In-vitro rapid synthesis of bovine bezoar based on multi-enzyme linkage catalysis technology

Abstract

The present application relates to the technical field of cow-bezoar synthesis, in particular to a rapid in-vitro cow-bezoar synthesis process based on multi-enzyme linkage catalysis technology, comprising the following steps: S1, substrate system preparation; S2, segmented self-adaptive multi-enzyme linkage catalysis; S3, in-situ biomimetic hierarchical mineralization; S4, purification, drying and shaping; the process route of the present application is constructed by avoiding light and oxygen in the substrate system, time-sequenced segmented multi-enzyme linkage catalysis, in-situ biomimetic hierarchical mineralization and gradient purification and shaping, which optimizes each step and component layer by layer from molecular catalysis to microstructure formation, so that each step and component can play a synergistic role, and the synthesis efficiency, product purity and quality stability of in-vitro cow-bezoar are systematically improved.

Description

Technical Field

[0001] This invention relates to the field of bezoar synthesis technology, specifically to a rapid in vitro bezoar synthesis process based on multi-enzyme linkage catalysis technology. Background Technology

[0002] Calculus bezoar, a traditional and precious Chinese medicinal material, has irreplaceable application value in clinical emergencies and traditional Chinese medicine prescriptions due to its effects of clearing the heart, resolving phlegm, opening the orifices, cooling the liver, calming wind, and detoxifying. Natural calculus bezoar comes from bovine gallstones, and its resources are scarce, costly to obtain, and of inconsistent quality, making it difficult to meet the needs of large-scale drug use. For a long time, in vitro synthesis of calculus bezoar has been considered the core route to replace natural calculus bezoar. Current synthetic processes mostly use chemical oxidation or simple enzymatic reactions to prepare bilirubin, and then complete the loading of inorganic components through physical blending. However, the overall technology still has significant limitations.

[0003] Existing enzymatic synthesis methods mostly employ single enzymes or simple mixed enzymes for catalysis, failing to design for the temporal and environmental adaptation of the conversion pathways of heme, biliverdin, and bilirubin. This easily leads to problems such as intermediate product accumulation, low catalytic efficiency, and mixed product configurations. Furthermore, natural bilirubin oxidase is easily inactivated and exhibits poor selectivity in aqueous systems, resulting in a low proportion of effective bilirubin configurations in the final product. In the mineralization stage, conventional processes often involve the direct co-precipitation of calcium salts and bilirubin, lacking a covalent coupling mechanism between the organic and inorganic phases. The resulting product has a loose structure, poor stability, and is prone to loss of effective components, significantly differing from the hierarchical and ordered structure of natural bezoar.

[0004] In addition, existing in vitro bezoar synthesis technologies suffer from problems such as low catalytic efficiency, insufficient product purity, poor biomimicry of structure, and weak product stability, making it difficult to simultaneously balance quality, efficiency, and cost in large-scale production. A comprehensive synthesis process is urgently needed. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and propose a rapid in vitro synthesis process for bezoar based on multi-enzyme linkage catalysis technology.

[0006] The specific technical solution is as follows: A rapid in vitro synthesis process for bezoar based on multi-enzyme linkage catalysis technology includes the following steps:

[0007] S1. Substrate system preparation: In a closed, constant-temperature bioreactor protected from light and continuously purged with nitrogen, a homogeneous substrate solution containing heme, buffer, nonionic phase transfer agent, and metal ion shielding agent was prepared. The molar concentration of heme was 0.8–1.2 mmol / L. After homogenization by stirring at 120 rpm for 10 min at constant temperature, the system was degassed under vacuum at -0.06–-0.08 MPa for 10–15 min to remove dissolved oxygen and heavy metal interfering ions.

[0008] S2. Segmented Adaptive Multi-Enzyme Catalysis: A time-gradient enzyme addition combined with a segmented pH and temperature adaptive regulation strategy is used for multi-enzyme coupled catalysis: In the first stage, the system pH is adjusted to 7.2–7.4 and the temperature to 32–35℃, and heme oxygenase is added for pre-catalysis for 2–3 hours; in the second stage, the system pH is adjusted to 7.0–7.2 and the temperature to 30–32℃, and biliverdin reductase is added for catalysis for 3–4 hours; in the third stage, the system pH is adjusted to 7.1 and the temperature to 31–33℃, and a chemically hydrophobically modified bilirubin oxidase complex is added for reconfiguration and purification. The mixture is gently stirred at 80–150 rpm throughout the process to prepare the bilirubin organic reaction solution.

[0009] S3. In-situ biomimetic hierarchical mineralization: A soluble calcium salt solution and a calcium phosphate / calcium carbonate composite nano precursor suspension modified with a silane coupling agent are simultaneously and uniformly added to the bilirubin organic reaction solution. Through the covalent coupling between the active functional groups on the precursor surface and the hydroxyl groups of bilirubin, organic-inorganic composite crystal nuclei are formed in an orderly manner and grow in a directional manner, thereby achieving hierarchical in-situ biomimetic mineralization.

[0010] S4. Purification, drying and shaping: After the mineralized mixture is subjected to segmented gradient induced sedimentation, solid-liquid separation and alternating washing, it is dried and shaped in a low temperature, vacuum and low humidity environment to obtain in vitro synthesized bezoar.

[0011] As a further technical solution: In step S1, the buffer is 0.08-0.12 mol / L phosphate buffer, the nonionic phase transfer agent is Tween 80, and the amount added is 0.4%-0.6% of the total mass of the substrate solution; the metal ion shielding agent is disodium EDTA, and the amount added is 0.2%-0.4% of the total mass of the substrate solution.

[0012] As a further technical solution, the preparation method of the chemically hydrophobically modified bilirubin oxidase complex in step S2 includes:

[0013] S201. Enzyme solution pretreatment: Select natural bilirubin oxidase with a specific activity ≥120U / mg, and purify it by dialysis with phosphate buffer at pH 6.8-7.2 to remove impurities such as proteins and stabilizers, and obtain homogeneous pure enzyme solution.

