A 7α-HSDH enzyme mutant and a 7β-HSDH enzyme mutant, their encoding genes and applications

Abstract

This invention provides a 7α-HSDH enzyme mutant and a 7β-HSDH enzyme mutant, their encoding genes, and their applications. Through artificial mutation screening, 7α-HSDH and 7β-HSDH enzyme mutants with specific amino acid sequences were identified, resulting in improved enzyme activity and catalytic efficiency compared to wild-type 7α-HSDH and wild-type 7β-HSDH. This invention also provides a method for preparing ursodeoxycholic acid using the aforementioned 7α-HSDH and 7β-HSDH enzyme mutants, specifically using NADP... + A one-pot catalytic method for the conversion of chenodeoxycholic acid to ursodeoxycholic acid using NADPH-dependent 7α-HSDH enzyme mutants and NADPH-dependent 7β-HSDH enzyme mutants is employed. This method is simple, yields high product conversion, and can also achieve the conversion of the coenzyme NADP. + / NADPH's self-circulating regeneration eliminates the need for auxiliary enzymes and raw materials used in coenzyme regeneration, greatly reducing production costs and environmental pressure, and possessing enormous potential for industrial applications.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a 7α-HSDH enzyme mutant and a 7β-HSDH enzyme mutant, their encoding genes, and their applications. Background Technology

[0002] Ursodeoxycholic acid (UDCA), chemically known as 3α,7β-dihydroxy-5β-cholic acid, possesses potent properties that promote the hydrolysis of fats and fatty acids. Therefore, UDCA has been used for centuries to treat hepatobiliary diseases, particularly cholesterol gallstones. To date, UDCA is the only drug approved by the US FDA for the treatment of primary biliary cirrhosis, and the clinical demand for UDCA is increasingly significant.

[0003] Currently, UDCA can be obtained through chemical synthesis or biosynthesis. Chemical synthesis, such as the synthesis method using androstenedione as a raw material or total synthesis method, has a complicated route, requires multiple steps of hydroxyl protection and deprotection, has high cost and low overall yield, and the chemical reagents used (such as pyridine, CrO3 and hydrazine) pollute the environment, making it unsuitable for industrial production. Biosynthetic methods, such as enzymatic catalysis, are green and environmentally friendly, in line with the current trend of green development and environmental protection, and have great application potential.

[0004] Traditional enzymatic synthesis of UDCA requires two catalytic steps. The first step involves oxidizing chenodeoxycholic acid (CDCA) to 7-ketolithocholic acid (7-KLCA) using 7α-hydroxysteroid dehydrogenase (7α-HSDH). The second step involves reducing the 7-KLCA obtained in the first step to UDCA using 7β-hydroxysteroid dehydrogenase (7β-HSDH). This process requires purification of the intermediate 7-KLCA, making the catalytic process relatively complex. Furthermore, the 7α-HSDH and 7β-HSDH currently used for UDCA synthesis each require the coenzyme NAD+. + The reaction also requires the participation of NADPH, therefore the catalytic reaction also requires the addition of coenzymes and excipients such as lactate dehydrogenase and glucose dehydrogenase to facilitate NADPH coenzyme ... + The regeneration of NADPH leads to a complex catalytic system and higher production costs.

[0005] Chinese invention patent with publication number "CN105861613A" discloses a one-step method for preparing ursodeoxycholic acid. The synthetic route is relatively simple, but the 7-αHSDH and 7-βHSDH used in the synthesis process are both lyophilized powders, which results in excessively high costs for enzyme-protein separation and purification, leading to excessively high catalytic costs. Furthermore, the conversion rate of the preparation method provided in this patent is low, with the highest conversion rate recorded in the examples being only 81%.

[0006] In summary, since the existing 7α-HSDH and 7β-HSDH have low conversion rates in the synthesis of ursodeoxycholic acid, in order to seek higher conversion rates, this invention proposes to artificially mutate the existing 7α-HSDH and 7β-HSDH amino acid sequences to screen out 7α-HSDH and 7β-HSDH with high conversion rates to participate in the synthesis reaction of ursodeoxycholic acid. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a 7α-HSDH enzyme mutant and a 7β-HSDH enzyme mutant, their encoding genes, and their applications. 7α-HSDH enzyme mutants and 7β-HSDH enzyme mutants with specific amino acid sequences are screened through artificial mutation. A fusion tag is added to the end of the 7β-HSDH enzyme mutant, enabling one-pot catalysis of CDCA to UDCA. This achieves in-situ recycling and regeneration of the coenzyme, reduces catalytic steps, improves catalytic stability and efficiency, and significantly saves production costs.

