A novel 7α-hydroxysteroid dehydrogenase gene R-α-1 and its application in the production of converted bear bile powder.

Abstract

This invention relates to the field of artificial bear bile powder preparation technology, specifically to a novel 7α-hydroxysteroid dehydrogenase gene R-α-1 and its application in the production and conversion of bear bile powder. This method isolates a novel gene catalyzing asymmetric carbonyl reduction reactions from the feces of Sichuan brown bears. This gene encodes a 7α-hydroxysteroid dehydrogenase, which catalyzes the formation of the intermediate tauroursodeoxycholic acid from tauroursodeoxycholic acid, which is further catalyzed by a 7β-hydroxysteroid dehydrogenase to produce tauroursodeoxycholic acid, the main component of bear bile powder. The nucleotide sequence of this gene is shown in SEQ ID NO.1, and its encoded amino acid sequence is shown in SEQ ID NO.2. This technical solution can solve the technical problem that existing types of 7α-hydroxysteroid dehydrogenases cannot meet the needs of large-scale production due to unsatisfactory enzyme activity and thermostability, and has ideal prospects for widespread application.

Description

Technical Field

[0001] This invention relates to the field of artificial bear bile powder preparation technology, specifically to a novel 7α-hydroxysteroid dehydrogenase gene R-α-1 and its application in the production of converted bear bile powder. Background Technology

[0002] Tauroursodeoxycholic acid (TUDCA) has excellent liver and gallbladder protection effects, and is very effective in treating inflammation, gallstones, and convulsions, exhibiting a variety of physiological and pharmacological functions.

[0003] In recent years, with the advancement of biotechnology, many people have utilized bio-enzymes to catalyze the conversion of tauroursodeoxycholic acid (TCDCA, a substance enriched in poultry bile powder) into tauroursodeoxycholic acid (TUDCA, one of the active ingredients in bear bile powder). Tauroursodeoxycholic acid, a bile acid widely found in poultry bile, can be converted into the intermediate tauroursodeoxycholic acid (T-7-KLCA) through the action of 7α-hydroxysteroid dehydrogenase (7α-HSDH). T-7-KLCA can then be further catalyzed by 7β-hydroxysteroid dehydrogenase (7β-HSDH) to obtain tauroursodeoxycholic acid. Compared with chemical synthesis methods, tauroursodeoxycholic acid obtained through biocatalysis is both environmentally friendly and ensures the safety of the pharmaceutical product.

[0004] Currently, the main method for preparing tauroursodeoxycholic acid (TCDCA) using biocatalysis is to use poultry bile as raw material. Poultry bile is rich in tauroursodeoxycholic acid. After water precipitation, alcohol precipitation, and extraction, chicken bile can be used to extract high levels of TCDCA. Then, TCDCA can be converted into TUDCA under the co-catalysis of 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase. The conversion process is shown in Formula I.

[0005]

[0006] Currently, many researchers have screened out microorganisms that can produce 7α-HSDH, which are derived from Clostridium absonum, Bacteroides fragilis, Escherichia coli, Clostridium scindens, Clostridium sordellii, etc. The sources of wild-type 7α-HSDH are becoming more and more widespread, but their enzyme activity and thermal stability are relatively weak, making it difficult to meet the requirements when used for production catalysis. There is an urgent need to research and develop a new 7α-hydroxysteroid dehydrogenase that can meet the needs of large-scale production. Summary of the Invention

[0007] The purpose of this invention is to provide a novel 7α-hydroxysteroid dehydrogenase gene, R-α-1, to solve the technical problem that existing types of 7α-hydroxysteroid dehydrogenases cannot meet the needs of large-scale production due to their unsatisfactory enzyme activity and thermostability.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A novel 7α-hydroxysteroid dehydrogenase R-α-1 gene, the sequence of which is shown in SEQ ID NO.1.

[0010] This technical solution also provides an expression cassette, vector, or engineered cell containing the 7α-hydroxysteroid dehydrogenase R-α-1 gene.

