifically adsorbed impurities and proteins with weak affinity. Subsequently, tandem cation exchange chromatography is used to achieve deep separation by utilizing the slight isoelectric point difference between bromelain and residual homologous proteins, further improving the impurity removal effect. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the process steps of the present invention. Detailed Implementation

[0020] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products: Ferrous chloride tetrahydrate: CAS No. 13478-10-9, purity 98%, iron content 44.1-45.3%.

[0022] Ferric chloride hexahydrate: CAS No. 10025-77-1, purity 98%, iron content 20.1-20.9%.

[0023] Ethyl orthosilicate: CAS No. 78-10-4, purity 99%, Theoretical content: 28.4%.

[0024] 3-(isobutenoyloxy)propyltrimethoxysilane: CAS No. 2530-85-0, purity 97%, double bond content 5.2-5.6 mmol / g.

[0025] Methyl acrylate: CAS No. 96-33-3, polymerization grade, purity 99.5%, polymerization inhibitor hydroquinone monomethyl ether 10ppm.

[0026] Ethylene glycol dimethacrylate: CAS No. 97-90-5, purity 98%, double bond functionality 2.0-0.1.

[0027] Epichlorohydrin: CAS No. 106-89-8, epoxy value 0.48 mol / 100g, hydrolytic chlorine 0.01%.

[0028] Heptapeptide H-Leu-Cys-Trp-Glu-Ile-Ala-Val-OH: solid-phase synthesis, HPLC purity 95%, D-isomer content 1%, verified by chiral HPLC, molecular weight 868.99 g / mol.

[0029] Ethylenediaminetetraacetic acid (EDTA): CAS No. 60-00-4, analytical grade, purity 99.5%, heavy metal content 10 ppm.

[0030] Ethanolamine: CAS No. 141-43-5, purity 99%, moisture 0.2%.

[0031] Reduced glutathione: CAS No. 70-18-8, pharmaceutical grade, 98% purity.

[0032] CMSepharoseFF cation exchange resin: The group is carboxymethyl, the exchange capacity is 0.12-0.18 mmol / mL, and the particle size is 45-165 μm.

[0033] Citric acid monohydrate: CAS No. 5949-29-1, for buffer solution, purity 99.5%, heavy metal content 5ppm.

[0034] The heptapeptide ligand used in this invention was obtained through precise screening using computer-aided molecular design based on the three-dimensional structural characteristics of the active site of bromelain.

[0035] Specifically, this invention utilizes the crystal structure of bromelain as a docking target. The active site of this enzyme consists of a catalytic triplet composed of Cys25 and his159, along with surrounding S1 / S2 and other subsites.

[0036] Molecular docking simulations revealed that the Leu and Val side chains in the sequence possess strong hydrophobicity, enabling them to form a tight cavity complementarity with the S2 hydrophobic pocket of the enzyme active site; the large conjugated indole ring of Trp can interact with aromatic residues on the enzyme surface via... Stacking enhances binding strength, and the carboxyl groups on the side chains of Glu are negatively charged in a near-neutral pH environment, which can form a directional hydrogen bond network with basic residues near the enzyme's active site.

[0037] Obtaining biomimetic ligands The heptapeptide ligand described in this invention is prepared using the standard Fmoc solid-phase synthesis method, and the specific steps are as follows: 2-Chlorotriphenylmethyl chloride resin (2-CTCResin) was selected, and the grafting rate was controlled at about 0.5 mmol / g. An appropriate amount of resin was placed in a solid-phase synthesis tube, anhydrous dichloromethane (DCM) was added to swell for 30 min, and the solvent was removed by filtration.

[0038] The operation is carried out cyclically from the C-terminus to the N-terminus. First, 3 molar amounts of Fmoc-protected amino acid, 3 molar amounts of condensing agent HBTU, and 6 molar amounts of DIPEA are dissolved in DMF and added to the reaction tube. The reaction is carried out at room temperature with shaking for 2 hours. A negative result for ninhydrin detection indicates complete coupling. Then, a 20% (v / v) piperidine / DMF solution is used to react for 20 minutes to remove the Fmoc protecting group at the amino terminus. The resin is then washed multiple times with DMF. After synthesis, cleavage reagents were added to the reaction tube. The volume ratio of cleavage reagents used in this study was TFA:EDT:water:TIS = 94:2.5:2.5:1.0. The mixture was then shaken at room temperature in the dark for 3 hours. This process simultaneously cleaved the peptide chain from the resin and removed the side chain protecting groups. The cutting fluid was filtered out and precipitated in icy diethyl ether, collecting a white solid crude peptide. Reversed-phase high-performance liquid chromatography (RP-HPLC) was used for fractionation purification. The target fraction was collected and freeze-dried to obtain a white powder with a purity greater than 95%. Mass spectrometry analysis showed that its experimental molecular weight of 868.99 g / mol was consistent with the theoretical value.

[0039] Preparation Example 1: Preparation of Basic Epoxy Magnetic Silica Support This preparation example provides a basic monolayer silica-coated magnetic carrier for use as a baseline control for subsequent purification tests.

