Method and device for generating ursodesoxycholic acid through continuous flow catalysis of chenodeoxycholic acid

Abstract

The invention relates to the field of biological catalysis, and discloses a method and a device for generating ursodesoxycholic acid through continuous flow catalysis of chenodeoxycholic acid. Comprising the following steps: (1) in the presence of first immobilized cells, carrying out a first catalytic reaction on a reaction system containing chenodeoxycholic acid to obtain a first product, the first immobilized cells expressing 7 alpha-hydroxysteroid dehydrogenase and lactic dehydrogenase; the concentration of the chenodeoxycholic acid in the reaction system is 40 to 70 mM; (2) adjusting the pH value of the first product to 8-9, and then in the presence of a second immobilized cell, carrying out a second catalytic reaction on the first product after the pH value is adjusted to obtain ursodesoxycholic acid, and the second immobilized cell expresses 7 beta-hydroxysteroid dehydrogenase and glucose dehydrogenase. According to the method, the stable activity of a catalytic system is kept, efficient conversion of high-concentration CDCA is achieved, meanwhile, precipitation of 7-KLCA is thoroughly avoided, and the synthesis efficiency, the product purity and the process continuity of UDCA are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis, and more specifically to a method and apparatus for the continuous flow catalytic generation of chenodeoxycholic acid from ursodeoxycholic acid. Background Technology

[0002] Ursodeoxycholic acid (UDCA) is a hydrophilic secondary bile acid with significant cell protection, choleretic, anti-inflammatory, immunomodulatory, and cholesterol metabolism-improving effects. It is an important clinical drug for the treatment of primary biliary cholangitis (PBC), cholesterol gallstones, various cholestatic liver diseases, and other hepatobiliary diseases.

[0003] Chemical synthesis methods typically involve multi-step reactions involving the inversion of the 7-hydroxyl group configuration, which presents problems such as numerous steps, poor stereoselectivity, harsh reaction conditions, high energy consumption, use of toxic reagents, and serious environmental pollution.

[0004] In contrast, biocatalytic methods (including free enzyme catalysis, immobilized enzyme catalysis, and whole-cell catalysis) use chenodeoxycholic acid (CDCA) as a cheap and readily available starting substrate, achieving conversion through a two-step enzymatic reaction: first, 7α-hydroxysteroid dehydrogenase (7α-HSDH) catalyzes the oxidation of the 7α-hydroxy group of CDCA to the intermediate 7-ketolithocholic acid (7-KLCA), a step dependent on NAD+. + As a cofactor, cofactor regeneration is typically achieved by coupling with lactate dehydrogenase (LDH); subsequently, 7β-hydroxysteroid dehydrogenase (7β-HSDH) catalyzes the stereoselective reduction of the 7-keto group at the 7-position of 7-KLCA to a 7β-hydroxy group, generating the target product UDCA. This step relies on NADPH as the reducing power and is typically coupled with glucose dehydrogenase (GDH) for cofactor regeneration. This method features mild reaction conditions, is environmentally friendly, and exhibits high stereoselectivity, representing the future development direction of UDCA industrial production.

[0005] Current biocatalysis processes mostly employ batch or semi-continuous operation. In recent years, some studies have explored continuous flow production to improve efficiency and scalability. Typical existing technologies primarily utilize immobilized enzymes (…). immobilized enzymesConstructing a two-stage series continuous flow reactor (e.g., using co-immobilized 7α-HSDH / LDH and 7β-HSDH / GDH on a support) typically limits the substrate CDCA concentration to around 5-20 mM, achieving relatively high conversion rates. However, it still suffers from the following significant drawbacks: 1. Continuous flow catalytic conversion of CDCA to 7-KLCA primarily utilizes immobilized enzymes, resulting in low substrate concentrations; 2. During the continuous flow catalytic conversion, after the first catalytic conversion, the intermediate product 7-KLCA precipitates from the substrate solution, affecting the second catalytic step; 3. Byproducts are easily generated during the reaction, impacting overall catalytic efficiency; 4. Most continuous flow processes rely on pure enzyme immobilization, which is costly, has relatively poor stability, and rarely reports the use of immobilized whole cells (…). immobilized whole cells This enables efficient two-step cascaded transformation of continuous flow.

