7 beta-HSDH enzyme mutant derived from ruminococcus torsionosus and application of 7 beta-HSDH enzyme mutant

Abstract

The invention discloses a ruminococcus torsionosus-sourced 7beta-HSDH enzyme mutant and application thereof, the ruminococcus torsionosus-sourced 7beta-HSDH enzyme is taken as a research object, a series of single-point mutation and combined mutation of the 7beta-HSDH enzyme are provided, and compared with wild type 7beta-HSDH enzyme, the thermal stability of the 7beta-HSDH enzyme mutant is improved by 1-20 DEG C, and the 7beta-HSDH enzyme mutant has the advantages that the 7beta-HSDH enzyme mutant has the advantages that the 7beta-HSDH enzyme mutant can be used for preparing the 7beta-HSDH enzyme mutant; compared with a wild type 7beta-HSDH enzyme, the 7 beta-HSDH enzyme has the advantages that the Tm value of the (C217I and E252K) mutant is improved by 1.2-4.2 times, the enzyme activity of the (C217I and E252K) mutant is 4.2 times that of the wild type 7beta-HSDH enzyme, and the (T103I, D251K, L229K and S161A) mutant has ultrahigh heat stability, the Tm value is close to 68 DEG C, and 50% of enzyme activity is maintained, so that the 7 beta-HSDH enzyme can be used for preparing the 7 beta-HSDH enzyme. Compared with a wild type 7 beta-HSDH enzyme, the 7 beta-HSDH enzyme mutant provided by the invention has wider application conditions, is more suitable for efficiently producing ursodesoxycholic acid from 3 alpha-hydroxy-7-oxo-5beta-cholanic acid (7-ketolithocholic acid, 7-KLCA) by a biological conversion method, and is beneficial to large-scale production and industrial application.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a 7β-HSDH enzyme mutant derived from Ruminococcus torques and application thereof. BACKGROUND

[0002] Ursodeoxycholic acid (UDCA) as a first-line choleretic drug has a huge and growing demand in the global pharmaceutical market. Traditional chemical synthesis and animal extraction methods have inherent defects such as complicated steps, low yield, serious environmental pollution and high cost. In contrast, the biocatalytic synthesis route based on 7β-hydroxysteroid dehydrogenase (7β-HSDH enzyme) is recognized as the most promising technology to replace traditional processes due to its mild reaction conditions, stereoselectivity and environmental friendliness.

[0003] However, in the process of pushing this advanced technology from the laboratory to large-scale industrial production, a fatal bottleneck has always failed to be effectively solved: the 7β-HSDH enzyme of natural origin is extremely fragile under industrial production conditions. The problems of insufficient inherent thermal stability and low catalytic efficiency (specific activity) of the enzyme lead to rapid deactivation of the enzyme catalyst in actual reactions, especially in scenarios where the temperature needs to be raised to improve the reaction rate and substrate solubility. As a result, the enzyme catalyst has to be added in excess or replaced frequently, resulting in a sharp rise in production costs, making it impossible to achieve economic efficiency. This has become a key barrier to the industrialization of this green technology.

[0004] Wild-type 7βHSDH enzymes are derived from a wide range of sources. At present, domestic and foreign scientific researchers have screened a large number of 7βHSDH enzyme-producing microorganisms and cloned their encoding genes, such as Collinsella aerofaciens, Clostridium absonum, Ruminococcus torques, etc. At the same time, many scientific researchers use protein engineering methods to mutate and transform wild-type 7βHSDH enzymes to improve their industrial application ability. Some researches on wild-type 7βHSDH enzymes derived from Ruminococcus torques have also reported the transformation of 7β-HSDH enzymes based on this species, including machine learning (ML), error-prone PCR, etc. Some mutation points such as G39D / T17A have been developed. These mutants have improved some activities to some extent, but their thermal stability has not been improved. The Tm value of the mutant with the highest Tm value is only 50.7℃ (Mu Qiang W, Zhi Neng Y, Bing Yi Y, et al. Machine-Learning-Guided Engineering of an NADH-Dependent 7β-Hydroxysteroid Dehydrogenase for Economic Synthesis of Ursodeoxycholic Acid. [J]. Journal of agricultural and food chemistry, 2023). In fact, the 7β-HSDH enzyme with this Tm value cannot meet the requirements of industrial production process.