[0040] Weigh 2.0 g of magnetite (Fe3O4) magnetic nanoparticles with a particle size of approximately 50 nm, disperse them in 100 mL of purified water, and sonicate for 30 min to ensure uniform suspension. Add 50 mL of anhydrous ethanol and 5 mL of 25% ammonia solution to the system sequentially, and heat to 40 °C.

[0041] 10 mL of tetraethyl orthosilicate (TEOS) was slowly added dropwise to the suspension under mechanical stirring. The reaction was maintained at a constant temperature of 40 °C and stirred for 12 h to induce a sol-gel reaction. After the reaction was completed, the solid product was separated using a magnetic field, washed three times successively with anhydrous ethanol and purified water, and dried in a vacuum drying oven at 60 °C for 8 h to obtain silica-coated magnetic microspheres (Fe3O4@SiO2).

[0042] Take 1.0 g of the above microspheres and suspend them in 50 mL of 1 mol / L sodium hydroxide solution, then add 10 mL of epichlorohydrin. Place the reaction system in a 60 °C constant temperature water bath and shake for 6 h.

[0043] After the reaction, the support was collected by magnetic separation, washed with a large amount of purified water until the eluent was neutral, and then dried under vacuum. The density of epoxy groups on the support surface was determined by sodium thiosulfate-hydrochloric acid titration, and the measured value was 14010 mol / g.

[0044] Preparation Example 2: Preparation of a highly cross-linked polymer hybrid epoxy-based magnetic support In this preparation example, a highly cross-linked polymer network is further coated on the outside of the silica layer to improve the mechanical strength and epoxy grafting density of the medium.

[0045] Take 2.0 g of the Fe3O4@SiO2 microspheres obtained in step 2 of Preparation Example 1, disperse them in 100 mL of anhydrous toluene, and add 5 mL of silane coupling agent KH-570 (-methacryloyloxypropyltrimethoxysilane). Reflux at 80 °C under nitrogen protection for 12 h. After the reaction, magnetic separation is performed, and the microspheres are washed repeatedly with anhydrous ethanol to obtain a magnetic carrier with double bonds on its surface.

[0046] The above-mentioned double-bonded support was dispersed in 80 mL of ethanol-water (volume ratio 1:1) mixed solvent, and 4.0 g of methyl acrylate (monomer), 1.5 g of glycidyl methacrylate (providing epoxy monomer), and 1.7 wt% of dimethacrylate (crosslinking agent) were added sequentially.

[0047] After purging the system with nitrogen for 30 minutes to remove oxygen, 0.1 g of azobisisobutyronitrile (AIBN) was added as an initiator. Surface-initiated polymerization was initiated by mechanical stirring at a constant temperature of 70°C for 24 hours.

[0048] The microspheres were separated using a magnetic field and washed five times alternately with ethanol and purified water to thoroughly remove amorphous free polymer, followed by vacuum drying. The epoxy group density on the surface of the polymer hybrid support was determined by sodium thiosulfate-hydrochloric acid titration, and the measured value was 16010 mol / g.

[0049] Preparation Example 3: Conventional Coupling Preparation of Bionic Affinity Media In this preparation example, the selected biomimetic heptapeptide was covalently coupled to the hybrid magnetic carrier obtained in Preparation Example 2.

[0050] Weigh 1.0 g of the highly cross-linked polymer hybrid epoxy magnetic support obtained in Preparation Example 2, wash and equilibrate it three times with 0.1 mol / L carbonate buffer (pH 8.3), 10 min each time.

[0051] The equilibrated carrier was suspended in 20 mL of the same buffer containing 2.0 mg / mL of the biomimetic heptapeptide (sequence h-leu-cys-trp-glu-ile-ala-val-oh). EDTA solution was added to the system to a final concentration of 1 mmol / L to chelate trace heavy metal ions and protect the cysteine ​​thiol groups in the peptide chain from oxidative cross-linking.

[0052] The mixed reaction system was placed in a constant temperature shaker at 28℃ and shaken at 150 r / min for 10 h. During the reaction, the pH of the system was continuously monitored and maintained at 8.3-0.1 using dilute alkali solution, so that the peptide could undergo a ring-opening addition reaction with the epoxy groups on the surface of the carrier through the thiol groups of the N-terminal amino or cysteine ​​side chains.

[0053] After the reaction, the solid phase was collected by magnetic separation and washed three times alternately with 0.1 mol / L phosphate buffer and purified water to remove uncovalently bound free peptides. The final covalently coupled amount of the ligand was determined to be 45.3-1.5 mol / g by ninhydrin colorimetric assay combined with supernatant differential method. This data indicates that approximately 28.3% of the surface epoxy groups completed ligand coupling, which is consistent with the reasonable conversion rate under the steric hindrance of macromolecules.

[0054] Preparation Example 4: Optimized Coupling Preparation of Biomimetic Affinity Media Based on Preparation Example 3, this preparation example improves the stability of the medium in an acidic environment by adjusting the pH conditions of the coupling reaction and optimizing the ligand binding sites.