[0011] This technical solution also provides a novel 7α-hydroxysteroid dehydrogenase, the sequence of which is shown in SEQ ID NO.2.

[0012] This technical solution also provides a method for preparing a novel 7α-hydroxysteroid dehydrogenase, which involves cloning the coding gene of the 7α-hydroxysteroid dehydrogenase as shown in SEQ ID NO.1; constructing an expression vector; transforming the protein expression host bacteria; inducing protein expression and purification.

[0013] Furthermore, the coding gene of the 7α-hydroxysteroid dehydrogenase, as shown in SEQ ID NO.1, was cloned using primers including an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.4.

[0014] Furthermore, the expression vector is a plasmid formed by integrating the sequence of 7α-hydroxysteroid dehydrogenase into the multiple cloning site of the pET28 empty vector.

[0015] This technical solution also provides the application of a novel 7α-hydroxysteroid dehydrogenase in the production of converted bear bile powder. Tauroursodeoxycholic acid is one of the active ingredients in bear bile powder. The 7α-hydroxysteroid dehydrogenase in this solution can be used to convert tauroursodeoxycholic acid into an intermediate product, which can then be converted to tauroursodeoxycholic acid using conventional methods. Therefore, the 7α-hydroxysteroid dehydrogenase in this solution can be applied in the production practice of converted bear bile powder.

[0016] Furthermore, the 7α-hydroxysteroid dehydrogenase is used to catalyze the asymmetric reduction reaction of carbonyl groups in bear bile powder.

[0017] Furthermore, the 7α-hydroxysteroid dehydrogenase is used to catalyze the production of taurine chenodeoxycholic acid from taurine 7-ketolithocholic acid.

[0018] Furthermore, the conditions for the 7α-hydroxysteroid dehydrogenase-catalyzed reaction are: 50-100mM Gly-NaOH buffer solution with pH 8.0-9.5 as the reaction solvent, and a reaction temperature of 25-35℃.

[0019] The technical principle and beneficial effects of this technical solution are as follows:

[0020] This technical solution utilizes genetic engineering technology to isolate and extract a novel gene from the feces of Sichuan brown bears, naming it the 7α-hydroxysteroid dehydrogenase gene R-α-1 (7α-HSDH R-α-1). The 7α-hydroxysteroid dehydrogenase (7α-HSDH R-α-1) protein obtained by fermentation induction of the 7α-HSDH R-α-1 of this invention exhibits approximately three times higher catalytic efficiency for tauroursodeoxycholic acid (TCDCA) compared to the currently disclosed 7α-hydroxysteroid dehydrogenase (7α-HSDH) activity of *E. coli* HB101. It effectively catalyzes the formation of tauroursodeoxycholic acid (T-7-KLCA) intermediate from TCDCA. Further catalytic conversion using conventional methods yields tauroursodeoxycholic acid (TUDCA), which has significant value for industrial production and can effectively reduce production costs. In addition, the 7α-hydroxysteroid dehydrogenase R-α-1 in this formulation has the advantage of high thermostability. After being placed at a relatively high temperature of 50°C for 20 minutes, the enzyme in this formulation still has 59.3% enzyme activity, which is higher than that of other types of wild-type 7α-hydroxysteroid dehydrogenases. Attached Figure Description

[0021] Figure 1 This is a sequence comparison diagram of the 7α-hydroxysteroid dehydrogenase gene R-α-1 from Example 1 and the 7α-hydroxysteroid dehydrogenase from Escherichia coli HB101.

[0022] Figure 2 This is a diagram showing the electrophoresis results of the 7α-HSDH R-α-1 gene fragment from Example 2. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially, including: fecal DNA extraction kit, gel extraction and purification kit, Tiangen plasmid extraction kit, pET28 vector, endonucleases EcoRI and SacI, Escherichia coli DH5α cells, Escherichia coli BL21 cells, TCDCA standard, NADPH, NADP. + And some common reagents, etc.

[0024] The term "engineered cell" refers to cells that have been modified through genetic engineering techniques. These cells can be bacteria, yeast, mammalian cells, or even insect cells, designed to achieve specific biological functions or produce specific biological products.

