Continuous enzymatic preparation method of ursodeoxycholic acid and continuous production process

Abstract

The application discloses a continuous enzymatic preparation method and a continuous production process of ursodeoxycholic acid, and belongs to the technical field of biological catalysis and drug synthesis. The core of the method is as follows: a carrier with an isoelectric point greater than 9.5 is used to covalently fix a carbonyl reductase and a coenzyme regeneration enzyme, the surface of the carrier is positively charged under the reaction condition, the large-molecule coenzyme with negative electricity is locally concentrated near the enzyme activity center through electrostatic adsorption, the oil-in-water microemulsion reaction system is constructed to efficiently solubilize the substrate, the dynamic pH gradient regulation is implemented in the continuous flow reaction bed to cooperatively optimize the double-enzyme activity, the online membrane separation and extraction unit is integrated to realize the efficient interception and reflux of the unreacted coenzyme and the immediate removal of the product, and the process realizes the efficient and high-selectivity continuous synthesis of the UDCA, the coenzyme utilization efficiency and the production space-time yield are significantly improved, the catalyst operation stability is good, and the process is suitable for the large-scale production of the medical-grade UDCA.

Description

Technical Field

[0001] This invention relates to the fields of biocatalysis and drug synthesis technology, specifically to a continuous enzymatic catalytic preparation method and continuous production process for ursodeoxycholic acid. Background Technology

[0002] Ursodeoxycholic acid (UDCA) is a hydrophilic dihydroxy bile acid. As an important drug for the treatment of cholesterol gallstones and cholestatic liver diseases such as primary biliary cholangitis, it has been widely used in clinical practice for many years. With the continuous growth of market demand, the development of efficient, green and economical UDCA production processes has become a research hotspot.

[0003] Currently, the industrial production routes of UDCA mainly include chemical synthesis and biocatalysis. Chemical synthesis usually uses inexpensive cholic acid or chenodeoxycholic acid as starting materials and prepares UDCA through multiple chemical transformations, such as oxidation, reduction, and epimerization. This route is complicated, requires a large amount of organic solvents and chemical reagents, and often involves highly toxic chemicals. It has inherent defects such as serious environmental pollution, poor atom economy, and difficulty in controlling the optical purity of the product. Biocatalysis, especially enzyme catalysis, is considered a more promising green synthetic route due to its advantages such as mild conditions, high selectivity, and environmental friendliness. Among them, the selective reduction of 7-ketolithocholic acid 7-KLCA to UDCA using 7β-hydroxysteroid dehydrogenase 7β-HSDH is one of the most attractive routes. However, this reaction depends on the expensive coenzyme nicotinamide adenine dinucleotide NADH, and its in-situ regeneration is the key to achieving economic feasibility. Existing technologies usually adopt an enzyme coupling strategy that couples coenzyme regeneration enzymes, such as glucose dehydrogenase GDH and formate dehydrogenase FDH, with the main enzyme. Despite the significant advantages of enzyme catalysis, developing it into a continuous, stable, and efficient industrial-scale production process still faces a series of key technological challenges: The cost and recycling of coenzymes are issues. Coenzyme NAD(P)H is expensive, and its non-renewable, one-time addition would make production costs unbearable. Current technologies often use covalent coupling of coenzymes with macromolecules such as polyethylene glycol (PEG), i.e., macromolecularization, in order to achieve separation and recovery from products through size exclusion. However, in continuous flow systems, how to confine water-soluble macromolecular coenzymes within the enzyme reaction area for long-term, efficient recycling and prevent them from being lost with the product stream is the core bottleneck to ensuring the economic efficiency of the process. Simple physical retention is difficult to achieve high concentration enrichment of coenzymes at the reaction interface, resulting in low reaction efficiency and a limited actual turnover number (TTN) of coenzymes. The poor water solubility of the substrate 7-KLCA severely limits the actual substrate concentration in the reaction system, resulting in low reactor volume space-time yield and low equipment utilization. Although some studies have tried to use organic solvents or surfactants to construct two-phase systems to improve substrate solubility, how to form a stable, homogeneous dispersion system that is easy to flow continuously in a fixed-bed reactor without affecting enzyme activity and stability is an engineering problem. Enzyme-coupled systems involve the synergistic action of two or more enzymes, such as carbonyl reductase and coenzyme regenerase. The optimal pH, temperature and other conditions often differ. In traditional batch reactions or continuous reactions under constant conditions, it is difficult to optimize the reaction conditions at the same time. This may lead to the inhibition of the activity of one enzyme or an increase in side reactions, affecting the overall reaction efficiency, the optical purity of the product and the long-term operational stability of the catalyst. Most existing biocatalysis processes are still in the intermittent or batch feeding stage, which has problems such as low production efficiency, high labor intensity, and batch-to-batch product quality differences. Achieving continuous and automated operation from catalytic reaction and coenzyme cycle to product separation is an inevitable direction to improve production efficiency, reduce costs, and ensure product quality stability. However, the seamless integration and stable operation of multiple unit operations such as immobilized enzyme catalysis, two-phase mass transfer, online separation, and material circulation is technically complex.

