7beta-hydroxysteroid dehydrogenase mutants and uses thereof

Abstract

The application discloses a 7beta-hydroxysteroid dehydrogenase mutant and application thereof, relates to the field of bioengineering technology, and is designed and reformed according to the amino acid sequence of wild-type 7beta-hydroxysteroid dehydrogenase derived from Collinsella aerofaciens, that is, 17 single-mutation sites or 7 combined-mutation sites of 7beta-hydroxysteroid dehydrogenase are provided, the obtained 7beta-hydroxysteroid dehydrogenase mutant has the thermal stability increased by 2-21.5 DEG C and the activity increased by nearly 1.2-2.24 times compared with the wild-type 7beta-hydroxysteroid dehydrogenase. According to the mutation sites with obvious increase in thermal stability and activity, three preferred mutant proteins are further provided, the three preferred mutant proteins have higher yield, activity and thermal stability compared with the wild-type 7beta-hydroxysteroid dehydrogenase, have wider application conditions, are more suitable for the efficient production of ursodeoxycholic acid by the bioconversion method, and are favorable for large-scale production and industrial application.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a 7β-hydroxysteroid dehydrogenase mutant and its applications. Background Technology

[0002] Ursodeoxycholic acid (UDCA) is the main medicinal component of bear bile. It is an FDA-approved bile acid derivative for the treatment of cholesterol gallstones and is currently the only alternative to surgical intervention. Bear bile resources are scarce, therefore, there is a need to find efficient in vitro preparation methods. In vitro preparation methods mainly include chemical synthesis and biotransformation. However, only 30% of ursodeoxycholic acid is efficiently obtained through chemical synthesis. Furthermore, chemical synthesis is cumbersome, involves many side reactions, often involving hazardous and toxic reagents, and generates significant environmental pollution. Therefore, biotransformation is currently the mainstream method. It uses cholic acid and chenodeoxycholic acid as raw materials to produce ursodeoxycholic acid through microbial enzymatic conversion. Specifically, 7α-hydroxysteroid dehydrogenase (7α-HSDH) in the presence of NAD(P)+ forms an intermediate from chenodeoxycholic acid, and then 7β-hydroxysteroid dehydrogenase (7β-HSDH) reduces and aminations the intermediate to produce ursodeoxycholic acid, thus achieving green and efficient production of ursodeoxycholic acid.

[0003] 7β-HSDH is a key enzyme in the synthesis of UDCA. Currently, the number of reported 7β-HSDH enzymes is limited. The main sources of reported 7β-HSDH enzymes, both domestically and internationally, include Clostridium absonum, Eubacterium aerofaciens, Ruminococcus torques, Ruminococcus gnavus, Collinsella aerofaciens, and Xanthomonas maltophilia. These enzymes all suffer from low activity or poor thermostability. While there is considerable research on the activity of 7β-HSDH derived from Collinsella aerofaciens, its poor thermostability—losing activity after 5 minutes at 50°C and maintaining activity for only 400 minutes at 40°C—significantly limits its application conditions, hindering large-scale production and industrial use. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a 7β-hydroxysteroid dehydrogenase mutant and its application.

[0005] The present invention achieves the above objectives through the following technical solutions:

[0006] As a first aspect of the present invention, a 7β-hydroxysteroid dehydrogenase mutant is provided, which is obtained by single-point mutation or combination mutation of the sequence of wild-type 7β-hydroxysteroid dehydrogenase as shown in SEQ ID NO.1;

[0007] The single-point mutation site includes at least one of I13V, K27A, T55K, A110K, Q155F, C217A, E182L, L164T, W152Y, D251E, C172R, L164R, C217V, L229R, L229K, S230E, or T103I;

[0008] The combined mutation sites include at least one of the following: (I13V, F87L), (K27R, C217A, F222L, E223A), (K27R, C217A, F222L), (K27R, E182L, I184V, C217A, F222L), (H108I, W152Y, Q155F), (L164Q, E182Q, F222L), or (L164D, E182L, I184V, C217A).

[0009] As a further optimization of the present invention, the wild-type 7β-hydroxysteroid dehydrogenase is derived from Collins aerogenes.

[0010] As a further optimization of the present invention, the mutation sites of the 7β-hydroxysteroid dehydrogenase mutant are T103I, L229K or (H108I, W152Y, Q155F), and the amino acid sequences of the 7β-hydroxysteroid dehydrogenase mutant obtained by the above mutation are shown in SEQ ID NO.2-SEQ ID NO.4.

