A steroid dehydrogenase mutant and its application in the preparation of steroid drugs

Abstract

The present invention discloses a steroid dehydrogenase mutant and its use in the preparation of steroidal drugs. The present invention improves the enzyme activity by screening the steroid dehydrogenase mutant, increasing it from 54.3 U / mg of wet bacterial cells to 67.7 U / mg of wet bacterial cells. The present invention connects the steroid dehydrogenase mutant with catalase via a linker peptide to form a fusion protein, which is then expressed and used to catalyze AD to produce ADD. This not only improves the substrate conversion rate, reaching as high as 98.6%, but also eliminates the problem of hydrogen peroxide toxicity to wet bacterial cells during the reaction, reducing production costs and promising industrial application prospects.

Description

(1) Technical field

[0001] The invention belongs to the technical field of enzyme engineering and genetic engineering, and particularly relates to a steroid dehydrogenase mutant with high catalytic activity and its application in preparing steroid drugs. (2) Background technology

[0002] Steroids are a class of compounds with a specific chemical structure, whose core is composed of a cyclopentaphenanthrene. Steroid drugs have a wide range of therapeutic effects in medicine, including anti-tumor, anti-inflammatory, antiviral, antibacterial, anticonvulsant, and anti-allergic effects. Their global sales volume is second only to antibiotics. To date, over 400 steroid drugs have been produced worldwide, and global sales of steroid hormones reached $100 billion in 2017.

[0003] Steroid biotransformation technology involves the conversion of steroid compounds into steroidal derivatives with pharmaceutical or industrial value through biocatalysis. It is an important method for structural modification of steroids and other complex natural products. Biotransformation offers significant advantages, including mild reaction conditions, minimal byproducts, simple procedures, and environmental friendliness. Therefore, it is widely used in the research and production of steroidal drugs.

[0004] Androstane-1,4-diene-3,17-dione (ADD) is a key steroidal derivative produced during sterol degradation and is widely used as a precursor for various steroidal drugs, including synthetic contraceptives, estrogens, and progestins. ADD can be biocatalyzed by the dehydrogenation of androstane-4-ene-3,17-dione (AD). KstD or KSDD, a key enzyme catalyzing the 1,2 dehydrogenation of steroids, effectively introduces a double bond at the 1,2 position of the steroidal A ring, enabling the synthesis of ADD. However, during the biotransformation of AD, the FAD regeneration system produces hydrogen peroxide (H2O2). H2O2, a strong oxidant, is toxic during the whole-cell catalytic conversion and may affect conversion efficiency.

[0005] Therefore, it is necessary to find a method for efficiently synthesizing ADD.

[0006] The error-prone PCR technology is used to obtain the preferred mutant of 3-sterol-Δ1-dehydrogenase, which can further improve the biotransformation efficiency and promote its application in the conversion of sterols in the biomedical field. (3) Summary of the invention

[0007] The present invention aims to provide a steroid dehydrogenase mutant and its use in the preparation of steroid drugs. The present invention utilizes error-prone PCR screening to obtain a steroid dehydrogenase mutant with significantly improved enzyme activity, and then couples the steroid dehydrogenase mutant with catalase to eliminate the effect of hydrogen peroxide on bioconversion, thereby improving the bioconversion rate and solving the problem of the toxic effect of hydrogen peroxide generated by the FAD regeneration system on bioconversion during whole-cell catalytic conversion.

[0008] The technical solution adopted in the present invention is:

[0009] The present invention provides a steroid dehydrogenase mutant, which is obtained by single or double mutation of positions 167 and 405 of the amino acid sequence of steroid dehydrogenase (KstD2) shown in SEQ ID NO.1.

[0010] Furthermore, the steroid dehydrogenase mutant is obtained by mutating glycine at position 167 to aspartic acid and / or glutamine at position 405 to arginine in the amino acid sequence shown in SEQ ID NO.1.

[0011] Furthermore, it is preferred that the steroid dehydrogenase mutant is a mutant wherein the glycine at position 167 of the amino acid sequence shown in SEQ ID NO.1 is mutated to aspartic acid and the glutamine at position 405 is mutated to arginine, and is designated as KstD2. G167D / Q405R , the amino acid sequence is shown in SEQ ID NO.3.

[0012] The present invention also relates to the coding gene, recombinant expression vector and recombinant genetic engineering bacteria of the steroid dehydrogenase mutant. The recombinant expression vector uses plasmid pET-28a(+) as a basic vector, and the recombinant genetic engineering bacteria uses Escherichia coli BL21 (DE3) as a host bacteria.

