Oriented synthesis process of albendazole side chain based on immobilized enzyme catalyst

Abstract

The invention relates to the technical field of biological manufacturing of veterinary bulk drugs, and discloses an immobilized enzyme catalyst-based albendazole side chain oriented synthesis process, which comprises a magnetic core; the proton channel array functional layer is coated on the substrate and is made of a metal organic framework material; the flexible molecular arm is connected to the outer surface of the functional layer; and a primary catalytic enzyme and a coenzyme regenerating enzyme covalently bonded by a flexible molecular arm. In a reaction medium, the catalyst, a substrate 5-(propylthio)-1H-benzo [d] imidazole-2-amine, glucose and a coenzyme NADP < + > are subjected to a catalytic reaction under mild conditions. The pH value of a double-enzyme catalysis microenvironment is stabilized through a proton channel constructed by a ZIF-8 layer, the activity loss in the enzyme immobilization process is reduced through the design of a flexible molecular arm, and the catalyst is conveniently recycled through a magnetic core. The process has the advantages of mild reaction conditions, high product selectivity, good catalyst operation stability and repeated use.

Description

Technical Field

[0001] This invention relates to the field of biomanufacturing technology for veterinary active pharmaceutical ingredients, specifically a process for the directional synthesis of propylthiobenzamide side chains based on immobilized enzyme catalysts. Background Technology

[0002] Benzimidazole compounds are widely used veterinary anthelmintics. As a leading enterprise in this field, the applicant has long been committed to the large-scale production and technological innovation of related active pharmaceutical ingredients. Methyl 5-propanethio-2-benzimidazole carbamate is one of its core products, with an annual production capacity of 1,000 tons.

[0003] In the process of continuous technological upgrades and process optimization of the albendazole product line, the development of its main active metabolite—albendazole (albendazole sulfoxide)—has become an important direction for enhancing product added value and technological competitiveness. Direct synthesis and use of albendazole, compared to its parent drug albendazole, achieves effective therapeutic concentrations in animals more quickly, demonstrating superior bioavailability.

[0004] Currently, the industrial synthesis of albendazole from its thioether precursor (5-(propylthio)-1H-benzo[d]imidazole-2-amine) mainly relies on chemical oxidation. However, for large-scale production enterprises like the applicant, the drawbacks of this method are becoming increasingly apparent: the selectivity of the chemical oxidant is difficult to control precisely, and over-oxidation is prone to occur during the reaction, generating sulfone byproducts that are difficult to separate, which directly affects the yield and purity of the final product. Furthermore, the chemical oxidation process is usually accompanied by the discharge of waste gas, wastewater, and solid waste, which is inconsistent with the current trend of green pharmaceutical development and places continuous pressure on the company's environmental protection operations.

[0005] Therefore, introducing biocatalysis technology and utilizing the high selectivity of enzymes for targeted oxidation has become an ideal approach to overcome the aforementioned process bottlenecks. Theoretically, constructing a dual-enzyme synergistic catalytic system using monooxygenase and glucose dehydrogenase can precisely complete the target conversion and achieve the recycling and regeneration of coenzymes. However, new technical challenges arise when this technology is transferred from the laboratory to industrial production. The inherent poor stability and difficulty in recovery of free enzyme systems make them unsuitable for large-scale production. Existing immobilized enzyme technologies, after simply immobilizing the two enzymes on a carrier, generally face problems such as significantly reduced enzyme activity and insufficient operational stability due to enzyme protein conformational limitations and pH imbalance in the catalytic microenvironment.