[0006] In summary, facing problems such as low yield, poor operational stability, and high cost, there is an urgent need to develop a new, efficient, and stable continuous flow catalytic system for the conversion of chenodeoxycholic acid to ursodeoxycholic acid. Summary of the Invention

[0007] The purpose of this invention is to overcome the problems of low volume yield due to low substrate concentration, easy precipitation of intermediate product 7-KLCA, easy generation of by-products, resulting in reduced catalytic efficiency and poor stability in existing technologies. This invention provides a method and apparatus for the continuous flow catalytic conversion of chenodeoxycholic acid to ursodeoxycholic acid. This method and apparatus achieve efficient conversion of high-concentration CDCA to UDCA in continuous flow catalysis, significantly improving volume yield and apparatus utilization efficiency. It solves the problems of easy precipitation of product 7-KLCA and easy generation of by-products during the CDCA conversion process, and achieves stable operation of the entire continuous flow process, resulting in significant economic and social benefits.

[0008] To achieve the above objectives, the present invention provides a method for the continuous flow catalytic conversion of chenodeoxycholic acid to ursodeoxycholic acid, the method comprising the following steps: (1) In the presence of the first immobilized cells, a first catalytic reaction is carried out on a reaction system containing chenodeoxycholic acid to obtain a first product, wherein the first immobilized cells express 7α-hydroxysteroid dehydrogenase and lactate dehydrogenase; the concentration of chenodeoxycholic acid in the reaction system is 40-70 mM. (2) Adjust the pH of the first product to 8-9, and then, in the presence of the second immobilized cell, perform a second catalytic reaction on the pH-adjusted first product to obtain ursodeoxycholic acid; the second immobilized cell expresses 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase.

[0009] A second aspect of the present invention provides an apparatus for the continuous flow catalytic conversion of chenodeoxycholic acid to ursodeoxycholic acid, the apparatus comprising a first reactor, a second reactor, a pumping system, and a pH adjustment device; wherein, the first reactor is loaded with a first immobilized cell; the second reactor is loaded with a second immobilized cell; the pumping system is connected between the first reactor and the second reactor for pumping reactants from the first reactor to the second reactor; the pH adjustment device is disposed in the flow path between the first reactor and the second reactor for adjusting the pH value of the first product flowing out of the first reactor; the first immobilized cell and the second immobilized cell are as described above.

[0010] Through the above technical solution, this invention, by setting up first immobilized cells expressing 7α-hydroxysteroid dehydrogenase and lactate dehydrogenase in the first reactor and second immobilized cells expressing 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase in the second reactor, combined with a continuous flow design of "adding alkaline substances to adjust the pH to alkalinity after the first step of catalysis before entering the second step of catalysis," not only fills the gap in immobilized cell systems for the conversion of CDCA to UDCA, maintaining the stable activity of the catalytic system and effectively replacing the costly and unstable immobilized enzyme system, but also achieves efficient conversion of high-concentration CDCA. Simultaneously, it completely avoids the impact of the intermediate product 7-KLCA precipitation on the second step of catalysis, suppresses the problems of decreased catalytic efficiency and increased byproducts caused by a drop in system pH, and significantly improves the synthesis efficiency, product purity, and process continuity of UDCA, providing reliable support for industrial production. Attached Figure Description

[0011] Figure 1 This is a flowchart of the UDCA biocatalytic synthesis process in Example 1; Figure 2 This is a comparison diagram of the temperature gradient between whole cells and immobilized cells in Example 1; Figure 3 This is a comparison of the acid and alkali resistance of whole cells and immobilized cells in Example 1; Figure 4 This is a flowchart of the continuous flow catalytic conversion of CDCA to UDCA in Example 2.

[0012] Explanation of reference numerals in the attached figures 1. Reaction system storage unit: Loads pre-prepared reaction system (containing CDCA, KPB buffer, pyruvate, glucose, NAD) + NADP + (pH 7-8), the snowflake symbol on the outside of the bottle indicates that the system needs to be stored at low temperature (0℃-30℃).