[0013] The present invention also provides an application of the steroid dehydrogenase mutant in the dehydrogenation of androst-4-ene-3,17-dione (AD) to generate androst-1,4-diene-3,17-dione (ADD). The application method comprises the following steps: using wet bacteria cultured by fermentation of an engineered bacterium expressing a fusion of the steroid dehydrogenase mutant and catalase as a catalyst, using androst-4-ene-3,17-dione as a substrate, and using a buffer solution with a pH of 5-10 as a reaction medium to form a conversion system, reacting at 25-55°C and 180 rpm, separating and purifying the reaction solution, and obtaining androst-1,4-diene-3,17-dione.

[0014]

[0015] Furthermore, in the transformation system, the substrate is added at a concentration of 20-60 g / L, preferably 40 g / L; the wet bacteria are added at a concentration of 20-60 g / L, preferably 40 g / L.

[0016] Furthermore, a cosolvent is added to the conversion system, wherein the cosolvent is hydroxypropyl-β-cyclodextrin (HP-β-CD), and the molar ratio of the added amount to the substrate is 1:1.

[0017] Furthermore, the buffer solution is 50 mM Tris-HCl buffer solution with a pH of 8.0.

[0018] Furthermore, the amino acid sequence of the catalase is shown in SEQ ID NO.4.

[0019] Furthermore, the method for constructing the engineered bacteria is as follows: a steroid dehydrogenase mutant and a catalase are connected via a connecting peptide to form a fusion protein, which is then inserted into a plasmid pET-28a(+) to construct a recombinant expression vector, which is then introduced into a host bacterium; the amino acid sequence of the connecting peptide is GGGGS.

[0020] Furthermore, the connection order of the fusion protein is: steroid dehydrogenase mutant-connector peptide-catalase or catalase-connector peptide-steroid dehydrogenase mutant.

[0021] Furthermore, the preferred engineering bacteria are constructed as follows: the synthetic recombinant plasmid pET-28a(+)-katA-L-KstD2 G167D / Q405R As a template, katA fragment with connecting peptide and KstD2 were obtained by PCR amplification. G167D / Q405R , and then seamlessly cloned with pET-28a (+) that was also digested with BamHI and HindIII to obtain the fusion protein KstD2 G167D / Q405R -L-katA.

[0022] Furthermore, the wet bacteria were prepared as follows: a glycerol tube of an engineered bacterium expressing a steroid dehydrogenase mutant and a catalase was inoculated into a TB medium containing 50 μg / mL kanamycin, and cultured at 37°C and 180 rpm for 12 h to serve as a seed solution; the seed solution was inoculated into a TB medium containing 50 μg / mL kanamycin at an inoculum concentration of 3% by volume, and cultured in a shaking incubator at 37°C and 180 rpm until the OD 600 When the pH value reached 0.6-0.8, IPTG (isopropyl-β-D-thiogalactoside) was added at a final concentration of 0.1 mM, and the mixture was induced on a shaker at 20°C and 180 rpm for 16 h. After the induction, the mixture was centrifuged at 8000 rpm for 10 min, and the precipitate was washed once with 0.9% (w / v) saline to remove residual culture medium components and other impurities to obtain wet bacteria.

[0023] Compared with the existing methods, the beneficial effects of the present invention are mainly reflected in:

[0024] (1) The present invention improves the enzyme activity by screening steroid dehydrogenase mutants, increasing it from the original 54.3 U / mg wet bacteria to 67.7 U / mg wet bacteria.

[0025] (2) The present invention connects the steroid dehydrogenase mutant and catalase through a connecting peptide to form a fusion protein, which is then expressed for catalyzing AD to prepare ADD. This not only improves the substrate conversion rate, which is as high as 98.6%, but also solves the problem of hydrogen peroxide toxicity to wet bacterial cells during the reaction, reduces production costs, and has good industrial application prospects. (IV) Description of the accompanying drawings

[0026] Figure 1 Diagram of the reaction mechanism of whole-cell biocatalysis of AD dehydrogenation to ADD using a dual-enzyme coupling system.

[0027] Figure 2 This is a map of the fusion expression plasmid.

[0028] Figure 3 The SDS-PAGE electrophoresis diagram of the fusion expression protein, where M is the protein molecular weight standard (Marker); Lane 1 is E. coli BL21-pET28a-KstD2 G167D / Q405R Lane 2 is E. coli BL21-pET28a-katA-L-KstD2 G167D / Q405R Lane 3 is E. coli BL21-pET28a-KstD2 G167D / Q405R -L-katA.

[0029] Figure 4 The conversion rate of AD to ADD by recombinant engineering bacteria in Example 4. G167D / Q405R 2 is the conversion rate of E.coli BL21 (DE3) -pET-28a (+) -katA-L-KstD2 G167D / Q405R 3 for E.coli BL21 (DE3) -pET-28a (+) KstD2 G167D / Q405R -L-katA conversion rate; 4 is E.coli BL21 (DE3) -pET-28a (+) KstD2 under optimized conditions G167D / Q405R -Conversion rate of L-katA. (V) Specific implementation methods

[0030] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0031] The culture medium involved in the embodiments of the present invention comprises:

[0032] LB liquid medium: 10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract, pH 7.0, sterilized at 121°C for 20 min. LB solid medium is LB liquid medium supplemented with 20 g / L agar powder.