[0006] In conclusion, for large-scale albendazole producers, there is an urgent need to develop a novel biocatalytic process that can overcome the shortcomings of existing immobilization technologies in order to achieve efficient, green, and economical industrial production of albendazole. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a directional synthesis process for the side chain of propylthiobenzamide based on an immobilized enzyme catalyst. This aims to solve the combined technical problems in existing immobilized dual-enzyme catalysis technologies, such as the loss of catalytic activity caused by the direct immobilization of enzyme proteins on the carrier surface, and the poor synergistic catalytic efficiency and insufficient overall operational stability caused by pH fluctuations in the dual-enzyme catalytic microenvironment.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0009] The first aspect of this invention provides a multi-level magnetic synergistic catalyst, the structure of which, from the inside out, comprises:

[0010] S1, magnetic core;

[0011] S2, a proton channel array functional layer made of metal-organic framework material covering the surface of the magnetic core;

[0012] S3, a flexible molecular arm with one end connected to the outer surface of the proton channel array functional layer and the other end free;

[0013] S4. The main catalytic enzyme and coenzyme regenerating enzyme are covalently bonded through the free end of the flexible molecular arm.

[0014] In one specific technical solution, the magnetic core is iron oxide nanoparticles with an average particle size of 50-200 nanometers.

[0015] In one specific technical solution, the proton channel array functional layer is a ZIF-8 thin shell with a thickness of 10–30 nanometers. The ZIF-8 thin shell has a regular pore structure, the pore size of which allows small molecules such as coenzymes, protons, and water to pass through, but blocks large molecules such as the main catalytic enzyme and coenzyme regeneration enzyme.

[0016] In one specific technical solution, the flexible molecular arm is a polyethylene glycol molecular chain with a molecular weight of 2000-5000 Da, which provides a reaction space away from the surface of the proton channel array functional layer for the main catalytic enzyme and the coenzyme regenerating enzyme.

[0017] In one specific technical solution, the main catalytic enzyme is a monooxygenase, and the coenzyme regenerating enzyme is glucose dehydrogenase. The main catalytic enzyme and the coenzyme regenerating enzyme are covalently bonded to the free end of the flexible molecular arm at an asymmetric molar ratio of 1:(2.0-4.0).

[0018] The second aspect of this invention provides a directed synthesis process for the side chain of propylthiobenzylimidazole based on an immobilized enzyme catalyst, comprising the following steps: adding the multi-level magnetic synergistic catalyst described in the first aspect, 5-(propylthio)-1H-benzo[d]imidazole-2-amine, glucose, and coenzyme NADP to a reaction medium. + The catalytic reaction is carried out at 25–45°C.

[0019] In the process described in this invention, the catalytic reaction mechanism includes: the coenzyme regenerating enzyme (glucose dehydrogenase) uses glucose as a substrate to convert the coenzyme NADP... + The reduction to NADPH releases protons (H) in the process. + The main catalytic enzyme (monooxygenase) uses the generated NADPH and oxygen in the reaction medium to oxidize the propylthio group side chain of 5-(propylthio)-1H-benzo[d]imidazol-2-amine to propylsulfinyl group, generating propylthiobenzylimidazole. This process consumes protons.

[0020] The proton channel array functional layer in the multi-level magnetic synergistic catalyst has a regular pore structure that forms a proton transport channel. During the catalytic reaction, protons released by the coenzyme regenerating enzyme or protons required by the main catalytic enzyme can be rapidly transferred between the two enzymes or exchanged with the bulk reaction medium through this channel, thereby maintaining the pH stability of the microenvironment where the main catalytic enzyme and the coenzyme regenerating enzyme are located.

[0021] In one specific technical solution, the preparation method of the multi-level structure magnetic synergistic catalyst includes: adding a methanol solution of zinc nitrate and a methanol solution of 2-methylimidazole dropwise to a methanol suspension of Fe3O4@APTES magnetic core, reacting at room temperature for 8 to 16 hours, and growing a ZIF-8 thin shell in situ on the surface of the magnetic core to form the proton channel array functional layer.

[0022] In one specific technical solution, the reaction medium is a phosphate buffer solution with a pH of 7.0 to 8.0.

[0023] In a specific technical solution, the process parameters of the catalytic reaction are further defined as follows: the reaction temperature is 30-40℃; air is introduced into the reaction medium at a flow rate of 0.2-0.6 vvm.

