Directionally modified 7beta-hydroxysteroid dehydrogenase iteration mutant as well as coding gene and application thereof

Abstract

The invention provides a directionally modified 7beta-hydroxysteroid dehydrogenase iteration mutant as well as a coding gene and application thereof. According to the invention, the expression vector or recombinant bacterium containing the 7beta-hydroxysteroid dehydrogenase iterative mutant gene is successfully constructed. According to the invention, the 7beta-hydroxysteroid dehydrogenase double mutants are directionally modified by an enzyme modification technology, and alanine at the 203th site of the 7beta-hydroxysteroid dehydrogenase double mutants is mutated into glutamine, so that the pH tolerance and catalytic activity of the enzyme are remarkably improved. The 7beta-hydroxysteroid dehydrogenase iteration mutant disclosed by the invention can be used for preparing UCDA (Urban Cyclin Dehydrogenase).

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of industrial biotechnology, and relates to a directedly reformed 7β-hydroxysteroid dehydrogenase iterative mutant, a coding gene thereof and application thereof. BACKGROUND

[0002] Ursodeoxycholic acid (UDCA), with a chemical name of 3α, 7β-dihydroxy-5β-cholestane-24-oic acid, is a drug component widely used in clinical treatment. The clinical application of UDCA mainly includes obvious improvement or treatment effect in diseases such as cholelithiasis, intrahepatic cholestasis of pregnancy, primary sclerosing cholangitis, infantile intestinal failure-related liver disease, primary biliary cholangitis, acquired immunodeficiency syndrome-related cholangiopathy and cystic fibrosis.

[0003] At present, the annual demand of UDCA in the global market is about 45-50 tons, and it continues to expand at an annual growth rate of ≥10%. However, whether it is the chemical synthesis route used for decades or the biosynthesis route that has emerged in the past decade, there are respective difficulties when it really goes to large-scale industrial production. The disadvantages of chemical synthesis route: low efficiency of chiral center inversion, high-toxicity / high-pollution reagents, long protection / deprotection steps, difficulty in recycling of noble metals and special solvents, etc.; the bottlenecks of biosynthesis: poor stability of key enzyme 7β-hydroxysteroid dehydrogenase (7β-HSDH), insufficient supply of cofactor regeneration, enzyme preparation scaling, substrate inhibition or product inhibition, etc. Specifically, the raw material 7-ketolithocholic acid (7-KLCA) for UDCA synthesis needs to be completely dissolved in alkaline conditions, and even the pH needs to reach 11 to meet the requirements, so the optimum pH of the reaction system is alkaline. However, the 7β-HSDH dimer is prone to depolymerization under alkaline conditions for a long time, resulting in enzyme inactivation and ineffective catalysis of the reaction. This phenomenon is a very tricky problem in many reactions and scale-up production, especially in scale-up reaction process, it is difficult to control the reaction to be completely reacted in a short time or to end the operation immediately, therefore, it is of great significance to develop 7β-HSDH enzyme that can tolerate alkaline conditions for application in industrial production. SUMMARY

[0004] In view of the problems of low substrate feeding amount, long reaction time, harsh pH condition and low yield caused by poor stability of the main enzyme 7β-HSDH in the biosynthesis process of UCDA, the present application obtains 7β-HSDH iterative mutant with higher catalytic efficiency and stronger pH tolerance through enzyme engineering technology, thereby greatly improving the synthesis efficiency of UDCA and reducing the production cost.

[0005] To address the aforementioned technical problems, the first objective of this invention is to provide an iterative mutant of 7β-hydroxysteroid dehydrogenase (7β-HSDH-M3). This 7β-hydroxysteroid dehydrogenase iterative mutant is based on a double mutant of 7β-hydroxysteroid dehydrogenase (7β-HSDH-M2) derived from Ruminococcus torques, with the alanine at position 203 of the double mutant mutated to glutamine. The amino acid sequence of this 7β-hydroxysteroid dehydrogenase iterative mutant is SEQ ID NO. 2: MNLREKYGEWGIILGATEGVGKAFAEKIASEGMSVVLVGRREEKLQELGKSISETYGVDHMVIRADFAQSDCTDKIFEATKDLDMGFMSYVACFHTFGKLQDTPWEKHEQMINVNVMTFLKCFYHYMGIFA KQDRGAVINVSSLTAISSSPYNAQYGAGKSYIKKLTEAVAAECESTNVDVEVITLGTVITPSLLSNLPGGPQGEAMMKTAMTPEACVEEAFDNLGKSLSVIAGEHNKANVHNWQANKTDDEYIRYMGSFYSNN.