[0014] S202, Mild surface hydrophobic modification: Under constant temperature conditions of 4-10℃, in the dark, and pH 6.8-7.2, a hydrophobic esterification modifier is added to the pure enzyme solution, and the reaction is carried out with low-speed stirring for 4-6 hours; the hydrophobic esterification modifier is octanoic anhydride or acetic anhydride, and the amount of modifier added is 1.5%-3.0% of the enzyme protein mass in the pure enzyme solution, thereby constructing local hydrophobic microregions in situ in the non-catalytic region of the enzyme molecule;

[0015] S203, self-assembly complex: Add an amphiphilic block copolymer solution dropwise to the modified enzyme solution and stir continuously at low speed for 18-22 hours to form a core-shell structure complex of an enzyme core and a polymer shell. After centrifugation and purification, a stable bilirubin oxidase complex is obtained.

[0016] As a further technical solution: the amphiphilic block copolymer is a polyethylene glycol-polylactic acid block copolymer, and the mass ratio of the amphiphilic block copolymer to the modified bilirubin oxidase is 1:10-15.

[0017] As a further technical solution, in step S2, the mass ratio of the heme oxygenase, biliverdin reductase, and modified bilirubin oxidase complex is 2:1:3.5 to 2:1:4.5, and the total catalytic time of the multi-enzyme linkage is 10 to 14 hours.

[0018] As a further technical solution, the preparation method of the calcium phosphate / calcium carbonate composite nano-precursor suspension modified with a silane coupling agent in step S3 includes:

[0019] S301. Preparation of composite nanoparticles: Amorphous calcium phosphate / calcium carbonate composite nanoparticles were synthesized by low-temperature liquid-phase coprecipitation method under nitrogen protection conditions with a volume ratio of 1:3 ethanol-water mixed solvent.

[0020] S302, Surface functionalization modification: The composite nanoparticles are dispersed in anhydrous ethanol at a solid-liquid ratio of 1g:40mL~50mL, a silane coupling agent is added, and the mixture is refluxed at 70℃ and 180rpm for 5h under nitrogen protection throughout the process to graft amino or epoxy active functional groups onto the surface of the particles.

[0021] S303. Preparation of stable suspension: The reaction product is purified by centrifugation at 9000 rpm for 10 min to remove free impurities. Then, it is homogenized by ultrasonication at 200W and 40kHz for 12 min and dispersed in sterile deionized water to prepare a nano-precursor suspension with a solid content of 5% to 10%.

[0022] As a further technical solution: the silane coupling agent is 3-aminopropyltriethoxysilane or 3-glycidyl etheroxypropyltrimethoxysilane, and the amount of silane coupling agent is 2% to 3% of the total mass of the composite nanoparticles; the molar ratio of calcium phosphate to calcium carbonate is 2 to 3:1.

[0023] As a further technical solution: In step S3, the soluble calcium salt is calcium chloride or calcium gluconate, the molar concentration of the calcium salt solution is 0.15-0.25 mol / L, and the mass ratio of the calcium salt to the composite nano precursor solid is 1.2-1.5:1; the calcium salt solution and the nano precursor suspension are added dropwise simultaneously at a uniform rate of 0.8-1.2 mL / min.

[0024] As a further technical solution, the in-situ biomimetic mineralization conditions are: temperature 30-34℃, oscillation speed 30-50rpm, and mineralization time 36-60h.

[0025] As a further technical solution: In step S4, the segmented gradient induced sedimentation is to first allow the sediment to settle at room temperature for 3-5 hours, and then centrifuge at a low speed of 3500-4500 rpm for 10-14 minutes; the alternating washing is to wash twice with anhydrous ethanol and twice with deionized water; the low-temperature vacuum drying conditions are: temperature 33-37℃, relative humidity 18%-22%, vacuum degree -0.085 to -0.095 MPa, and drying time 5-7 hours.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention utilizes a process route that involves constructing a light-shielding and oxygen-removing substrate system, sequential segmented multi-enzyme catalysis, in-situ biomimetic hierarchical mineralization, and gradient purification and shaping. This route optimizes the process layer by layer from molecular catalysis to microstructure formation, enabling each step and component to work synergistically, thereby systematically improving the synthesis efficiency, product purity, and quality stability of bezoar in vitro.

[0028] During the substrate preparation stage, continuous nitrogen purging and vacuum degassing, combined with the metal ion shielding agent disodium EDTA, can quickly remove dissolved oxygen and heavy metal interfering ions from the system, avoiding enzyme protein oxidation and inactivation and side reactions. Phosphate buffer provides a stable pH environment, and Tween 80, as a non-ionic phase transfer agent, enhances the compatibility between the substrate and the enzyme. Due to the synergistic effect of the above components and conditions, the substrate system is homogeneous and stable, and interfering factors are significantly suppressed, thus providing a safe and efficient reaction environment for subsequent multi-enzyme catalysis. In the segmented adaptive multi-enzyme linkage catalysis, heme oxygenase, biliverdin reductase, and modified bilirubin oxidase complex are added sequentially and stepwise, matched with corresponding pH and temperature ranges, so that each catalysis step is carried out in the optimal activity range, avoiding the accumulation of intermediate products. After hydrophobic modification and core-shell self-assembly, the catalytic selectivity and stability of bilirubin oxidase are significantly improved. Due to the segmented temperature and pH control and the synergistic catalysis of modified enzymes, high conversion rate and high single configuration of bilirubin are achieved for the targeted preparation, thereby solving the problems of low efficiency, mixed configurations, and easy inactivation of traditional enzyme catalysis.