[0013] 2. First pumping unit: a peristaltic pump, connected to the reaction system storage unit and the first reactor, used to pump the reaction system into the first reactor at a uniform speed and control the feed rate to match the efficiency of the first catalytic reaction.

[0014] 3. First reactor: The reactor is loaded with the first immobilized cells (E. coli expressing 7-HSDH and LDH) on agar as a carrier to realize the oxidation reaction of CDCA to 7-KLCA; the outer side of the reactor is marked with the type of immobilized cells loaded with it.

[0015] 4. pH Adjustment Unit: Includes NaOH storage bottle, pH sensor (electronic detection device), and mixing tank (container with stirring function): The NaOH storage bottle is connected to the mixing tank via a pipeline to provide the alkalinity regulator; The pH sensor monitors the pH value of the first product in the mixing tank in real time, and controls the amount of NaOH injected after receiving the feedback signal, so as to precisely adjust the pH to 8-9. The snowflake symbol on the outside of the mixing tank indicates that the first product needs to be kept in a low-temperature environment.

[0016] 5. Second pumping unit: Connects the first reactor and the pH adjustment unit, used to pump the first product from the first reactor into the mixing tank for pH adjustment.

[0017] 6. Third pumping unit: Connects the pH adjustment unit to the second reactor and is used to pump the first product after pH adjustment into the second reactor.

[0018] 7. Second reactor: The second reactor is loaded with agar-based immobilized cells (E. coli expressing 7β-HSDH and GDH) to achieve the reduction reaction of 7-KLCA to UDCA.

[0019] 8. Product collection unit: Connected to the outlet of the second reactor, used to collect the final product (UDCA). The product components marked on the bottom of the bottle are the final components of the reaction system after two-step catalysis.

[0020] 9. Flow path identification: The solid black line represents the main channel, corresponding to the main flow paths of the reaction system, the first product, and the final product. The blue dashed line represents the auxiliary channel, corresponding to the injection path of the pH adjuster (NaOH). Detailed Implementation

[0021] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0022] The first aspect of this invention provides a method for the continuous flow catalytic conversion of chenodeoxycholic acid to ursodeoxycholic acid, the method comprising the following steps: (1) In the presence of the first immobilized cells, a first catalytic reaction is carried out on a reaction system containing chenodeoxycholic acid to obtain a first product, wherein the first immobilized cells express 7α-hydroxysteroid dehydrogenase and lactate dehydrogenase; the concentration of chenodeoxycholic acid in the reaction system is 40-70 mM. (2) Adjust the pH of the first product to 8-9, and then, in the presence of the second immobilized cell, perform a second catalytic reaction on the pH-adjusted first product to obtain ursodeoxycholic acid; the second immobilized cell expresses 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase.

[0023] This invention, by setting up a first immobilized cell expressing 7α-hydroxysteroid dehydrogenase and lactate dehydrogenase in a first reactor and a second immobilized cell expressing 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase in a second reactor, combined with a continuous flow design of "adding alkaline substances to adjust the pH to alkalinity after the first step of catalysis before proceeding to the second step of catalysis," not only fills the gap in immobilized cell systems for the conversion of CDCA to UDCA, maintaining stable catalytic activity and effectively replacing the costly and unstable immobilized enzyme systems, but also achieves efficient conversion of high-concentration CDCA. Simultaneously, it completely avoids the impact of the intermediate product 7-KLCA precipitation on the second step of catalysis, suppresses the problems of decreased catalytic efficiency and increased byproducts caused by pH drops, and significantly improves the synthesis efficiency, product purity, and process continuity of UDCA, providing reliable support for industrial production.

[0024] According to the present invention, preferably, the first immobilized cell and the second immobilized cell are each independently selected from immobilized *Escherichia coli* and / or immobilized yeast. The inventors have found that using immobilized *Escherichia coli* or yeast as carrier cells can significantly reduce enzyme purification costs, while the whole-cell system provides a natural cofactor regeneration environment and better enzyme stability.

[0025] More preferably, the first immobilized cell and the second immobilized cell are each independently immobilized *E. coli*. The inventors discovered in their research that immobilized *E. coli* further improves enzyme expression efficiency and cell activity, achieving rapid conversion at high substrate concentrations (>50 mM) with significantly increased yield, while also facilitating large-scale fermentation preparation of immobilized cells.