[0006] Furthermore, the 7β-hydroxysteroid dehydrogenase double mutant has a mutation at position 189 (threonine) to valine and position 207 (valine) to methionine, relative to the natural enzyme. The amino acid sequence of the 7β-hydroxysteroid dehydrogenase double mutant is SEQ ID NO. 3: MNLREKYGEWGIILGATEGVGKAFAEKIASEGMSVVLVGRREEKLQELGKSISETYGVDHMVIRADFAQSDCTDKIFEATKDLDMGFMSYVACFHTFGKLQDTPWEKHEQMINVNVMTFLKCFYHYMGIFAKQDRGAVINVSSLTAISSSPYNAQYGAGKSYIKKLTEAVAAECESTNVDVEVITLGTVITPSLLSNLPGGPAGEAMMKTAMTPEACVEEAFDNLGKSLSVIAGEHNKANVHNWQANKTDDEYIRYMGSFYSNN.

[0007] The second objective of this invention is to provide a gene encoding an iterative mutant of 7β-hydroxysteroid dehydrogenase as described above, the nucleotide sequence of which is SEQ ID NO. 1: and the nucleotide sequence can be obtained by gene synthesis or genetic engineering.

[0008] A third objective of this invention is to provide an expression vector or recombinant bacteria containing the aforementioned genes, both of which are common and frequently used vectors and cells in the field and can be purchased from reputable companies. Preferably, the expression vector is pET28a(+), and the recombinant bacteria uses Escherichia coli BL21(DE3) as the host bacterium.

[0009] The fourth objective of this invention is to provide the application of the above-mentioned 7β-hydroxysteroid dehydrogenase iterative mutant in the preparation of ursodeoxycholic acid.

[0010] The fifth objective of this invention is to provide a method for improving the catalytic efficiency of 7β-hydroxysteroid dehydrogenase. This method achieves this by improving the stability of the enzyme, thereby giving it greater pH tolerance. The improvement in enzyme stability is achieved by increasing the binding stability of the 7β-hydroxysteroid dehydrogenase dimer. 7β-HSDH typically functions in the form of a dimer to catalyze substrate reactions. Unsuitable conditions such as temperature or pH can easily lead to the depolymerization of the dimer, resulting in enzyme inactivation or even degradation of the monomer, which then loses its catalytic activity. Therefore, the binding stability of the dimer is crucial to the stability of the 7β-HSDH enzyme, and improving the binding stability of the dimer can improve the stability of the enzyme.

[0011] Further steps include: using a rumen-derived 7β-hydroxysteroid dehydrogenase double mutant as a template, homology modeling analysis was performed using Uniprot, NCBI, and BLAST. The analysis identified and mutated key sites affecting the binding stability of the 7β-HSDH dimer, and then directed these sites to be modified. Through enzyme activity detection and enzyme transfection, iterative mutants of 7β-hydroxysteroid dehydrogenase with stronger pH tolerance and stability were screened out.

[0012] Furthermore, the 7β-hydroxysteroid dehydrogenase double mutant mutates threonine at position 189 to valine and valine at position 207 to methionine relative to the natural enzyme, with the key site being alanine at position 203, and the directed modification involving mutating alanine at position 203 to glutamine.

[0013] The sixth objective of this invention is to provide a method for preparing ursodeoxycholic acid, comprising the following steps: using the above-mentioned 7β-hydroxysteroid dehydrogenase iterative mutant to catalyze a substrate mixture to obtain ursodeoxycholic acid.

[0014] Furthermore, the substrate mixture includes 7-ketolithocholic acid, NADPH, and potassium phosphate, where 7-KLCA is reduced to UDCA, while NADPH is oxidized to NADP. + NADPH has a maximum absorption peak at 340 nm, so the rate of the reaction can be reflected by detecting the change in absorbance at 340 nm.

[0015] Alternatively, the substrate mixture may include 7-ketolithocholic acid, NADP + In addition to potassium phosphate, glucose dehydrogenase (GDH) is added to form a "coupling system" with GDH. GDH uses inexpensive glucose to convert NADP... + By reducing it to NADPH, the expensive coenzyme NADPH can be continuously "regenerated". The 7β-hydroxysteroid dehydrogenase iterative mutant immediately uses NADPH on 7-KLCA to generate the target product UDCA, thereby reducing production costs.