[0025] The term "engineered bacteria" refers to microorganisms, usually bacteria, that have been modified through genetic engineering techniques to achieve specific functions or produce specific products.

[0026] The term "competent cell" refers to a cell that, after special treatment, is able to absorb and integrate exogenous DNA under natural conditions.

[0027] In the fields of molecular biology and genetic engineering, the term "expression cassette" refers to a DNA sequence that contains all the necessary genetic information and regulatory elements for the efficient transcription and translation of foreign genes in host cells.

[0028] The term "expression vector" is a tool in molecular biology used to express specific genes within host cells. They are typically based on plasmids, viruses, or other types of DNA molecules that are engineered to carry foreign genes into the host cell, where they are efficiently transcribed and translated to produce the desired protein.

[0029] The following detailed description illustrates the specific implementation method:

[0030] Example 1: Discovery of the 7α-HSDH R-α-1 gene

[0031] This technical solution utilizes genetic engineering technology to isolate and extract a novel gene from the feces of Sichuan brown bears, naming it the 7α-hydroxysteroid dehydrogenase gene R-α-1 (7α-HSDH R-α-1). The 7α-hydroxysteroid dehydrogenase (7α-HSDH R-α-1) protein obtained by fermentation induction of the 7α-HSDH R-α-1 of this invention exhibits approximately three times higher catalytic efficiency for tauroursodeoxycholic acid (TCDCA) compared to the currently disclosed 7α-hydroxysteroid dehydrogenase (7α-HSDH) activity of *E. coli* HB101. It effectively catalyzes the formation of tauroursodeoxycholic acid (T-7-KLCA) intermediate from TCDCA, which, upon further catalytic conversion, yields tauroursodeoxycholic acid (TUDCA). This has significant value for industrial production and can effectively reduce production costs.

[0032] Total DNA was extracted from brown bear feces stored at -80℃ using a fecal DNA extraction kit (fecal DNA extraction kit), and sent to a bio-sequencing company for gene sequencing. The obtained gene sequence was compared with the existing 7α-HSDH gene sequence of E. coli HB101, and the similarity was found to be 48.68%. Figure 1 As shown, the new gene was named 7α-HSDH R-α-1, and its encoded nucleotide sequence is shown in SEQ ID NO.1:

[0033] ATGGGCAGCAGCCATCATCATCATCATCATAAAAAACTGGAAGATAAAGTGGCGATTATTACCGCGGCGACCAAAGGCATTGGCGAAGCGAGCGCGGAAGTGCTGGCGGAAAACGGCGCGACCGTGTATATTGCGGCGCGCAGCGAAGAACTGGCGCGCGAAGTGATTAGCAACATTGAAAGCAACGGCGGCCGCGCGAAATTTGTGTATTTTAACGCGCGCGAACCGCAGACCTATACCACCATGGTGGAAACCGTGGCGCAGAACATGGGCCGCCTGGATATTCTGGTGAACAACTATGGCGAAACCAACGTGAAACTGGATCGCGATCTGGTGAACATTGATACCGAAGAATTTTTTCGCATTGTGCAGGATAACCTGCAGAGCGTGTATCTGCCGAGCAAAGCGGCGATTCCGCGCATGGCGAAAAACGGCGGCGGCAGCATTGTGAACATTAGCACCATTGGCAGCGTGGTGCCGGATCTGGGCCGCATTCGCTATTGCGTGAGCAAAGCGGCGATTCGCAGCCTGACCCAGAACATTGCGCTGCAGTATGCGCGCCAGGGCGTGCGCTGCAACGCGGTGCTGCCGGGCCTGATTGGCACCAAAGCGGCGATGGAAAACATGACCGATCCGTTTCGCGATAGCTTTCTGCGCCATGTGGATATTAACCGCGTGGGCAAACCGGAAGATATTGCGAAAGCGGTGCTGTATTATGCGAGCGATGATAGCGATTATGTGACCGGCATGATTCATGAACAGGCGGGCGGCTATGCGCTGGGCAGCCCGCAGTATGCGCTGTTTAGCGCGATGATGGAACGCAGCCGCTAA。