[0055] Weigh 1.0 g of the highly cross-linked polymer hybrid epoxy magnetic support obtained in Preparation Example 2, wash and equilibrate 3 times with 0.1 mol / L phosphate buffer (pH 7.8).

[0056] The carrier was suspended in 20 mL of the same buffer (pH 7.8) containing 2.0 mg / mL of the above-mentioned biomimetic heptapeptide, and EDTA was added to a final concentration of 1 mmol / L.

[0057] The reaction system was placed in a constant-temperature shaker at 28°C and shaken for 14 hours. The pH was strictly controlled at 7.8-0.1 throughout the reaction. These conditions effectively reduced the rate of hydrolysis side reactions of epoxy groups under alkaline conditions, while also promoting the preferential reaction of cysteine ​​thiols with epoxy groups in the peptides, forming a thioether bond conformation with less steric hindrance and higher chemical bond energy.

[0058] After the reaction, magnetic separation was performed, and the support was washed using the method described in Preparation Example 3. The covalent coupling amount of the ligand was determined to be 42.8-1.2 mol / g. The solution was sealed in purified water and stored at 4°C for later use.

[0059] Example 1: Purification of bromelain See appendix Figure 1 This embodiment provides a method for purifying bromelain, including the following steps: (1) Take the pineapple pomace extract, centrifuge at 8000g for 15min, and collect the supernatant; adjust the pH of the supernatant to 7.00.1 with 0.1mol / L phosphate buffer, and add 1mmol / L ethylenediaminetetraacetic acid to obtain crude enzyme solution.

[0060] (2) Add the recombinant biomimetic affinity medium prepared in Preparation Example 3 to the above crude enzyme solution. The mass of the affinity medium added is 1.5% of the total mass of the crude enzyme solution. Adsorb the enzyme by constant temperature oscillation at 150 r / min at 4°C for 40 min. Then apply an external magnetic field of 0.5T for magnetic separation and remove the supernatant to obtain the enzyme-carrying magnetic medium.

[0061] (3) Add 0.1 mol / L citrate buffer at pH 5.8 to the enzyme-carrying magnetic medium for pre-elution, with an elution volume of 2 column volumes; then perform main elution with 0.1 mol / L phosphate buffer containing reduced glutathione, controlling the pH of the eluent to 7.2, and perform 10-step gradient elution of reduced glutathione concentration from 0 to 6 mmol / L (each step concentration increment 0.6 mmol / L), with 2 BV elution per step, controlling the flow rate to 1.0 mL / min, and collect the main eluent.

[0062] (4) The affinity chromatography main eluent collected in step (3) was dialyzed and desalted, and then loaded into a pre-equilibrated CMSepharose FF cation exchange chromatography column. Taking advantage of the slight difference in isoelectric point between bromelain and residual homologous proteins (such as papain), linear gradient elution was performed using 0.1 mol / L phosphate buffer (pH 7.2) containing 0 to 0.5 mol / L sodium chloride. The main elution peak of the target bromelain was collected by online monitoring of the 280 nm UV absorption spectrum. After dialyzing and desalting, the product was freeze-dried to obtain high-purity bromelain lyophilized powder.

[0063] (5) Immerse the eluted affinity medium in a regeneration solution containing 0.05 mol / L sodium hydroxide and 1 mol / L sodium chloride for 30 min. After magnetic separation, wash with deionized water until neutral to complete regeneration.

[0064] Example 2: Purification of bromelain This embodiment provides a method for purifying bromelain, including the following steps: (1) Take the pineapple pomace extract, centrifuge at 8000g for 15min, and collect the supernatant; adjust the pH of the supernatant to 6.9-0.1 with 0.1mol / L phosphate buffer, and add ethylenediaminetetraacetic acid (EDTA) to a final concentration of 1mmol / L to obtain crude enzyme solution.

[0065] (2) The recombinant biomimetic affinity medium prepared in Preparation Example 3 was added to the crude enzyme solution. The mass of the affinity medium added was 1.0% of the total mass of the crude enzyme solution. The solution was oscillated at 150 r / min at 4 °C for 55 min. Then, an external magnetic field of 0.5 T was applied for magnetic separation. The supernatant was removed to obtain the enzyme-carrying magnetic medium.

[0066] (3) Add 0.1 mol / L citrate buffer with pH 5.8 to the enzyme-carrying magnetic medium for pre-elution, with an elution volume of 2 column volumes (BV); then perform main elution with 0.1 mol / L phosphate buffer containing reduced glutathione, control the pH of the eluent to 7.2, and perform 8-step gradient elution with the concentration of reduced glutathione from 0 to 5 mmol / L (each step concentration increment 0.625 mmol / L), with 2 BV of elution per step, control the flow rate to 1.0 mL / min, and collect the main eluent.

[0067] (4) The steps are the same as in Example 1.