[0016] Furthermore, the temperature of the catalytic reaction is 30-40 °C, preferably 37 °C, and the pH is 6.5-8.5, preferably 8.0-8.2.

[0017] Furthermore, hexanol is added to the catalytic reaction to improve substrate solubility.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention provides a targeted modified iterative mutant of 7β-hydroxysteroid dehydrogenase, its encoding gene, and its applications. Through iterative mutation of the 7β-hydroxysteroid dehydrogenase double mutant (7β-HSDH-M2) using enzyme modification technology, the binding stability of the enzyme's dimer is improved, significantly enhancing the enzyme's pH tolerance and stability, thereby increasing catalytic activity. These improvements enhance reaction stability, shorten production cycles, increase production efficiency, and reduce production costs, providing an economically feasible solution for the large-scale industrial production of UCDA. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The structure analysis diagram of the 7β-HSDH-M3 dimer is shown below;

[0022] Figure 2 Gel images of 7β-HSDH-M2 and 7β-HSDH-M3;

[0023] Figure 3 A comparison of pH-tolerant enzyme activities of 7β-HSDH-M2 and 7β-HSDH-M3;

[0024] Figure 4This is an HPLC chromatogram of 7β-HSDH-M3 enzyme transfection. Detailed Implementation

[0025] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The present invention will be specifically described below with reference to specific embodiments.

[0026] Example 1 Iterative Mutation of 7β-HSDH

[0027] Previous research (see Chinese patent publication number CN119432787A) has revealed that the 7β-HSDH-M2 double mutant can enhance protein expression and stability. The specific mutation sites are T189V and V207M. Therefore, this study used this mutant as a template to improve the binding stability of the 7β-HSDH dimer. Homology modeling analysis was performed using Uniprot, NCBI, and BLAST. The analysis showed that the mutation of alanine at position 203 to glutamine resulted in a tighter dimer binding. Structural analysis is as follows: Figure 1 As shown in the figure, after the alanine at position 203 is mutated to glutamine, the nitrogen atom of glutamine forms a hydrogen bond with the threonine at position 188 and the oxygen atom of the asparagine side chain at position 237 of the other subunit of the dimer, which improves the stability of the dimer.

[0028] The specific steps are as follows:

[0029] First, the 3D structure of the enzyme was downloaded from the PDB database. Homologous sequences were aligned using Clustal Omega to identify conserved regions and variable sites. Stability data of existing mutants were collected to establish a baseline dataset. Analysis and prediction showed that iterative mutation of A203Q on the basis of the T189V and V207M double mutation could enhance the hydrogen bond force between the two monomers. Subsequently, based on 7β-hydroxysteroid dehydrogenase (PDB: 5FYD), the basic template protein was imported into Pymol software, and mutations were performed at three sites: T189V, V207M, and A203Q. Hydrogen bonds were observed to form between Q203 and T188 and N237 in the dimers. In summary, mutations were made at position 189 (threonine to valine), position 207 (valine to methionine), and position 203 (alanine to glutamine).

[0030] Example 2: Construction and Induced Expression of Recombinant Engineered Bacteria

[0031] The above-mentioned iterative three-point mutant was constructed in the pET28a(+) expression vector. The specific construction steps are as follows:

[0032] Based on the mutation site, the following site-directed mutagenesis primers were designed and synthesized:

[0033] M3-FP (SEQ ID NO. 4):

[0034] 5'-CCCGCAAGGTGAAGCAATGATGAAAACCG-3';

[0035] M3-RP (SEQ ID NO. 5):

[0036] 5'-ACCTTGCGGGCCGCCCGGCAGATTGCTCA-3'.

[0037] Using the pET28a-7β-HSDH-M2 double mutant plasmid as a template, the target fragment was amplified using the primers described above. The specific PCR conditions were as follows:

[0038]

[0039] The amplified PCR products were recovered by agarose gel electrophoresis and then digested with Dpn I restriction endonuclease before being transformed into TOP10 *E. coli*. The transformed products were plated on LB agarose gel containing 50 μg / ml kanamycin and incubated overnight at 37°C. Single clones were picked for sequencing, and the correctly mutated plasmid was transformed into BL21(DE3) *E. coli* to induce expression.

