A method for synthesizing ursodeoxycholic acid

Abstract

The application provides a synthesis method of ursodeoxycholic acid, and belongs to the field of biological medicines. The application provides a synthesis method of ursodeoxycholic acid, which comprises the following steps: mixing a 7-carbonyl lithocholic acid solution, glucose, coenzyme NADP, a bacterial body suspension and ethyl acetate to perform a reduction reaction, so as to obtain the ursodeoxycholic acid; the bacterial bodies in the bacterial body suspension comprise Escherichia coli expressing 7beta-steroid dehydrogenase and Escherichia coli expressing glucose dehydrogenase. In the process of synthesizing ursodeoxycholic acid from 7-carbonyl lithocholic acid, ethyl acetate is used for auxiliary catalysis, so that the conversion rate and yield of a substrate are improved.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a method for synthesizing ursodeoxycholic acid. Background Technology

[0002] Ursodeoxycholic acid (UDCA) is a natural bile acid with significant medicinal value. It has demonstrated remarkable efficacy in treating cholesterol gallstones and hepatobiliary diseases; therefore, its demand in the pharmaceutical field continues to grow, and its prospects are promising.

[0003] Currently, the bio-based preparation of ursodeoxycholic acid using Escherichia coli as a host faces significant bottlenecks. It is not only cumbersome and costly, involving complex operations such as cell disruption and protein refolding, but also poses potential risks to large-scale production due to endotoxin issues within the bacteria. In contrast, whole-cell catalysis technology eliminates the need for cell disruption, directly utilizing intracellular enzyme systems for catalysis. This significantly simplifies the process, reduces production costs, and effectively avoids the endotoxin problem, although the substrate conversion rate is lower. Summary of the Invention

[0004] This invention provides a method for synthesizing ursodeoxycholic acid, which has a high substrate conversion rate.

[0005] This invention provides a method for synthesizing ursodeoxycholic acid, comprising the following steps: A solution of 7-carbonyllithocholic acid, glucose, coenzyme NADP, bacterial suspension and ethyl acetate were mixed and subjected to a reduction reaction to obtain ursodeoxycholic acid; The bacterial suspension contains Escherichia coli expressing 7β-steroid dehydrogenase and Escherichia coli expressing glucose dehydrogenase.

[0006] Preferably, the method for preparing the 7-carbonyllithocholic acid includes the following steps: The 7-carbonyllithocholic acid was obtained by oxidizing a solution of chenodeoxycholic acid, Escherichia coli expressing 7α-steroid dehydrogenase, Escherichia coli expressing lactate dehydrogenase, coenzyme NAD, and sodium pyruvate.

[0007] Preferably, the mass ratio of chenodeoxycholic acid to Escherichia coli expressing 7α-steroid dehydrogenase is 80:56~71; The mass ratio of chenodeoxycholic acid to Escherichia coli expressing lactate dehydrogenase is 20:4~9; The mass ratio of chenodeoxycholic acid to coenzyme NAD is 200:0.8~1; The mass ratio of chenodeoxycholic acid to sodium pyruvate is 20:9~11; The oxidation reaction is carried out at a temperature of 20~30℃.

[0008] Preferably, after the oxidation reaction, the method further includes: adjusting the pH of the system obtained from the oxidation reaction to 12-13 using a pH adjuster, then performing solid-liquid separation on the resulting mixture to obtain a liquid phase; mixing the liquid phase with an acid and then precipitating and separating the liquid phase, and then drying the resulting wet solid to obtain the 7-carbonyllithocholic acid.

[0009] Preferably, the mixing includes: first mixing a solution of 7-carbonyllithocholic acid, glucose, coenzyme NADP, and bacterial suspension to obtain a first mixed solution; and second mixing the first mixed solution with ethyl acetate. The volume of the ethyl acetate is 0.5 to 1 times the volume of the solvent in the first mixed solution.

[0010] Preferably, the mass ratio of glucose to 7-carbonyllithocholic acid is 24:16~36; The mass ratio of glucose to coenzyme NADP is 90~110:1; The mass ratio of 7-carbonyllithocholic acid to Escherichia coli expressing 7β-steroid dehydrogenase is 24:14~18; The mass ratio of 7-carbonyllithocholic acid to Escherichia coli expressing glucose dehydrogenase is 24:7~10.

[0011] Preferably, the reduction reaction is carried out at a temperature of 25~35℃ for 18 hours.