[0008] The technical solution of this invention is as follows:

[0009] A 7α-HSDH enzyme mutant, comprising any one of NADP-1A, NADP-2A, NADP-3A, NADP-4A, and NADP-5A, wherein the amino acid sequence of NADP-1A is shown in SEQ ID NO.1, the amino acid sequence of NADP-2A is shown in SEQ ID NO.2, the amino acid sequence of NADP-3A is shown in SEQ ID NO.3, the amino acid sequence of NADP-4A is shown in SEQ ID NO.4, and the amino acid sequence of NADP-5A is shown in SEQ ID NO.5.

[0010] The NADP-1A is obtained by mutating the wild-type 7α-HSDH (NADP-1a) amino acid sequence as shown in SEQ ID NO.12, with combined mutations at positions 44-47, 103-104, 123-126, and 190-191 of the amino acid sequence shown in SEQ ID NO.12 (A44G, R45V, A46I, D47A, N103A, T104G, A123P, S124A, Q125K, T126A, T190I, A191G).

[0011] The NADP-2A is obtained by mutating the wild-type 7α-HSDH (NADP-2a) amino acid sequence as shown in SEQ ID NO.13, specifically by a combined mutation at positions 46-48, 86, 118-119, 141, 155, 191-192, 225, and 250 of the amino acid sequence shown in SEQ ID NO.13 (V46R, I47A, A48D, I86V, V118L, Q119K, V141I, R155Q, I191T, G192A, N225A, A250G).

[0012] The NADP-3A and NADP-4A are obtained through DNA shuffling technology. The target sequences of the 7α-HSDH-related gene family are randomly fragmented and assembled into full-length chimeric genes by primerless PCR and primer-assisted PCR. The chimeric genes are then screened in high-throughput mode to select mutants with improved or novel functions as templates for the next round of DNA shuffling. The above steps are repeated for multiple rounds of shuffling and high-throughput screening to finally obtain NADP-3A and NADP-4A.

[0013] The NADP-5A is obtained by mutating the wild-type 7α-HSDH (NADP-5a) amino acid sequence as shown in SEQ ID NO.14, with combined mutations at positions 24, 104-105, 111-112, and 135 of the amino acid sequence shown in SEQ ID NO.14 (E24G, A104N, G105T, F111I, R112A, V135N).

[0014] A 7β-HSDH enzyme mutant, which is any one of NADP-1B, NADP-2B, and NADP-3B, wherein the amino acid sequence of NADP-1B is shown in SEQ ID NO.6, the amino acid sequence of NADP-2B is shown in SEQ ID NO.7, and the amino acid sequence of NADP-3B is shown in SEQ ID NO.8.

[0015] The NADP-1B is obtained by mutating the wild-type 7β-HSDH (NADP-1b) amino acid sequence as shown in SEQ ID NO.15, with combined mutations at positions 26-27, 132-133, and 207 of the amino acid sequence shown in SEQ ID NO.15 (F26E, E27K, E132K, R133Q, A207T).

[0016] The NADP-2B is obtained by mutating the wild-type 7β-HSDH (NADP-2b) amino acid sequence as shown in SEQ ID NO.16, with combined mutations at positions 42, 87-88, 176, and 232 of the amino acid sequence shown in SEQ ID NO.16 (E42K, F87V, M88I, S176N, I232L).

[0017] The NADP-3B is obtained by mutating the wild-type 7β-HSDH (NADP-3b) amino acid sequence as shown in SEQ ID NO.17, with combined mutations at positions 42, 87-88, 176, and 232 of the amino acid sequence shown in SEQ ID NO.17 (E42K, F87V, M88I, S176N, I232L).

[0018] The 7α-HSDH enzyme mutant is NADP. + The 7β-HSDH enzyme mutant is NADPH-dependent.

[0019] A method for preparing ursodeoxycholic acid using the above-mentioned 7α-HSDH enzyme mutant and 7β-HSDH enzyme mutant includes the following steps:

[0020] S1, Add chenodeoxycholic acid to phosphate buffer and adjust the pH until the chenodeoxycholic acid is completely dissolved.