[0024] In a specific technical solution, the concentrations of each component in the reaction medium are limited as follows: the initial concentration of 5-(propylthio)-1H-benzo[d]imidazole-2-amine is 20–80 mM; the coenzyme NADP… + The initial concentration is 0.2 to 0.8 mM; the molar amount of glucose is 1.2 to 1.8 times the molar amount of 5-(propylthio)-1H-benzo[d]imidazole-2-amine.

[0025] In one specific technical solution, after the catalytic reaction is completed, the process further includes the step of: separating and recovering the multi-level magnetic synergistic catalyst by applying a magnetic field outside the reaction system.

[0026] This invention provides a process for the directional synthesis of propylthiobenzamide side chains based on immobilized enzyme catalysts.

[0027] It has the following beneficial effects:

[0028] 1. This invention constructs a proton channel array functional layer formed by a metal-organic framework material, utilizing its regular pore structure to rapidly conduct and buffer protons generated during catalytic reactions. This design can maintain the pH stability of the microenvironment of the dual-enzyme synergistic catalytic system, avoiding enzyme protein inactivation caused by local pH fluctuations, thereby improving the operational stability of the catalyst and extending its service life.

[0029] 2. This invention effectively isolates the enzyme protein from the carrier surface by introducing flexible molecular arms with a specific molecular weight range as spacers. This structure provides the enzyme protein with ample three-dimensional space, reducing conformational changes caused by steric hindrance during immobilization, allowing the enzyme to retain its natural catalytic activity to a large extent, thereby ensuring the efficient operation of the entire co-catalytic system.

[0030] 3. This invention employs a magnetic core as the catalyst's core, enabling the entire catalyst to be rapidly and non-destructively separated and recovered from the reaction system after the reaction is complete by applying an external magnetic field. This feature not only simplifies the post-processing and purification of the product but also enables convenient recovery and reuse of the catalyst, reducing process costs. Detailed Implementation

[0031] The main raw materials and reagents used in the following examples and comparative examples are sourced and specified as follows. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0032] Iron oxide nanoparticles: average particle size 100 nm, CAS: 1317-61-9;

[0033] (3-Aminopropyl)triethoxysilane: Purity ≥98%, CAS: 919-30-2;

[0034] Zinc nitrate hexahydrate: purity ≥99%, CAS: 10196-18-6;

[0035] 2-Methylimidazole: Purity ≥99%, CAS: 693-98-1;

[0036] α-Amino-ω-carboxylated polyethylene glycol: molecular weight 3000 Da;

[0037] N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride: purity ≥98.5%, CAS: 25952-53-8;

[0038] N-Hydroxysuccinimide: Purity ≥98%, CAS: 6066-82-6;

[0039] Monooxygenase: EC1.14.14.1, recombinantly expressed and purified in Escherichia coli (E. coli);

[0040] Glucose dehydrogenase: EC1.1.1.47, recombinantly expressed and purified in Escherichia coli (E. coli);

[0041] 5-(propylthio)-1H-benzo[d]imidazol-2-amine: purity ≥ 98%;

[0042] Oxidized Coenzyme II Sodium Salt: Purity ≥95%, CAS: 24292-60-2;

[0043] D-glucose: anhydrous, purity ≥99.5%, CAS: 50-99-7;

[0044] Methanol: chromatographic grade, CAS: 67-56-1;

[0045] Phosphate buffer: 0.1M, pH 7.4.

[0046] Example:

[0047] Example 1: Multi-level magnetic synergistic catalyst (Fe3O4@ZIF-8@PEG) 3000 Preparation of dual enzymes

[0048] 1.1 Surface Amination of the Magnetic Core (Preparation of Fe3O4@APTES) 1.0 g of iron(III) oxide nanoparticles were dispersed in a mixed solution of 100 mL ethanol and 25 mL water, and sonicated for 15 minutes. 3.0 mL of (3-aminopropyl)triethoxysilane (APTES) was added to the suspension, and the mixture was mechanically stirred at 60 °C for 8 hours. After the reaction, the product was separated by an external magnetic field, washed three times each with ethanol and deionized water, and then dried in a vacuum drying oven at 60 °C for 12 hours to obtain Fe3O4@APTES.