[0012] Preferably, after the reduction reaction, the method further includes: mixing the system obtained from the reduction reaction with an extractant for extraction and separation to obtain an organic liquid phase; The organic liquid phase was mixed with water, precipitated, and separated. The resulting solid was then dried to obtain the crude product. The crude product was mixed with a mixed solvent of acetone and ethanol, and then mixed with triethylamine to carry out a salt formation reaction and filter to obtain ursodeoxycholic acid triethylamine salt. The ursodeoxycholic acid triethylamine salt was sequentially mixed with an aqueous solution of acetone, a weak organic acid, and a strong acid to induce crystallization. The resulting crystallized system was then subjected to solid-liquid separation to obtain a solid. The solid was then washed and dried to obtain the ursodeoxycholic acid.

[0013] Preferably, the extractant comprises methanol; The volume of the mixed solvent is 6 to 12 times the volume of the crude product; the volume ratio of acetone to ethanol in the mixed solvent is 6 to 20:1. The volume ratio of acetone to water in the aqueous solution of acetone is 10:2~4.

[0014] Preferably, the pH of the system obtained after mixing with an organic weak acid is 4 to 5, wherein the organic weak acid includes glacial acetic acid.

[0015] This invention utilizes ethyl acetate as an auxiliary catalyst in the synthesis of ursodeoxycholic acid from 7-carbonyllithocholic acid, thereby improving the conversion and yield of the substrate.

[0016] Furthermore, the synthesis process of this invention is mild and has high production efficiency.

[0017] Furthermore, this invention uses four bacteria expressing 7α-steroid dehydrogenase (7α-HSDH), lactate dehydrogenase (LDH), 7β-steroid dehydrogenase (7β-HSDH), and glucose dehydrogenase (GDH) respectively to catalyze the generation of ursodeoxycholic acid in series with high efficiency. Ethyl acetate is added during the catalytic process to obtain a crude product with a conversion rate of over 90% for chenodeoxycholic acid.

[0018] Furthermore, the present invention uses a mixed solvent of acetone and ethanol in the purification process to improve the purity of ursodeoxycholic acid. Compared with using a single solvent, the amount of mixed solvent is reduced by 8 times, which is 50 times less than that of a single solvent. Attached Figure Description

[0019] Figure 1 The HPLC chromatogram of crude ursodeoxycholic acid obtained in Comparative Example 1 is shown. Figure 2 The HPLC chromatogram of crude ursodeoxycholic acid obtained in Comparative Example 2 is shown. Figure 3 The HPLC chromatogram of crude ursodeoxycholic acid obtained in Comparative Example 3 is shown. Figure 4 The HPLC chromatogram of ursodeoxycholic acid obtained in Comparative Example 4 is shown. Figure 5 The HPLC chromatogram of ursodeoxycholic acid obtained in Comparative Example 5 is shown. Figure 6 The HPLC chromatogram of ursodeoxycholic acid obtained in Example 1 is shown below. Figure 7 The HPLC chromatogram of ursodeoxycholic acid obtained in Example 2 is shown below. Figure 8 The HPLC chromatogram of ursodeoxycholic acid obtained in Example 3 is shown below. Figure 9 The HPLC chromatogram of ursodeoxycholic acid obtained in Example 4 is shown below. Figure 10 The image shows the HPLC chromatogram of ursodeoxycholic acid obtained in Example 5. Detailed Implementation

[0020] This invention provides a method for synthesizing ursodeoxycholic acid, comprising the following steps: A solution of 7-carbonyllithocholic acid, glucose, coenzyme NADP, bacterial suspension and ethyl acetate were mixed and subjected to a reduction reaction to obtain ursodeoxycholic acid; The bacterial suspension contains Escherichia coli expressing 7β-steroid dehydrogenase and Escherichia coli expressing glucose dehydrogenase.

[0021] In this invention, the mixing preferably includes: first mixing a solution of 7-carbonyllithocholic acid, glucose, coenzyme NADP and bacterial suspension to obtain a first mixed solution; and second mixing the first mixed solution with ethyl acetate.

[0022] In this invention, the preferred method for preparing the solution of 7-carbonyllithocholic acid includes: mixing 7-carbonyllithocholic acid, potassium phosphate buffer, and a strong base to obtain the solution of 7-carbonyllithocholic acid.

[0023] In this invention, the preferred ratio of 7-carbonyllithocholic acid to potassium phosphate buffer is 4-9 g: 50 mL; the preferred concentration of the potassium phosphate buffer is 100 mM.

[0024] In this invention, the pH value of the 7-carbonyllithocholic acid solution is preferably 8.3; the strong base preferably includes a sodium hydroxide solution.