[0068] Example 3: Purification of bromelain This embodiment provides a method for purifying bromelain, including the following steps: (1) Take the pineapple pomace extract, centrifuge at 8000g for 15min, and collect the supernatant; adjust the pH of the supernatant to 7.1-0.1 using 0.1mol / L phosphate buffer, and add ethylenediaminetetraacetic acid to a final concentration of 1mmol / L to obtain crude enzyme solution.

[0069] (2) The recombinant biomimetic affinity medium (with optimized acid stability) prepared in Preparation Example 4 was added to the crude enzyme solution. The mass of the affinity medium added was 3.0% of the total mass of the crude enzyme solution. The solution was adsorbed by constant temperature oscillation at 150 r / min at 4 °C for 30 min. Then, an external magnetic field of 0.5 T was applied for magnetic separation to remove the supernatant.

[0070] (3) Add 0.1 mol / L citrate buffer with pH 5.8 to the enzyme-carrying magnetic medium for pre-elution, with an elution volume of 2 BV; then perform main elution with 0.1 mol / L phosphate buffer containing reduced glutathione, control the pH of the eluent to 7.2, and perform 12-step gradient elution with the concentration of reduced glutathione from 0 to 6 mmol / L (each step concentration increment 0.5 mmol / L), each step elution 1.5 BV, control the flow rate to 1.0 mL / min, and collect the main eluent.

[0071] (4) The steps are the same as in Example 1.

[0072] Comparative Example 1: Compared with Example 1, the difference is that after the main eluent is collected in step (3), the CMSepharoseFF cation exchange chromatography column purification in step (4) is not performed. Instead, the main eluent is directly desalted, concentrated and freeze-dried to obtain the bromelain product. The remaining steps are the same as in Example 1.

[0073] Comparative Example 2: Compared with Example 1, the difference is that in the main elution in step (3), instead of using a 10-step gradient elution of 0 to 6 mmol / L, a single-step elution is performed directly using 0.1 mol / L phosphate buffer containing 6 mmol / L reduced glutathione, with an elution volume of 20 column volumes. The remaining steps are the same as in Example 1.

[0074] Comparative Example 3: Compared with Example 1, the difference is that in the elution process of step (3), the pre-elution step of pH 5.8 citrate buffer is cancelled, and the GSH main elution stage is directly entered after adsorption is completed. The remaining steps are the same as in Example 1.

[0075] Comparative Example 4: Compared with Example 1, the difference is that in step (2), the microspheres with a crosslinking degree of 1.0 wt% obtained in Preparation Example 1 are used instead of the microspheres in Preparation Example 3. Based on Preparation Example 2, the crosslinking degree is 1.7 wt%, and the other steps are the same.

[0076] Test Example 1: Biomimetic Affinity and Magnetic Response Characteristics Test This test was used to evaluate the ability of the affinity medium obtained in Preparation Example 3 to capture bromelain, its adsorption kinetics, and its magnetic field response efficiency, and to analyze the scientific validity of the binding between the ligand and the enzyme active site through thermodynamic parameters.

[0077] Prepare a series of bromelain standard solutions with concentrations ranging from 0.2 mg / mL to 2.5 mg / mL, using 0.1 mol / L phosphate buffer (pH 7.0) as the buffer system. Transfer 10 mL of each enzyme solution to a centrifuge tube and add 50 mg of the affinity medium described in Example 3. Place the centrifuge tubes in a 4°C constant-temperature shaker and shake at 150 rpm for 2 hours to ensure adsorption equilibrium is reached. Collect the supernatant using a magnetic separation device and determine the residual protein concentration in the supernatant using the BCA method.

[0078] The equilibrium adsorption capacity was calculated according to the formula, and the Langmuir model was used for linear fitting to calculate the maximum adsorption capacity and dissociation constant.

[0079] Take 100 mL of a 1.5 mg / mL bromelain solution prepared with 0.1 mol / L phosphate buffer (pH 7.0), add 500 mg of affinity medium, and stir continuously at 4 °C. Take 0.5 mL samples at 1, 3, 5, 10, 15, 20, 30, 45, 60, and 90 min, immediately perform magnetic separation, and determine the protein concentration of the supernatant. Record the change in adsorption capacity over time, calculate the time required to reach adsorption equilibrium, and fit a kinetic model.

[0080] 100 mg of the affinity medium was dispersed in 50 mL of purified water and sonicated for 2 min to form a homogeneous suspension. The suspension was placed in a cuvette, and the absorbance change was monitored at a wavelength of 600 nm. A permanent magnet with a surface magnetic field strength of 0.5 T was placed on one side of the cuvette, and the time required for the absorbance of the suspension to drop to below 10% of its initial value was recorded. After removing the magnetic field, manual shaking was performed, and the consistency of the magnetic separation time was recorded by repeating the test three times to evaluate the magnetic response stability of the medium.