[0026] According to the present invention, preferably, the immobilization carriers for the first and second immobilized cells are each independently selected from at least one of agar, alginate, calcium alginate, and polyacrylamide. These natural or synthetic polymer carriers have good biocompatibility, mechanical strength, and low toxicity, and can effectively protect cells from shear forces and substrate toxicity. The inventors have found that this preferred carrier improves the mechanical stability and reusability of the immobilized cells, reduces cell leakage, extends catalyst lifetime, and supports the long-term stable operation of continuous flow reactors.

[0027] More preferably, the immobilization carrier for the first and second immobilized cells is agar. Agar, as a thermogel carrier, offers a gentle immobilization process, uniform pore structure, minimal damage to cell viability, and is inexpensive. The inventors discovered in their research that agar carriers further improve cell embedding efficiency and diffusion performance, resulting in more efficient substrate / product mass transfer, higher catalytic activity retention, and suitability for continuous transformation of high-concentration substrates.

[0028] According to the present invention, preferably, in step (1), the reaction system comprises buffer solution, chenodeoxycholic acid, pyruvate, glucose, and NAD+. + and NADP + The pH of the reaction system is 7-8. The inventors discovered in their research that this optimized system design significantly reduces the cost of cofactors, improves reaction economy, and maintains high conversion rates and low byproduct formation.

[0029] More preferably, based on the reaction system, the concentration of pyruvate is 80-120 mM; the concentration of glucose is 80-120 mM; the concentration of NAD+ is 0.05-0.5 mM; and the concentration of NADP... + The concentration of the buffer is 0.1-1 mM; the buffer solution is KPB buffer; more preferably, it is 80-12 mM KPB buffer. The inventors discovered in their research that this preferred concentration range achieves high substrate loading, efficient cofactor regeneration, and reaction equilibrium. High concentrations of CDCA (far exceeding the conventional 5-20 mM) achieved a 3-5 fold increase in yield per unit volume, while avoiding intermediate precipitation and inhibition, significantly improving industrial production efficiency.

[0030] According to the present invention, preferably, in step (2), the pH adjuster for adjusting the pH is an alkaline substance, preferably selected from at least one of sodium hydroxide, potassium hydroxide, and sodium carbonate. The inventors have found in their research that the preferred alkaline substance effectively prevents the precipitation of 7-KLCA under acidic conditions, ensuring the smooth progress of the second catalytic step and improving the overall conversion rate and product purity.

[0031] According to the present invention, preferably, all reactions are carried out at 0°C-30°C. The inventors have found that this preferred operating temperature improves the long-term stability of immobilized cells, reduces byproduct formation, extends catalyst lifespan, and supports the stability of continuous flow processes and the acquisition of high-purity products.

[0032] A second aspect of this invention provides an apparatus for the continuous flow catalytic conversion of chenodeoxycholic acid to ursodeoxycholic acid. The apparatus includes a first reactor, a second reactor, a pumping system, and a pH adjustment device. The first reactor is loaded with first immobilized cells; the second reactor is loaded with second immobilized cells; the pumping system is connected between the first and second reactors for pumping reactants from the first reactor to the second reactor; the pH adjustment device is disposed in the flow path between the first and second reactors for adjusting the pH value of the first product exiting the first reactor; the first and second immobilized cells are as described above. The inventors have found that this apparatus design achieves seamless connection of the two-step cascade reaction and precise pH control, avoiding efficiency losses caused by intermediate precipitation and pH drift, improving the stability and automation of the continuous flow process, and is suitable for industrial-scale production.

[0033] According to the present invention, preferably, the pH adjustment device includes a pH adjuster injection system, a pH sensor, and a controller; the pH sensor is disposed in the flow path and is used to monitor the pH value of the first product; the controller is electrically connected between the pH sensor and the pH adjuster injection system, and is used to receive the monitoring signal from the pH sensor and control the pH adjuster injection system to inject alkaline substances. The inventors have found that by adopting the above-mentioned preferred method, the lag and error of manual pH adjustment are avoided, ensuring the complete dissolution of the intermediate product 7-KLCA. Furthermore, automated and precise control reduces the excessive use of alkaline adjusters, lowers the risk of pH fluctuations in subsequent reaction systems, and provides a stable environmental guarantee for the smooth operation of the continuous flow process.