[0029] In the in-situ biomimetic hierarchical mineralization process, the calcium phosphate / calcium carbonate composite nano-precursor modified with a silane coupling agent can form a covalent coupling with the hydroxyl groups of bilirubin. Using organic components as templates, it guides the orderly growth of crystal nuclei, forming a hierarchical and dense structure similar to natural bezoar. Soluble calcium salts are added simultaneously and uniformly with the precursor, ensuring a uniform and controllable mineralization process. Due to the synergistic effect of organic-inorganic covalent coupling and uniform mineralization, the product's structural integrity and retention rate of active ingredients are improved, thus solving the problems of loose structure, poor stability, and easy loss of active ingredients in traditional co-precipitation products. The purification, drying, and shaping process employs segmented gradient-induced sedimentation and low-temperature vacuum low-humidity drying. Solid-liquid separation and dehydration are completed under mild conditions. The gentle treatment and precise environmental control throughout the process maximize the protection of active ingredients such as bilirubin from damage, achieving the overall goal of high efficiency, high purity, high stability, and high biomimeticity in the in vitro synthesis of bezoar. This allows for large-scale replacement of natural bezoar, meeting pharmaceutical needs. Attached Figure Description

[0030] Figure 1 This is a flowchart of the in vitro rapid synthesis process of bezoar based on multi-enzyme linkage catalysis technology. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0032] This invention provides a rapid in vitro synthesis process for bezoar based on multi-enzyme catalytic technology, comprising the following steps:

[0033] S1. Substrate system preparation: In a closed, temperature-controlled bioreactor protected from light and continuously purged with nitrogen, a homogeneous substrate solution containing heme, buffer, nonionic phase transfer agent, and metal ion shielding agent was prepared. After homogenization by stirring at a constant temperature, the system was degassed under vacuum to remove dissolved oxygen and heavy metal interfering ions.

[0034] S2. Segmented adaptive multi-enzyme linkage catalysis: A time-gradient enzyme addition combined with a segmented pH and temperature adaptive control strategy is adopted for multi-enzyme coupling catalysis. The corresponding enzyme preparations are added in stages to carry out the catalytic reaction. The reaction is carried out with gentle stirring throughout the process to prepare bilirubin organic reaction solution.

[0035] S3. In-situ biomimetic hierarchical mineralization: A soluble calcium salt solution and a calcium phosphate / calcium carbonate composite nano-precursor suspension modified with a silane coupling agent are simultaneously and uniformly added to the bilirubin organic reaction solution. Through the covalent coupling between the active functional groups on the precursor surface and the hydroxyl groups of bilirubin, organic-inorganic composite crystal nuclei are formed in an orderly manner and grow in a directional manner, thus achieving hierarchical in-situ biomimetic mineralization.

[0036] S4. Purification, drying and shaping: After the mineralized mixture is subjected to segmented gradient induced sedimentation, solid-liquid separation and alternating washing, it is dried and shaped in a low temperature, vacuum and low humidity environment to obtain in vitro synthesized bezoar.

[0037] In this invention, the molar concentration of heme in step S1 is preferably 0.8–1.2 mmol / L. The stirring speed for homogenization is preferably 120 rpm, and the stirring time is preferably 10 min. The vacuum degree for vacuum degassing is preferably -0.06 to -0.08 MPa, and the vacuum degassing time is preferably 10–15 min.

[0038] The buffer in step S1 is preferably a 0.08–0.12 mol / L phosphate buffer solution, and the nonionic phase transfer agent is preferably Tween 80, with the addition amount preferably being 0.4%–0.6% of the total mass of the substrate solution. The metal ion shielding agent is preferably disodium EDTA, with the addition amount preferably being 0.2%–0.4% of the total mass of the substrate solution.

[0039] In this invention, the segmented adaptive multi-enzyme linkage catalysis in step S2 is divided into three stages. In the first stage, the pH of the system is preferably adjusted to 7.2–7.4, the temperature to 32–35°C, and the pre-catalytic time for heme oxygenase is preferably 2–3 hours. In the second stage, the pH of the system is preferably adjusted to 7.0–7.2, the temperature to 30–32°C, and the catalytic time for biliverdin reductase is preferably 3–4 hours. In the third stage, the pH of the system is adjusted to 7.1, the temperature to 31–33°C, and a chemically hydrophobically modified bilirubin oxidase complex is added for conformational remodeling and purification. The stirring speed throughout the process is preferably 80–150 rpm.

[0040] The method for preparing the chemically hydrophobically modified bilirubin oxidase complex described in step S2 includes:

[0041] S201. Enzyme pretreatment: Select natural bilirubin oxidase with a specific activity ≥120U / mg, and purify it by dialysis with phosphate buffer at pH 6.8-7.2 to remove impurities such as proteins and stabilizers, and obtain homogeneous pure enzyme solution.

[0042] S202. Mild surface hydrophobic modification: Under constant temperature conditions of 4–10℃, in the dark, and pH 6.8–7.2, a hydrophobic esterification modifier is added to the pure enzyme solution, and the reaction is carried out with low-speed stirring for 4–6 hours. The hydrophobic esterification modifier is preferably octanoic anhydride or acetic anhydride, and the amount of modifier added is preferably 1.5%–3.0% of the enzyme protein content in the pure enzyme solution, thereby constructing local hydrophobic microregions in situ in the non-catalytic region of the enzyme molecule.

[0043] S203, self-assembly complex: Add an amphiphilic block copolymer solution dropwise to the modified enzyme solution and stir continuously at low speed for 18-22 hours to form a core-shell structure complex of an enzyme core and a polymer shell. After centrifugation and purification, a stable bilirubin oxidase complex is obtained.

[0044] The amphiphilic block copolymer is preferably a polyethylene glycol-polylactic acid block copolymer, and the mass ratio of the amphiphilic block copolymer to the modified bilirubin oxidase is preferably 1:10-15.

[0045] In step S2, the preferred mass ratio of the heme oxygenase, biliverdin reductase, and modified bilirubin oxidase complex is 2:1:3.5 to 4.5, and the preferred total catalytic time for the multi-enzyme linkage is 10 to 14 hours.

[0046] In this invention, the preparation method of the calcium phosphate / calcium carbonate composite nano-precursor suspension modified with a silane coupling agent in step S3 includes:

[0047] S301. Preparation of composite nanoparticles: Amorphous calcium phosphate / calcium carbonate composite nanoparticles were synthesized by low-temperature liquid-phase coprecipitation method under nitrogen protection conditions with a volume ratio of 1:3 in an ethanol-water mixed solvent.