[0005] Therefore, based on the 7β-HSDH enzyme derived from Ruminococcus torques as the research object, further improving its thermal stability and activity has greater research value for developing 7β-HSDH enzymes suitable for industrial production process. SUMMARY

[0006] The purpose of the present application is to solve the existing technical problems and provide a Ruminococcus torques-derived 7β-HSDH enzyme mutant and its application.

[0007] The present application achieves the above-mentioned purpose through the following technical solutions: As a first aspect of the present application, a Ruminococcus torques-derived 7β-HSDH enzyme mutant is provided, which is obtained by single mutation or combined mutation of the protein sequence of wild-type 7β-HSDH enzyme derived from Ruminococcus torques as shown in SEQ ID NO. 1; The single mutation site includes at least one of E18G, H60T, T103V, T103I, R202R, L229R, L229Q, L229K, E235P, N237W, Q245M or D251K; The combined mutation sites include at least one of the following (1)-(11): (1) T103I, L229R; (2) T103I, E18G; (3) T103I, D251K; (4) T103I, E252K; (5) C217I, L229R; (6) C217I, E18G; (7) C217I, D251K; (8) C217I, E252K; (9) S161A, E18G; (10) T103I, D251K, L229K, S161A; (11) T103I, E18G, L229K, S161A.

[0008] As a further optimization of the present invention, the 7β-HSDH enzyme mutant is based on a combination of mutation sites C217I and E252K on the protein sequence of the wild-type 7β-HSDH enzyme, and the amino acid sequence of the 7β-HSDH enzyme mutant is shown in SEQ ID NO.2.

[0009] As a further optimization of the present invention, the 7β-HSDH enzyme mutant is based on a combination of mutation sites T103I, D251K, L229K and S161A on the protein sequence of the wild-type 7β-HSDH enzyme, and the amino acid sequence of the 7β-HSDH enzyme mutant is shown in SEQ ID NO.3.

[0010] As a second aspect of the invention, a polynucleotide encoding a 7β-HSDH enzyme mutant as described above is also provided.

[0011] As a further optimization of the present invention, the sequence of the polynucleotide is as shown in SEQ ID NO.4 or SEQ ID NO.5.

[0012] As a third aspect of the invention, a recombinant plasmid is also provided, which is an expression vector containing any of the polynucleotides described above and capable of translating and expressing the 7β-HSDH enzyme mutant as described above.

[0013] As a further optimization of the present invention, the expression vector is a pET-28a vector, and the T7 promoter of the expression vector is fused with a StrepII-8His-TEV-GG tag, the sequence of which is shown in SEQ ID NO.6.

[0014] As a fifth aspect of the invention, the use of any of the above-described 7β-HSDH enzyme mutants, any of the above-described polynucleotides, or any of the above-described recombinant plasmids in the catalytic production of ursodeoxycholic acid from 7-ketolithocholic acid is also provided.

[0015] As a further optimization of the present invention, the application involves adding the 7β-hydroxysteroid dehydrogenase mutant to the catalytic reaction system, wherein the catalytic reaction system also includes the coenzyme NADPH.

[0016] The present invention has the following beneficial effects: This invention focuses on 7β-HSDH enzymes derived from *Ruminococcus truncatula*, providing a series of single-point and combined mutations of these 7β-HSDH enzymes. These mutants exhibit 1-20°C higher thermostability and 1.2-4.2 times higher activity compared to wild-type 7β-HSDH enzymes. Among them, the (C217I, E252K) mutant not only shows improved thermostability (Tm value) compared to wild-type 7β-HSDH enzymes, but also demonstrates significantly higher enzyme activity. The (T103I, D251K, L229K, S161A) mutant exhibits 4.2 times the activity of the wild-type 7β-HSDH enzyme and possesses extremely high thermal stability, with a Tm value approaching 68°C. Furthermore, it maintains 50% of its enzyme activity. Therefore, the 7β-HSDH enzyme mutant provided by this invention has a wider range of applications than the wild-type 7β-HSDH enzyme and is more suitable for the efficient production of ursodeoxycholic acid from 3α-hydroxy-7-oxo-5β-cholanic acid (7-ketolithocholic acid) via biotransformation, which is beneficial for large-scale production and industrial applications. Attached Figure Description