[0081] Experimental data Table 1: Statistical Table of Adsorption Performance and Magnetic Separation Parameters of Affinity Media for Bromelain According to the data in Table 1, the affinity medium obtained in Preparation Example 3 exhibited a high adsorption capacity and good magnetic response characteristics for bromelain. The experimentally measured equilibrium adsorption capacity increased with increasing initial enzyme concentration, and the theoretical maximum adsorption capacity obtained by fitting the Langmuir model reached 143.56 mg / g. This value indicates that the highly cross-linked surface formed by adjusting the ratio of methyl acrylate to ethylene glycol dimethacrylate in Preparation Example 2, combined with the high-density epoxy coupling process in Preparation Example 3, can provide sufficient grafting sites for the biomimetic heptapeptide, thereby ensuring a high loading capacity for bromelain. The calculated dissociation constant was 3.25. This indicates that the biomimetic heptapeptide sequence forms a strong and stable interaction with the active site of bromelain through residues such as leu, trp, and glu. This affinity is sufficient to support the specific capture of the target enzyme from fruit pomace extracts with complex impurity components.

[0082] In terms of adsorption kinetics, the adsorption capacity of the medium rapidly increased within the first 15 minutes after contacting the enzyme solution, completing approximately 68% of the theoretical adsorption capacity, and finally reaching equilibrium at 37.4 minutes. This rapid adsorption rate reflects the good mass transfer efficiency of the macroporous structure and loosely grafted peptide chains on the surface of the magnetic composite microspheres, reducing the adsorption delay caused by steric hindrance.

[0083] Regarding the magnetic response performance, the medium can be separated within 20 seconds under an external magnetic field of 0.5T, and the magnetic loss rate after 50 regeneration cycles is only 0.089%. This proves that the silica layer and polymer layer described in Preparation Example 1 and Preparation Example 2 provide a tight coating for the Fe3O4 magnetic core, preventing the magnetic core from peeling off during the acid-base regeneration process.

[0084] Test Example 2: Comparative Test of Overall Purification Process Efficiency This test examines the actual effectiveness of the purification process described in this invention in improving the specific activity, purity and recovery rate of bromelain by comparing the experimental results of Examples 1-3 and Comparative Examples 1-3, and verifies the necessity of the process step design.

[0085] Crude extracts of pineapple pomace from the same batch were taken, and the initial protein concentration and initial enzyme activity were determined. The crude extracts were divided into 6 groups, 500 mL each, and the purification processes described in Examples 1, 2, and 3, as well as Comparative Examples 1, 2, and 3, were performed respectively.

[0086] During the purification process, the time and reagent consumption for each group in the adsorption, elution, and purification stages were recorded. For the example groups, a gradient elution procedure was strictly followed; for Comparative Example 2, a single-step high-concentration GSH solution was used for elution; for Comparative Example 3, the pre-elution process with pH 5.8 buffer was omitted.

[0087] Collect the purified enzyme solutions obtained from each group and record the total volume. The protein concentration of the products from each group was determined using the BCA method; the enzyme activity was determined using the benzoyl-L-arginine ethyl ester substrate method, and one activity unit was defined as the amount of enzyme required to catalyze the hydrolysis of 1 mol BAEE per minute at 25℃ and pH 7.0.

[0088] The purity of the product was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). Chromatographic conditions: C18 column, mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: acetonitrile, gradient elution, detection wavelength: 280 nm. The mass percentage of bromelain was calculated using the peak area normalization method.

[0089] Based on the experimental data, the specific activity (U / mg), purification factor (product specific activity / crude liquid specific activity), and total enzyme activity recovery rate (product total activity / crude liquid total activity) of each group were calculated.

[0090] Experimental data Table 2: Comparison of purification efficiency test results between different examples and comparative examples According to the data in Table 2, the purification method provided by this invention is superior to the comparative examples in all core biochemical indicators, proving the rationality and efficiency of the process combination.

[0091] The specific activity of the final products in Examples 1-3 remained above 4100 u / mg. Example 3, through optimization of the added medium and elution steps, achieved the highest specific activity of 4312.5 u / mg, with a purification factor reaching 22.7, representing a significant improvement compared to the original crude solution. HPLC purity testing showed that the purity of the products in the example groups all exceeded 97%, reaching electrophoretic purity. This demonstrates the design of this invention... The biomimetic heptapeptide ligand can accurately identify the active site of bromelain and has strong selectivity in complex fruit pomace extract environments.

[0092] The comparative data further revealed the technical contributions of each key step. In Comparative Example 1, although the total enzyme activity recovery remained at a high level of 89.21% after the cation exchange chromatography purification step was omitted, its HPLC purity was only 85.12%, and its specific activity decreased by approximately 18.8% compared to Example 1. This indicates that while single-step affinity chromatography can enrich the target enzyme, it cannot completely remove homologous proteins with weak affinity for the ligand. A subsequent ion exchange step is necessary to utilize differences in charge distribution for deep purification.

[0093] Comparative Example 2, which used a single-step elution instead of a 10-step gradient elution, resulted in a significant decrease in specific activity to 3125.4 u / mg and a drop in recovery rate to 74.56%. The single addition of a high-concentration competing agent caused impurity proteins adsorbed on the medium to be simultaneously displaced into the elution buffer along with the target enzyme, leading to peak broadening and severely impacting separation resolution. In contrast, Comparative Example 3, by eliminating the pH 5.8 pre-elution step, showed a decrease in product purity to 89.47%, and the product color was darker, indicating that the pre-elution step effectively removed impurities such as polysaccharides, polyphenols, and pigments that are non-specifically adsorbed onto the carrier surface by electrostatic attraction.