[0034] In the following examples, Escherichia coli BL21 was purchased from Tolo Harbour; chenodeoxycholic acid (CDCA, purity ≥98%) was purchased from Maclean's; and the remaining raw materials and reagents were commercially available products.

[0035] Example 1: Preparation of immobilized cells Figure 1 The flowchart of the biocatalytic synthesis of UDCA illustrates the core principle of the two-step enzymatic reaction of this invention: the substrate CDCA (chenodeoxycholic acid) is catalyzed by 7α-hydroxysteroid dehydrogenase (7α-HSDH) and the cofactor NAD. +With the participation of [unclear], oxidation produces the intermediate product 7-KLCA (7-ketolithocholic acid). Lactate dehydrogenase (LDH) coupled with sodium pyruvate can achieve NAD+ [unclear]. + The cells undergo cyclic regeneration; subsequently, 7-KLCA is reduced to the target product UDCA (ursodeoxycholic acid) under the catalysis of 7β-hydroxysteroid dehydrogenase (7β-HSDH) and the participation of the cofactor NADPH. Glucose dehydrogenase (GDH) coupled with glucose enables the cyclic regeneration of NADPH. The first and second immobilized cells prepared in this embodiment correspond to the two catalytic steps in this reaction pathway, laying the foundation for continuous flow processes.

[0036] 1. Construction of recombinant bacteria: The plasmid pRSFDuet-1-Sm7α-HSDH was transformed into the first sample of E. coli BL21 competent cells, and screened on LB tread plates to obtain recombinant E. coli expressing only 7α-hydroxysteroid dehydrogenase; the plasmid pRSFDuet-1-LDH was transformed into the second sample of E. coli BL21 competent cells, and screened to obtain recombinant E. coli expressing only lactate dehydrogenase. These two types of recombinant E. coli were the core recombinant engineered bacteria of the first immobilized cells; the plasmid pRSFDuet-1-7β-HSDH was transformed into the third sample of E. coli BL21 competent cells, and screened on LB tread plates to obtain recombinant E. coli expressing only 7β-hydroxysteroid dehydrogenase; the plasmid pRSFDuet-1-GDH was transformed into the fourth sample of E. coli BL21 competent cells, and screened to obtain recombinant E. coli expressing only glucose dehydrogenase. These two types of recombinant E. coli were the core recombinant engineered bacteria of the second immobilized cells.

[0037] All the above plasmids were synthesized by GenScript and were previously described in the literature: Boosting the Biosynthesis of Ursodeoxycholic Acid via a Novel 7β-Hydroxysteroid Dehydrogenase from Olsenella sp. and Biotransformation Systematic Optimization.

[0038] 2. Bacterial culture: The four recombinant Escherichia coli strains were inoculated into LB liquid medium (5 g yeast extract, 10 g NaCl, 10 g peptone dissolved in 1 L water) and cultured at 37°C with shaking at 200 rpm until the logarithmic growth phase. The cells were collected by centrifugation at 8000 rpm for 10 min, washed twice with 100 mM KPB buffer, and resuspended to a concentration of 1 × 10⁻⁶ cells / mL. 8 CFU / mL, for later use.

[0039] 3. Agar Immobilization: Precisely prepare a 5.5% (v / v) agar aqueous solution, autoclave at 121°C for 20 min, and then cool to 50°C to avoid high temperature damage to cell viability; mix equal volumes of resuspensions of two recombinant E. coli strains expressing 7α-hydroxysteroid dehydrogenase and lactate dehydrogenase, and then rapidly mix with the cooled agar aqueous solution at a 1:1 (v / v) ratio. Pour the mixture into a sterile spherical mold (5 mm in diameter), allow it to cool and solidify naturally at room temperature for 30 min, and then demold to obtain the first immobilized cells; mix equal volumes of resuspensions of two recombinant E. coli strains expressing 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase, and then rapidly mix with the cooled agar aqueous solution at a 1:1 (v / v) ratio. After the same molding, solidification, and demolding steps, obtain the second immobilized cells; at the same time, retain equal volumes of unimmobilized resuspensions of each recombinant E. coli, mix them in the corresponding proportions, and use them as a free cell control group.