[0017] Figure 1 The purification results of 14 single-point mutant proteins of 7β-HSDH provided by this invention; Figure 2 Purification results of 11 combinatorial mutant proteins of 7β-HSDH provided by this invention; Figure 3 The activity assay results of 14 single-point mutant proteins of 7β-HSDH provided by this invention; Figure 4 The activity assay results of 11 combined mutant proteins of 7β-HSDH provided by this invention; Figure 5 The affinity purification results of the wild-type 7β-HSDH enzyme and two high-quality 7β-HSDH enzyme mutant proteins provided by this invention; Figure 6 QC test results for wild-type 7β-HSDH and two high-quality 7β-HSDH enzyme mutant proteins provided for this invention. Detailed Implementation

[0018] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0019] 1. Materials Unless otherwise specified, all methods used in this invention are conventional methods known to those skilled in the art. Where specific conditions are not specified, they shall be performed according to conventional conditions or conditions recommended by the manufacturer. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0020] 2. Method 2.1 Construction of wild-type 7β-HSDH enzyme and its mutant protein grains The gene sequences of wild-type 7β-HSDH enzyme and its mutants were obtained through gene synthesis methods.

[0021] The protein sequence of the wild-type 7β-HSDH enzyme derived from Ruminococcus torques is shown in SEQ ID NO.1 (Uniprot: D4M4P2). All mutants were constructed using corresponding mutant primers designed based on the wild-type 7β-HSDH enzyme sequence, and constructed using molecular cloning methods. Single-point mutations include 14 mutations: E18G; H60T; A65V; T103V; T103I; R202R; L229R; L229Q; L229K; E235P; N237W; Q245M; D251K; and E252K. The combined mutations include (T103I, L229R); (T103I, E18G); (T103I, D251K); (T103I, E252K); (C217I, L229R); (C217I, E18G); (C217I, D251K); (C217I, E252K); (S161A, E18G); (T103I, D251K, L229K, S161A) and (T103I, E18G, L229K, S161A), totaling 11.

[0022] Wild-type 7β-HSDH enzyme and its mutants were constructed on the modified pET-28a vector (GenScript). The T7 promoter of this vector is fused with a StrepII-8His-TEV-GG tag sequence, which is shown in SEQ ID NO.6 (where Strep II and 8His are tag sequences used for affinity purification, "TEV" is the TEV protease cleavage site used for tag removal during subsequent purification, and "GG" is the tag sequence). The gene sequences of the recombinant proteins were verified to be correct by the sequencing company.

[0023] 2.2. Small-scale expression and purification of 7β-HSDH enzyme mutant 2.2.1 Low-level expression of wild-type 7β-HSDH enzyme and its mutants The small-scale expression of the 7β-HSDH enzyme mutant was performed using a cell-free expression method. Cell-free reagents were purchased from Biortus. Specific expression protocols were described in [Levine, MZ, et al. (2019). Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology]. The main steps included: (1) Preparation of crude extract of Escherichia coli: BL21(DE3) bacterial culture was inoculated into 2×YT medium and cultured at 37℃ until OD 600 When the concentration reaches 0.6-0.8, add isopropyl β-D-1-thiogalactopyranoside (IPTG). OD 600 At approximately 3 minutes, centrifuge to collect the bacteria. Wash each gram of wet bacterial cells three times with S30 buffer (10 mM Tris-acetate, pH 8.2, 14 mM magnesium acetate, 50 mM potassium acetate, 2 mM DTT) at 4 °C. Resuspend the bacterial cells in 1 mL of S30 buffer at a ratio of 1 g of bacterial cells. After sonicating the cells with an ultrasonic disruptor, centrifuge at 13000×g for 10 minutes. Transfer the supernatant to a nuclease-free tube, flash freeze in liquid nitrogen, and store at -80 °C.

[0024] (2) Expression of protein in cell-free system: Plasmid, cell extract, reaction buffer (phosphoenolpyruvate, PEG mixture, potassium glutamate, magnesium glutamate, amino glutamate, 20 amino acids, reaction energy substances (nicotinamide adenine dinucleotide, adenosine 5'-triphosphate disodium salt, cytidine disodium salt, guanosine 5'-monophosphate disodium hydrate and uridine disodium salt, leucovorin calcium salt and tRNA), glucose, spermidine and 1,4-diaminobutane, etc., were added to a 15 mL nuclease-free centrifuge tube according to the proportions in the literature. The reaction system was 200 μL. The reaction conditions were 37 ℃, 200 rpm. After 4 hours, the reaction solution was centrifuged at 12000 rpm for 10 minutes. The supernatant and precipitate were collected and the samples from each step were fixed with loading.