[0011] As a second aspect of the invention, the use of any of the above-described 7β-hydroxysteroid dehydrogenase mutants in enhancing 7β-hydroxysteroid dehydrogenase activity or thermal stability is also provided.

[0012] As a third aspect of the invention, a polynucleotide is also provided, said polynucleotide encoding a 7β-hydroxysteroid dehydrogenase mutant as described in any of the above descriptions.

[0013] As a further optimization of the present invention, the polynucleotide sequences encoding the 7β-hydroxysteroid dehydrogenase mutant as described above are shown in SEQ ID NO.6-SEQ ID NO.8 in sequence.

[0014] As a third aspect of the invention, a recombinant plasmid is also provided, the recombinant plasmid being an expression vector containing an expression vector capable of translating and expressing any of the 7β-hydroxysteroid dehydrogenase mutants described above.

[0015] As a further optimization of the present invention, the expression vector is the pET-28a vector.

[0016] The present invention has the following beneficial effects:

[0017] This invention uses 7β-HSDH derived from *Colinella aerogenes* as the research object. Using the computer design software ProteinMPNN, numerous effective mutation sites were designed, resulting in a series of single-point and combined mutations of 7β-HSDH. These mutant proteins exhibit improved thermostability (2-21.5℃) and activity (approximately 1.2-2.24 times) compared to wild-type 7β-HSDH. The 7β-HSDH mutants demonstrate higher yield, activity, and thermostability than wild-type 7β-HSDH, offering broader application possibilities and making them more suitable for the efficient biotransformation production of ursodeoxycholic acid, thus facilitating large-scale production and industrial applications. Attached Figure Description

[0018] Figure 1 The image shows the modified pET-28a vector.

[0019] Figure 2A , 2B 2C represents the small-scale purification results of the 7β-HSDH single mutant protein in a cell-free expression system;

[0020] Figure 3A , 3B 3C represents the small-scale purification results of the 7β-HSDH combined mutant protein in a cell-free expression system;

[0021] Figure 4 The results of the activity assay for 7β-HSDH mutant proteins are shown in the figure (numbers 1-48 in the figure correspond to the 7β-HSDH mutant proteins shown in Table 1-2).

[0022] Figure 5 The results are from affinity chromatography purification of the preferred 7β-HSDH mutant protein;

[0023] Figure 6 The results show the protein content of the preferred 7β-HSDH mutant. Detailed Implementation

[0024] 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.

[0025] 1. Materials and Reagents

[0026] 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.

[0027] 2. Method

[0028] 2.1 Construction of the 7β-HSDH mutant plasmid

[0029] The gene sequences of wild-type 7β-HSDH and its mutants provided in this embodiment were obtained through gene synthesis. The protein sequence of wild-type 7β-HSDH is shown in SEQ ID NO.1. All mutants were constructed using molecular cloning methods based on corresponding mutant primers designed from the wild-type mutants. These mutants included 30 single-point mutations: I13V, C172K, C217V, D251E, M85L, A131R, S230H, K121A, H108K, A110K, L120K, C217A, E182Q, E182L, C172R, F222L, L164D, T55R, L164T, T55K, Q155L, L229R, L164R, W152Y, T103I, Q155F, V117T, K27A, C172A, and L229K.

[0030] Combinatorial mutations include: (I13V, F87L), (I13V, M85L, F87L), (K27R, C217A, F222L, E223A), (K27R, C217A, F222L), (K27R, E182L, I184V, C217A, F222L), (K27R, E182Q, F222L, E223A), (F87L, A131K, C172R, E182L), (T103I, K107T, H108I, Q155F), (H108I, W152Y, Q155F), (L164Q, E182Q, F2 A total of 18 combined mutant proteins were identified, including (L164D, E182L, I184V, C217A, F222L), (F87L, A131K, C172R, L164D, C172R, E182L, I184V, F222L), (L164D, E182L, I184V, C217A), (L164D, C172R, E182L), (L164D, C172R, E182L, I184V), (L164Q, E182Q), (E182Q, F222L, E223A), and (I184V, C217A, F222L).

[0031] Wild-type 7β-HSDH and its mutant proteins were constructed into the modified pET-281 vector (GenScript). This vector has an 8His-strepII-TEV-GG tag sequence fused to the T7 promoter. The tag sequence is shown in SEQ ID NO. 5 (where 8His and strep II 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 constructed recombinant proteins were verified to be correct by the sequencing company. The vector map is shown below. Figure 1 .