[0004] Therefore, there is an urgent need in the field for a new method for producing UDCA that can overcome the above-mentioned defects, especially an enzyme catalytic preparation process that can achieve efficient in-situ recycling of coenzymes, overcome substrate mass transfer limitations, take into account the synergistic effect of multiple enzymes, and operate stably for a long time in an integrated continuous flow device, so as to meet the requirements of modern pharmaceutical industry for green manufacturing and process intensification. Summary of the Invention

[0005] The purpose of this invention is to provide a continuous enzymatic catalytic preparation method and continuous production process for ursodeoxycholic acid; In this invention, macromolecular coenzymes refer to coenzymes whose molecular weight has been increased through chemical modification, such as polyethylene glycol modification, for example, polyethylene glycol-modified oxidized nicotinamide adenine dinucleotide PEG-NAD. + It is used to transfer hydrogen in enzyme-catalyzed reactions, and its macromolecular properties facilitate subsequent separation and recycling. One method provides a continuous enzymatically catalyzed preparation method for ursodeoxycholic acid, comprising the following steps: S1, in the microemulsion reaction system, the substrate 7-ketolithocholic acid, macromolecular coenzyme, and an auxiliary substrate for coenzyme regeneration matched with coenzyme regeneration enzyme are mixed with two-phase solvents to form an oil-in-water microemulsion. S2, the microemulsion is passed through a reaction bed filled with a bifunctional immobilized carrier. The bifunctional immobilized carrier is covalently immobilized with carbonyl reductase and coenzyme regeneration enzyme. The isoelectric point of the carrier is greater than 9.5, so that the surface Zeta potential measured at 25°C and a reaction system conductivity of 1-5 mS / cm is +15mV to +50mV. Through electrostatic adsorption, the adsorption capacity of the macromolecular coenzyme on the carrier reaches more than 0.02 mmol / g. The enzymatic reduction reaction and coenzyme regeneration reaction are carried out simultaneously in the bed. S3, the microemulsion after reaction is subjected to online membrane separation. The molecular weight cutoff of the online membrane separation is set to be less than the molecular weight of the macromolecular coenzyme, so that the unadsorbed free macromolecular coenzyme is retained and refluxed to step S1, and the product ursodeoxycholic acid is discharged with the permeate. Within the reaction bed, the pH value of the microemulsion reaction system dynamically changes along the flow direction from 7.8-8.2 at the inlet to 6.5-7.0 at the outlet, forming a dynamic pH gradient.

[0006] Preferably, the carbonyl reductase is a 7β-hydroxysteroid dehydrogenase mutant; The combination of coenzyme regenerating enzyme and auxiliary substrate is selected from the combination of glucose dehydrogenase and glucose, or the combination of formate dehydrogenase and formate.

[0007] Preferably, the macromolecular coenzyme is polyethylene glycol-modified nicotinamide adenine dinucleotide, with a polyethylene glycol molecular weight of 10kDa-20kDa; the molecular weight cutoff for online membrane separation is 5kDa-8kDa.

[0008] Preferably, the volume ratio of the aqueous phase to the organic phase in the microemulsion reaction system is 3:1 to 5:1, and the volume percentage of n-hexanol in the organic phase of the two solvent phases is ≥60%. The two-phase solvent contains hydroxypropyl-β-cyclodextrin at a concentration of 8%-12% w / v, and the resulting microemulsion droplets have an average particle size of 200-500 nm.

[0009] Preferably, the bifunctional immobilization carrier is prepared by cross-linking amino-functionalized silica microspheres with glutaraldehyde, and the average pore size is 50nm-80nm.

[0010] Preferably, the liquid hourly space velocity (LHSV) in the reaction bed is 0.8 h⁻¹. -1 -2.0h -1 .

[0011] Preferably, the dynamic pH gradient is actively controlled by 3-5 buffer supply ports set along the reaction bed; The pH setting of the buffer solution added at each feed inlet decreases sequentially along the flow direction, so that the pH of the reaction system continuously decreases from the inlet value to the outlet value.

[0012] Preferably, the online membrane separation adopts a tangential flow filtration mode, and the membrane module is regularly backwashed to maintain separation performance.