[0021] S2, continue adding coenzyme NADP + The 7α-HSDH enzyme mutant and the 7β-HSDH enzyme mutant were used to carry out a catalytic reaction, and finally the finished product ursodeoxycholic acid was obtained.

[0022] The 7α-HSDH enzyme mutant and the 7β-HSDH enzyme mutant can be crude enzyme solutions, which can greatly reduce catalytic costs compared to pure enzymes.

[0023] Among them, the 7α-HSDH enzyme mutant mentioned in S2 is NADP. + The 7β-HSDH enzyme mutant is NADPH-dependent.

[0024] Preferably, a fusion tag is added to the "N-terminus" or "C-terminus" of the 7β-HSDH enzyme mutant to form a 7β-HSDH fusion protein; by adding a fusion tag to the end of the 7β-HSDH enzyme mutant, the catalytic stability and catalytic efficiency are greatly improved.

[0025] Preferably, the fusion tag is any one of SUMO, GST, or MBP; the amino acid sequence of SUMO is shown in SEQ ID NO.9, the amino acid sequence of GST is shown in SEQ ID NO.10, and the amino acid sequence of MBP is shown in SEQ ID NO.11.

[0026] Preferably, the chenodeoxycholic acid and NADP + The mass ratio between the 7α-HSDH enzyme mutant and the 7β-HSDH enzyme mutant / 7β-HSDH fusion protein is 1:0.0002~0.003:0.1~0.5:0.2~1.0.

[0027] Preferably, the concentration of the phosphate buffer solution is 10–100 mM and the pH is 6.5–8.5.

[0028] Preferably, the temperature of the catalytic reaction in S2 is 25–40°C, the pH of the reaction is 7.0–8.5, and the reaction time is 1–15 h.

[0029] The catalytic reaction in the above method for preparing ursodeoxycholic acid is as follows:

[0030]

[0031] This invention uses NADP + Both the NADPH-dependent 7α-HSDH enzyme mutant and the NADPH-dependent 7β-HSDH enzyme mutant co-catalyze the production of UDCA from CDCA. Specifically, the 7α-HSDH enzyme mutant catalyzes the conversion of CDCA to 7-ketolithocholic acid (7-KLCA), while simultaneously converting NADP... + The 7β-HSDH enzyme mutant converts 7-KLCA to NADPH, catalyzing the production of UDCA, while simultaneously converting NADPH to NADP. + This method can achieve the coenzyme NADP. + / NADPH's self-circulating regeneration eliminates the need for additional coenzymes and auxiliary materials used for coenzyme regeneration.

[0032] The beneficial effects of this invention are:

[0033] 1) This invention screens out 7α-HSDH enzyme mutants and 7β-HSDH enzyme mutants with specific amino acid sequences, and adds fusion tags SUMO, GST or MBP to the end of the 7β-HSDH enzyme mutant, which greatly improves catalytic efficiency and catalytic stability.

[0034] 2) This invention uses NADP. +The NADPH-dependent 7α-HSDH enzyme mutant and the NADPH-dependent 7β-HSDH enzyme mutant co-catalyze the production of UDCA from CDCA, thus enabling the production of the coenzyme NADP. + / NADPH’s self-circulating regeneration does not require the addition of auxiliary enzymes and auxiliary raw materials for coenzyme regeneration, and uses crude enzyme solution to participate in the catalytic reaction, which greatly saves production costs.

[0035] 3) This invention uses a one-pot catalytic method to prepare UDCA from CDCA. Compared with the traditional two-step catalytic method, it reduces the crude extraction step of intermediate products, simplifies the process, saves production costs, and is suitable for large-scale industrial application. Attached Figure Description

[0036] Figure 1 This is the HPLC detection chromatogram in Example 1 of the present invention. Detailed Implementation

[0037] The following description is based on specific embodiments:

[0038] During the screening of 7α-HSDH enzyme mutants and 7β-HSDH enzyme mutants, enzyme activity assays revealed that the activity of the enzymes after joint mutation at certain sites was significantly improved compared to the wild-type enzymes. Based on the enzyme activity assay results, we identified the sites of joint mutations and successfully screened out the 7α-HSDH enzyme mutants NADP-1A, NADP-2A, NADP-3A, NADP-4A, and NADP-5A, and the 7β-HSDH enzyme mutants NADP-1B, NADP-2B, and NADP-3B.