[0034] The amino acid sequence corresponding to SEQ ID NO.1 is shown in SEQ ID NO.2:

[0035] MGSSHHHHHHKKLEDKVAIITAATKGIGEASAEVLAENGATVYIAARSEELAREVISNIESNGGRAKFVYFNAREPQTYTTMVETVAQNMGRLDILVNNYGETNVKLDRDLVNIDTEEFFRIVQDNLQSVYLPSKAAI PRMAKNGGGSIVNISTIGSVVPDLGRIRYCVSKAAIRSLTQNIALQYARQGVRCNAVLPGLIGTKAAMENMTDPFRDSFLRHVDINRVGKPEDIAKAVLYYASDDSDYVTGMIHEQAGGYALGSPQYALFSAMMERSR.

[0036] Example 2:

[0037] (1) PCR gene cloning

[0038] Using extracted total DNA from brown bears as a template, cloning was performed using the following primers (SEQ ID NO.3 and SEQ ID NO.4), 5 pmol each, 50 μl volume. PCR amplification was performed using Prime STAR Max Premix (2x) fidelity enzyme to obtain the 7α-HSDH R-α-1 gene fragment. Nucleic acid electrophoresis was performed, and the fragment was recovered from the gel. The results of the 7α-HSDH R-α-1 gene fragment nucleic acid electrophoresis are shown below. Figure 2 As shown in Table 1. The gel extraction process followed the Takara gel extraction kit to recover the target gene fragment, and the PCR cloning system was performed according to the Prime STAR Max Premix (2x) fidelity enzyme instructions. The gene amplification program is shown in Table 1.

[0039] 7α-HSDH R-α-1 upstream primer-F: GAATTCatgggcagcagccatcatcatca (SEQ ID NO.3);

[0040] 7α-HSDH R-α-1 downstream primer-R: GAGCTTCttagcggctgcgttccatcatcg (SEQ ID NO.4).

[0041] Table 1: 7α-HSDH R-α-1 gene amplification program (50 μL)

[0042]

[0043]

[0044] (2) Carrier construction

[0045] The pET28 vector and the gene fragment obtained in (1) were double-digested according to the instructions for use of the restriction enzymes. Then, the 7α-HSDH R-α-1 gene fragment was ligated to the vector using DNA ligase to obtain a recombinant plasmid.

[0046] (3) Transformation and positive identification of Escherichia coli DH5α competent cells

[0047] Remove the DH5α competent cells stored at -80℃ and freeze-thaw them on ice. Then, add the constructed recombinant plasmid to the DH5α competent cells, incubate on ice for 30 min, heat shock at 42℃ for 90 s, incubate on ice again for 2 min, and finally add 800 μl of SOC or LB medium and incubate on a shaker at 37℃ and 180 rpm for 50 min.

[0048] Spread 200 μL of the cultured bacterial solution onto kanamycin-resistant LB agar plates (final concentration 50 μg / ml), and incubate overnight at 37°C. Select approximately 10 smooth, white single colonies and inoculate them into LB liquid medium containing kanamycin (final concentration 50 μg / ml), and incubate at 37°C for 8–12 h at 220 rpm.

[0049] Bacterial culture PCR verification: Using the cultured bacterial cultures as templates, PCR verification was performed according to the PCR gene cloning steps described above to confirm whether the target gene fragment was inserted into the pET28 vector (more specifically, the vector used was the conventional pET28a(+)). Agarose gel electrophoresis was used for detection. Bacterial cultures that showed positive bands were used to extract plasmids according to the instructions of the Tiangen plasmid extraction kit. The recombinant plasmids were then sent to Sangon Biotech for sequencing. Finally, the recombinant plasmid pET28-7α-HSDH R-α-1, which successfully aligned with the sequencing data, was stored as an expression vector for later use.

[0050] (4) Transformation of Escherichia coli BL21 competent cells and expression of 7α-HSDH R-α-1 gene.