[0040] The specific steps for inducing expression are as follows: The recombinant plasmid is transformed into *E. coli* BL21(DE3) competent cells, plated on LB agar containing 50 μg / ml kanamycin, and incubated overnight at 37°C. Single colonies are picked and inoculated into 30 ml of LB agar containing 50 μg / ml kanamycin, and cultured at 37°C and 220 rpm for 6-12 h on a shaker. OD is then measured. 600 To achieve an inoculum size of 0.5-1.0, transfer a 1% inoculum to 200 ml of LB medium containing kanamycin. Use a 1000 ml Erlenmeyer flask, filling the flask to 1 / 5 of its capacity with the medium. Incubate at 37°C and 220 rpm on a shaker until the bacterial OD of the culture reaches 0.5-1.0. 600Once the pH reached 0.5-1.0, IPTG was added to a final concentration of 0.1 mM, and the mixture was incubated overnight at 16°C and 200 rpm on a shaker to induce *E. coli* to express the target protein. After induction, the bacterial culture was centrifuged at low temperature to collect the bacterial cell pellet. The cells were washed with potassium phosphate buffer, centrifuged again, and the supernatant was discarded. The cells were then resuspended in phosphate buffer at a weight-to-volume ratio of 30% (w / v). The cells were then homogenized using a homogenizer or sonicator, and the supernatant was collected after centrifugation; this was the crude 7β-HSDH enzyme solution. The protein expression was analyzed by SDS-PAGD electrophoresis of the homogenized supernatant and pellet.

[0041] The liquid culture medium was LB medium, which included yeast extract (1%), tryptone (2%), sodium chloride (1%), and kanamycin (50 μg / ml). The solid culture medium was formulated based on the liquid culture medium with the addition of agar powder (1.5%).

[0042] Both the template mutant 7β-HSDH-M2 and the iterative mutant 7β-HSDH-M3 were obtained from crude enzyme solutions using the above steps. Protein expression was as follows: Figure 2 As shown in the figure, the 7β-HSDH protein is about 29kD in size and is expressed in soluble form. The expression level of the iterative mutant 7β-HSDH-M3 is slightly higher than that of the template mutant 7β-HSDH-M2.

[0043] Example 3 Enzyme activity stability of 7β-HSDH iterative mutant at different pH values

[0044] Specific operations:

[0045] 1) Under normal conditions, measure the enzyme activity of the control mutant 7β-HSDH-M2 enzyme solution and the iterative mutant 7β-HSDH-M3 enzyme solution to determine the dilution factor of the crude enzyme solution. The actual dilution can be 200-300 times.

[0046] 2) Prepare five different pH 100 mM potassium phosphate buffer solutions (0.09348M K2HPO4 and 0.006523M KH2PO4 added to purified water to prepare 1L): pH 6.5, pH 7.0, pH 7.5, pH 8.0, and pH 8.5, and filter them through a 0.22um filter membrane;

[0047] 3) Dilute the control mutant 7β-HSDH-M2 enzyme solution and the iterative mutant 7β-HSDH-M3 enzyme solution with potassium phosphate buffer of different pH values, respectively, for a total of 10 portions, each diluted 200 times;

[0048] 4) Take 200 μl of each of the 10 enzyme solutions and put them into 1.5 ml centrifuge tubes, and incubate them in a water bath at 37°C for two hours;

[0049] 5) Ten enzyme solutions were prepared according to the enzyme activity assays of the control mutant 7β-HSDH-M2 and the iterative mutant 7β-HSDH-M3.

[0050] 6) Repeat the above operation three times and statistically analyze the enzyme activity data.

[0051] The results are as follows Figure 3 As shown, the data indicates that 7β-HSDH-M3 exhibits minimal enzyme activity loss in environments with gradually increasing pH, retaining 80% of its activity at pH 8.0; in contrast, the enzyme activity of 7β-HSDH-M2 decreases sharply with increasing pH, with a 66% loss at pH 8.0. This suggests that 7β-HSDH-M3 has stronger pH tolerance.

[0052] The method for measuring 7β-HSDH enzyme activity is as follows:

[0053] Highly efficient and accurate enzyme activity determination was performed using a UV2510 micro-volume ultraviolet spectrophotometer. The activity detection method involved treating the crushed and centrifuged crude enzyme solution under specific conditions, then mixing it with substrate, coenzyme, buffer system, and other substances before detection.