[0032] 2.2 Expression and purification of 7β-HSDH mutant protein

[0033] 2.2.1 Low-level expression of 7β-HSDH mutant protein

[0034] The low-level expression of the 7β-HSDH mutant protein was performed using a cell-free expression method. The cell-free expression protocol is described in [Levine, MZ, et al. (2019). Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology]. It mainly includes the following two steps:

[0035] (1) Preparation of crude extract of Escherichia coli: BL21(DE3) bacterial culture was inoculated into 2×YT medium and cultured at 37℃ until D 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.

[0036] (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.

[0037] 2.2.2 Small-scale purification of 7β-HSDH mutant protein

[0038] (A) Small-scale purification of 7β-HSDH single mutant protein

[0039] The supernatant from the expression in the cell-free system was added to 50 μL of Strep-1200-dose buffer solution (25 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM TCEP). XT packing material was incubated at 4°C for 30 minutes. After incubation, the sample was centrifuged at 12000 rpm for 10 minutes at 4°C. 1 mL of buffer was added, and the sample was washed three times. Then, 100 μL of elution buffer (25 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM TCEP, 75 mM biotin) was added, and the sample was centrifuged at 12000 rpm for 5 minutes at 4°C. The eluted sample was collected. A small amount of sample from each step was fixed with loading buffer and analyzed by SDS-PAGE. Experimental results are shown below. Figure 2A , Figure 2B and Figure 2C Of the 30 single-point mutations, all single-point mutant proteins were significantly expressed. FSEC analysis of the reacted samples revealed that, except for the A131R and L164D single-point mutant proteins which exhibited a monomeric aggregation state, the other mutant proteins were dimeric proteins, similar to the wild-type. Purification of all expressed samples showed that, except for the L120K and H108K mutant proteins which were insoluble, all other proteins could be eluted with high purity.

[0040] (B) Small-scale purification of 7β-HSDH combined mutant protein

[0041] The purification method for combined mutants was the same as that for single-point mutants. Small-scale purification results showed that all 18 combined mutant proteins were clearly expressed in a cell-free system. FSEC results of the expressed samples showed that all combined mutant proteins aggregated in a dimer state. Further purification was performed on all expressed samples; the purification results are shown below. Figure 3A , Figure 3B and Figure 3C All samples can be eluted and are of high purity.

[0042] 2.3. Detection of thermal stability of 7β-HSDH mutant protein

[0043] The thermal stability of the 7β-HSDH mutant protein was tested using protein thermal shift (ThermoFluor). Utilizing the protein's structural characteristics, the protein possesses hydrophobic regions hidden internally. As temperature rises, this structure opens up, exposing the hydrophobic regions. The fluorescent dye SYPRO Orange binds to these regions, stimulating fluorescence. Changes in fluorescence intensity form a melting curve, and the temperature corresponding to the maximum derivative of the melting curve is the melting point (Tm). The more stable the protein, the higher the measured Tm value.

[0044] The specific steps are as follows:

[0045] 5 μg of 7β-HSDH mutant protein was added to each well of a 96-well PCR plate, followed by 10×SYPRO Orange fluorescent dye. The 96-well PCR plate was placed in a qPCR instrument, and the instrument parameters were set to increase the temperature from 25℃ to 99℃ at a gradient of 1℃ per minute. The protein melting curves were calculated. The specific Tm values ​​for all single mutant proteins are shown in Table 1.

[0046] Table 17. Tm values ​​of β-HSDH single mutant proteins

[0047]

[0048]

[0049] As shown in Table 1, among the 28 single mutant proteins, the Tm values ​​of C217A, E182L, L164T, W152Y, C172R, and L164R increased by 1-4℃; the Tm value of C217V increased by 4.79℃; the Tm value of D251E increased by 6.89℃; the Tm value of L229R increased by 4.82℃; the Tm value of L229K increased by 8.37℃; and the Tm value of T103I increased by 9.08℃. Therefore, this invention screened out 11 mutants (C217A, E182L, L164T, D251E, W152Y, C172R, L164R, C217V, L229R, L229K, and T103I) that can improve the thermal stability of 7β-HSDH.

[0050] The Tm values ​​of the specific combined mutant proteins tested are shown in Table 2.

[0051] Table 27. Tm values ​​of β-HSDH combined mutant proteins

[0052]

[0053]

[0054] As shown in Table 2, except for the (L164Q, E182Q) combination mutant protein for which no Tm value was detected, the Tm values ​​of other combination mutants were significantly higher than those of the wild type, with an increase of approximately 5-21.5℃. Among them, the Tm values ​​of the three combination mutant proteins (H108I, W152Y, Q155F), (I13V, M85L, F87L), and (T103I, K107T, H108I, Q155F) all exceeded 60℃.