[0040] 4. Performance verification of immobilized cells: 4.1 Temperature Stability Verification Experiment Design: The first immobilized cells, the second immobilized cells, and the corresponding free cells were incubated at constant temperatures of 25℃, 30℃, 35℃, and 40℃ for 2 hours, respectively. Then, each group of cells was added to a standard reaction system (100 mM KPB buffer, 10 mM CDCA, 100 mM sodium pyruvate, 100 mM glucose, 0.15 mM NAD). + 0.6 mM NADP + The reaction was carried out at 37°C and 200 rpm for 1 hour in pH 7. The conversion rate of CDCA was detected by HPLC, and the results are as follows: Figure 2 As shown, within the temperature range of 25℃–40℃, the catalytic conversion efficiency of the first immobilized cell and the second immobilized cell is comparable to that of the corresponding free cell, both remaining above 90% (91% conversion rate for immobilized cells and 92% for free cells at 25℃; 90% conversion rate for immobilized cells and 91% for free cells at 40℃).

[0041] 4.2 Acid-base stability verification experiment design: The first immobilized cells, the second immobilized cells, and the corresponding free cells were immersed in 100 mM KPB buffer solutions at pH 5, 6, 7, 8, and 9, respectively, and incubated at 25°C for 12 hours. Subsequently, a catalytic reaction was carried out in the above standard reaction system. The CDCA conversion rate was detected by HPLC. The results are as follows: Figure 3 As shown, by Figure 3 As shown in Figures A and C, after incubation for 12 hours within a pH range of 5–9, the catalytic conversion rate of the first and second immobilized cells remained above 85% (86% at pH 5, 94% at pH 7, and 88% at pH 9). Figure 3As shown in Figures B and D, the conversion rate of free cells decreased to 58% and 55% at pH 5 and pH 9, respectively, indicating a significant loss of cell viability. These results confirm that agar immobilization significantly enhances the acid and alkali resistance of cells, enabling them to withstand the effects of pH decreases in continuous flow reactions.

[0042] Example 2: Method for Continuous Flow Catalytic Generation of UDCA from CDCA Figure 4 This is a flowchart of the continuous flow catalytic conversion of CDCA to UDCA. The diagram clearly shows the component connections, material flow direction, and core reaction nodes of the continuous flow device used in this embodiment. The corresponding meanings of the reference numerals in the diagram are as follows: 1-First reactor, 2-Second reactor, 3-Peristaltic pump 1 (reaction system transport), 4-Peristaltic pump 2 (product transport), 5-Peristaltic pump 3 (NaOH transport), 6-pH adjustment device, 7-First immobilized cell, 8-Second immobilized cell, 9-Reaction system storage bottle, 10-NaOH storage bottle, 11-Intermediate product adjustment section, 12-Final product collection bottle.

[0043] 1. The experimental setup (pH of the reaction system adjusted to 7.5) uses a customized continuous flow reactor, mainly including: Reaction system storage bottle: 2L capacity, to contain the optimized reaction system, placed in an ice bath to maintain low temperature; First reactor: 50 mL in volume, filled with the first immobilized cells prepared in Example 1, used to catalyze the oxidation of CDCA to 7-KLCA; Collection and pH adjustment device: Located at the outlet of the first reactor, it includes a 10 mL collection tube, a 1 M NaOH storage bottle and a peristaltic pump 3 (for precise addition of NaOH solution), and is equipped with an online pH monitor to monitor the pH of the collected solution in real time; Second reactor: 50 mL in volume, filled with the second immobilized cells prepared in Example 1, used to catalyze the reduction of 7-KLCA to UDCA; Pumping system: Peristaltic pump 1 (transports the reaction system to the first reactor) and peristaltic pump 2 (transports the pH-adjusted product to the second reactor), both pump speeds can be precisely adjusted; Final product collection bottle: 2 L volume, placed in an ice bath, to collect the product liquid flowing out of the second reactor.