[0049] 1.2 In-situ growth of the proton channel array functional layer (preparation of Fe3O4@ZIF-8): 0.2 g of the above Fe3O4@APTES was accurately weighed and dispersed in 50 mL of methanol to form suspension A. Separately, 1.48 g of zinc nitrate hexahydrate was dissolved in 25 mL of methanol to form solution B; 3.28 g of 2-methylimidazole was dissolved in 25 mL of methanol to form solution C. At room temperature, solutions B and C were simultaneously and slowly added dropwise to the vigorously stirred suspension A. After the addition was complete, the reaction was continued at room temperature for 12 hours. After the reaction was completed, the product was separated by an external magnetic field, washed three times with methanol, and dried in a vacuum drying oven at 60 °C for 12 hours to obtain Fe3O4@ZIF-8.

[0050] 1.3 Grafting of Flexible Molecular Arms (Preparation of Fe3O4@ZIF-8@PEG) 0.1 g of the above Fe3O4@ZIF-8 was dispersed in 50 mL of phosphate buffer (0.1 M, pH 7.4). 0.2 g of α-amino-ω-carboxylated polyethylene glycol (molecular weight 3000 Da) was added, followed by 0.1 g of EDC·HCl and 0.06 g of NHS. The reaction was carried out at 25 °C with stirring in the dark for 24 hours. After the reaction was completed, the product was separated by an external magnetic field and washed five times with phosphate buffer to remove unreacted polyethylene glycol and coupling agent, yielding Fe3O4@ZIF-8@PEG.

[0051] 1.4 Covalent bonding of the main catalytic enzyme and the coenzyme regenerating enzyme (Preparation of Fe3O4@ZIF-8@PEG dual enzyme) All the Fe3O4@ZIF-8@PEG obtained in step 1.3 was dispersed in 20 mL of phosphate buffer (0.1 M, pH 7.4). 0.1 g EDC·HCl and 0.06 g NHS were added, and the mixture was stirred and activated at 4 °C for 2 hours. Subsequently, 10 mg of monooxygenase and 35 mg of glucose dehydrogenase (the molar ratio of the two enzymes was approximately 1:3.0) were added. The reaction was continued at 4 °C in the dark for 24 hours. After the reaction, the product was separated by an external magnetic field, washed five times with phosphate buffer to remove unbound enzymes, and the final product was dispersed in phosphate buffer and stored at 4 °C, which is the multi-level magnetic synergistic catalyst prepared in this example.

[0052] Example 2: Synthesis of propoxurazole using the catalyst prepared in Example 1

[0053] In a 100 mL isothermal glass reactor equipped with a magnetic stirrer and an air inlet tube, 50 mL of phosphate buffer (0.1 M, pH 7.4) was added. Subsequently, the entire catalyst suspension prepared in Example 1, 133.5 mg of 5-(propylthio)-1H-benzo[d]imidazol-2-amine (substrate starting concentration 10 mM), and 9.3 mg of NADP were added. +Sodium salt (initial concentration 0.2 mM) and 216.2 mg of D-glucose (molar amount 1.2 times that of the substrate) were used. The reaction system temperature was set to 35 °C, magnetic stirring was turned on (300 rpm), and air was introduced into the reaction solution at a flow rate of 0.4 vvm. The reaction was stopped after 24 hours. The catalyst was completely adsorbed onto the reactor wall by applying a magnetic field outside the reactor, and the supernatant was collected for subsequent analysis.

[0054] Example 3: Catalysts using flexible molecular arms of different molecular weights (PEG) 2000 Preparation and application of )

[0055] The catalyst preparation steps are the same as in Example 1, except that the molecular weight of the α-amino-ω-carboxylated polyethylene glycol used in step 1.3 is 2000 Da. The process steps for synthesizing propylthiobenzamide using this catalyst are the same as in Example 2.

[0056] Example 4: Catalysts using flexible molecular arms of different molecular weights (PEG) 5000 Preparation and application of )

[0057] The catalyst preparation steps are the same as in Example 1, except that the molecular weight of the α-amino-ω-carboxylated polyethylene glycol used in step 1.3 is 5000 Da. The process steps for synthesizing propylthiobenzamide using this catalyst are the same as in Example 2.