[0051] Remove BL21 competent cells stored at -80℃ and freeze-thaw them on ice. Then add 1-2 μl of successfully sequenced recombinant plasmid, incubate on ice for 30 min, heat shock at 42℃ for 90 s, incubate on ice again for 2 min, and finally add 800 μl of SOC or LB medium. Incubate on a shaker at 37℃ and 180 rpm for 50 min. Spread 200 μl of the cultured bacterial solution onto kanamycin-resistant LB plates (final concentration 50 μg / ml) and incubate overnight at 37℃ to complete the transformation of competent cells.

[0052] Single colonies were selected and inoculated into LB liquid medium, and kanamycin was added to a final concentration of 50 μg / ml. The medium was then incubated on a shaker at 37°C and 220 rpm. The OD of the bacterial culture was measured periodically. 600 When the OD600 value of the bacterial culture is between 0.6 and 1.0, add IPTG to a final concentration of 1 mM to the shake flask for induction at 20°C and 220 rpm overnight for 12–16 hours, and then collect the bacterial cells by centrifugation.

[0053] Example 3: Assay of 7α-hydroxysteroid dehydrogenase R-α-1 enzyme activity

[0054] The enzyme activity of the induced products was detected using a UV spectrophotometer. The detection method is as follows:

[0055] The bacterial cells collected in Example 2 were diluted with 100mM pH 9.0 Gly-NaOH buffer at a certain ratio (ensuring the detection OD value is between 0.2-0.6A; for example, 1g of bacterial cells added to 100mL of buffer is a 100-fold dilution). The cells were then disrupted using an ultrasonic cell disruptor to obtain expression cell lysate. Enzyme activity was detected using this expression cell lysate. For each test, to ensure the parallelism of the experimental results, 3g of bacterial cells were diluted with buffer at a certain ratio to obtain a bacterial suspension, which was then disrupted to obtain the expression cell lysate. Enzyme activity detection of the expression cell lysate obtained after disrupting the bacterial suspension reflects the final result of bacterial fermentation (further conversion according to the dilution ratio reflects the overall enzyme activity of the bacteria), which is a more important issue in actual industrial production. The main factors determining the enzyme activity of the expression cell lysate are: the expression level of the target protein by the bacteria and the enzyme activity of the target protein itself. The two factors mentioned above determine the overall enzyme activity of the bacterial cells. Only when both factors are ideal will the overall enzyme activity of the expression cell lysate be higher. The expression cell lysate will be directly used for biotransformation of TCDCA, etc., rather than purifying the enzymes in the expression cell lysate before application. Enzyme purification is time-consuming and labor-intensive, unsuitable for industrial applications, while the process of obtaining the expression cell lysate is simpler and suitable for direct industrial production. The detection method (enzyme activity measurement) used in this scheme is more meaningful for actual production. If the pure enzyme itself has high activity but the expression level is too low, or if the enzyme expression level is high but the pure enzyme activity is too low, the overall enzyme activity of the expression cell lysate will be unsatisfactory to some extent, rendering it unusable for production.

[0056] Add 2890 μl of 100 mM pH 9.0 Gly-NaOH buffer (in practice, 50-100 mM pH 8.0-9.5 Gly-NaOH buffer can be used), 50 μl of 50 mM TCDCA solution, and 50 μl of 50 mM NADP to a 3 ml cuvette. + After placing the solution in a UV spectrophotometer, add 10 μl of the test product diluent (using an equal volume of 100 mM pH 9.0 Gly-NaOH buffer as a blank). Start timing immediately upon adding the test product diluent, mix thoroughly by pipetting several times, and record the absorbance at 340 nm after 1 minute. If the absorbance is not between 0.2-0.6 Å, re-dilute the test product concentration and repeat the measurement steps until the absorbance is between 0.2-0.6 Å. Calculate the enzyme activity of the expressed product based on the NADPH standard curve. The entire reaction process can be carried out at room temperature, i.e., 25-35℃.