[0044] 2. Process parameters Reaction system: 100 mM KPB buffer, 50 mM CDCA, 100 mM sodium pyruvate, 100 mM glucose, 0.15 mM NAD + 0.6 mM NADP + ; Operating conditions: The reaction system and the collected liquid were kept on ice throughout the process, maintaining a temperature of 0-10℃; the flow rates of peristaltic pumps 1 and 2 were both set to 1 mL / min; the addition rate of NaOH solution was adjusted by peristaltic pump 3 to keep the pH of the collected liquid at the outlet of the first reactor stable at 9.0±0.1. Pretreatment of immobilized cells: Before use, the first and second immobilized cells prepared in Example 1 were incubated at 37°C for 3 hours and then incubated at pH 7 for 12 hours. Running time: 72 hours of continuous operation, with 1 mL samples taken from the outlets of the first and second reactors every 12 hours for detection and analysis.

[0045] 3. Solution to the problem of 7-KLCA precipitation: The state of the collected liquid at the outlet of the first reactor was observed throughout the process. No precipitate was visible to the naked eye, which confirms that the method provided by the present invention can solve the problem of easy precipitation of 7-KLCA in the prior art and ensure the smooth operation of the continuous flow process. High-concentration substrate conversion efficiency: At a CDCA concentration of 50 mM (far exceeding the 10 mM commonly used in existing continuous flow processes), the average CDCA conversion rate in the first reactor reached 94.5%, and the average yield of 7-KLCA was 94.2%; in the second reactor, the average conversion rate of 7-KLCA reached 92.3%, and the average yield of UDCA was 91.8%, with a byproduct content of less than 2%, confirming that the present invention can achieve efficient conversion of high-concentration CDCA. Process continuity and stability: During 72 hours of continuous operation, the UDCA yield decreased from the initial 92.5% to 89.3%, a decrease of only 3.2%, and the catalytic activity of the immobilized cells remained stable. Moreover, the pH of the system did not drift significantly during the reaction process, and remained within the range of 7-9. This solves the problems of "gradual decrease in system pH affecting catalytic efficiency" and "easy generation of by-products" in the prior art, and fully verifies the superiority of the immobilized cell system and the stability of the continuous flow process provided by this invention.

[0046] Example 3 The first and second immobilized cells were prepared according to the method provided in Example 1, except that the agar in Example 1 was replaced with alginate (3% by weight), calcium alginate (4% by weight), and polyacrylamide (8% by weight), respectively, while the rest remained unchanged. Then, the continuous flow catalytic generation of UDCA from CDCA was carried out according to the method provided in Example 2, and the yield of UDCA was detected.

[0047] Results: The yield of UDCA was 76.2% in the alginate carrier group, 78.5% in the calcium alginate carrier group, and 75.7% in the polyacrylamide carrier group. Although these yields were lower than those in the 5.5% agar carrier group (91.8%), they were all superior to the existing immobilized enzyme system (≤80%).

[0048] Example 4 The continuous flow catalytic conversion of CDCA to UDCA was carried out according to the method provided in Example 2, except that the alkaline substance NaOH was replaced with potassium hydroxide (KOH) and sodium carbonate (Na2CO3), respectively. The pH of the collected liquid at the outlet of the first reactor was adjusted to 9, while other parameters remained unchanged, and the UDCA yield was measured.

[0049] Results: The KOH group achieved a 100% dissolution rate of 7-KLCA and a UDCA conversion rate of 81.2%; the Na2CO3 group achieved a 7-KLCA dissolution rate of 85.3% and a UDCA conversion rate of 69.2%, both demonstrating the ability to dissolve and efficiently convert 7-KLCA.

[0050] Example 5 The continuous flow catalytic conversion of CDCA to UDCA was carried out according to the method provided in Example 2, except that the CDCA concentration was set to 30 mM, 50 mM and 70 mM respectively, while the rest remained unchanged, and the UDCA conversion rate was detected.

[0051] Results: The UDCA conversion rates were 96.3% in the 30 mM group, 91.8% in the 50 mM group, and 78.9% in the 70 mM group, all of which were significantly higher than the 5-20 mM concentrations commonly used in existing processes (average conversion rate ≤85%).