[0058] Example 5: Synthesis of albendazole at 25°C

[0059] The catalyst prepared in Example 1 was used. The process steps for the synthesis of propylthiobenzamide were the same as in Example 2, except that the reaction temperature was set to 25°C.

[0060] Example 6: Synthesis of propylthiobenzamide at 45°C

[0061] The catalyst prepared in Example 1 was used. The process steps for the synthesis of propylthiobenzamide were the same as in Example 2, except that the reaction temperature was set to 45°C.

[0062] Comparative example:

[0063] Comparative Example 1: Compared with Examples 1 and 2, the difference is that the preparation of its catalyst does not include the in-situ growth process of the proton channel array functional layer in step 1.2, i.e., the flexible molecular arm is directly grafted onto the Fe3O4@APTES intermediate and the two enzymes are bonded. All other aspects are the same.

[0064] Comparative Example 2: Compared with Examples 1 and 2, the difference is that the preparation of its catalyst does not include the grafting process of the flexible molecular arm in step 1.3, that is, the activation and covalent bonding of the two enzymes are performed directly on the surface of the Fe3O4@ZIF-8 product. All other aspects are the same.

[0065] Comparative Example 3: Compared with Example 2, the difference is that no immobilized catalyst was added to the reaction system; instead, free monooxygenase and free glucose dehydrogenase with the same loading as those on the catalyst in Example 1 were directly added. All other aspects were the same.

[0066] Test example:

[0067] Test Example 1: Determination of Product Conversion and Selectivity

[0068] 1.1 High-Performance Liquid Chromatography (HPLC) Analysis Conditions Quantitative analysis of the reaction products was performed using a high-performance liquid chromatograph (HPLC). The chromatographic column was a C18 reversed-phase column (4.6 mm × 250 mm, 5 μm). Mobile phase A was 0.1% (v / v) formic acid aqueous solution, and mobile phase B was methanol. Gradient elution was used, with the following elution program: 0–10 min, 10–90% B; 10–15 min, 90% B. The mobile phase flow rate was 1.0 mL / min, the column temperature was maintained at 30 °C, the detection wavelength was set to 292 nm, and the injection volume was 10 μL.

[0069] 1.2 Sample Processing and Detection 100 μL of the reaction supernatant from each of the reaction systems in Examples 2-6 and Comparative Examples 1-3 was precisely transferred and added to 900 μL of methanol to terminate the reaction and precipitate residual proteins. After centrifuging the mixture at 12000 rpm for 5 minutes, the supernatant was filtered through a 0.22 μm filter membrane, and the resulting filtrate was the sample to be tested. Quantitative analysis was performed using the external standard method. The conversion rate and selectivity were calculated by determining the residual concentration of the substrate 5-(propylthio)-1H-benzo[d]imidazole-2-amine and the concentration of the target product propylthiobenzamide formed in the sample.

[0070] 1.3 Conversion and Selectivity Calculation Conversion (%) = [(Initial moles of substrate - Remaining moles of substrate) / Initial moles of substrate] × 100% Selectivity (%) = [Moles of target product generated / (Initial moles of substrate - Remaining moles of substrate)] × 100%.

[0071] 1.4 Results The catalytic reaction results of each example and the comparative example after 24 hours of reaction are summarized in Table 1.

[0072] Table 1. Catalytic reaction results of each example and comparative example.

[0073]

[0074]

[0075] Analysis of results based on test case 1

[0076] The data in Table 1 show that the product conversion rate of the multi-level magnetic synergistic catalyst described in Example 2 is significantly higher than that of Comparative Examples 1, 2, and 3. The catalyst in Comparative Example 1 lacks a proton channel array functional layer, resulting in a significant decrease in its conversion rate. This indicates that the presence of the ZIF-8 layer is the structural basis for maintaining high catalytic efficiency. The regular channels of this ZIF-8 layer provide a pathway for the transfer of protons released from the coenzyme regenerating enzyme (glucose dehydrogenase) to the region required for the main catalytic enzyme (monooxygenase) catalytic reaction, maintaining the pH stability of the microenvironment in which the two enzymes reside, thereby ensuring the continuous catalytic activity of the enzymes.