[0048] Specifically, a mixed solvent of ethanol and deionized water in a volume ratio of 1:3 was prepared and pre-cooled to 4°C. Dissolved oxygen and carbon dioxide in the system were continuously removed by nitrogen purging. Anhydrous calcium chloride source solution and a mixed anionic solution of sodium dihydrogen phosphate and sodium bicarbonate were prepared according to the molar ratio of the final products of calcium phosphate and calcium carbonate, respectively. The two solutions were simultaneously and slowly added dropwise at a rate of 0.5 mL / min to the low-temperature mixed solvent, and stirred at a constant speed of 180 rpm to maintain the system in a low supersaturated state. After the addition was completed, the system was continuously reacted under low temperature and sealed for 30 min to lock the amorphous amorphous structure using low-temperature thermodynamics. After the reaction was completed, the system was immediately centrifuged at 4°C and 9000 rpm for 10 min to separate the solid and liquid phases, obtaining amorphous calcium phosphate / calcium carbonate composite nanoparticles.

[0049] S302, Surface functionalization modification: The composite nanoparticles are dispersed in anhydrous ethanol at a solid-liquid ratio of 1g:40mL~50mL, a silane coupling agent is added, and the mixture is refluxed at 70℃ and 180rpm for 5h under nitrogen protection throughout the process to graft amino or epoxy active functional groups onto the particle surface.

[0050] S303. Preparation of stable suspension: The reaction product is purified by centrifugation at 9000 rpm for 10 min to remove free impurities. Then, it is homogenized by ultrasonication at 200W and 40kHz for 12 min and dispersed in sterile deionized water to prepare a nano-precursor suspension with a solid content of 5% to 10%.

[0051] The silane coupling agent is preferably 3-aminopropyltriethoxysilane or 3-glycidyl etheroxypropyltrimethoxysilane, and the amount of silane coupling agent used is preferably 2% to 3% of the total mass of the composite nanoparticles. The molar ratio of calcium phosphate to calcium carbonate is preferably 2 to 3:1.

[0052] The soluble calcium salt mentioned in step S3 is preferably calcium chloride or calcium gluconate, the molar concentration of the calcium salt solution is preferably 0.15–0.25 mol / L, and the mass ratio of the calcium salt to the composite nano-precursor solid is preferably 1.2–1.5:1. The synchronous dropping acceleration rate of the calcium salt solution and the nano-precursor suspension is preferably 0.8–1.2 mL / min.

[0053] The preferred in-situ biomimetic mineralization conditions are: temperature 30–34℃, oscillation speed 30–50 rpm, and mineralization time 36–60 h.

[0054] In this invention, the segmented gradient induced sedimentation in step S4 involves first allowing the mixture to settle at room temperature for 3–5 hours, followed by centrifugation at a low speed of 3500–4500 rpm for 10–14 minutes. Alternating washing consists of two washes with anhydrous ethanol and two washes with deionized water. The preferred low-temperature vacuum drying conditions are: temperature 33–37°C, relative humidity 18%–22%, vacuum degree -0.085–-0.095 MPa, and drying time 5–7 hours.

[0055] The rapid in vitro synthesis process for bezoar provided by this invention employs multi-enzyme catalysis combined with in-situ biomimetic graded mineralization, enabling efficient and targeted synthesis of bilirubin and organic-inorganic composite mineralization. This significantly shortens the bezoar synthesis cycle and improves product purity and quality. Segmented adaptive enzyme catalysis ensures maximum activity of each enzyme, resulting in high catalytic conversion rates. In-situ biomimetic mineralization makes the bezoar structure closer to that of natural bezoar, leading to superior product stability and medicinal properties. Simultaneously, this process enables large-scale in vitro synthesis, addressing the scarcity of natural bezoar resources and reducing production costs.

[0056] Example 1:

[0057] S1. Substrate System Preparation: In a closed, temperature-controlled bioreactor protected from light and continuously purged with nitrogen, a homogeneous substrate solution containing heme, 0.08 mol / L phosphate buffer, Tween 80, and disodium EDTA was prepared. The molar concentration of heme was 0.8 mmol / L, the amount of Tween 80 added was 0.4% of the total mass of the substrate solution, and the amount of disodium EDTA added was 0.2% of the total mass of the substrate solution. After homogenization by stirring at 120 rpm for 10 min at a constant temperature, the system was degassed under vacuum at -0.06 MPa for 10 min to remove dissolved oxygen and heavy metal interfering ions.

[0058] S2. Segmented Adaptive Multi-Enzyme Catalysis: In the first stage, the system pH was adjusted to 7.2 and the temperature to 32℃, and heme oxygenase was added for pre-catalysis for 2 hours. In the second stage, the system pH was adjusted to 7.0 and the temperature to 30℃, and biliverdin reductase was added for catalysis for 3 hours. In the third stage, the system pH was adjusted to 7.1 and the temperature to 31℃, and a chemically hydrophobically modified bilirubin oxidase complex was added for reconfiguration and purification, with gentle stirring at 80 rpm throughout. The mass ratio of heme oxygenase, biliverdin reductase, and modified bilirubin oxidase complex was 2:1:3.5, and the total multi-enzyme catalysis time was 10 hours, yielding an organic bilirubin reaction solution.

[0059] Preparation of chemically hydrophobically modified bilirubin oxidase complex:

[0060] S201. Select natural bilirubin oxidase with a specific activity ≥120U / mg, and purify it by dialyzing with pH 6.8 phosphate buffer to remove impurities such as proteins and stabilizers, and obtain homogeneous pure enzyme solution.

[0061] S202. Under constant temperature conditions of 4℃, protected from light, and pH 6.8, add octanoic anhydride to the pure enzyme solution and stir at low speed for 4 hours. The amount of octanoic anhydride added is 1.5% of the enzyme protein content in the pure enzyme solution.