[0057] Calculation formula: Enzyme activity (U / mL) = OD 340 ×V t ×df / (k×4×1.0×V s );

[0058] Where: OD 340 Vt is the absorbance of the UV spectrophotometer at 340 nm; df is the total reaction volume (3 ml); k is the dilution factor of the test product (converted according to a 1:4 dissolution ratio); k is the slope of the NADPH standard curve; 1.0 is the measurement distance; Vs is the volume of the test product added (0.01 ml). See Table 2 for detailed test results.

[0059] The 7α-HSDH R-α-1 sample (expression cell lysate) was heated in a 50℃ water bath for 20 min, and the residual enzyme activity was then detected according to the enzyme activity detection method described above to reflect the enzyme's thermal stability. The experimental results are detailed in Table 3. The enzyme activity retention of 7α-HSDH R-α-1 was 59.3% (residual enzyme activity = (enzyme activity before water bath - enzyme activity after water bath) / enzyme activity before water bath × 100%).

[0060] Comparative Example 1: Enzyme Activity Comparison

[0061] Following the same method described above, an engineered bacterium overexpressing the 7α-HSDH gene was constructed, and protein expression and enzyme activity of the existing *E. coli* HB101 7α-HSDH gene were measured. The sequence of the *E. coli* HB101 7α-HSDH gene is SEQ ID NO.5, and its amino acid sequence is shown in SEQ ID NO.6. The test results are detailed in Table 2.

[0062] SEQ ID NO.5:

[0063] ATGTTTAACAGCGATAACCTGCGCCTGGATGGCAAATGCGCGATTATTACCGGCGCGGGCGCGGGCATTGGCAAAGAAATTGCGATTACCTTTGCGACCGCGGGCGCGAGCGTGGTGGTGAGCGATATTAACGCGGATGCGGCGAACCATGTGGTGGATGAAATTCAGCAGCTGGGCGGCCAGGCGTTTGCGTGCCGCTGCGATATTACCAGCGAACAGGAACTGAGCGCGCTGGCGGATTTTGCGATTAGCAAACTGGGCAAAGTGGATATTCTGGTGAACAACGCGGGCGGCGGCCCGAAACCGTTTGATATGCCGATGGCGGATTTTCGCCGCGCGTATGAACTGAACGTGTTTAGCTTTTTTCATCTGAGCCAGCTGGTGGCGCCGGAAATGGAAAAAAACGGCGGCGGCGTGATTCTGACCATTACCAGCATGGCGGCGGAAAACAAAAACATTAACATGACCAGCTATGCGAGCAGCAAAGCGGCGGCGAGCCATCTGGTGCGCAACATGGCGTTTGATCTGGGCGAAAAAAACATTCGCGTGAACGGCATTGCGCCGGGCGCGATTCTGACCGATGCGCTGAAAAGCGTGATTACCCCGGAAATTGAACAGAAAATGCTGCAGCATACCCCGATTCGCCGCCTGGGCCAGCCGCAGGATATTGCGAACGCGGCGCTGTTTCTGTGCAGCCCGGCGGCGAGCTGGGTGAGCGGCCAGATTCTGACCGTGAGCGGCGGCGGCGTGCAGGAACTGAACTAA;

[0064] SEQ ID NO.6:

[0065] MFNSDNLRLDGKCAIITGAGAGIGKEIAITFATAGASVVVSDINADAANHVVDEIQQLGGQAFACRCDITSEQELSALADFAISKLGKVDILVNNAGGGPKPFDMPMADFRRAYELNVFSFFHLSQL VAPEMEKNGGGVILTITSMAAENKNINMTSYASSKAAASHLVRNMAFDLGEKNIRVNGIAPGAILTDALKSVITPEIEQKMLQHTPIRRLGQPQDIANAALFLCSPAASWVSGQILTVSGGGVQELN.