[0025] Wild-type 7β-HSDH enzyme was expressed in small quantities using the same method.

[0026] 2.2.2 Small-scale purification of the 7β-HSDH enzyme mutant After expression in the cell-free system, 50 μL of Strep-Tactin®XT packing material, which had been treated with buffer (25 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM TCEP), was added to the supernatant. The mixture was incubated at 4°C for 30 minutes. The incubated samples were then centrifuged at 12000 rpm at 4°C for 10 minutes. After washing three times with 1 mL of buffer, 100 μL of elution buffer (25 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM TCEP, 75 mM biotin) was added, followed by washing twice with 100 μL of elution buffer (25 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM TCEP, 75 mM biotin). The mixture was then centrifuged at 12000 rpm at 4°C for 5 minutes. The eluted samples were collected. A small amount of the samples from each step was fixed with loading buffer and analyzed by SDS-PAGE.

[0027] The experimental results are shown in Figure 1 All 14 single mutant proteins were clearly expressed.

[0028] 2.3 Detection of the thermostability of wild-type 7β-HSDH enzyme and its mutants The thermal stability of wild-type 7β-HSDH enzyme and its mutants was determined using protein thermal shift (ThermoFluor). This technique utilizes the structural characteristics of proteins: hydrophobic regions are hidden internally. As temperature rises, the protein structure opens up, exposing the hydrophobic regions. The fluorescent dye SYPRO Orange can then bind to these regions, exciting fluorescence. A melting curve is formed based on the change in fluorescence signal intensity. The temperature corresponding to the maximum value of the derivative of the melting curve is the melting point (Tm). The more stable the protein, the higher the measured Tm value.

[0029] The specific steps are as follows: Take 5 μg of wild-type 7β-HSDH enzyme and its mutant and add them to a 96-well PCR plate. Add 10×SYPROOrange fluorescent dye to the corresponding wells. Place the 96-well PCR plate in a qPCR instrument, set the instrument parameters, and increase the temperature from 25 °C to 99 °C at a gradient of 1 °C per minute. Calculate the protein melting curve.

[0030] The Tm values ​​of wild-type 7β-HSDH enzyme and all its single mutants are shown in Table 1.

[0031] Table 1. Results of Tm value determination for wild-type 7β-HSDH enzyme and its single mutant. ; As shown in Table 1, among the 14 single mutant proteins, except for the A65V single mutant which had a reduced Tm value of 1℃ and the E252K single mutant which had the same Tm value as the wild-type 7β-HSDH enzyme, the Tm values ​​of the other single mutants were increased. Therefore, this invention screened out 12 mutants that can improve the thermostability of 7β-HSDH. Among them, the mutation sites of the single mutant proteins with Tm values ​​above 50℃ were T103V and T103I. Among all single mutants, T103I had the highest Tm value, at 55.6℃.

[0032] In addition to single mutations, a series of combined mutations (including double, triple, and quadruple mutations) were designed. The expression and purification of the combined mutant proteins were the same as those of the single mutant proteins. The results are shown in […]. Figure 2 All the combined mutant proteins were clearly expressed. After obtaining the proteins, the Tm values ​​of the combined mutant proteins were also measured, and the results are shown in Table 2.

[0033] Table 2. Results of Tm value determination for 7β-HSDH combined mutants ; As shown in Table 2, the Tm values ​​of all 11 combined mutant proteins were increased, ranging from 1.6 to 20.7 °C. Among them, the Tm values ​​of the (T103I, D251K, L229K, S161A) and (T103I, E18G, L229K, S161A) mutant proteins were increased by more than 20 °C.

[0034] 2.4 Activity assay of wild-type 7β-HSDH enzyme and its mutants The activity assay of the 7β-HSDH enzyme mutant used 3α-hydroxy-7-oxo-5β-cholanic acid (7-Ketolithocholic acid, 7-KLCA) as a substrate and NADPH as a coenzyme. The activity of the 7β-HSDH enzyme was calculated by detecting the amount of NADPH converted into NADP+.