[0055] 2.4 Detection of 7β-HSDH mutant protein activity

[0056] 7β-HSDH uses chenodeoxycholic acid (CDCA) as a substrate to convert NADP+ to NADPH. The activity assay system provided in this invention uses CDCA and NADP+ as substrates. Since NADP+ is colorless, while NADPH exhibits fluorescence absorption at 340 nm, the fluorescence signal can be detected at 340 nm after NADP+ is converted to NADPH in the reaction system. This invention expresses enzyme activity parameters as the fluorescence signal intensity per second produced by each nanomolar of 7β-HSDH and its mutant protein. Specific experimental procedures are as follows:

[0057] Preparation buffer: 50 mM Glycine-NaOH, pH 9.0; substrates: 0.5 mM CDCA and 0.5 mM NADP+; reaction temperature: 30℃. 7β-HSDH and its mutant proteins were serially diluted 2-fold from 200 nM using the buffer, resulting in 12 concentrations. 50 μL of CDCA dissolved in 50 mM Glycine-NaOH was transferred to a 384-well plate with two replicates. 40 μL of NADP+ was added to each well, followed by 10 μL of each diluted 7β-HSDH and its mutant protein 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 GraphPadPrism9 software to obtain the enzyme activity parameters of the tested proteases. Results are shown below. Figure 4 As shown.

[0058] Among them, the activities of 12 single mutants, namely I13V, K27A, T55K, T103I, A110K, W152Y, Q155L, Q155F, C217A, L229R, L229K and S230E, were all increased to a certain extent, by about 1.2-2.24 times. The combined mutations (I13V, F87L), (K27R, C217A, F222L, E223A), (K27R, C217A, F222L), (K27R, E182L, I184V, C217A, F222L), (H108I, W152Y, Q155F), (L164Q, E182Q, F222L), and (L164D, E182L, I184V, C217A) all showed an activity increase of 1.4-1.6 times.

[0059] Based on the data on thermal stability and activity, it can be seen that among single mutations, T103I, W152Y, L164R, C172R, L229R, and L229K are high-quality mutation sites that improve both thermal stability and activity; among combined mutations, (I13V, F87L), (K27R, C217A, F222L, E223A), (K27R, C217A, F222L), (K27R, E182L, I184V, C217A, F222L), (H108I, W152Y, Q155F), (L164Q, E182Q, F222L), and (L164D, E182L, I184V, C217A) are high-quality mutation sites that improve both thermal stability and activity.

[0060] 2.5 Expression and purification of high-quality 7β-HSDH mutant proteins

[0061] To further investigate the function of high-quality 7β-HSDH mutant proteins, single mutant proteins T103I and L229K, which showed significant improvements in Tm value and activity, were selected. Their amino acid sequences are shown in SEQ ID NO.1-2 and their nucleotide sequences are shown in SEQ ID NO.6-7, respectively. A combined mutant protein (H108I, W152Y, Q155F), with its amino acid sequence shown in SEQ ID NO.3 and its nucleotide sequence shown in SEQ ID NO.8, was also selected. These mutant proteins were heterologously expressed in E. coli, and the expressed proteins were then purified.

[0062] 2.5.1 Expression of high-quality 7β-HSDH mutant proteins

[0063] After transforming the above-mentioned 7β-HSDH(T103I), 7β-HSDH(L229K), and 7β-HSDH(108I, W152Y, Q155F) and wild-type 7β-HSDH recombinant plasmids into BL21(DE3) strain, the strains were inoculated into 50 ml of LB liquid medium and cultured overnight at 37°C. The overnight cultured bacteria were then inoculated into 1 L of LB liquid medium at a ratio of 1:100 and cultured at 37°C until the bacterial culture reached OD. 600 When the concentration is 0.6-0.8, add 0.5mM IPTG, incubate overnight at 15℃, and collect the bacterial cells by centrifugation at 5000rpm for purification.