[0077] A comparison of the results of Example 2 and Comparative Example 2 shows that the placement of the flexible molecular arm plays a decisive role in catalyst performance. In Comparative Example 2, the enzyme was directly immobilized on the ZIF-8 surface, and its conversion rate was only 41.7%, indicating that the activity of the enzyme protein was severely inhibited. In Examples 2, 3, and 4, by introducing polyethylene glycol molecular chains with a molecular weight of 2000-5000 Da as flexible molecular arms, the enzyme protein was spatially isolated from the rigid surface of the support, providing it with the necessary conformational freedom and reducing the adverse effects of steric hindrance on enzyme activity. A comparison of the data from Examples 2, 3, and 4 further demonstrates that the length (i.e., molecular weight) of the flexible molecular arm is a parameter affecting catalytic efficiency.

[0078] Data from Examples 2, 5, and 6 collectively confirm the operability of the synthesis process provided by this invention within a temperature range of 25-45°C. This multi-level magnetic synergistic catalyst, through the structural synergy of the proton channel array functional layer and the flexible molecular arm, jointly solves the technical problem of reduced activity in immobilized two-enzyme systems caused by microenvironmental pH fluctuations and enzyme conformational limitations, ultimately achieving high substrate conversion and high selectivity for the target product.

[0079] Test Example 2: Determination of Catalyst Operational Stability

[0080] 2.1 Reusability Test Procedure: The catalysts prepared in Example 2 and Comparative Examples 1 and 2 were tested for reusability stability. The first round of catalytic reaction was carried out according to the process steps described in Example 2. After 24 hours of reaction, the catalyst was separated and recovered by applying a magnetic field outside the reactor. The recovered catalyst was washed three times with 0.1M, pH 7.4 phosphate buffer solution, with solid-liquid separation performed by magnetic separation each time. The washed catalyst was directly added to a fresh reaction system with the same composition and volume as the first round of reaction to start the next round of catalytic reaction. The above "reaction-magnetic separation-washing-reuse" cycle was repeated 10 times. After each round of reaction, a sample of the supernatant was taken, and its product conversion rate was determined according to the method described in Test Example 1.

[0081] 2.2 Relative Activity Calculation: The catalytic activity of each catalyst in the first round of use is defined as 100%. The relative activity in each subsequent round is calculated using the following formula: Relative Activity (%) = [(Product Conversion Rate in Round N) / (Product Conversion Rate in Round I)] × 100%

[0082] 2.3 Results The relative activity data of the catalysts of Example 2, Comparative Example 1 and Comparative Example 2 in 10 consecutive use cycles are summarized in Table 2.

[0083] Table 2. Stability results of different catalysts after repeated use

[0084]

[0085] Analysis of results based on test case 2

[0086] Table 2 shows that the catalyst described in Example 2 retained 83.6% of its initial activity after 10 cycles of reuse, while the catalysts in Comparative Examples 1 and 2 showed activities decreasing to 25.2% and 19.3% of their initial activities, respectively. The only structural difference between the catalysts in Example 2 and Comparative Example 1 is the presence or absence of a ZIF-8 proton channel array functional layer. The rapid decline in the activity of the catalyst in Comparative Example 1 indicates that the lack of a pH microenvironment regulation mechanism during repeated catalysis leads to irreversible inactivation of the enzyme protein. The ZIF-8 layer in this invention, through its inherent pore structure, effectively buffers local proton concentration fluctuations in the two-enzyme reaction system during each catalytic cycle, thereby protecting the structural stability of the enzyme. This is a prerequisite for the long-term reuse of the catalyst.