[0094] Test Example 3: Specific Removal Efficiency Test of Homologous Impurities This test is used to verify the specific removal ability of the purification process provided by this invention for homologous proteins with highly similar structures and physicochemical properties, taking papain as an example, and to examine the separation mechanism of gradient elution and purification steps using biomimetic affinity media.

[0095] Experimental steps Take crude extract of pineapple pomace from the same batch, add a certain amount of papain standard to it, and prepare a crude extract with an initial papain content of about 8500 ng / mg (that is, 8500 nanograms of papain per milligram of total protein). Mix well and let stand for 30 minutes.

[0096] The simulated crude solution was divided into four groups. The first and second groups were purified according to the process steps described in Examples 1 and 2, respectively. The third group was operated according to the steps of Comparative Example 1, that is, after completing the affinity main elution, it was directly desalted and freeze-dried without cation exchange chromatography purification. The fourth group used the traditional purification process as a control, that is, ammonium sulfate was added to 60% saturation for salting out precipitation, centrifuged and reconstituted, and then purified by CMSepharose FF cation exchange column.

[0097] The target protein solutions from each group were collected after the purification process, desalted, and brought to the same volume. The residual concentration of papain in the final products of each group was quantitatively detected using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). The detection process strictly followed the kit instructions, with absorbance read at 450 nm and absolute content calculated based on the standard curve.

[0098] Take a small amount of the final product from each group and determine the total proportion of non-bromelain proteins in the product using reverse-phase hplc. Combine the determination results from step (3) to calculate the papain removal rate of each process.

[0099] Experimental data Table 3: Statistical table of the removal effects of different purification processes on homologous contaminants According to the data in Table 3, the purification process described in this invention can remove homologous impurities with similar physicochemical properties to bromelain with extremely high efficiency.

[0100] Bromelain and papain belong to the same cysteine ​​protease family and share high homology in catalytic mechanism, molecular weight, and three-dimensional folding structure. The traditional salting-out method only achieved a 62.83% clearance rate for papain, with the final product containing as much as 26.41% impurities. This is because the surface hydrophobicity and solubility curves of the two enzymes overlap, making effective separation impossible using ammonium sulfate precipitation with a concentration gradient. Subsequent single ion exchange chromatography also lost its separation capability due to excessive column load.

[0101] Both Examples 1 and 2 achieved a papain removal rate of over 99%, with the residual amount reduced to below 25 ng / mg after purification. This result validates the structural specificity of the heptapeptide ligand h-lcweiav-oh. This ligand is designed for the specific spatial and charge distribution of the S2 subsite of the bromelain active site. Although papain possesses a similar active pocket, the hydrophobic volume of its S2 subsite is small, preventing it from forming a stable conformational fit with the leu and val residues in the ligand. Therefore, during the affinity adsorption phase, papain can only adsorb onto the medium surface through non-specific electrostatic interactions or competitive adsorption at its very weak active site. Combined with the 0-6 mmol / L reduced glutathione gradient elution program used in the examples, contaminating proteins with varying affinity levels were preemptively replaced in the lower concentration elution layers, thus ensuring the purity of the main elution peak.

[0102] Comparing the data from Example 1 and Comparative Example 1, the residual amount of papain in the final product of Comparative Example 1 reached 486.3 ng / mg, and the total amount of impurities in the product increased to 14.88%. This indicates that relying solely on the difference in binding force of the biomimetic affinity medium cannot achieve complete impurity removal. The trace amounts of homologous proteins co-eluted must be separated again by subsequent CMSepharoseFF cation exchange chromatography. The isoelectric point of bromelain is approximately 9.5, while that of papain is approximately 8.7. In the elution environment of pH 7.2, there is a slight difference in the positive charge density carried on the surfaces of the two. Example 1 utilized this charge difference to retain or differentially elute the residual papain during the purification stage, ultimately achieving electrophoretic purity for the target product. The tandem mechanism of affinity chromatography and ion exchange chromatography in the purification system fills the gap in cross-binding defects of single affinity ligands in homologous protein recognition.

[0103] Test Example 4: Cyclic Stability and Mechanical Strength Test of Affinity Media This test is used to examine the performance degradation of the magnetic hybrid affinity medium used in the purification process under multiple adsorption, elution, and regeneration cycles, as well as the effects of different degrees of crosslinking and coupling conditions on the mechanical strength and chemical bond stability of the carrier.