[0064] 2.5.2 Purification of high-quality 7β-HSDH mutant protein

[0065] (1) Affinity chromatography

[0066] The collected bacterial blocks were weighed and added to the appropriate volume of lysis buffer (50mM Tris-HCl (pH 7.5), 500mM NaCl, 5% glycerol) at a 1:10 ratio. The bacterial cells were homogenized using an autoclave, and the supernatant was collected by centrifugation at 16,000 rpm. All recombinant 7β-HSDH mutant proteins were StrepII-tagged, and the proteins were enriched and purified using a Strep-Tactin XT affinity chromatography column. The specific procedure was as follows: the Strep-Tactin XT affinity chromatography column was first washed and equilibrated with lysis buffer to a volume of 10. Then, the lysis supernatant was loaded onto a Strep-Tactin XT FF affinity chromatography column and eluted with lysis buffer containing 75mM biotin. The eluted protein was collected for SDS-PAGE analysis, and the protein concentration was determined using Nanodrop to calculate the protein yield. The protein purification results are shown below. Figure 6 The yield of the wild type was 12.5 mg / L, while the yields of T103I and L229K were 32.9 mg / L and 34.3 mg / L, respectively; the yield of (108I, W152Y, Q155F) was 23.1 mg / L. The yields of T103I and L229K were 2.6 and 2.7 times higher than those of the wild type, respectively, and the yield of (108I, W152Y, Q155F) was also 1.85 times higher than that of the wild type.

[0067] (2) Enzyme digestion and reverse affinity chromatography

[0068] To obtain a protein with higher purity, a certain amount of TEV enzyme was added to the sample after affinity chromatography. After overnight digestion at 4°C, the supernatant was further purified using a HisFF chromatography column. Since the 7β-HSDH wild-type and its mutant protease do not have an affinity tag after digestion, they will not bind to the affinity column and will flow out of the column (denoted as the permeate). Therefore, the permeate was collected.

[0069] (3) Gel filtration chromatography and QC detection

[0070] The permeate was concentrated to approximately 2 mL and then subjected to gel filtration chromatography. The gel chromatography column was a Superdex 200Increase 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.

[0071] SDS-PAGE results showed that the purity of both the wild-type and 7β-HSDH mutant proteins was greater than 99%. Mass spectrometry analysis also indicated that the molecular weight of the tested samples was essentially consistent with the target protein, confirming that the purified protein was the target protein. Furthermore, analytical molecular sieve analysis showed that all proteins were in a near-dimer state in solution. Detection results are as follows... Figure 5 As shown.

[0072] 3. Conclusion

[0073] The above description shows that the 7β-hydroxysteroid dehydrogenase mutant protein provided by the present invention has higher protein yield, higher enzyme activity and better thermal stability, has broader application conditions and stronger practical application value, and is more suitable for large-scale production and industrial use.

[0074] 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-hydroxysteroid dehydrogenase mutant, characterized in that, The 7β-hydroxysteroid dehydrogenase mutant was obtained by single-point mutation or combination mutation of the sequence of wild-type 7β-hydroxysteroid dehydrogenase as shown in SEQ ID NO.1; The single mutation site includes at least one of I13V, K27A, T55K, A110K, Q155F, C217A, E182L, L164T, W152Y, D251E, C172R, L164R, C217V, L229R, L229K, S230E, or T103I; The combined mutation sites include at least one of the following: (I13V, F87L), (K27R, C217A, F222L, E223A), (K27R, C217A, F222L), (K27R, E182L, I184V, C217A, F222L), (H108I, W152Y, Q155F), (L164Q, E182Q, F222L), or (L164D, E182L, I184V, C217A).

2. The 7β-hydroxysteroid dehydrogenase mutant according to claim 1, characterized in that, The wild-type 7β-hydroxysteroid dehydrogenase is derived from Collins aerogenes.

3. A 7β-hydroxysteroid dehydrogenase mutant according to claim 1, characterized in that, The mutation sites of the 7β-hydroxysteroid dehydrogenase mutant are T103I, L229K or (H108I, W152Y, Q155F), and the amino acid sequences of the 7β-hydroxysteroid dehydrogenase mutant obtained by the above mutations are shown in SEQ ID NO.2-SEQ ID NO.

4.

4. The use of a 7β-hydroxysteroid dehydrogenase mutant as described in any one of claims 1-3 in enhancing the activity or thermal stability of 7β-hydroxysteroid dehydrogenase.

5. A polynucleotide, characterized in that, The polynucleotide encodes the 7β-hydroxysteroid dehydrogenase mutant as described in any one of claims 1-3.

6. The polynucleotide according to claim 5, characterized in that, The polynucleotide sequence encoding the 7β-hydroxysteroid dehydrogenase mutant as described in claim 3 is shown sequentially as SEQ ID NO.6-SEQ ID NO.

8.

7. A recombinant plasmid, characterized in that, The recombinant plasmid is an expression vector containing the ability to translate and express the 7β-hydroxysteroid dehydrogenase mutant as described in any one of claims 1-3.

8. The recombinant vector according to claim 7, characterized in that, The expression vector is pET-28a.

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