[0087] The significant difference in operational stability between the catalyst of Example 2 and the catalyst of Comparative Example 2 demonstrates the role of the flexible molecular arm in this structure. In Comparative Example 2, the enzyme is directly immobilized on the support surface. During repeated physical operations such as magnetic separation, washing, and redispersion, the enzyme protein is susceptible to shear forces and rigid interaction with the support surface, leading to conformational disruption and detachment, resulting in a rapid decline in activity. The polyethylene glycol flexible molecular arm in Example 2 provides a buffer space for the enzyme protein, reducing the direct impact on the enzyme protein during physical operations, thereby maintaining the structural integrity and catalytic function of the enzyme during multiple cycles of use.

[0088] In summary, the high operational stability of the multi-level magnetic synergistic catalyst of this invention is a result of the synergistic effect of the proton channel array functional layer and the flexible molecular arms. The proton channel array functional layer provides chemical stability by stabilizing the pH value of the catalytic microenvironment; the flexible molecular arms provide physical stability by isolating the support surface and buffering physical stress. The combination of these two structural features jointly solves the problem of rapid deactivation of immobilized enzymes due to chemical and physical factors during repeated use, enabling the catalyst to be recycled multiple times.

Claims

1. A process for the directed synthesis of propylthiobenzimidazole side chains based on immobilized enzyme catalysts, characterized in that, The method comprises the following steps: In the reaction medium, a multi-level structure magnetic synergistic catalyst, 5- (propylthio) -1H-benzo [d] imidazole-2-amine, glucose and coenzyme NADP are added + The catalytic reaction is carried out at 25-45℃. The multi-level structure magnetic synergistic catalyst comprises the following structures from inside to outside: S1, a magnetic core; S2, a proton channel array functional layer composed of metal organic framework material coated on the surface of the magnetic core; S3, a flexible molecular arm with one end connected to the outer surface of the proton channel array functional layer and the other end free; S4, a main catalytic enzyme and a coenzyme regenerating enzyme covalently bonded through the free end of the flexible molecular arm, the molar ratio of the main catalytic enzyme to the coenzyme regenerating enzyme being an asymmetric molar ratio of 1: (2.0-4.0).

2. The process according to claim 1, characterized in that, The proton channel array functional layer is a ZIF-8 thin shell with a thickness of 10-30 nanometers prepared on the surface of the magnetic core by an in-situ growth method.

3. The process of claim 1, wherein, The magnetic core is a ferroferric oxide nanoparticle with an average particle size of 50-200 nanometers.

4. The process of claim 1, wherein, The flexible molecular arm is a polyethylene glycol molecular chain with a molecular weight of 2000-5000 Da.

5. The process of claim 1, wherein, The main catalytic enzyme is a monooxygenase, and the coenzyme regenerating enzyme is glucose dehydrogenase.

6. The process of claim 1, wherein, The reaction medium is a phosphate buffer with a pH value of 7.0-8.

0.

7. The process of claim 1, wherein, The process parameters of the catalytic reaction are further limited as follows: The reaction temperature is 30-40℃; Air is introduced into the reaction medium at a flow rate of 0.2-0.6 vvm.

8. The process of claim 1, wherein, The concentration of each component in the reaction medium is limited as follows: The initial concentration of the 5- (propylsulfanyl) -1H-benzo [d] imidazole-2-amine is 20-80 mM; The coenzyme NADP + has a starting concentration of 0.2-0.8 mM; The molar amount of glucose is 1.2-1.8 times the molar amount of 5- (propylsulfanyl) -1H-benzo [d] imidazole-2-amine.

9. The process of claim 1, wherein, The preparation method of the multi-level structure magnetic synergistic catalyst comprises the following steps: in the methanol suspension of the Fe3O4@APTES magnetic core, drop the methanol solution of zinc nitrate and the methanol solution of 2-methyl imidazole, react at room temperature for 8-16 hours, grow a ZIF-8 thin shell on the surface of the magnetic core in-situ to form the proton channel array functional layer.

10. The process of claim 1, wherein, After the catalytic reaction is completed, the method further comprises the step of separating and recovering the multi-level structure magnetic synergistic catalyst by applying a magnetic field outside the reaction system.

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