[0013] Secondly, a continuous production process for ursodeoxycholic acid is provided, comprising: The permeate containing ursodeoxycholic acid was obtained using the above preparation method; The permeate is introduced into an online extraction unit, where continuous liquid-liquid extraction is performed using an organic solvent that is immiscible with the aqueous phase in the permeate.

[0014] The beneficial effects of this invention are as follows: Compared with the prior art, the present invention has the following beneficial effects: This technology enables efficient in-situ enrichment and recycling of coenzymes by employing an immobilized carrier with a highly positively charged surface, targeting negatively charged macromolecular coenzymes such as PEG-NAD. + A strong electrostatic adsorption effect is generated, which can enrich the local concentration of coenzyme within the carrier pores by hundreds of times compared to the bulk concentration. Combined with online membrane separation technology that precisely matches the molecular weight cutoff, the unadsorbed free coenzyme is efficiently retained and refluxed into the reaction system, achieving in-situ efficient recycling of coenzyme. The coenzyme recovery rate is as high as 96.5%, and the total turnover number (TTN) can reach 7.5 × 10⁻⁶. 4 This reduces the cost of using coenzymes; Significantly improved production efficiency and reactor utilization. By using hydroxypropyl-β-cyclodextrin-stabilized oil-in-water microemulsion as the reaction medium, the operating concentration of the poorly soluble substrate 7-ketolithocholic acid was successfully increased to over 50 mM, breaking the mass transfer limitation. Combined with continuous flow process, production efficiency was increased by orders of magnitude. To ensure the long-term operational stability and high selectivity of the catalytic system, a dynamic gradient from inlet pH 7.8-8.2 to outlet pH 6.5-7.0 is constructed within the reaction bed, while taking into account the optimal pH environment required for both carbonyl reduction and coenzyme regeneration reactions. This design enables the dual-enzyme system to maintain a high conversion rate of over 99% and a product optical purity of over 99.5% even after continuous operation for more than 240 hours. By setting 3-5 buffer supply ports, the pH of the reaction system can be precisely and linearly controlled, avoiding local pH changes that may be caused by traditional two-stage regulation, and providing a high-quality synergistic working environment for the dual-enzyme system. This invention seamlessly integrates unit operations such as continuous microemulsion feeding, fixed-bed enzyme catalysis, online membrane separation, and continuous liquid-liquid extraction. The online extraction unit, such as a centrifugal extractor, can achieve instantaneous and efficient separation of the product UDCA, avoiding product inhibition. The entire system exhibits excellent operational stability, and the catalyst avoids mechanical shear deactivation caused by intermittent stirring in plug flow mode, providing a reliable technical solution for the industrial continuous production of UDCA. Attached Figure Description

[0015] Figure 1 This is a flowchart of the continuous enzyme-catalyzed preparation method of ursodeoxycholic acid according to the present invention; Figure 2 The key physicochemical properties of the prepared bifunctional immobilized carrier are characterized by graphs. A is a graph showing the surface Zeta potential of the carriers in Example 1 and Comparative Example 1 as a function of pH. B is a bar graph showing the pore size distribution of the carrier in Example 1. Figure 3 This is a graph showing the measured data of the axial pH gradient within the reaction bed in Example 2; Figure 4 This is a graph showing the membrane flux maintenance effect during the operation of the online membrane separation unit in Example 2; Figure 5 This is a graph comparing the long-term operational stability of a representative embodiment of the present invention with that of a comparative example. Detailed Implementation

[0016] To further illustrate the technical means and effects adopted by the present invention to achieve its intended purpose, exemplary embodiments will be described in detail below, examples of which are illustrated in the accompanying drawings. In the following description relating to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. Rather, they are merely examples of methods and systems consistent with some aspects of this application as detailed in the appended claims.

[0017] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The singular forms “a” and “the” as used in this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0018] To better illustrate the purpose, technical solutions, and advantages of this application, the following description, in conjunction with specific embodiments and comparative examples, aims to provide a detailed understanding of the content of this application, rather than limiting it. All other embodiments obtained by those skilled in the art without inventive effort are within the protection scope of this application. Unless otherwise specified, the experimental reagents and instruments involved in the implementation of this application are commonly used reagents and instruments. In this application, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0019] The following detailed description of the specific implementation methods, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided in detail.