[0062] S203. Polyethylene glycol-polylactic acid block copolymer solution was added dropwise to the modified enzyme solution, and the mixture was stirred at low speed for 18 hours for self-assembly. The mass ratio of polyethylene glycol-polylactic acid block copolymer to modified bilirubin oxidase was 1:10. The bilirubin oxidase complex was obtained by centrifugation purification.

[0063] S3, In-situ biomimetic graded mineralization: Preparation of calcium phosphate / calcium carbonate composite nano-precursor suspension modified with silane coupling agent:

[0064] S301. Amorphous calcium phosphate / calcium carbonate composite nanoparticles were synthesized by low-temperature liquid-phase coprecipitation under nitrogen protection conditions with a volume ratio of 1:3 in an ethanol-water mixed solvent. The molar ratio of calcium phosphate to calcium carbonate was 2:1.

[0065] S302. Disperse the composite nanoparticles in anhydrous ethanol at a solid-liquid ratio of 1g:40mL, add 3-aminopropyltriethoxysilane at 2% of the total mass of the composite nanoparticles, and reflux at 70℃ and 180rpm for 5h under nitrogen protection throughout.

[0066] S303. The reaction product was purified by centrifugation at 9000 rpm for 10 min, homogenized by ultrasonication at 200W and 40kHz for 12 min, and dispersed in sterile deionized water to prepare a nano-precursor suspension with a solid content of 5%.

[0067] A 0.15 mol / L calcium chloride solution and the above-mentioned nano-precursor suspension were simultaneously and uniformly added dropwise to the bilirubin organic reaction solution. The mass ratio of calcium chloride to the composite nano-precursor solids was 1.2:1, and the dropping rate was 0.8 mL / min. The in-situ biomimetic mineralization conditions were: temperature 30℃, shaking speed 30 rpm, and mineralization time 36 h.

[0068] S4. Purification, Drying, and Shaping: The mineralized mixture was first allowed to settle at room temperature for 3 hours, then centrifuged at 3500 rpm for 10 minutes. After solid-liquid separation, it was washed twice with anhydrous ethanol and twice with deionized water. It was then dried for 5 hours at 33℃, 18% relative humidity, and -0.085 MPa vacuum to obtain the in vitro synthesized bezoar.

[0069] Example 2:

[0070] S1. Substrate system preparation: In a closed, temperature-controlled bioreactor protected from light and continuously purged with nitrogen, a homogeneous substrate solution containing heme, 0.12 mol / L phosphate buffer, Tween 80, and disodium EDTA was prepared. The molar concentration of heme was 1.2 mmol / L, the amount of Tween 80 added was 0.6% of the total mass of the substrate solution, and the amount of disodium EDTA added was 0.4% of the total mass of the substrate solution. After homogenization by stirring at 120 rpm for 10 min at a constant temperature, the system was degassed under vacuum at -0.08 MPa for 15 min to remove dissolved oxygen and heavy metal interfering ions.

[0071] S2. Segmented Adaptive Multi-Enzyme Catalysis: In the first stage, the system pH was adjusted to 7.4 and the temperature to 35℃, and heme oxygenase was added for pre-catalysis for 3 hours. In the second stage, the system pH was adjusted to 7.2 and the temperature to 32℃, and biliverdin reductase was added for catalysis for 4 hours. In the third stage, the system pH was adjusted to 7.1 and the temperature to 33℃, and a chemically hydrophobically modified bilirubin oxidase complex was added for reconfiguration and purification, with gentle stirring at 150 rpm throughout. The mass ratio of heme oxygenase, biliverdin reductase, and modified bilirubin oxidase complex was 2:1:4.5, and the total multi-enzyme catalysis time was 14 hours, yielding an organic bilirubin reaction solution.

[0072] Preparation of chemically hydrophobically modified bilirubin oxidase complex:

[0073] S201. Select natural bilirubin oxidase with a specific activity ≥120U / mg, and purify it by dialysis with pH 7.2 phosphate buffer to remove impurities such as proteins and stabilizers, and obtain homogeneous pure enzyme solution.

[0074] S202. At 10℃, protected from light, and under constant temperature conditions of pH 7.2, acetic anhydride was added to the pure enzyme solution and the mixture was stirred at low speed for 6 hours. The amount of acetic anhydride added was 3.0% of the enzyme protein content in the pure enzyme solution.

[0075] S203. Polyethylene glycol-polylactic acid block copolymer solution was added dropwise to the modified enzyme solution, and the mixture was stirred at low speed for 22 hours for self-assembly. The mass ratio of polyethylene glycol-polylactic acid block copolymer to modified bilirubin oxidase was 1:15. The bilirubin oxidase complex was obtained by centrifugation purification.

[0076] S3, In-situ biomimetic graded mineralization: Preparation of calcium phosphate / calcium carbonate composite nano-precursor suspension modified with silane coupling agent:

[0077] S301. Amorphous calcium phosphate / calcium carbonate composite nanoparticles were synthesized by low-temperature liquid-phase coprecipitation under nitrogen protection conditions with a volume ratio of 1:3 in an ethanol-water mixed solvent. The molar ratio of calcium phosphate to calcium carbonate was 3:1.

[0078] S302. Disperse the composite nanoparticles in anhydrous ethanol at a solid-liquid ratio of 1g:50mL, add 3-glycidyl etheroxypropyltrimethoxysilane at a mass of 3% of the total mass of the composite nanoparticles, and reflux at 70℃ and 180rpm for 5h under nitrogen protection throughout.

[0079] S303. The reaction product was purified by centrifugation at 9000 rpm for 10 min, homogenized by ultrasonication at 200 W and 40 kHz for 12 min, and dispersed in sterile deionized water to prepare a nano-precursor suspension with a solid content of 10%.

[0080] A 0.25 mol / L calcium gluconate solution and the above-mentioned nano-precursor suspension were simultaneously and uniformly added dropwise to the bilirubin organic reaction solution. The mass ratio of calcium gluconate to the composite nano-precursor solids was 1.5:1, and the dropping rate was 1.2 mL / min. The in-situ biomimetic mineralization conditions were: temperature 34℃, shaking speed 50 rpm, and mineralization time 60 h.