[0025] In this invention, the method for preparing the bacterial suspension includes: mixing Escherichia coli expressing 7β-steroid dehydrogenase and Escherichia coli expressing glucose dehydrogenase with potassium phosphate buffer; the volume ratio of the Escherichia coli expressing 7β-steroid dehydrogenase to the potassium phosphate buffer is preferably 3g:25mL.

[0026] In this invention, the volume of ethyl acetate is preferably 0.5 to 1 times the volume of the solvent in the first mixed solution, and in specific embodiments of this invention, it can be 0.6 times, 0.7 times, 0.8 times, or 0.9 times; the solvent in the first mixed solution preferably includes potassium phosphate buffer solution used to prepare the bacterial suspension and potassium phosphate buffer solution used to prepare the 7-carbonyllithocholic acid solution.

[0027] In this invention, the preferred mass ratio of glucose to 7-carbonyllithocholic acid is 24:16 to 36. In specific embodiments of this invention, it can be 24:17, 24:18, 24:19, 24:20, 24:21, 24:22, 24:23, 24:24, 24:25, 24:26, 24:27, 24:28, 24:29, 24:30, 24:31, 24:32, 24:33, 24:34, or 24:35.

[0028] In this invention, the preferred mass ratio of glucose to coenzyme NADP is 90-110:1. In specific embodiments of this invention, it can be 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1, 101:1, 102:1, 103:1, 104:1, 105:1, 106:1, 107:1, 108:1, or 109:1. In this invention, the preferred mass ratio of 7-carbonyllithocholic acid to *E. coli* expressing 7β-steroid dehydrogenase is 24:14-18, and in specific embodiments of this invention, it can be 24:15, 24:16, or 24:17. 7-Carbonyllithocholic acid is also known as 3α-hydroxy-7-oxo-5β-cholanic acid, CAS number 4651-67-6. In specific embodiments of this invention, the *E. coli* expressing 7β-steroid dehydrogenase is derived from the bacteria prepared in Example 1 of CN120536393A.

[0029] In this invention, the preferred mass ratio of 7-carbonyllithocholic acid to *E. coli* expressing glucose dehydrogenase is 24:7 to 10, and in specific embodiments of this invention, it can be 24:8 or 24:9. In specific embodiments of this invention, the *E. coli* expressing glucose dehydrogenase is derived from the bacteria prepared in Example 1 of CN120536393A.

[0030] In this invention, the method for preparing the 7-carbonyllithocholic acid preferably includes the following steps: The 7-carbonyllithocholic acid was obtained by oxidizing a solution of chenodeoxycholic acid, Escherichia coli expressing 7α-steroid dehydrogenase, Escherichia coli expressing lactate dehydrogenase, coenzyme NAD, and sodium pyruvate.

[0031] In this invention, the preferred method for preparing the solution of chenodeoxycholic acid includes mixing chenodeoxycholic acid, potassium phosphate buffer, and a strong base to obtain the solution of chenodeoxycholic acid.

[0032] In this invention, the preferred ratio of chenodeoxycholic acid to potassium phosphate buffer is 4g:50mL; the preferred concentration of potassium phosphate buffer is 100mM.

[0033] In this invention, the pH value of the chenodeoxycholic acid solution is preferably 8; the strong base preferably includes a sodium hydroxide solution.

[0034] In this invention, the mass ratio of chenodeoxycholic acid to Escherichia coli expressing 7α-steroid dehydrogenase is 80:56~71. In specific embodiments of this invention, it can be 80:56, 80:57, 80:58, 80:59, 80:60, 80:61, 80:62, 80:63, 80:64, 80:65, 80:66, 80:67, 80:68, 80:69, or 80:70. In specific embodiments of this invention, the Escherichia coli expressing 7α-steroid dehydrogenase is derived from the bacteria prepared in Example 1 of CN120485142A.

[0035] In this invention, the preferred mass ratio of chenodeoxycholic acid to *E. coli* expressing lactate dehydrogenase is 20:4 to 9, and in specific embodiments of this invention, it can be 20:4, 20:5, 20:6, 20:7, or 20:8. In specific embodiments of this invention, the *E. coli* expressing lactate dehydrogenase is derived from the bacteria prepared in Example 1 of CN120485142A.

[0036] In this invention, the mass ratio of chenodeoxycholic acid to coenzyme NAD is preferably 200:0.8~1, and in a specific embodiment of this invention it can be 200:0.9.

[0037] In this invention, the preferred mass ratio of chenodeoxycholic acid to sodium pyruvate is 20:9 to 11. In specific embodiments of this invention, it can be 20:9, 20:9.2, 20:9.5, 20:9.8, 20:10, 20:10.2, 20:10.5 or 20:10.8.