[0020] Example 1 This embodiment provides a complete preparation process for a bifunctional immobilized carrier, including the synthesis of amino-functionalized silica microspheres, the co-immobilization of 7β-hydroxysteroid dehydrogenase 7β-HSDH and glucose dehydrogenase GDH, and the preliminary confirmation of the immobilized enzyme activity. The preparation method of amino-functionalized silica microspheres is as follows: Weigh 2.0 g of monodisperse silica microspheres with an average particle size of 5 micrometers, place them in a 100 mL round-bottom flask, add 40 mL of anhydrous toluene, and sonicate for 10 minutes to ensure full dispersion. Under nitrogen protection, 2.0 mL of 3-aminopropyltriethoxysilane APTES was slowly added dropwise to the system. The mixture was refluxed and stirred at 110 °C for 8 hours. After the reaction was completed, the system was cooled to room temperature. The product was washed by centrifugation at 8000 rpm for 5 minutes each time with anhydrous toluene and anhydrous ethanol three times to completely remove unreacted silane reagent. The washed solid was placed in a vacuum drying oven and dried at 60°C for 6 hours to obtain amino-functionalized silica microspheres, denoted as SiO2-NH2.

[0021] Methods for co-immobilization of two enzymes: Take 200 mg of dried SiO2-NH2 microspheres and place them in 10 mL of phosphate buffer, 0.1 M, pH 7.5. Disperse them evenly by ultrasonication. Add 0.5 mL of 25% glutaraldehyde aqueous solution (from Sinopharm Chemical Reagent Co., Ltd.) to make the final concentration of glutaraldehyde in the system approximately 1.25% v / v. Place the mixture in a constant temperature shaker at 25℃ and crosslink it by shaking at 150 rpm for 2 hours. After cross-linking, the microspheres were collected by centrifugation at 8000 rpm for 5 minutes and washed three times with the above phosphate buffer to remove residual glutaraldehyde. The washed microspheres were then redispersed in 10 mL of phosphate buffer, 0.1 M, pH 7.5. To prepare the mixed enzyme solution, the laboratory-made recombinant 7β-HSDH mutant with a specific activity of 35 U / mg was mixed with commercial glucose dehydrogenase GDH (Beijing Solarbio Science & Technology Co., Ltd.) at a protein mass ratio of 1:1, with a total protein content of 20 mg, i.e., 10 mg of each enzyme was added, and dissolved in the same buffer solution. Add the mixed enzyme solution to the well dispersed microsphere suspension and react slowly at 100 rpm in a 4°C cold room for 12 hours to ensure that the enzyme and the activation carrier are fully combined. After immobilization, the solid was collected by centrifugation and washed repeatedly with phosphate buffer until the absorbance of the supernatant at 280 nm reached a stable baseline, indicating that the unfixed free protein had been washed away. The resulting wet immobilized enzyme microspheres are bifunctional immobilization carriers, denoted as SiO2-NH2@7β-HSDH / GDH, which can be used immediately for subsequent catalytic reactions or stored for a short period of time in a buffer at 4 °C.

[0022] Example 2 This embodiment provides a continuous enzymatic catalytic preparation method and continuous production process for ursodeoxycholic acid; The catalyst was prepared using the bifunctional immobilized support SiO2-NH2@7β-HSDH / GDH prepared by the method in Example 1. The main equipment is an integrated continuous flow experimental setup, which includes: Precision constant flow pump, constant temperature reaction column, jacketed glass column with an inner diameter of 10 mm and a bed volume of 15.7 mL, online pH monitor, multi-channel buffer replenishment system, tangential flow ultrafiltration membrane packaged with PES membrane with a molecular weight cutoff of 6 kDa, CINCV02 centrifugal extractor, and cascade product collection system. Reactor loading: 15.0g of wet immobilized carrier was wet-packed into the reaction column to form a uniform fixed bed. Please refer to Figure 1 The continuous enzymatic catalytic preparation method of ursodeoxycholic acid is as follows: S1, Preparation and feeding of microemulsion feed solution; In the aqueous phase, 10% w / v hydroxypropyl-β-cyclodextrin (HP-β-CD), 50 mM 7-ketolithocholic acid (7-KLCA), and 0.5 mM PEG-NAD were dissolved in 0.1 M phosphate buffer. + The PEG molecular weight was 20 kDa and the auxiliary substrate was 1.0 M glucose. The conductivity of the solution was adjusted to 3.2 mS / cm using NaCl. The organic phase consists of n-hexanol and ethyl acetate mixed at a volume ratio of 7:3, wherein n-hexanol accounts for 70% v / v; Emulsification involves mixing the aqueous phase and the organic phase at a volume ratio of 4:1, followed by high-speed shear emulsification to form an oil-in-water (W / O) microemulsion. The average droplet size was measured to be 320 nm. The prepared microemulsion was placed in the feed tank and pumped into the system at a flow rate of 15.7 mL / h using a constant flow pump. The initial pH of the feed microemulsion was adjusted to 8.2. S2, a continuous enzyme-catalyzed reaction, is dynamically regulated by pH gradient. The microemulsion enters the reaction bed filled with immobilized carrier. Four buffer supply ports are set along the flow direction of the bed for active control. The added buffer is 0.1M phosphate buffer, and its pH set values ​​along the flow direction are 7.9, 7.6, 7.3 and 7.0 respectively. The pH decrease value between adjacent supply ports is 0.3 units. By using online pH sensor monitoring and acceleration rate linkage control, a dynamic pH gradient is created within the bed, smoothly changing from approximately pH 8.2 at the inlet to approximately pH 7.0 at the outlet. The reaction column jacket is circulated with water at a constant temperature of 30°C. Under these conditions, 7-KLCA in the microemulsion is reduced to UDCA under the catalysis of immobilized 7β-HSDH, while GDH uses glucose to regenerate coenzymes, and the reaction proceeds continuously. S3, Online membrane separation and material circulation of the reaction solution; After the microemulsion flows out of the reaction bed, it immediately enters the tangential flow ultrafiltration membrane separation unit; The membrane module has a molecular weight cutoff of 6 kDa, which is less than that of the PEG-NAD used. + With a molecular weight of approximately 20 kDa, the unadsorbed free macromolecular coenzyme is effectively retained, and the retained liquid is returned to the feed tank in step S1 through the pipeline, thus realizing the continuous recycling of the coenzyme. The permeate containing the product UDCA then enters the subsequent process. To maintain separation performance, the membrane module is backwashed once every 24 hours for 5 minutes each time. Online extraction involves immediately introducing the permeate obtained in step S3, which is mainly aqueous, into an online extraction unit, namely a CINCV02 centrifugal extractor. Ethyl acetate, which is immiscible with the permeate, is used as the extractant. Three-stage countercurrent continuous extraction is carried out under the condition of an oil-water volume ratio of 1:3, i.e., organic phase: aqueous phase, at an operating temperature of 25°C. This step achieves immediate and efficient separation of the product UDCA from the reaction system. After the system was started, it ran continuously and stably for 240 hours.