[0054] Enzyme activity assay method and principle: The mixed system contains 0.276 mg / ml 7-ketolithocholic acid (7-KLCA) substrate, 8 mM NADPH coenzyme, and 100 mM pH 7.0 potassium phosphate solution as buffer. The reaction temperature is 37 ℃, and the reaction time is 1 min. 7-KLCA is reduced to UDCA, while NADPH is oxidized to NADP+. NADPH has a maximum absorption peak at 340 nm; therefore, the reaction rate can be reflected by detecting the change in absorbance at 340 nm. One unit of enzyme activity (1 IU) refers to the amount of enzyme required to consume 1 μmol of NADPH within 1 min at 37 ℃.

[0055] The specific steps are as follows: 1) Prepare Reagent I: 100 mM pH 7.0 potassium phosphate buffer (0.09348M K2HPO4 and 0.006523M KH2PO4 added to purified water to prepare 1L), filter through a 0.22um filter membrane; 2) Prepare Reagent II: 7-ketolithocholic acid (7-KLCA, 0.276mg / ml): Weigh 276mg of 7-KLCA and dissolve it in 1L of Reagent I until there are no particles. If necessary, sonicate to dissolve. After preparation, aliquot and store at -20℃ for later use; 3) Prepare Reagent III: 8.0 mM NADPH, with 100 mM pH 7.0 buffer. 7.0% potassium phosphate solution is dissolved and prepared. It can be aliquoted and stored at -20℃. 4) Dilute the test samples (control mutant 7β-HSDH-M2 enzyme solution and iterative mutant 7β-HSDH-M3 enzyme solution): dilute with reagent I to a suitable range (control the slope change of the sample per second within 0.001-0.003. If it does not meet this range, change the sample dilution factor). 5) Add 2.9 ml of reagent II and 50 μl of reagent III to a 3 ml cuvette, mix well, and preheat at 37℃ for 5 min. 6) Add 50 μl of the test enzyme solution, mix well, and start the reaction. 7) Measure the rate of change of absorbance per second at 37℃ within 1 min at 340 nm.

[0056] Vitality calculation formula:

[0057]

[0058] Vt: Total volume of the reaction solution (3.00 ml)

[0059] Vs: Enzyme solution volume (0.05 ml)

[0060] 1.0: Optical path length (cm)

[0061] df: Dilution factor

[0062] 6.22: Extinction coefficient of NADPH at 340nm wavelength

[0063] 60: 1min = 60s

[0064] Example 4: Catalysis of 7-KLCA to UDCA under conditions of sufficient 7β-HSDH-M2 and 7β-HSDH-M3 enzymes

[0065] Weigh out 8g of 7-KLCA, 5g of glucose, and NADP. +10 mg of hexanol and 5 ml of 7β-HSDH enzyme solution were dissolved completely in 92 ml of potassium phosphate buffer (pH 8.0). 1 ml of GDH enzyme solution and 2 ml of 7β-HSDH enzyme solution were added, and the mixture was stirred at 37 °C for 8 h, maintaining the pH between 8.0 and 8.2 during the reaction. Two portions of the reaction system were prepared: one containing 7β-HSDH-M3 enzyme solution and the other containing 7β-HSDH-M2 enzyme solution. Results showed that the 7β-HSDH-M2 mutant achieved a conversion rate of over 95% after 6 h of reaction, but further reaction did not significantly improve the conversion rate. This is because the enzyme is not very stable under alkaline conditions; prolonged reaction leads to decreased or even inactivated enzyme activity, and extending the reaction time does not completely catalyze the conversion of the raw materials. The 7β-HSDH-M3 iterative mutant achieved a conversion rate of over 99% after 6 h of reaction. The conversion rate comparison data are shown in Table 1. The high-performance liquid chromatography (HPLC) chromatogram of 7β-HSDH-M3 catalyzing the conversion of 7-KLCA to UDCA is shown in Table 1. Figure 4 As shown in the figure, under the catalysis of 7β-HSDH-M3, the substrate 7-KLCA (retention time 7.187 min) was almost completely consumed, with the vast majority being converted into the target product UDCA (retention time 5.418 min). The conversion rate mentioned above refers to the proportion of substrate converted to product in the reaction, calculated using the following formula:

[0066] Conversion rate = (Product peak area ratio / Product peak area ratio + Substrate peak area ratio)

[0067] The peak area ratio needs to be determined by quantitative analysis (such as HPLC).

[0068] Table 1. Conversion rates of 7β-HSDH-M3 and 7β-HSDH-M2 enzyme solutions at different reaction times.

[0069]

[0070] Example 5: Catalysis of UDCA from 7-KLCA under reduced amounts of 7β-HSDH-M2 and 7β-HSDH-M3 enzymes.