[0038] In this invention, the temperature of the oxidation reaction is preferably 20~30℃, and the time is preferably 18~24h. In specific embodiments of this invention, it can be 19h, 20h, 21h, 22h or 23h.

[0039] After the oxidation reaction, the present invention preferably further includes: adjusting the pH of the system obtained from the oxidation reaction to 12-13 with a pH adjuster, then performing solid-liquid separation on the obtained mixture to obtain a liquid phase; mixing the liquid phase with an acid and then precipitating and separating the liquid phase, and then drying the obtained wet solid to obtain the 7-carbonyllithocholic acid.

[0040] In this invention, the pH adjuster preferably comprises a sodium hydroxide solution.

[0041] In this invention, the acid includes sulfuric acid.

[0042] In this invention, the pH value during precipitation is preferably 1.5.

[0043] In this invention, the temperature of the reduction reaction is preferably 25~35℃, and the time is preferably 18h.

[0044] After the reduction reaction, the present invention preferably further includes: mixing the system obtained from the reduction reaction with an extractant for extraction and separation to obtain an organic liquid phase; The organic liquid phase was mixed with water, precipitated, and separated. The resulting solid was then dried to obtain the crude product. The crude product was mixed with a mixed solvent of acetone and ethanol, and then mixed with triethylamine to carry out a salt formation reaction, followed by cooling, filtration, and washing to obtain ursodeoxycholic acid triethylamine salt. The ursodeoxycholic acid triethylamine salt was sequentially mixed with an aqueous solution of acetone, a weak organic acid, and a strong acid to induce crystallization. The resulting crystallized system was then subjected to solid-liquid separation to obtain a solid. The solid was then washed and dried to obtain the ursodeoxycholic acid.

[0045] In this invention, the volume of the extractant is preferably 1 to 1.5 times the total volume of the solvent in the solution of 7-carbonyllithocholic acid and the suspending agent in the bacterial suspension in the system obtained by the reduction reaction; the extractant preferably includes methanol.

[0046] The volume of the mixed solvent is preferably 6 to 12 times the volume of the crude product, and in specific embodiments of the present invention, it can be 7, 8, 9, 10, or 11 times; the volume ratio of acetone to ethanol in the mixed solvent is preferably 6 to 20:1, and in specific embodiments of the present invention, it can be 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, or 19:1.

[0047] In this invention, the molar ratio of ursodeoxycholic acid to triethylamine in the crude product is preferably 1:2~3.

[0048] In this invention, the preferred temperature for the salt formation reaction is 45°C, and the preferred time is 1 to 1.5 hours.

[0049] In this invention, the cooling process preferably includes: naturally cooling the system obtained from the salt formation reaction to room temperature, then cooling it to 15°C, stirring for 1 hour, and then continuing to cool it to 5°C and stirring for 3 hours.

[0050] In this invention, the detergent used for washing preferably includes acetone; the temperature of the detergent is preferably 2~8°C.

[0051] In this invention, the volume ratio of the filter cake obtained by filtration to the acetone aqueous solution is preferably 1:13; the volume ratio of acetone to water in the acetone aqueous solution is preferably 10:3.

[0052] The preferred temperature for mixing with the acetone aqueous solution is 45°C, and the preferred time is 1 hour.

[0053] In this invention, the pH value of the system obtained after mixing with an organic weak acid is preferably 4-5, and the organic weak acid preferably includes glacial acetic acid. Glacial acetic acid can complex metal ions, reducing the residue on ignition of the finished product.

[0054] The preferred temperature for mixing with the weak organic acid is 45°C, and the preferred time is 30 minutes.

[0055] In this invention, mixing with acid preferably includes first performing a third mixing with a portion of the acid, followed by crystallization, and then performing a fourth mixing with the remaining acid in the resulting system.

[0056] The preferred temperature for the third mixing is 15~35℃.

[0057] In this invention, the crystallization temperature is preferably 3~6℃ and the time is preferably 1h.

[0058] In this invention, the pH value of the fourth mixture is preferably below 2.

[0059] In this invention, the concentration of the acid is preferably 0.1M, and the acid preferably includes sulfuric acid.

[0060] The following detailed description of the synthesis method of ursodeoxycholic acid provided by the present invention, with reference to the embodiments, should not be construed as limiting the scope of protection of the present invention.