[0023] Example 3 This embodiment provides a continuous enzymatic catalytic preparation method and continuous production process for ursodeoxycholic acid with different parameters; The system loading, catalyst, and core steps are the same as in Example 2, with the main parameter changes as follows: The microemulsion composition was adjusted with the aqueous phase to organic phase volume ratio to 5:1; the HP-β-CD concentration was adjusted to 8% w / v. Macromolecularized coenzymes use PEG-NAD with a molecular weight of 10 kDa. + The average particle size of the microemulsion after preparation is 380 nm. The reaction operation was carried out with the microemulsion feed having an initial pH of 7.8. The target outlet pH is 7.0, actively regulated through three buffer supply ports. The pH setpoints for the three ports along the flow direction are 7.5, 7.2, and 6.8, with pH decreases of 0.3 and 0.4 units between adjacent ports, respectively. The liquid hourly space velocity (LHSV) is 0.8 h⁻¹. -1 ; Membrane separation was performed using a PES membrane with a molecular weight cutoff of 5 kDa to ensure the separation of 10 kDa PEG-NAD. + Effective interception; The system operated continuously for 120 hours under these mild boundary conditions.

[0024] Example 4 This embodiment provides a continuous enzymatic catalytic preparation method and continuous production process for ursodeoxycholic acid with different parameters; The system and catalyst are the same as in Example 3, with the main parameter changes as follows: The microemulsion composition was adjusted to an aqueous phase to organic phase volume ratio of 3:1. The volume percentage of n-hexanol in the organic phase is 80%. The HP-β-CD concentration was adjusted to 12% w / v; Macromolecularized coenzymes using PEG-NAD + 20kDa, the average particle size of the microemulsion after preparation is 240nm; The reaction operation was carried out with the microemulsion feed initially at pH 8.2 and the target outlet pH at 6.5, which was adjusted by using three buffer replenishment ports at pH 7.9, 7.5, and 6.5. Liquid time space velocity (LHSV) increased to 2.0 h⁻¹. -1 ; The system ran continuously for 120 hours under these harsh conditions.

[0025] Example 5 This embodiment provides a continuous enzymatic catalytic preparation method for ursodeoxycholic acid using an FDH / formate system; The carrier was prepared according to the method in Example 1. A bifunctional immobilized carrier loaded with 7β-HSDH and formate dehydrogenase FDH with a specific activity of 120 U / mg was prepared and denoted as SiO2-NH2@7β-HSDH / FDH. The reaction was carried out in a continuous flow using the same system as in Example 2, with the auxiliary substrate replaced by 1.0 M sodium formate in the microemulsion aqueous phase, initial pH 7.9, and LHSV of 1.2 h. -1 The dynamic pH gradient was constructed using three feed ports at pH 7.6, 7.2, and 6.8, with the online membrane separation conditions the same as in Example 2. The system ran continuously for 120 hours.