[0066] Table 2: Comparison of protease activity encoded by the 7α-HSDH R-α-1 gene of this invention and the 7α-HSDH gene of Escherichia coli HB101

[0067]

[0068] The experimental results show that the enzyme activity of the engineered bacteria constructed using the 7α-HSDH R-α-1 gene obtained through this technical scheme, after induction expression and cell disruption, is significantly higher than that obtained using the 7α-HSDH gene from *E. coli* HB101, approximately three times higher. Therefore, the novel gene discovered in this scheme is more beneficial for the biotransformation of TCDCA and other bacteria, and is more suitable for industrial applications. Furthermore, it is important to emphasize that the enzyme activity of 7α-HSDH from wild-type *E. coli* HB101 is at a similar level to that of other wild-type 7α-HSDH enzymes (from other bacterial species and types) in existing technologies. For example, the 7α-HSDH enzymes of *Clostridium absonum*, *Bacteroides fragilis*, *Closteridium scindens*, and *Clostridium sordellii*. That is, the enzyme activity of the expression cell lysate obtained by constructing engineered bacteria according to the process of this scheme and the expression cell lysate obtained by other wild-type 7α-HSDH at the same cell suspension concentration is in the range of 500-1500 U / ml, which is different from the enzyme activity of the expression cell lysate obtained by this scheme from 7α-HSDH R-α-1.

[0069] Comparative Example 2: Thermal Stability Comparison

[0070] The thermal stability of wild-type 7α-HSDH was tested using the method described in Example 3, and the results are shown in Table 3. The data in the table below show that the enzyme activity of wild-type 7α-HSDH decreased significantly, retaining only 32.6% of its activity, while 7α-HSDHR-α-1 retained 59.3% of its activity, demonstrating better thermal stability compared to the wild type.

[0071] Table 3: Comparison of thermal stability of proteins encoded by the 7α-HSDH R-α-1 gene of this invention and the wild-type 7α-HSDH gene.

[0072]

[0073] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A novel 7α-hydroxysteroid dehydrogenase R-α-1 gene, characterized in that, Its sequence is shown in SEQ ID NO.

1.

2. An expression cassette, vector, or engineered cell comprising the 7α-hydroxysteroid dehydrogenase R-α-1 gene of claim 1.

3. A novel 7α-hydroxysteroid dehydrogenase, characterized in that: Its sequence is shown in SEQ ID NO.

2.

4. A method for preparing a novel 7α-hydroxysteroid dehydrogenase as described in claim 3, characterized in that: The coding gene of the 7α-hydroxysteroid dehydrogenase was cloned as shown in SEQ ID NO.1; an expression vector was constructed; the protein expression host bacteria were transformed; protein expression was induced and purified.

5. The method for preparing 7α-hydroxysteroid dehydrogenase according to claim 4, characterized in that: The cloning of the coding gene of the 7α-hydroxysteroid dehydrogenase, as shown in SEQ ID NO.1, was performed using primers including an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.

4.

6. The method for preparing 7α-hydroxysteroid dehydrogenase according to claim 5, characterized in that: The expression vector is a plasmid formed by integrating the sequence of 7α-hydroxysteroid dehydrogenase into the multiple cloning site of the pET28 empty vector.

7. The application of the novel 7α-hydroxysteroid dehydrogenase according to claim 3 in the production of converted bear bile powder.

8. The application of a novel 7α-hydroxysteroid dehydrogenase according to claim 7 in the production of converted bear bile powder, characterized in that: The 7α-hydroxysteroid dehydrogenase is used to catalyze the asymmetric reduction reaction of carbonyl groups in the conversion of bear bile powder.

9. The application of a novel 7α-hydroxysteroid dehydrogenase according to claim 8 in the production of converted bear bile powder, characterized in that: The 7α-hydroxysteroid dehydrogenase is used to catalyze the conversion of taurine chenodeoxycholic acid to taurine 7-ketolithocholic acid.

10. The application of a novel 7α-hydroxysteroid dehydrogenase according to claim 9 in the production of converted bear bile powder, characterized in that: The conditions for the 7α-hydroxysteroid dehydrogenase-catalyzed reaction are: 50-100mM Gly-NaOH buffer solution with pH 8.0-9.5 as the reaction solvent, and a reaction temperature of 25-35℃.

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