[0061] Comparative Example 1 80g of chenodeoxycholic acid was weighed and suspended in 1000mL of 100mM potassium phosphate buffer. The pH was adjusted to 8.0 using 5mol / L sodium hydroxide solution. 0.4g of NAD and 44g of sodium pyruvate were added, along with 71g of Escherichia coli expressing 7α-steroid dehydrogenase (7α-HSDH) and 36g of Escherichia coli expressing lactate dehydrogenase (LDH). The mixture was stirred at 30℃ for 18h. The pH was then adjusted to 13 using 5mol / L NaOH solution. After centrifugation, the supernatant was titrated to pH 1.5 using 5% H2SO4 solution. The mixture was then filtered and dried to obtain crude 7-KLCA with a conversion rate of 99.5%.

[0062] 1.8 g of crude 7-KLCA was dissolved in 10 mL of 100 mM potassium phosphate buffer, and the pH was adjusted to 8.3 with 5 mol / L sodium hydroxide solution. Then, 2.4 g of glucose and 0.025 g of NADP were added to obtain a mixed substrate solution. 1.2 g of Escherichia coli expressing 7β-steroid dehydrogenase (7β-HSDH) and 0.69 g of Escherichia coli expressing glucose dehydrogenase (GDH) were resuspended in 10 mL of 100 mM potassium phosphate buffer to obtain a bacterial suspension. The above mixed substrate solution and bacterial suspension were mixed and reacted at 25°C for 18 h. Then 20 mL of methanol was added, the supernatant was collected by centrifugation, 120 mL of pure water was added dropwise to the supernatant, and the mixture was filtered and dried to obtain crude UDCA with a conversion rate of 89.86%.

[0063] Figure 1 The image shows the HPLC chromatogram of crude ursodeoxycholic acid obtained in Comparative Example 1.

[0064] Test results: UDCA: 87.027% 7-KLCA: 12.113% CDCA: 0.329% Other impurities: 0.531% Total miscellaneous: 12.973% Comparative Example 2 1.8 g of crude 7-KLCA obtained in Comparative Example 1 was dissolved in 10 mL of 100 mM potassium phosphate buffer, and the pH was adjusted to 8.3 with 5 mol / L sodium hydroxide solution. Then, 2.4 g of glucose and 0.025 g of NADP were added to obtain a mixed substrate solution. 1.2 g of Escherichia coli expressing 7β-steroid dehydrogenase and 0.69 g of Escherichia coli expressing glucose dehydrogenase were resuspended in 10 mL of 100 mM potassium phosphate buffer to obtain a bacterial suspension. The above mixed substrate solution and bacterial suspension were mixed to obtain a mixed solution. Then, 0.5 times the volume of potassium phosphate buffer used for the mixed solution was added to ethanol and the mixture was reacted at 25°C for 18 hours. Then, 20 mL of methanol was added, the mixture was centrifuged and the supernatant was collected. 120 mL of pure water was added to the supernatant, and the mixture was filtered and dried to obtain crude UDCA with a conversion rate of 82.23%.

[0065] Figure 2 The image shows the HPLC chromatogram of crude ursodeoxycholic acid obtained in Comparative Example 2.

[0066] Test results: UDCA: 86.197% 7-KLCA: 12.778% CDCA: 0.182% Other impurities: 0.843% Total miscellaneous: 13.803% Comparative Example 3 1.8 g of crude 7-KLCA obtained in Comparative Example 1 was dissolved in 10 mL of 100 mM potassium phosphate buffer, and the pH was adjusted to 8.3 with 5 mol / L sodium hydroxide solution. Then, 2.4 g of glucose and 0.025 g of NADP were added to obtain a mixed substrate solution. 1.2 g of Escherichia coli expressing 7β-steroid dehydrogenase and 0.69 g of Escherichia coli expressing glucose dehydrogenase were resuspended in 10 mL of 100 mM potassium phosphate buffer to obtain a bacterial suspension. The above mixed substrate solution and bacterial suspension were mixed to obtain a mixed solution. Then, ethanol with a volume equal to that of potassium phosphate buffer used for the mixed solution was added and the mixture was reacted at 25°C for 18 hours. Then, 20 mL of methanol was added, the mixture was centrifuged and the supernatant was collected. 120 mL of pure water was added to the supernatant, and the mixture was filtered and dried to obtain crude UDCA with a conversion rate of 48.01%.

[0067] Figure 3 The image shows the HPLC chromatogram of crude ursodeoxycholic acid obtained in Comparative Example 3.