[0026] Comparative Example 1 This comparative example uses unmodified pristine silica microspheres with an isoelectric point pI≈2-3 as a carrier. Glutaraldehyde crosslinking and dual-enzyme 7β-HSDH / GDH immobilization are performed using the same steps as in Example 1. The resulting carrier is denoted as SiO2@Enzymes. Continuous reaction testing is conducted under the same process conditions as in Example 2.

[0027] Comparative Example 2 Using the same reaction system and the same batch of the present invention carrier SiO2-NH2@7β-HSDH / GDH as in Example 2, the multi-buffered slurry feeding system was turned off, and the entire reaction bed was controlled at a constant pH of 7.5. All other conditions, such as microemulsion composition, LHSV, temperature, membrane separation, etc., were kept consistent with those in Example 2. The system was continuously run for 120 hours under constant pH conditions.

[0028] Comparative Example 3 To prepare a pure aqueous reaction solution, 7-KLCA was directly dissolved in 0.1 M phosphate buffer (pH 8.0). Due to its extremely low water solubility, the substrate concentration was only ~12 mM. The reaction solution contained PEG-NAD... + The glucose concentration was the same as that of the aqueous phase in Example 2. Under the conditions of 30°C and constant pH 8.0, 200 mL of this substrate solution was mixed with 15.0 g wet weight of the immobilized carrier from the same batch as in Example 2 in a stirred tank and the mixture was subjected to intermittent stirring reaction for 24 hours.

[0029] Comparative Example 4 Using a conventional mechanically stirred tank reactor, 15.0 g of the same immobilized carrier as in Example 2 (wet weight) and 500 mL of microemulsion reaction solution with the same composition as in step S1 of Example 2 were added. The reaction was carried out intermittently at 30°C, with a constant pH of 8.0 maintained by an automatic titrator, and a stirring speed of 200 rpm. Each batch was reacted for 20 hours. After the reaction was completed, the carrier was recovered by centrifugation and used for the reaction of the next batch of fresh reaction solution to examine the catalyst reuse performance.

[0030] Please refer to Figure 2 Key characterization and performance were tested: The surface zeta potential and isoelectric point were determined by dispersing the SiO2-NH2 support prepared in Example 1 in a simulated microemulsion aqueous phase with a conductivity of 3.0 mS / cm at 25°C. The zeta potential was +38 mV at pH 7.8 and +22 mV at pH 7.0, with an isoelectric point pI of 9.8. The zeta potential of the SiO2@Enzymes support in Comparative Example 1 was -25 mV under the same conditions at pH 7.8. Pore ​​size and specific surface area were determined using the BET nitrogen adsorption method. The specific surface area of ​​the SiO2-NH2 support was 118 m². 2 / g, the most probable pore size calculated by the BJH model is 66nm, and the main pore distribution is about 90% between 55-78nm; Electrostatic adsorption enrichment effect verification, in a solution containing 0.2 mM PEG-NAD + In a simulated microemulsion aqueous phase with pH 7.8 and conductivity 3.0 mS / cm, adsorption equilibrium experiments were conducted at 25℃. The experiments showed that the SiO2-NH2 support exhibited strong adsorption for PEG-NAD. + The adsorption capacity was 0.025 mmol / g, confirming the carrier's ability to efficiently enrich coenzymes via electrostatic adsorption, based on a carrier pore volume of 0.75 cm³. 3 Based on / g calculation, the theoretical local concentration of coenzyme in the pores reached 33.3mM, which is about 775 times higher than the bulk concentration of 0.043mM after adsorption equilibrium. To quantitatively evaluate the impact of the immobilization process on enzyme activity, the activity recovery rate of the immobilized enzyme was calculated. The activity recovery rate was defined as the percentage of the total activity exhibited by the immobilized carrier relative to the total activity of the corresponding free enzyme before immobilization. The calculation formula is as follows: Activity recovery rate = [Total activity of immobilized carrier / (Input free enzyme * specificity of the free enzyme)] * 100%; The activity recovery rate of 7β-HSDH was calculated. During the immobilization operation, a total of 10 mg of free enzyme, namely 7β-HSDH protein, was added. Its specific activity was 35 U / mg. The theoretical total activity of the added 7β-HSDH was 10 mg × 35 U / mg = 350 U. For the determination of immobilized enzyme activity, accurately weigh 200 mg, which is all of the above wet immobilized carrier, and load it into a small glass reaction column with a bed volume of about 0.5 mL. Dissolve 0.5 mM 7-ketolithocholic acid in an aqueous solution containing 5% hydroxypropyl-β-cyclodextrin and 0.15 mM NADH in Tris-HCl buffer (50 mM, pH 8.0), and pass it through the reaction column at a constant flow rate at 30 °C. The initial effluent was collected, and the NADH consumption rate was immediately determined using a UV spectrophotometer at a wavelength of 340 nm. The total 7β-HSDH activity exhibited by the 200 mg immobilized carrier was calculated to be 227.5 U. The activity recovery rate of 7β-HSDH is 65% when substituted into the formula.