[0081] S4. Purification, Drying, and Shaping: The mineralized mixture was first allowed to settle at room temperature for 5 hours, then centrifuged at 4500 rpm for 14 minutes. After solid-liquid separation, the mixture was washed twice with anhydrous ethanol and twice with deionized water. It was then dried for 7 hours at 37℃, 22% relative humidity, and -0.095 MPa vacuum to obtain the in vitro synthesized bezoar.

[0082] Example 3:

[0083] S1. Substrate system preparation: In a closed, temperature-controlled bioreactor protected from light and continuously purged with nitrogen, a homogeneous substrate solution containing heme, 0.10 mol / L phosphate buffer, Tween 80, and disodium EDTA was prepared. The molar concentration of heme was 1.0 mmol / L, the amount of Tween 80 added was 0.5% of the total mass of the substrate solution, and the amount of disodium EDTA added was 0.3% of the total mass of the substrate solution. After homogenization by stirring at 120 rpm for 10 min at a constant temperature, the system was degassed under vacuum at -0.07 MPa for 12 min to remove dissolved oxygen and heavy metal interfering ions.

[0084] S2. Segmented Adaptive Multi-Enzyme Catalysis: In the first stage, the system pH was adjusted to 7.3 and the temperature to 33℃, and heme oxygenase was added for pre-catalysis for 2.5 h. In the second stage, the system pH was adjusted to 7.1 and the temperature to 31℃, and biliverdin reductase was added for catalysis for 3.5 h. In the third stage, the system pH was adjusted to 7.1 and the temperature to 32℃, and a chemically hydrophobically modified bilirubin oxidase complex was added for reconfiguration and purification, with gentle stirring at 120 rpm throughout. The mass ratio of heme oxygenase, biliverdin reductase, and modified bilirubin oxidase complex was 2:1:4.0, and the total multi-enzyme catalysis time was 12 h, yielding an organic bilirubin reaction solution.

[0085] Preparation of chemically hydrophobically modified bilirubin oxidase complex:

[0086] S201. Select natural bilirubin oxidase with a specific activity ≥120U / mg, and purify it by dialysis with pH7.0 phosphate buffer to remove impurities such as proteins and stabilizers, and obtain homogeneous pure enzyme solution.

[0087] S202. Under constant temperature conditions of 7℃, protected from light, and pH 7.0, add octanoic anhydride to the pure enzyme solution and stir at low speed for 5 hours. The amount of octanoic anhydride added is 2.2% of the enzyme protein content in the pure enzyme solution.

[0088] S203. Polyethylene glycol-polylactic acid block copolymer solution was added dropwise to the modified enzyme solution, and the mixture was stirred at low speed for 20 hours for self-assembly. The mass ratio of polyethylene glycol-polylactic acid block copolymer to modified bilirubin oxidase was 1:12. The bilirubin oxidase complex was obtained by centrifugation purification.

[0089] S3, In-situ biomimetic graded mineralization: Preparation of calcium phosphate / calcium carbonate composite nano-precursor suspension modified with silane coupling agent:

[0090] S301. Amorphous calcium phosphate / calcium carbonate composite nanoparticles were synthesized by low-temperature liquid-phase coprecipitation under nitrogen protection conditions with a volume ratio of 1:3 in an ethanol-water mixed solvent. The molar ratio of calcium phosphate to calcium carbonate was 2.5:1.

[0091] S302. Disperse the composite nanoparticles in anhydrous ethanol at a solid-liquid ratio of 1g:45mL, add 3-aminopropyltriethoxysilane at a rate of 2.5% of the total mass of the composite nanoparticles, and reflux at 70℃ and 180rpm for 5 hours under nitrogen protection throughout.

[0092] S303. The reaction product was purified by centrifugation at 9000 rpm for 10 min, homogenized by ultrasonication at 200W and 40kHz for 12 min, and dispersed in sterile deionized water to prepare a nano-precursor suspension with a solid content of 7%.

[0093] A 0.20 mol / L calcium chloride solution and the above-mentioned nano-precursor suspension were simultaneously and uniformly added dropwise to the bilirubin organic reaction solution. The mass ratio of calcium chloride to the composite nano-precursor solids was 1.3:1, and the dropping rate was 1.0 mL / min. The in-situ biomimetic mineralization conditions were: temperature 32℃, shaking speed 40 rpm, and mineralization time 48 h.

[0094] S4. Purification, Drying, and Fixation: The mineralized mixture was first allowed to settle at room temperature for 4 hours, then centrifuged at 4000 rpm for 12 minutes. After solid-liquid separation, the mixture was washed twice with anhydrous ethanol and twice with deionized water. It was then dried for 6 hours at 35℃, 20% relative humidity, and -0.090 MPa vacuum to obtain the in vitro synthesized bezoar.

[0095] Comparative Example 1:

[0096] Compared with Example 3, segmented adaptive multi-enzyme linkage catalysis was not used. Only unmodified natural bilirubin oxidase was added for one-step catalysis. The remaining steps and parameters were the same as in Example 3.

[0097] Comparative Example 2:

[0098] Compared with Example 3, no calcium phosphate / calcium carbonate composite nano precursor modified with silane coupling agent was added. Only soluble calcium salt was added for mineralization. The remaining steps and parameters were the same as in Example 3.

[0099] Comparative Example 3:

[0100] Compared with Example 3, the substrate system did not contain the metal ion shielding agent disodium EDTA, and the remaining steps and parameters were the same as in Example 3.

[0101] Comparative Example 4:

[0102] Compared with Example 3, in-situ biomimetic graded mineralization was not performed; the bilirubin reaction solution was directly purified and dried, and the remaining steps and parameters were the same as in Example 3.