[0068] Test results: UDCA: 53.167% 7-KLCA: 46.129% CDCA: 0.069% Other impurities: 0.635% Total miscellaneous: 46.833% Comparative Example 4 1.8 g of crude 7-KLCA obtained in Comparative Example 1 was dissolved in 10 mL of 100 mM potassium phosphate buffer, and the pH was adjusted to 8.3 with 5 mol / L sodium hydroxide solution. Then, 2.4 g of glucose and 0.025 g of NADP were added to obtain a mixed substrate solution. 1.2 g of Escherichia coli expressing 7β-steroid dehydrogenase and 0.69 g of Escherichia coli expressing glucose dehydrogenase were resuspended in 10 mL of 100 mM potassium phosphate buffer to obtain a bacterial suspension. The above mixed substrate solution and bacterial suspension were mixed to obtain a mixed solution. Then, 0.5 times the volume of the potassium phosphate buffer used for the mixed solution was added to methanol and the mixture was reacted at 25°C for 18 hours. Then, 20 mL of methanol was added, the mixture was centrifuged and the supernatant was collected. 120 mL of pure water was added to the supernatant, and the mixture was filtered and dried to obtain crude UDCA with a conversion rate of 80.07%.

[0069] Figure 4 The image shows the HPLC chromatogram of crude ursodeoxycholic acid obtained in Comparative Example 4.

[0070] Test results: UDCA: 75.709% 7-KLCA: 24.002% CDCA: Not detected Other impurities: 0.289% Total miscellaneous: 24.291% Comparative Example 5 1.8 g of crude 7-KLCA obtained in Comparative Example 1 was dissolved in 10 mL of 100 mM potassium phosphate buffer, and the pH was adjusted to 8.3 with 5 mol / L sodium hydroxide solution. Then, 2.4 g of glucose and 0.025 g of NADP were added to obtain a mixed substrate solution. 1.2 g of Escherichia coli expressing 7β-steroid dehydrogenase and 0.69 g of Escherichia coli expressing glucose dehydrogenase were resuspended in 10 mL of 100 mM potassium phosphate buffer to obtain a bacterial suspension. The above mixed substrate solution and bacterial suspension were mixed to obtain a mixed solution. Then, methanol with a volume equal to that of potassium phosphate buffer used for the mixed solution was added and the mixture was reacted at 25°C for 18 hours. Then, 20 mL of methanol was added, the mixture was centrifuged and the supernatant was collected. 120 mL of pure water was added to the supernatant, and the mixture was filtered and dried to obtain crude UDCA with a conversion rate of 61.98%.

[0071] Figure 5 The image shows the HPLC chromatogram of crude ursodeoxycholic acid obtained in Comparative Example 5.

[0072] Test results: UDCA: 60.940% 7-KLCA: 37.910% CDCA: 0.282% Other impurities: 0.868% Total miscellaneous items: 39.06% Example 1 1.8 g of crude 7-KLCA obtained in Comparative Example 1 was dissolved in 10 mL of 100 mM potassium phosphate buffer, and the pH was adjusted to 8.3 with 5 mol / L sodium hydroxide solution. Then, 2.4 g of glucose and 0.025 g of NADP were added to obtain a mixed substrate solution. 1.2 g of Escherichia coli expressing 7β-steroid dehydrogenase and 0.69 g of Escherichia coli expressing glucose dehydrogenase were resuspended in 10 mL of 100 mM potassium phosphate buffer to obtain a bacterial suspension. The above mixed substrate solution and bacterial suspension were mixed to obtain a mixed solution. Then, 0.5 times the volume of potassium phosphate buffer used for the mixed solution was added to ethyl acetate and the mixture was reacted at 25°C for 18 h. Then, 20 mL of methanol was added, the mixture was centrifuged and the supernatant was collected. 120 mL of pure water was added dropwise to the supernatant, and the mixture was filtered and dried to obtain crude UDCA with a conversion rate of 98.02%.

[0073] Figure 6 The image shows the HPLC chromatogram of crude ursodeoxycholic acid obtained in Example 1.

[0074] Test results: UDCA: 98.982% 7-KLCA: 0.362% CDCA: 0.195% Other impurities: 0.461% Total miscellaneous: 1.018% Example 2 1.8 g of crude 7-KLCA obtained in Comparative Example 1 was dissolved in 10 mL of 100 mM potassium phosphate buffer, and the pH was adjusted to 8.3 with 5 mol / L sodium hydroxide solution. Then, 2.4 g of glucose and 0.025 g of NADP were added to obtain a mixed substrate solution. 1.2 g of Escherichia coli expressing 7β-steroid dehydrogenase and 0.69 g of Escherichia coli expressing glucose dehydrogenase were resuspended in 10 mL of 100 mM potassium phosphate buffer to obtain a bacterial suspension. The above mixed substrate solution and bacterial suspension were mixed to obtain a mixed solution. Then, ethyl acetate with a volume equal to that of potassium phosphate buffer used for the mixed solution was added and the mixture was reacted at 25°C for 18 hours. Then, 20 mL of methanol was added, the mixture was centrifuged and the supernatant was collected. 120 mL of pure water was added dropwise to the supernatant, and the mixture was filtered and dried to obtain crude UDCA with a conversion rate of 99.67%.