[0031] The specific activity of GDH added is 250 U / mg, the amount of protein added is 10 mg, and the theoretical total activity of GDH added is 10 mg × 250 U / mg = 2500 U. Using the same batch of 200 mg immobilized carrier, 1.0 M glucose and 0.5 mM NAD were added. + Phosphate buffer, 50 mM, pH 7.5, was passed through a reaction column at a constant temperature of 30 °C. The eluent was collected, and the NADH generation rate at 340 nm was measured. The total GDH activity exhibited by the 200 mg immobilized carrier was calculated to be 1750 U. The GDH activity recovery rate, when substituted into the formula, is 70%. Example 5 was tested according to the method described in Example 1. The SiO2-NH2@7β-HSDH / FDH support of Example 5 showed a 7β-HSDH activity recovery rate of 62% and an FDH activity recovery rate of 68%.

[0032] For performance testing of continuous process operation, please refer to [reference needed]. Figure 5 : Table 1. Results of Example 2 (GDH / glucose system, LHSV=1.0h) -1 (240 hours) ; Table 2 Results of Example 3 (mild conditions, LHSV=0.8h) -1 (120 hours) ; Table 3. Results of Example 4 (Demanding conditions, LHSV=2.0h) -1 (120 hours) ; Table 4 Results of Example 5 (FDH / formate system, LHSV=1.2h) -1 (120 hours) ; Table 5 Comparative Example 1 Operating Results (Negative Charger, 24 Hours) ; Table 6 Results of Comparative Example 2 (constant pH 7.5, 120 hours) ; Please refer to Figure 3 In Example 2, the axial pH gradient of the bed after stabilization was measured. The measured pH values ​​from different locations on the bed were: 8.18 at the inlet, 7.85 at 25%, 7.52 at 50%, 7.15 at 75%, and 6.81 at the outlet, forming a smoothly decreasing gradient. Please refer to Figure 4 Regarding membrane separation performance, during 240 hours of operation in Example 2, the average membrane flux decay rate was 12%, and after backwashing (5 minutes) every 24 hours, the flux could be restored to more than 97% of the initial value. In Example 2, step S4, the online extraction efficiency, after three-stage countercurrent continuous extraction, showed a total extraction yield greater than 99%, and the residual concentration of UDCA in the aqueous phase was less than 0.5 mM. Coenzyme cycling and consumption, PEG-NAD was introduced at the start of the operation in Example 2. + The total mass of the 20kDa enzyme was 152.0mg, and 146.7mg was recovered after the experiment, with a coenzyme recovery rate of 96.5%. A total of 18.6g of UDCA (0.0474mol) was generated, and only 0.40mg of coenzyme was consumed during the operation. + Approximately 6.3 × 10 -7 The calculated total coenzyme turnover (TTN) was as high as 7.5 × 10⁻⁶ mol. 4 ; Spacetime productivity calculation: Spacetime productivity is a key engineering indicator for measuring production efficiency, and its calculation formula is as follows: Spacetime yield = (product yield) / (reactor volume × time); Calculations based on data from Example 2: The reactor volume, i.e. the bed volume, is 0.0157 L; Product formation rate, outlet UDCA concentration ≈ 50 mM × 99% ≈ 49.5 mM; The outflow volume is 15.7 mL per hour, and the LHSV is 1.0 h. -1 Therefore, the hourly UDCA production is 49.5 mmol / L × 0.0157 L / h ≈ 0.777 mmol / h; Substituting into the spacetime production formula, the calculated spacetime production rate is 19.4 g·L. -1 ·h -1 .

[0033] The aqueous intermittent reaction in Comparative Example 3, limited by substrate solubility, had a theoretical maximum space-time yield of only about 2.50 g·L⁻¹, calculated using the method described in Example 2. -1 ·h -1 ; Catalyst stability comparison: In the intermittent stirring process of Comparative Example 4, the activity of the immobilized support was severely reduced after one batch was reused, with a residual relative enzyme activity of <20%, which is in stark contrast to the stable operation of the continuous flow process of Example 2 for more than 200 hours.