[0104] Experimental steps 2.0 g each of the dry affinity media from Example 1 (using the medium obtained in Preparation Example 2), Example 3 (using the medium obtained in Preparation Example 4), and Comparative Example 4 (using the medium obtained in Preparation Example 1) were weighed. Each group of media was added to a reactor containing 50 mL of crude pineapple pomace enzyme solution and subjected to adsorption by shaking at 4°C for 60 min. After magnetic separation, unbound proteins were washed away with 0.1 mol / L phosphate buffer. Regeneration was then performed for 30 min with a mixed solution of 0.05 mol / L sodium hydroxide and 1 mol / L sodium chloride, followed by washing with water until neutral. This process was defined as one complete operating cycle. Fifty consecutive cycles were performed. During the adsorption phase of the first and 50th cycles, a quantitative amount of media was taken to determine its static saturation adsorption capacity for bromelain.

[0105] Weigh 0.5 g of each of the three groups of fresh, dry media and suspend them in 20 mL of 0.1 mol / L citrate buffer solution (pH 4.5). Place the suspensions in a 25°C constant-temperature shaker and continuously shake at 200 rpm for 24 h. Allow to stand and apply a magnetic field to separate the solid phase media, then collect the supernatant. Detect the concentration of free heptapeptide ligands in the supernatant using reversed-phase high-performance liquid chromatography (RP-HPLC). Calculate the ligand detachment rate based on the theoretical ligand loading of the initial media and the total amount of detached ligands in the supernatant.

[0106] Samples of the swollen media microspheres from each group were placed on the testing platform of a universal testing machine. Uniaxial compression tests were conducted at room temperature with a loading rate of 1.0 mm / min. The critical pressure values ​​at which the microspheres underwent macroscopic rupture or yield deformation were recorded, and the compressive strength of the medium was calculated. Ten parallel tests were performed for each group, and the average value was taken.

[0107] Experimental data Table 4: Statistical Table of Mechanical Strength and Cyclic Stability Test Results for Different Affinity Media Experimental conclusions According to the data in Table 4, the degree of crosslinking of the dielectric polymer layer and the fine-tuning of the ligand coupling process conditions have a decisive impact on the industrial application life of the material.

[0108] The compressive strengths of the media used in Examples 1 and 3 reached 3.16 MPa and 3.22 MPa, respectively, and the adsorption capacity retention rate was above 92% after 50 strongly alkaline regeneration cycles. Although the initial adsorption capacity of the media used in Comparative Example 4 reached 145.1 mg / g, the adsorption capacity sharply decreased to 89.6 mg / g after 50 cycles, with a capacity retention rate of only 61.75%, and its compressive strength test value was only 1.43 MPa. This difference stemmed from the increase in the amount of ethylene glycol dimethacrylate added in Preparation Example 2 from 1.0 wt% to 1.7 wt%. In the hybrid coating system of polymethyl methacrylate and silica, the 1.7 wt% crosslinking degree constructed a high-density three-dimensional network structure around the magnetic core, limiting the excessive swelling of the polymer long chains in the 0.05 mol / L sodium hydroxide regeneration solution. In Comparative Example 4, with low cross-linking degree, the polymer matrix underwent microscopic tearing and even local peeling under repeated osmotic pressure changes and mechanical shearing, resulting in a large loss of epoxy groups and ligands grafted onto it, which macroscopically manifested as irreversible decay of adsorption capacity.

[0109] In Example 3, the ligand shedding rate in an acidic environment was as low as 1.45%, significantly lower than the 3.12% in Example 1 and 8.74% in Comparative Example 4. Example 3 used the optimized parameters of Preparation Example 4, adjusting the pH value of the peptide coupling stage from 8.3 to 7.8. During the ring-opening addition reaction between the epoxy group and the terminal amino group or free thiol group of the heptapeptide, pH 7.8 ensures both the degree of nucleophilic dissociation and inhibits the side reaction hydrolysis of the epoxy ring under alkaline conditions, thereby generating secondary amine bonds or thioether bonds with less steric hindrance and higher covalent bond energy.

[0110] This optimized bonding structure exhibits strong resistance to hydrolysis and dissociation in acidic citrate buffer at pH 4.5. In practical applications where pineapple pomace crude enzyme solutions are generally acidic and contain a large amount of endogenous hydrolytic enzymes, reducing the ligand shedding rate not only maintains the long-term adsorption capacity of the medium but also prevents detached peptide fragments from contaminating the target enzyme product with the eluent, thus avoiding a decrease in purity.

[0111] The carrier material formulation and coupling parameters provided by this invention can meet the physical and chemical stability requirements of high-frequency industrial purification.

[0112] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A recombinant biomimetic affinity medium for purifying bromelain, characterized in that, It is prepared from the following raw materials in parts by weight: Double-bonded silica-coated magnetic microspheres: 100 parts; Monomer methyl acrylate: 150-250 parts; Glycidyl methacrylate containing epoxy monomer: 50-100 parts; Crosslinking agent: ethylene glycol dimethacrylate: 3-6 parts; Bionic affinity heptapeptide: 10-20 parts; The amino acid sequence of the biomimetic affinity heptapeptide is H-Leu-Cys-Trp-Glu-Ile-Ala-Val-OH.