[0075] Figure 7 The image shows the HPLC chromatogram of crude ursodeoxycholic acid obtained in Example 2.

[0076] Test results: UDCA: 99.155% 7-KLCA: 0.320% CDCA: 0.156% Other impurities: 0.369% Total impurities: 0.845% Example 3 Take 9.26 g of the crude UDCA obtained in Example 2 and add it to 10 times its volume of acetone-ethanol solution (acetone to ethanol volume ratio 6:1). Stir at 45°C for 1.5 h, then slowly add 2 times the amount of UDCA triethylamine. Reflux at 45°C for 1 h, then stop heating and allow to cool naturally to room temperature. The next day, slowly cool to 5°C, stir for 2 h, filter, and wash with cold acetone at 4°C to obtain 7.92 g of ursodeoxycholic acid triethylamine salt. Add 13 times its volume of acetone aqueous solution (acetone to water volume ratio 10:3), stir and heat to 45°C, maintaining for 1 h. Slowly add glacial acetic acid to adjust the pH to 4-5, maintain stirring for 30 min, turn off heating, allow to cool naturally to room temperature, then slowly add 45 mL of 0.1 M H2SO4, then cool to 5°C, crystallize for 1 h, and continue adding 0.1 M H2SO4. H2SO4 was used to adjust the pH to 1.5-1.7, followed by filtration. The filter cake was washed with cold water at 4°C and dried to obtain 6.13g of high-purity ursodeoxycholic acid, with a yield of 72%.

[0077] Figure 8 The image shows the HPLC chromatogram of ursodeoxycholic acid obtained in Example 3.

[0078] Test results: UDCA: 99.949% 7-KLCA: 0.051% CDCA: Not detected Other impurities: Not detected Total miscellaneous: 0.051% Example 4 Take 9.33 g of the crude UDCA obtained in Example 2 and add it to 10 times its volume of acetone-ethanol solution (acetone to ethanol volume ratio of 12:1). Stir at 45°C for 1.5 h, then slowly add 2 times the amount of UDCA triethylamine. Reflux at 45°C for 1.5 h, then stop heating and allow to cool naturally. The next day, slowly cool to 15°C, stir for 1 h, continue cooling to 5°C, stir for 3 h, filter, wash with cold acetone at 4°C to obtain 8.15 g of ursodeoxycholic acid triethylamine salt. Add 13 times its volume of acetone aqueous solution (acetone to water volume ratio of 10:3), stir and heat to 45°C, maintain for 30 min, slowly add glacial acetic acid to adjust the pH to 4.54, maintain stirring for 30 min, turn off heating, cool to 15°C, slowly add 45 mL of 0.1 M H2SO4, then cool to 5°C, crystallize for 1 h, and continue adding 0.1 M H2SO4. The pH was adjusted to 1.53 with H2SO4, filtered, and the filter cake was washed with cold water at 4℃ and dried to obtain 6.95g of high-purity ursodeoxycholic acid, with a yield of 81%.

[0079] Figure 9 The image shows the HPLC chromatogram of ursodeoxycholic acid obtained in Example 4.

[0080] Test results: UDCA: 99.940% 7-KLCA: 0.060% CDCA: Not detected Other impurities: Not detected Total miscellaneous substances: 0.060% Example 5 Take 9.15g of the crude UDCA obtained in Example 2 and add it to 10 times its volume of acetone-ethanol solution (acetone to ethanol volume ratio of 20:1). Stir at 45°C for 1.5h, then slowly add 2 times the amount of UDCA triethylamine. Reflux at 45°C for 1.5h, then stop heating and allow to cool naturally. The next day, slowly cool to 15°C, stir for 1h, continue cooling to 5°C, stir for 3h, filter, wash with cold acetone at 4°C to obtain 8.09g of wet ursodeoxycholic acid triethylamine salt. Add 13 times its volume of acetone aqueous solution (acetone to water volume ratio of 10:3), stir and heat to 45°C, maintain for 30min, slowly add glacial acetic acid to adjust the pH to 4.52, maintain stirring for 30min, turn off heating, cool to 15°C, slowly add 45mL of 0.1M H2SO4, then cool to 5°C, crystallize for 1h, and continue adding 0.1M H2SO4. The pH was adjusted to 1.51 using H2SO4, filtered, and the filter cake was washed with cold water at 4°C and dried to obtain 7.2g of high-purity ursodeoxycholic acid, with a yield of 86%.