[0052] Example 6 The method provided in Example 2 was used to catalyze the conversion of CDCA to UDCA in a continuous flow, except that the first immobilized cells and the second immobilized cells were not incubated before use, while the rest remained the same, and the conversion rate of UDCA was found to be 80.7%.

[0053] Comparative Example 1 The first and second immobilized cells were prepared according to the method provided in Example 1, except that the second immobilized cells did not express 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase. Then, the continuous flow catalysis of CDCA to UDCA was performed according to the method provided in Example 2, and the yield of UDCA was detected to be 0%.

[0054] Comparative Example 2 The continuous flow catalytic conversion of CDCA to UDCA was carried out according to the method provided in Example 2, except that the pH was not adjusted using the alkaline substance NaOH, while other parameters remained unchanged, and the UDCA yield was found to be 38.7%.

[0055] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for the continuous flow catalytic conversion of chenodeoxycholic acid to ursodeoxycholic acid, characterized in that, The method includes the following steps: (1) In the presence of the first immobilized cells, a first catalytic reaction is carried out on a reaction system containing chenodeoxycholic acid to obtain a first product, wherein the first immobilized cells express 7α-hydroxysteroid dehydrogenase and lactate dehydrogenase; the concentration of chenodeoxycholic acid in the reaction system is 40-70 mM. (2) Adjust the pH of the first product to 8-9, and then, in the presence of the second immobilized cell, perform a second catalytic reaction on the pH-adjusted first product to obtain ursodeoxycholic acid; the second immobilized cell expresses 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase.

2. The method according to claim 1, characterized in that, The first immobilized cell and the second immobilized cell are each independently selected from immobilized Escherichia coli and / or immobilized yeast; Preferably, the first immobilized cell and the second immobilized cell are each independently immobilized Escherichia coli.

3. The method according to claim 1, characterized in that, The immobilization carriers for the first and second immobilized cells are each independently selected from at least one of agar, alginate, calcium alginate, and polyacrylamide.

4. The method according to claim 3, characterized in that, The immobilization carriers for the first and second immobilized cells are each independently agar.

5. The method according to claim 1, characterized in that, In step (1), the reaction system comprises buffer solution, chenodeoxycholic acid, pyruvate, glucose, and NAD+. + and NADP + The pH of the reaction system is 7-8.

6. The method according to claim 5, characterized in that, Based on the reaction system, the concentration of the pyruvate is 80-120 mM; And / or, based on the reaction system, the concentration of glucose is 80-120 mM; And / or, based on the reaction system, the NAD + The concentration is 0.05-0.5 mM; And / or, based on the reaction system, the NADP + The concentration is 0.1-1 mM; And / or, based on the reaction system, the buffer solution is KPB buffer; Preferably, the concentration of the KPB buffer solution is 80-120 mM.

7. The method according to any one of claims 1-6, characterized in that, In step (2), the pH adjuster is an alkaline substance, preferably selected from at least one of sodium hydroxide, potassium hydroxide and sodium carbonate.

8. The method according to any one of claims 1-7, characterized in that, The first catalytic reaction and the second catalytic reaction are carried out independently at 0°C-30°C.

9. An apparatus for the continuous flow catalytic conversion of chenodeoxycholic acid to ursodeoxycholic acid, characterized in that, The apparatus includes a first reactor, a second reactor, a pumping system, and a pH adjustment device; wherein, the first reactor is loaded with a first immobilized cell; the second reactor is loaded with a second immobilized cell; the pumping system is connected between the first reactor and the second reactor for pumping reactants from the first reactor to the second reactor; the pH adjustment device is disposed in the flow path between the first reactor and the second reactor for adjusting the pH value of the first product flowing out of the first reactor; the first immobilized cell and the second immobilized cell are as described in claim 1.

10. The apparatus according to claim 9, characterized in that, The pH adjustment device includes a pH adjustment agent injection system, a pH sensor, and a controller; the pH sensor is disposed in the flow path and is used to monitor the pH value of the first product; the controller is electrically connected between the pH sensor and the pH adjustment agent injection system and is used to receive the monitoring signal from the pH sensor and control the pH adjustment agent injection system to inject alkaline substances.

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