2. The recombinant biomimetic affinity medium for purifying bromelain according to claim 1, characterized in that: The double-bonded silica-coated magnetic microspheres are prepared by the following method: magnetic nanoparticles of iron oxide are suspended in a system containing ammonia water and reacted with tetraethyl orthosilicate in a sol-gel reaction to obtain silica-coated magnetic microspheres; then they are dispersed in anhydrous toluene and reacted with the silane coupling agent methacryloyloxypropyltrimethoxysilane under reflux and nitrogen protection conditions to achieve surface double bonding.

3. The recombinant biomimetic affinity medium for purifying bromelain according to claim 1, characterized in that: The epoxy group density on the surface of the affinity medium is 150-170 mol / g, the covalent coupling amount of the biomimetic affinity heptapeptide on the surface of the affinity medium is 41.6-46.8 mol / g, and the biomimetic affinity heptapeptide is bonded to the support through a secondary amine bond or thioether bond formed by the ring-opening addition of the free thiol group of its N-terminal amino or cysteine ​​side chain to the epoxy group.

4. A method for preparing a recombinant biomimetic affinity medium for purifying bromelain, characterized in that, Includes the following steps: S1. After coating the surface of the magnetite nanoparticles with a silica layer, the surface is modified with a silane coupling agent to obtain double bonded silica-coated magnetic microspheres. S2. The magnetic microspheres coated with double-bonded silica are dispersed in a mixed solvent, and methyl acrylate, glycidyl methacrylate and ethylene glycol dimethacrylate are added in sequence. After nitrogen gas is introduced to remove oxygen, an initiator is added to carry out a surface-initiated polymerization reaction to obtain a highly cross-linked polymer hybrid epoxy magnetic carrier. S3. The highly cross-linked polymer hybrid epoxy magnetic carrier is suspended in a buffer solution containing a biomimetic affinity heptapeptide for coupling reaction. After the reaction is completed, the free polypeptide is washed away to obtain the recombinant biomimetic affinity medium.

5. The method for preparing a recombinant biomimetic affinity medium for purifying bromelain according to claim 4, characterized in that: In step S2, the mixed solvent is an ethanol-water mixture with a volume ratio of 1:1, the initiator is azobisisobutyronitrile, and the process parameters for the surface-initiated polymerization reaction are: mechanical stirring reaction at a constant temperature of 70°C for 24 hours.

6. The method for preparing a recombinant biomimetic affinity medium for purifying bromelain according to claim 4, characterized in that: In step S3, the reaction system was controlled in advance as follows: a 0.1 mol / L phosphate buffer solution with a pH of 7.8 was used, and ethylenediaminetetraacetic acid with a final concentration of 1 mmol / L was added to the coupling reaction system; the process parameters for the coupling reaction were: the system was placed in a constant temperature shaker at 28°C and shaken for 14 h, and the pH of the system was strictly maintained between 7.8 and 0.1 throughout the process using dilute alkali solution.

7. A method for purifying bromelain using a recombinant biomimetic affinity medium, wherein the recombinant biomimetic affinity medium for purifying bromelain according to any one of claims 1-3 is characterized in that, Includes the following steps: (1) Centrifuge the pineapple pomace extract to collect the supernatant, adjust the pH to 6.9-7.1, and add ethylenediaminetetraacetic acid to a final concentration of 1 mmol / L to obtain crude enzyme solution; (2) Add the recombinant biomimetic affinity medium to the crude enzyme solution, and oscillate and adsorb at 4°C. Then apply an external magnetic field for magnetic separation to obtain the enzyme-carrying magnetic medium. (3) Add pre-elution buffer to the enzyme-carrying magnetic medium for pre-elution, and then perform gradient main elution using main elution buffer containing a competing agent, and collect the main elution buffer; (4) After dialysis and desalting, the collected main eluent is loaded into a pre-equilibrated cation exchange chromatography column for linear gradient elution. The main elution peak of the target bromelain is collected and freeze-dried to obtain high-purity bromelain.

8. The method for purifying bromelain using a recombinant biomimetic affinity medium according to claim 7, characterized in that: In step (3), the pre-elution buffer is a 0.1 mol / L citrate buffer with pH 5.8 and the elution volume is 2 times the column volume; the main elution buffer is a 0.1 mol / L phosphate buffer containing reduced glutathione as a competing agent and its pH is controlled at 7.

2.

9. The method for purifying bromelain using a recombinant biomimetic affinity medium according to claim 7, characterized in that: The specific implementation method of the main elution in step (3) is as follows: control the flow rate to 1.0 mL / min, and perform 8 to 12 gradient elutions of the reduced glutathione concentration in the range of 0 to 6 mmol / L, with each elution being 1.5 to 2 column volumes.

10. The method for purifying bromelain using a recombinant biomimetic affinity medium according to claim 7, characterized in that: In step (4), the cation exchange chromatography column is a CMSepharoseFF resin column with carboxymethyl groups, and the linear gradient elution is carried out using a 0.1 mol / L phosphate buffer containing 0 to 0.5 mol / L sodium chloride, and the 280 nm ultraviolet absorption spectrum is monitored online during the elution process.

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