[0039] The specific mutation sites of the enzyme mutants obtained in the final screening and the comparison results of their activities with wild-type enzymes are shown in Table 1. The activity of 7α-HSDH enzyme is defined as the amount of enzyme required to generate 1 μmol of NADPH per minute; the activity of 7β-HSDH enzyme is defined as the amount of enzyme required to consume 1 μmol of NADPH per minute.

[0040] Table 1. Comparison of enzyme activities between enzyme mutants and wild-type enzymes

[0041]

[0042] Example 1:

[0043] In this embodiment, 7α-HSDH (NADP-5A) and 7β-HSDH fusion protein with a GST tag at the C-terminus (GST-NADP-1B) were used to co-catalyze the production of UDCA.

[0044] I. Preparation of crude enzyme solution:

[0045] Recombinant *E. coli* containing the gene encoding the 7α-HSDH or 7β-HSDH fusion protein was streaked onto LB agar plates (5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride) for activation. After overnight incubation at 37°C, single colonies were picked and inoculated into liquid LB agar. The cells were then cultured at 37°C and 220 rpm for 12 h on a shaker. Finally, a 1% (v / v) inoculation was carried out on TB agar (24 g / L yeast extract, 12 g / L tryptone, 4 mL / L glycerol, 2.2 g / L potassium dihydrogen phosphate, 9.4 g / L dipotassium hydrogen phosphate) and cultured at 37°C. Cells were cultured until they reached the OD value. 600 When the concentration of β-D-thiogalactoside (IPTG) was 0.6, 0.2 mM of IPTG was added to induce enzyme expression, and the cells were then cultured in a shaker at 20°C for 12 h. After the culture was completed, the cells were collected by centrifugation (8000 rpm, 5 min). The cells were washed twice with 50 mM phosphate buffer, and then resuspended in 100 mM phosphate buffer (1 / 15 volume of fermentation broth). The cells were then homogenized using a high-pressure homogenizer, and the cell lysate was centrifuged (12000 rpm, 5 min). The resulting supernatant was the crude enzyme solution.

[0046] II. Preparation of UDCA by Coenzyme Self-Circulation Catalysis:

[0047] Weigh 25.0 g of CDCA and add it to 20 mL of 50 mM phosphate buffer (pH 8.5). Adjust the pH with 4 M NaOH solution until the CDCA is completely dissolved. Then add 45.0 mg of NADP. + 6.0 g of crude 7α-HSDH enzyme (NADP-5A) and 14.6 g of crude 7β-HSDH fusion protein enzyme (GST-NADP-1B) were used to maintain the reaction solution at pH 8.5 and temperature 35℃. After timed sampling, the substrate and product contents were determined by liquid chromatography (HPLC). The chromatographic conditions were: Shimadzu LC-20A chromatograph, Phenomenx Gemini NX-C18 column (5 μm, 250 × 4.6 mm), RID detector, mobile phase: acetonitrile:sodium dihydrogen phosphate buffer (6.5 mM, pH 3.0):methanol = 30:37:40, flow rate 0.8 mL / min, column temperature 40℃. All reaction solution samples were first dissolved in methanol, then diluted with the mobile phase, filtered through a 0.45 μm filter membrane, and then analyzed by HPLC. After 8 h of catalytic reaction, the UDCA conversion rate was 98%. The HPLC chromatogram at this conversion rate is shown below. Figure 1 As shown.

[0048] Example 2:

[0049] In this embodiment, 7α-HSDH (NADP-2A) and 7β-HSDH fusion protein with an N-terminal SUMO tag (SUMO-NADP-3B) were used to co-catalyze the production of UDCA.

[0050] The preparation method of the crude enzyme solution is the same as in Example 1;

[0051] Weigh 18.4 g of CDCA and add it to 15 mL of 50 mM phosphate buffer (pH 8.0). Adjust the pH with 4 M NaOH solution until the CDCA is completely dissolved. Then add 14.2 mg of NADP. + 3.7 g of crude 7α-HSDH enzyme (NADP-2A) and 5.5 g of crude 7β-HSDH fusion protein enzyme (SUMO-NADP-3B) were added. The reaction solution was maintained at pH 8.0 and temperature 37°C. Samples were taken periodically, and the substrate and product contents were determined by liquid chromatography under the same chromatographic conditions as in Example 1. After 4 hours of catalytic reaction, the UDCA conversion rate was 92%.