[0035] When determining the activity of the 7β-HSDH enzyme mutant, the enzyme activity parameter is expressed as the intensity of the absorbance signal (340 nm) generated per nanomolar of the 7β-HSDH enzyme mutant per second. The specific experimental procedures are as follows: Preparation buffer: 50 mM Tris-HCl pH 7.0, substrate 1 mM T-7-KLCA, coenzyme 0.5 mM NADPH, reaction temperature 25 ℃, and 30 nM of different concentrations of 7β-HSDH mutants. 20 μL of dissolved T-7-KLCA was transferred to a 384-well plate with two replicates. 20 μL of NADPH was added to each well, followed by 20 μL of each different 7β-HSDH enzyme mutant to the corresponding well. The plates were immediately centrifuged and vortexed to mix. Fluorescence signals were collected using a TECAN F200 microplate reader. Data analysis was performed using GraphPad Prism9 software to obtain the enzyme activity parameters of the tested 7β-HSDH enzyme mutants.

[0036] The activity of wild-type 7β-HSDH enzyme was determined using the same method.

[0037] The results are as follows Figure 3 and Figure 4 As shown, all single mutants exhibited a certain degree of activity improvement compared to the wild-type 7β-HSDH enzyme, with an increase of approximately 1.1-1.9 times. Among the combinatorial mutants (T103I, D251K, L229K, S161A) and (T103I, E18G, L229K, S161A) mutants, the activity decreased, but still maintained 30%-50% of the enzyme activity. The activities of other combinatorial mutants increased by 1.2-4.2 times.

[0038] 2.5 Expression and purification of high-quality 7β-HSDH mutant proteins To further investigate the function of the superior 7β-HSDH mutant protein, two mutants with the most significant increase in activity (C217I, E252K) and the largest increase in Tm value (T103I, D251K, L229K, S161A) were selected. Their amino acid sequences are shown in SEQ ID NO. 2-3, and their nucleotide sequences are shown in SEQ ID NO. 4-5, respectively. Heterologous expression was performed in *E. coli*, and the expressed protein was purified.

[0039] 2.5.1 Expression of high-quality 7β-HSDH mutant proteins The 7β-HSDH (C217I, E252K), 7β-HSDH (T103I, D251K, L229K, S161A), and wild-type 7β-HSDH recombinant plasmids were transformed into BL21(DE3) strains, respectively. The strains were then inoculated into 50 ml LB broth and cultured overnight at 37°C. The overnight cultured bacteria were then inoculated into 1 L LB broth at a 1:100 ratio and cultured at 37°C until the bacterial culture reached its OD value. 600 When the concentration is 0.6-0.8, add 0.5 mM IPTG, incubate overnight at 15°C, and collect the bacterial cells by centrifugation at 5000 rpm for purification.

[0040] 2.5.2 Purification of high-quality 7β-HSDH mutant protein Affinity Chromatography The collected bacterial blocks were weighed separately, and lysis buffer (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5% glycerol) was added at a 1:10 ratio. The bacterial cells were lysed using a high-pressure homogenizer, and the supernatant was collected by centrifugation at 16,000 rpm. All recombinant 7β-HSDH mutants were tagged with StrepII, and the protein was enriched and purified using a Strep-Tactin XT affinity chromatography column.

[0041] The specific process is as follows: First, wash and equilibrate the Strep-Tactin XT affinity chromatography column with lysis buffer for 10 column volumes. Then, load the lysis supernatant onto the Strep-Tactin XT FF affinity chromatography column and elute with lysis buffer containing 75 mM biotin. Collect the eluted protein for SDS-PAGE analysis and use Nanodrop to determine the protein concentration and calculate the protein yield.

[0042] Protein purification results are as follows Figure 5Purification results of wild-type 7β-HSDH enzyme and 7β-HSDH (C217I, E252K) and 7β-HSDH (T103I, D251K, L229K, S161A) mutants showed that both had high purity.

[0043] (2) Enzyme digestion and reverse affinity chromatography To obtain a protein with higher purity, a certain amount of TEV enzyme was added to the sample after affinity chromatography. After digestion at 4°C overnight, the supernatant was further purified using a HisFF chromatography column. Since wild-type 7β-HSDH enzyme and its mutant protease do not have affinity tags after digestion, they will not bind to the affinity column and will flow out of the column (denoted as permeate). Therefore, the permeate was collected.

[0044] (3) Gel filtration chromatography and QC detection The permeate was concentrated to approximately 2 mL and then subjected to gel filtration chromatography. The gel chromatography column was a Superdex 200 Increase 10 / 300GL, and the buffer consisted of 20 mM Tris–HCl pH 7.5, 150 mM NaCl, and 1 mM DTT. The gel filtration samples were collected and subjected to protein content analysis, specifically SDS-PAGE purity determination, mass spectrometry analysis, and analytical molecular sieve detection.