[0103] test:

[0104] Experiment 1: Detection of bilirubin synthesis efficiency and purity:

[0105] Experimental methods:

[0106] The bilirubin concentration in each sample was determined by high performance liquid chromatography (HPLC), and the catalytic conversion rate was calculated. The proportion of bilirubin in a single configuration was determined by chiral column chromatography. Catalytic conversion rate = (actual moles of bilirubin produced / theoretical moles of bilirubin produced) × 100%, and proportion of a single configuration = (mass of target configuration bilirubin / mass of total bilirubin) × 100%.

[0107] Experimental data:

[0108] Table 1

[0109] Sample number Catalytic conversion rate (%) Percentage of bilirubin monoforms (%) Example 1 90.2 95.1 Example 2 91.5 96.3 Example 3 92.8 97.2 Comparative Example 1 72.3 81.5 Comparative Example 2 92.5 97.0 Comparative Example 3 81.6 88.3 Comparative Example 4 92.6 97.1

[0110] Examples 1-3 showed catalytic conversion rates ≥90% and single configuration percentages ≥95%. Comparative Example 1 used a one-step unmodified enzyme catalysis, resulting in poor enzyme activity stability and an uncontrollable catalytic pathway, leading to a significant decrease in catalytic conversion rate and single configuration percentage. Comparative Example 3 did not add a metal ion shielding agent; heavy metal ions in the system inhibited enzyme activity, reducing catalytic efficiency and product purity. Comparative Examples 2 and 4 did not affect the enzyme catalysis process, and their catalytic conversion rates and configuration percentages showed no significant difference from the examples.

[0111] Experiment 2: Determination of mineralized structure and physicochemical stability of bezoar:

[0112] Experimental methods:

[0113] The micro-mineralization structure of bezoar was observed using scanning electron microscopy. The bilirubin retention rate was detected by accelerated aging test (temperature 60℃, humidity 75%, placed for 7 days). The bilirubin retention rate = (mass of bilirubin after aging / mass of bilirubin before aging) × 100%.

[0114] Experimental data:

[0115] Table 2

[0116] Sample number Microstructural integrity Bilirubin retention rate (%) Example 1 Complete hierarchical mineralization structure 92.3 Example 2 Complete hierarchical mineralization structure 93.5 Example 3 Complete hierarchical mineralization structure 94.8 Comparative Example 1 Complete hierarchical mineralization structure 94.5 Comparative Example 2 Irregular mineralization structure 76.2 Comparative Example 3 Complete hierarchical mineralization structure 85.1 Comparative Example 4 Unmineralized bonded structure 68.5

[0117] Examples 1-3 formed a complete hierarchical mineralization structure with bilirubin retention ≥92% and excellent stability. Comparative Example 2 did not use a modified composite nano-precursor; its mineralization structure was loose and irregular, offering poor protection for bilirubin, resulting in significant bilirubin degradation after aging. Comparative Example 4 lacked an organic-inorganic mineralization structure, directly exposing bilirubin and exhibiting the worst stability. Differences in enzyme catalysis between Comparative Examples 1 and 3 did not affect the mineralization structure; stability was slightly affected by the purity of the enzyme-catalyzed product.

[0118] Experiment 3: In vitro detection of medicinal components in bezoar:

[0119] Experimental methods:

[0120] Referring to the quality testing method for natural bezoar, the total bilirubin content and bile acid content in the sample were determined, and the total percentage of effective ingredients was calculated.

[0121] Experimental data:

[0122] Table 3

[0123] Sample number Total bilirubin content (%) Bile acid content (%) Total percentage of active ingredients (%) Example 1 38.2 8.1 46.3 Example 2 39.5 8.3 47.8 Example 3 40.8 8.5 49.3 Comparative Example 1 29.5 6.2 35.7 Comparative Example 2 38.0 8.0 46.0 Comparative Example 3 33.6 7.1 40.7 Comparative Example 4 30.2 6.5 36.7

[0124] In Examples 1-3, the total proportion of active ingredients was ≥46%, approaching the quality of natural bezoar. In Comparative Example 1, due to low enzyme catalytic efficiency, the amount of bilirubin produced was low, resulting in a significantly reduced content of active ingredients. In Comparative Example 3, the presence of heavy metal ions led to incomplete enzyme catalysis, resulting in a decrease in the content of active ingredients. In Comparative Example 4, the lack of mineralized structure support resulted in easy loss of active ingredients, leading to a lower content. In Comparative Example 2, the mineralized structure did not affect the synthesis of active ingredients, and the content was close to that of the Examples.

[0125] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not describe all details exhaustively, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification.

Claims

1. A rapid in vitro synthesis process for bezoar based on multi-enzyme catalytic technology, characterized in that, Includes the following steps: S1. Substrate system preparation: In a closed, constant-temperature bioreactor protected from light and continuously purged with nitrogen, a homogeneous substrate solution containing heme, buffer, nonionic phase transfer agent, and metal ion shielding agent was prepared. The molar concentration of heme was 0.8–1.2 mmol / L. After homogenization by stirring at 120 rpm for 10 min at constant temperature, the system was degassed under vacuum at -0.06–-0.08 MPa for 10–15 min to remove dissolved oxygen and heavy metal interfering ions. S2. Segmented adaptive multi-enzyme linkage catalysis: Multi-enzyme coupling catalysis is carried out by using a time-gradient enzyme addition combined with a segmented pH and temperature adaptive regulation strategy: In the first stage, the pH of the system is regulated to 7.2-7.4 and the temperature to 32-35℃, and heme oxygenase is added for pre-catalysis for 2-3 hours. In the second stage, the pH of the system was adjusted to 7.0–7.2 and the temperature to 30–32°C. Biliverdin reductase was added and catalyzed for 3–4 hours. In the third stage, the pH of the system was adjusted to 7.1 and the temperature to 31–33°C. Biliverdin oxidase complex modified by chemical hydrophobicity was added for reconfiguration and purification. The mixture was gently stirred at 80–150 rpm throughout the process to prepare the bilirubin organic reaction solution. S3. In-situ biomimetic hierarchical mineralization: A soluble calcium salt solution and a calcium phosphate / calcium carbonate composite nano precursor suspension modified with a silane coupling agent are simultaneously and uniformly added to the bilirubin organic reaction solution. Through the covalent coupling between the active functional groups on the precursor surface and the hydroxyl groups of bilirubin, organic-inorganic composite crystal nuclei are formed in an orderly manner and grow in a directional manner, thereby achieving hierarchical in-situ biomimetic mineralization. S4. Purification, drying and shaping: After the mineralized mixture is subjected to segmented gradient induced sedimentation, solid-liquid separation and alternating washing, it is dried and shaped in a low temperature, vacuum and low humidity environment to obtain in vitro synthesized bezoar.