[0034] The above examples, comparative examples, and test data fully demonstrate that the continuous enzymatic catalytic preparation method, continuous production process, and application of ursodeoxycholic acid provided by this invention, through the synergistic effect of key technical features such as electrostatic adsorption of coenzyme by positively charged carriers, dynamic pH gradient control, microemulsion reaction system, and online separation and circulation, achieves the industrial production goals of high conversion rate, high selectivity, high stability, high production efficiency, and low coenzyme consumption. The produced ursodeoxycholic acid has an optical purity (de value) consistently higher than 99.3% and a chemical purity exceeding 99.0%, fully meeting the quality requirements for pharmaceutical raw materials.

[0035] When a positively charged carrier is present, dynamic pH regulation causes the surface charge density of the carrier to vary between +22 mV and +38 mV, which precisely matches the coenzyme PEG-NAD. + The charge distribution achieves adsorption-desorption equilibrium; If the pH is constant, the charge density is constant, and the excessive adsorption of coenzyme on the carrier surface leads to an increase in mass transfer resistance. The 6kDa molecular weight cutoff of the membrane cycle matches the 20kDa coenzyme. If there is no pre-enrichment of positively charged carrier, the membrane flux will decrease by 70% within 24 hours, proving that the three must work together.

[0036] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A continuous enzymatically catalyzed preparation method for ursodeoxycholic acid, characterized in that, Includes the following steps: S1, in the microemulsion reaction system, the substrate 7-ketolithocholic acid, macromolecular coenzyme, and an auxiliary substrate for coenzyme regeneration matched with coenzyme regeneration enzyme are mixed with two-phase solvents to form an oil-in-water microemulsion. S2, the microemulsion is passed through a reaction bed filled with a bifunctional immobilized carrier. The bifunctional immobilized carrier is covalently immobilized with carbonyl reductase and coenzyme regeneration enzyme. The isoelectric point of the carrier is greater than 9.5, so that its surface Zeta potential measured at 25°C and a reaction system conductivity of 1-5 mS / cm is +15mV to +50mV. Through electrostatic adsorption, the adsorption capacity of the macromolecular coenzyme on the carrier reaches more than 0.02 mmol / g. The enzymatic reduction reaction and the coenzyme regeneration reaction are carried out simultaneously in the bed. S3, the microemulsion after reaction is subjected to online membrane separation. The molecular weight cutoff of the online membrane separation is set to be less than the molecular weight of the macromolecular coenzyme, so that the unadsorbed free macromolecular coenzyme is retained and refluxed to step S1, and the product ursodeoxycholic acid is discharged with the permeate. Within the reaction bed, the pH value of the microemulsion reaction system dynamically changes along the flow direction from a pH value of 7.8-8.2 at the inlet to a pH value of 6.5-7.0 at the outlet, forming a dynamic pH gradient.

2. The method according to claim 1, characterized in that, The carbonyl reductase is a 7β-hydroxysteroid dehydrogenase mutant; The combination of coenzyme regenerating enzyme and auxiliary substrate is selected from the combination of glucose dehydrogenase and glucose, or the combination of formate dehydrogenase and formate.

3. The method according to claim 1, characterized in that, The macromolecular coenzyme is polyethylene glycol-modified nicotinamide adenine dinucleotide, with a polyethylene glycol molecular weight of 10kDa-20kDa; the molecular weight cutoff for online membrane separation is 5kDa-8kDa.

4. The method according to claim 1, characterized in that, The volume ratio of the aqueous phase to the organic phase in the microemulsion reaction system is 3:1 to 5:1, and the volume percentage of n-hexanol in the organic phase of the two-phase solvent is ≥60%. The two-phase solvent contains hydroxypropyl-β-cyclodextrin at a concentration of 8%-12% w / v, and the resulting microemulsion droplets have an average particle size of 200-500 nm.

5. The method according to claim 1, characterized in that, The bifunctional immobilized carrier is prepared by cross-linking amino-functionalized silica microspheres with glutaraldehyde, and has an average pore size of 50nm-80nm.

6. The method according to claim 1, characterized in that, The liquid hourly space velocity (LHSV) within the reaction bed is 0.8 h⁻¹. -1 -2.0h -1 .

7. The method according to claim 1, characterized in that, The dynamic pH gradient is actively controlled by 3-5 buffer supply ports set along the reaction bed. The pH setting of the buffer solution added at each feed inlet decreases sequentially along the flow direction, so that the pH of the reaction system continuously decreases from the inlet value to the outlet value.

8. The method according to claim 1, characterized in that, The online membrane separation adopts a tangential flow filtration mode, and the membrane module is regularly backwashed to maintain separation performance.

9. A continuous production process for ursodeoxycholic acid, characterized in that, include: A permeate containing ursodeoxycholic acid was obtained by the preparation method according to any one of claims 1-8; The permeate is introduced into an online extraction unit, where continuous liquid-liquid extraction is performed using an organic solvent that is immiscible with the aqueous phase in the permeate.

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