[0045] Test results as follows Figure 6 As shown in the SDS-PAGE results, the purity of both the wild-type 7β-HSDH enzyme and its mutants is greater than 99%. Mass spectrometry results also show that the molecular weight of the tested samples is basically consistent with the target protein, indicating that the purified protein is the target protein. Furthermore, analytical molecular sieve analysis shows that all proteins are in a near-dimer state in solution.

[0046] 3. Conclusion The above description indicates that this invention uses wild-type 7β-HSDH enzyme from *Ruminococcus truncatula* as the research object. Single or combined mutations were performed on the protein sequence of wild-type 7β-HSDH enzyme to obtain several mutant proteins. These 7β-HSDH enzyme mutants exhibit improved thermostability by 1-20°C and maintained activity at 30%-420% compared to wild-type 7β-HSDH enzyme. Furthermore, two superior 7β-HSDH enzyme mutant proteins, (C217I, E252K) mutant and (T103I, D251K, L229K, S161A) mutant, were selected. These mutants exhibit higher enzyme activity and better thermostability compared to wild-type 7β-HSDH enzyme, respectively. Therefore, compared to wild-type 7β-HSDH enzyme, they have broader application conditions and stronger practical application value, making them more suitable for large-scale production and industrial use.

[0047] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A 7β-HSDH enzyme mutant derived from *Ruminococcus tortifolius*, characterized in that, The 7β-HSDH enzyme mutant was obtained by single or combined mutations of the protein sequence of wild-type 7β-HSDH enzyme derived from Ruminococcus tortifolia, as shown in SEQ ID NO.

1. The single mutation site includes at least one of E18G, H60T, T103V, T103I, R202R, L229R, L229Q, L229K, E235P, N237W, Q245M or D251K; The combined mutation sites include at least one of the following (1)-(11): (1) T103I, L229R; (2) T103I, E18G; (3) T103I, D251K; (4) T103I, E252K; (5) C217I, L229R; (6) C217I, E18G; (7) C217I, D251K; (8) C217I, E252K; (9) S161A, E18G; (10) T103I, D251K, L229K, S161A; (11) T103I, E18G, L229K, S161A.

2. The 7β-HSDH enzyme mutant derived from *Ruminococcus tortifolia* according to claim 1, characterized in that, The 7β-HSDH enzyme mutant is based on the combined mutation sites C217I and E252K of the protein sequence of the wild-type 7β-HSDH enzyme, and the amino acid sequence of the 7β-HSDH enzyme mutant is shown in SEQ ID NO.

2.

3. The 7β-HSDH enzyme mutant derived from *Ruminococcus tortifolia* according to claim 1, characterized in that, The 7β-HSDH enzyme mutant is based on a combination of mutation sites T103I, D251K, L229K and S161A on the protein sequence of the wild-type 7β-HSDH enzyme, and the amino acid sequence of the 7β-HSDH enzyme mutant is shown in SEQ ID NO.

3.

4. A polynucleotide, characterized in that, The polynucleotide encodes the 7β-HSDH enzyme mutant as described in any one of claims 1-3.

5. A polynucleotide according to claim 4, characterized in that, The sequence of the polynucleotide is shown in SEQ ID NO.4 or SEQ ID NO.

5.

6. A recombinant plasmid, characterized in that, The recombinant plasmid is an expression vector containing the polynucleotides as described in any one of claims 4-5 and capable of translating and expressing the 7β-HSDH enzyme mutant as described in any one of claims 1-3.

7. A recombinant plasmid according to claim 6, characterized in that, The expression vector is the pET-28a vector, and the T7 promoter of the pET-28a vector is fused with a StrepII-8His-TEV-GG tag, the sequence of which is shown in SEQ ID NO.

6.

8. The use of a 7β-HSDH enzyme mutant as described in any one of claims 1-3, a polynucleotide as described in any one of claims 4-5, or a recombinant plasmid as described in any one of claims 6-7 in the catalytic production of ursodeoxycholic acid from 7-ketolithocholic acid.

9. The application according to claim 8, characterized in that, The application involves adding the 7β-hydroxysteroid dehydrogenase mutant to a catalytic reaction system, which also includes the coenzyme NADPH.

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