[0081] Figure 10 The image shows the HPLC chromatogram of ursodeoxycholic acid obtained in Example 5.

[0082] Test results: UDCA: 99.925% 7-KLCA: 0.055% CDCA: Not detected Other impurities: 0.02% Total impurities: 0.075%.

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for synthesizing ursodeoxycholic acid, characterized in that, Includes the following steps: A solution of 7-carbonyllithocholic acid, glucose, coenzyme NADP, bacterial suspension and ethyl acetate were mixed and subjected to a reduction reaction to obtain ursodeoxycholic acid; The bacterial suspension contains Escherichia coli expressing 7β-steroid dehydrogenase and Escherichia coli expressing glucose dehydrogenase.

2. The synthesis method according to claim 1, characterized in that, The preparation method of the 7-carbonyllithocholic acid includes the following steps: The 7-carbonyllithocholic acid was obtained by oxidizing a solution of chenodeoxycholic acid, Escherichia coli expressing 7α-steroid dehydrogenase, Escherichia coli expressing lactate dehydrogenase, coenzyme NAD, and sodium pyruvate.

3. The synthesis method according to claim 2, characterized in that, The mass ratio of chenodeoxycholic acid to Escherichia coli expressing 7α-steroid dehydrogenase is 80:56~71; The mass ratio of chenodeoxycholic acid to Escherichia coli expressing lactate dehydrogenase is 20:4~9; The mass ratio of chenodeoxycholic acid to coenzyme NAD is 200:0.8~1; The mass ratio of chenodeoxycholic acid to sodium pyruvate is 20:9~11; The oxidation reaction is carried out at a temperature of 20~30℃.

4. The synthesis method according to claim 2 or 3, characterized in that, After the oxidation reaction, the process further includes: adjusting the pH of the system obtained from the oxidation reaction to 12-13 using a pH adjuster, then performing solid-liquid separation on the resulting mixture to obtain a liquid phase; mixing the liquid phase with an acid and then precipitating and separating the liquid phase, and then drying the resulting wet solid to obtain the 7-carbonyllithocholic acid.

5. The synthesis method according to claim 1, characterized in that, The mixing process includes: first mixing a solution of 7-carbonyllithocholic acid, glucose, coenzyme NADP, and bacterial suspension to obtain a first mixed solution; and second mixing the first mixed solution with ethyl acetate. The volume of the ethyl acetate is 0.5 to 1 times the volume of the solvent in the first mixed solution.

6. The synthesis method according to claim 1 or 5, characterized in that, The mass ratio of glucose to 7-carbonyllithocholic acid is 24:16~36; The mass ratio of glucose to coenzyme NADP is 90~110:1; The mass ratio of 7-carbonyllithocholic acid to Escherichia coli expressing 7β-steroid dehydrogenase is 24:14~18; The mass ratio of 7-carbonyllithocholic acid to Escherichia coli expressing glucose dehydrogenase is 24:7~10.

7. The synthesis method according to claim 1, characterized in that, The reduction reaction is carried out at a temperature of 25-35°C for 18 hours.

8. The synthesis method according to claim 1, characterized in that, The reduction reaction is followed by: mixing the system obtained from the reduction reaction with an extractant for extraction and separation to obtain an organic liquid phase; The organic liquid phase was mixed with water, precipitated, and separated. The resulting solid was then dried to obtain the crude product. The crude product was mixed with a mixed solvent of acetone and ethanol, and then mixed with triethylamine to carry out a salt formation reaction and filter to obtain ursodeoxycholic acid triethylamine salt. The ursodeoxycholic acid triethylamine salt was sequentially mixed with an aqueous solution of acetone, a weak organic acid, and a strong acid to induce crystallization. The resulting crystallized system was then subjected to solid-liquid separation to obtain a solid. The solid was then washed and dried to obtain the ursodeoxycholic acid.

9. The synthesis method according to claim 8, characterized in that, The extractant includes methanol; The volume of the mixed solvent is 6 to 12 times the volume of the crude product; the volume ratio of acetone to ethanol in the mixed solvent is 6 to 20:

1. The volume ratio of acetone to water in the aqueous solution of acetone is 10:2~4.

10. The synthesis method according to claim 8, characterized in that, The pH of the system obtained after mixing with an organic weak acid is 4-5, wherein the organic weak acid includes glacial acetic acid.

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