[0052] Example 3:

[0053] In this embodiment, 7α-HSDH (NADP-1A) and 7β-HSDH fusion protein with an "N"-terminal MBP tag (MBP-NADP-3B) were used to co-catalyze the production of UDCA.

[0054] The preparation method of the crude enzyme solution is the same as in Example 1;

[0055] Weigh 12.0 g of CDCA and add it to 15 mL of 50 mM phosphate buffer (pH 7.5). Adjust the pH with 4 M NaOH solution until the CDCA is completely dissolved. Then add 12.0 mg of NADP. + 1.8 g of crude 7α-HSDH enzyme (NADP-1A) and 3.6 g of crude 7β-HSDH fusion protein enzyme (MBP-NADP-3B) were added. The reaction solution was maintained at pH 8.0 and temperature 35°C. Samples were taken periodically, and the substrate and product contents were determined by liquid chromatography under the same chromatographic conditions as in Example 1. After 3 hours of catalytic reaction, the UDCA conversion rate was 93%.

[0056] Example 4:

[0057] In this embodiment, 7α-HSDH (NADP-3A) and 7β-HSDH fusion protein with a GST tag at the C-terminus (GST-NADP-2B) were used to co-catalyze the production of UDCA.

[0058] The preparation method of the crude enzyme solution is the same as in Example 1;

[0059] Weigh 15.0 g of CDCA and add it to 20 mL of 50 mM phosphate buffer (pH 6.5). Adjust the pH with 4 M NaOH solution until the CDCA is completely dissolved. Then add 20.0 mg of NADP. + 5.3 g of crude 7α-HSDH enzyme (NADP-3A) and 6.8 g of crude 7β-HSDH fusion protein enzyme (GST-NADP-2B) were added. The reaction solution was maintained at pH 8.0 and temperature 30°C. Samples were taken periodically, and the substrate and product contents were determined by liquid chromatography under the same chromatographic conditions as in Example 1. After 6 hours of catalytic reaction, the UDCA conversion rate was 88%.

[0060] Example 5:

[0061] In this embodiment, 7α-HSDH (NADP-4A) and 7β-HSDH fusion protein with an N-terminal GST tag (GST-NADP-2B) were used to co-catalyze the production of UDCA.

[0062] The preparation method of the crude enzyme solution is the same as in Example 1;

[0063] Weigh 18.0 g of CDCA and add it to 25 mL of 50 mM phosphate buffer (pH 7.0). Adjust the pH with 4 M NaOH solution until the CDCA is completely dissolved. Then add 10.0 mg of NADP. + 3.5 g of crude 7α-HSDH enzyme (NADP-4A) and 4.6 g of crude 7β-HSDH fusion protein enzyme (GST-NADP-2B) were added. The reaction solution was maintained at pH 8.0 and temperature 35°C. Samples were taken periodically, and the substrate and product contents were determined by liquid chromatography under the same chromatographic conditions as in Example 1. After 4 hours of catalytic reaction, the UDCA conversion rate was 94%.

[0064] Example 6:

[0065] Unlike Example 1, this example uses 7β-HSDH (NADP-1B) without a fusion tag;

[0066] The preparation methods for the crude enzyme solution and UDCA were the same as in Example 1. After 8 hours of catalytic reaction, the conversion rate of UDCA was 87%.

[0067] Example 7:

[0068] Unlike Example 2, this example uses 7β-HSDH (NADP-3B) without a fusion tag;

[0069] The preparation methods for the crude enzyme solution and UDCA were the same as in Example 2. After 4 hours of catalytic reaction, the conversion rate of UDCA was 81%.

[0070] Example 8:

[0071] Unlike Example 3, this example uses 7β-HSDH (NADP-3B) without a fusion tag;

[0072] The preparation methods for the crude enzyme solution and UDCA were the same as in Example 3. After 3 hours of catalytic reaction, the conversion rate of UDCA was 84%.

[0073] The conversion rates of UDCA prepared in Examples 1-8 above are shown in Table 2 below:

[0074] Table 2. UDCA conversion rates in Examples 1–8

[0075]

[0076] As shown in Table 2, the conversion rate of UDCA is significantly reduced without the addition of the fusion protein. This invention significantly improves the catalytic efficiency and stability of UDCA by adding fusion tags SUMO, GST, or MBP to the end of 7β-HSDH.