2. The rapid in vitro synthesis process of bezoar based on multi-enzyme catalytic technology according to claim 1, characterized in that: In step S1, the buffer is 0.08-0.12 mol / L phosphate buffer, the nonionic phase transfer agent is Tween 80, and the amount added is 0.4%-0.6% of the total mass of the substrate solution; the metal ion shielding agent is disodium EDTA, and the amount added is 0.2%-0.4% of the total mass of the substrate solution.

3. The rapid in vitro synthesis process of bezoar based on multi-enzyme catalytic technology according to claim 1, characterized in that, The method for preparing the chemically hydrophobically modified bilirubin oxidase complex in step S2 includes: S201. Enzyme solution pretreatment: Select natural bilirubin oxidase with a specific activity ≥120U / mg, and purify it by dialysis with phosphate buffer at pH 6.8-7.2 to remove impurities such as proteins and stabilizers, and obtain homogeneous pure enzyme solution. S202, Mild surface hydrophobic modification: Under constant temperature conditions of 4-10℃, in the dark, and pH 6.8-7.2, a hydrophobic esterification modifier is added to the pure enzyme solution, and the reaction is carried out with low-speed stirring for 4-6 hours; the hydrophobic esterification modifier is octanoic anhydride or acetic anhydride, and the amount of modifier added is 1.5%-3.0% of the enzyme protein mass in the pure enzyme solution, thereby constructing local hydrophobic microregions in situ in the non-catalytic region of the enzyme molecule; S203, self-assembly complex: Add an amphiphilic block copolymer solution dropwise to the modified enzyme solution and stir continuously at low speed for 18-22 hours to form a core-shell structure complex of an enzyme core and a polymer shell. After centrifugation and purification, a stable bilirubin oxidase complex is obtained.

4. The rapid in vitro synthesis process of bezoar based on multi-enzyme catalytic technology according to claim 3, characterized in that: The amphiphilic block copolymer is a polyethylene glycol-polylactic acid block copolymer, and the mass ratio of the amphiphilic block copolymer to the modified bilirubin oxidase is 1:10-15.

5. The rapid in vitro synthesis process of bezoar based on multi-enzyme catalytic technology according to claim 1, characterized in that, In step S2, the mass ratio of the heme oxygenase, biliverdin reductase, and modified bilirubin oxidase complex is 2:1:3.5 to 2:1:4.5, and the total catalytic time of the multi-enzyme linkage is 10 to 14 hours.

6. The rapid in vitro synthesis process of bezoar based on multi-enzyme catalytic technology according to claim 1, characterized in that, The preparation method of the calcium phosphate / calcium carbonate composite nano-precursor suspension modified with silane coupling agent in step S3 includes: S301. Preparation of composite nanoparticles: Amorphous calcium phosphate / calcium carbonate composite nanoparticles were synthesized by low-temperature liquid-phase coprecipitation method under nitrogen protection conditions with a volume ratio of 1:3 ethanol-water mixed solvent. S302, Surface functionalization modification: The composite nanoparticles are dispersed in anhydrous ethanol at a solid-liquid ratio of 1g:40mL~50mL, a silane coupling agent is added, and the mixture is refluxed at 70℃ and 180rpm for 5h under nitrogen protection throughout the process to graft amino or epoxy active functional groups onto the surface of the particles. S303. Preparation of stable suspension: The reaction product is purified by centrifugation at 9000 rpm for 10 min to remove free impurities. Then, it is homogenized by ultrasonication at 200W and 40kHz for 12 min and dispersed in sterile deionized water to prepare a nano-precursor suspension with a solid content of 5% to 10%.

7. The rapid in vitro synthesis process of bezoar based on multi-enzyme catalytic technology according to claim 6, characterized in that: The silane coupling agent is 3-aminopropyltriethoxysilane or 3-glycidyl etheroxypropyltrimethoxysilane, and the amount of silane coupling agent used is 2% to 3% of the total mass of the composite nanoparticles; the molar ratio of calcium phosphate to calcium carbonate is 2 to 3:

1.

8. The rapid in vitro synthesis process of bezoar based on multi-enzyme catalytic technology according to claim 1, characterized in that: In step S3, the soluble calcium salt is calcium chloride or calcium gluconate, the molar concentration of the calcium salt solution is 0.15-0.25 mol / L, and the mass ratio of the calcium salt to the composite nano precursor solid is 1.2-1.5:1; the calcium salt solution and the nano precursor suspension are added dropwise simultaneously at a uniform rate of 0.8-1.2 mL / min.

9. The rapid in vitro synthesis process of bezoar based on multi-enzyme catalytic technology according to claim 1, characterized in that: The in-situ biomimetic mineralization conditions are: temperature 30-34℃, oscillation speed 30-50rpm, and mineralization time 36-60h.

10. The rapid in vitro synthesis process of bezoar based on multi-enzyme catalytic technology according to claim 1, characterized in that: In step S4, the segmented gradient induced sedimentation involves first allowing the sediment to settle at room temperature for 3–5 hours, followed by centrifugation at a low speed of 3500–4500 rpm for 10–14 minutes; the alternating washing involves washing twice with anhydrous ethanol and twice with deionized water; and the low-temperature vacuum drying conditions are: temperature 33–37°C, relative humidity 18%–22%, vacuum degree -0.085–-0.095 MPa, and drying time 5–7 hours.

Images