[0071] Weigh out 8g of 7-KLCA, 5g of glucose, and NADP. +10 mg of hexanol and 5 ml of glucose dehydrogenase were dissolved completely in 92 ml of potassium phosphate buffer (pH 8.0). 1 ml of glucose dehydrogenase (GDH) enzyme solution and 1 ml of 7β-HSDH enzyme solution were added, and the mixture was stirred at 37 °C for 24 h, maintaining the pH between 8.0 and 8.2 during the reaction. Two portions of the reaction system were prepared: one portion containing 7β-HSDH-M3 enzyme solution and the other containing 7β-HSDH-M2 enzyme solution. Results showed that the conversion rate of the 7β-HSDH-M2 mutant continuously increased from the start of the reaction until 4 h, reaching a maximum of 72%. Further reaction up to 24 h showed no significant increase in conversion rate, indicating that the reaction was stopped after 4 h. The conversion rate of the 7β-HSDH-M3 iterative mutant gradually increased during the reaction, reaching 92% after 12 h, and remained at 92% after 24 h, indicating that the reaction was stopped after 12 h. The conversion rate comparison data are shown in Table 2. The results further demonstrate that the 7β-HSDH-M3 iterative mutant can react for a longer time under alkaline conditions, indicating that it has stronger pH tolerance and can maintain enzyme activity stability for a longer period of time. The conversion rate was calculated according to the method in Example 4.

[0072] Table 2. Conversion rates of 7β-HSDH-M3 and 7β-HSDH-M2 enzyme solutions with reduced enzyme content at different reaction times.

[0073]

[0074] The above description is merely a description of preferred embodiments of the present invention and is not intended to limit the scope of the present invention in any way. Any changes or modifications made by those skilled in the art based on the above disclosure shall fall within the protection scope of the claims.

Claims

1. A 7β-hydroxysteroid dehydrogenase iterative mutant, characterized in that, The 7β-hydroxysteroid dehydrogenase iterative mutant is based on a rumenococcal-derived 7β-hydroxysteroid dehydrogenase double mutant, with the alanine at position 203 of the 7β-hydroxysteroid dehydrogenase double mutant mutated to glutamine.

2. The 7β-hydroxysteroid dehydrogenase iterative mutant according to claim 1, characterized in that, The 7β-hydroxysteroid dehydrogenase double mutant has a threonine mutation at position 189 to valine and a valine mutation at position 207 to methionine, compared to the natural enzyme.

3. A gene encoding an iterative mutant of the 7β-hydroxysteroid dehydrogenase as described in claim 1 or 2, characterized in that, Its nucleotide sequence is SEQ ID NO.

1.

4. An expression vector or recombinant bacteria containing the gene of claim 3.

5. The application of the 7β-hydroxysteroid dehydrogenase iterative mutant according to claim 1 or 2 in the preparation of ursodeoxycholic acid.

6. A method for improving the catalytic efficiency of 7β-hydroxysteroid dehydrogenase, characterized in that, The method achieves this by improving the stability of the enzyme, thereby giving it greater pH tolerance. This improvement in enzyme stability is achieved by enhancing the binding stability of the 7β-hydroxysteroid dehydrogenase dimer.

7. The method according to claim 6, characterized in that, Includes the following steps: Using a rumen-derived 7β-hydroxysteroid dehydrogenase double mutant as a template, homology modeling analysis was performed using Uniprot, NCBI, and BLAST. The analysis identified and mutated key sites affecting the binding stability of the 7β-HSDH dimer, and then directed these sites to be modified. Through enzyme activity detection and enzyme transfection, iterative mutants of 7β-hydroxysteroid dehydrogenase with stronger pH tolerance and stability were screened out.

8. The method according to claim 7, characterized in that, The 7β-hydroxysteroid dehydrogenase double mutant mutates threonine at position 189 to valine and valine at position 207 to methionine relative to the natural enzyme. The key site is alanine at position 203, and the directed modification involves mutating alanine at position 203 to glutamine.

9. A method for preparing ursodeoxycholic acid, characterized in that, The method includes the following steps: catalyzing a substrate mixture with the 7β-hydroxysteroid dehydrogenase iterative mutant of claim 1 or 2 to obtain ursodeoxycholic acid, wherein the substrate mixture comprises 7-ketolithocholic acid, NADPH and potassium phosphate.

10. The method according to claim 9, characterized in that, The catalytic reaction is carried out at a temperature of 30-40 °C and a pH of 6.5-8.5.

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