[0077] Comparative Example 1:

[0078] Unlike Example 6, this comparative example uses wild-type 7α-HSDH (NADP-5a) before mutation and wild-type 7β-HSDH (NADP-1b) without fusion tag before mutation to co-catalyze the production of UDCA.

[0079] The preparation methods for the crude enzyme solution and UDCA were the same as in Example 6. After 8 hours of catalytic reaction, the conversion rate of UDCA was 84%.

[0080] Comparative Example 2:

[0081] Unlike Example 7, this comparative example uses wild-type 7α-HSDH (NADP-2a) before mutation and wild-type 7β-HSDH (NADP-3b) without fusion tag before mutation to co-catalyze the production of UDCA.

[0082] The preparation methods for the crude enzyme solution and UDCA were the same as in Example 7. After 4 hours of catalytic reaction, the conversion rate of UDCA was 76%.

[0083] Comparative Example 3:

[0084] Unlike Example 8, this comparative example uses wild-type 7α-HSDH (NADP-1a) before mutation and wild-type 7β-HSDH (NADP-3b) without fusion tag to co-catalyze the production of UDCA.

[0085] The preparation methods for the crude enzyme solution and UDCA were the same as in Example 8. After 3 hours of catalytic reaction, the conversion rate of UDCA was 82%.

[0086] The conversion rates of UDCA prepared in Comparative Examples 1-3 above are shown in Table 3 below:

[0087] Table 3. UDCA conversion rates of Comparative Examples 1–3

[0088]

[0089] As can be seen from Table 3, the UDCA conversion rate was lower when wild-type 7α-HSDH and wild-type 7β-HSDH were used compared to Examples 6-8.

[0090] In summary, this invention significantly improves the catalytic efficiency and stability of UDCA by artificially mutagenesing the existing 7α-HSDH and 7β-HSDH amino acid sequences to screen for 7α-HSDH enzyme mutants with specific amino acid sequences, and by adding fusion tags SUMO, GST or MBP to the end of the 7β-HSDH enzyme mutant.

Claims

1. A 7α-HSDH enzyme mutant, characterized in that, The 7α-HSDH enzyme mutant is NADP-1A, and the amino acid sequence of NADP-1A is shown in SEQ ID NO.

1.

2. The use of the 7α-HSDH enzyme mutant of claim 1 in the preparation of ursodeoxycholic acid.

3. A method for preparing ursodeoxycholic acid using the 7α-HSDH enzyme mutant of claim 1, characterized in that, Includes the following steps: S1, Add chenodeoxycholic acid to phosphate buffer and adjust the pH until the chenodeoxycholic acid is completely dissolved. S2, continue adding coenzyme NADP + The 7α-HSDH enzyme mutant and the 7β-HSDH enzyme mutant are subjected to a catalytic reaction to obtain the finished product ursodeoxycholic acid; wherein the 7β-HSDH enzyme mutant is any one of NADP-1B, NADP-2B, and NADP-3B, the amino acid sequence of NADP-1B is shown in SEQ ID NO.6, the amino acid sequence of NADP-2B is shown in SEQ ID NO.7, and the amino acid sequence of NADP-3B is shown in SEQ ID NO.

8.

4. A method as described in claim 3, characterized in that, The 7β-HSDH enzyme mutant has a fusion tag added to its "N-terminus" or "C-terminus" to become a 7β-HSDH fusion protein; the fusion tag is any one of SUMO, GST or MBP; the amino acid sequence of SUMO is shown in SEQ ID NO.9, the amino acid sequence of GST is shown in SEQ ID NO.10, and the amino acid sequence of MBP is shown in SEQ ID NO.

11.

5. A method as described in claim 3, characterized in that, The chenodeoxycholic acid, NADP + The mass ratio between 7α-HSDH enzyme mutant and 7β-HSDH enzyme mutant / 7β-HSDH fusion protein is 1 : 0.0002~0.003 : 0.1~0.5 : 0.2~1.

0.

6. A method as described in claim 3, characterized in that, The concentration of the phosphate buffer solution is 10~100mM, and the pH is 6.5~8.

5.

7. A method as described in claim 3, characterized in that, The catalytic reaction is carried out at a temperature of 25-40°C, a pH of 7.0-8.5, and a reaction time of 1-15 h.

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