[0033] TB liquid medium: yeast extract 23.6 g / L, tryptone 11.8 g / L, glycerol 4 mL / L, dipotassium hydrogen phosphate 9.4 g / L, potassium dihydrogen phosphate 2.2 g / L, sterilize at 121°C for 20 min.

[0034] Example 1. Construction and expression of steroid dehydrogenase starting strain

[0035] 1. Construction of starting strain

[0036] The steroid dehydrogenase KstD2 (gene Bank: MG251736) from Mycolicibacterium neoaurum DMS1381 in the Gene Bank was codon-optimized according to the codon preference of Escherichia coli. The steroid dehydrogenase gene (amino acid sequence shown in SEQ ID NO.1, nucleotide sequence shown in SEQ ID NO.2) was commissioned to Beijing Qingke Biotechnology Co., Ltd. for synthesis and inserted between the restriction sites BamHI and HindIII of the plasmid pET-28a(+) to obtain the recombinant plasmid pET-28a(+)-KstD2.

[0037] The recombinant plasmid pET-28a(+)-KstD2 was introduced into the host Escherichia coli BL21(DE3) to obtain the strain E. coli BL21(DE3)-pET-28a(+)-KstD2, which was used as the starting strain.

[0038] 2. Expression of starting strain

[0039] A glycerol tube of E. coli BL21 (DE3) - pET-28a (+) -KstD2 was streaked onto a plate of LB solid medium (containing 50 μg / mL kanamycin) and cultured at 37 ° C overnight. A single colony was picked from the plate and inoculated into 20 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37 ° C, 180 rpm for 12 hours as a seed solution. The seed solution was inoculated into 100 mL of TB medium containing 50 μg / mL kanamycin at a volume concentration of 3%. The culture was shaken at 37 ° C, 180 rpm until the OD600 reached 0.6-0.8, and IPTG (isopropyl-β-D-thiogalactopyranoside) was added at a final concentration of 0.1 mM. The culture was placed in a shaker at 20 ° C, 180 rpm for induction for 16 hours. After induction, the mixture was centrifuged at 8000 rpm for 10 min, and the precipitate was washed once with 0.9% (w / v) saline to remove residual culture medium components and other impurities. The wet cells were collected, and the enzyme activity of the wet cells was 54.3 U / mg.

[0040] Example 2: Screening of steroid dehydrogenase mutants and construction of engineered bacteria

[0041] 1. Error-prone PCR

[0042] The plasmid pET-28a(+)-KstD2 of E. coli BL21(DE3)-pET-28a(+)-KstD2 constructed in Example 1 was used as a template, and random mutations were introduced by error-prone PCR.

[0043] Table 1. Error-prone PCR primers:

[0044] Primers Sequence (5'-3') Upstream primer gcaaatgggtcgcggatccGTGACCGACCAGAACAACATC Downstream primer tcgagtgcggccgcaagcttTTAGGTGTGACCTGCAGCG

[0045] Table 2. Error-prone PCR amplification system (50 μL)

[0046] Reaction components Dosage Taq enzyme 0.5μL dNTPs 1 μL 10×Taq Buffer 5μL Upstream primer 1 μL Downstream primer 1 μL template 0.5μL <![CDATA[25mM MgCl2]]> 4 μL <![CDATA[10mM MnCl2]]> 0.5μL <![CDATA[dd H2O]]> To 50μL

[0047] The PCR amplification program was as follows: pre-denaturation at 98°C for 5 min, denaturation at 98°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 1 min, 30 cycles, final extension at 72°C for 10 min, and storage at 4°C.

[0048] After PCR amplification, the product was detected and analyzed by agarose gel electrophoresis, and the target band was purified and recovered using a gel recovery kit.

[0049] Plasmid pET-28a(+) was linearized by double enzyme digestion with BamHI and HindIII. The digestion products were analyzed by agarose gel electrophoresis and recovered.

[0050] The recovered PCR product and the enzyme-digested product were seamlessly cloned and connected, and the connected product was introduced into the competent E. coli BL21 (DE3).

[0051] 2. High-throughput screening steps:

[0052] (1) Transformation and cultivation:

[0053] From the E. coli BL21 (DE3) strain containing the mutant plasmid introduced in step 1, randomly select single colonies and transfer them to a 96-well plate in TB medium containing 50 μg / mL kanamycin. Culture overnight at 37°C and 180 rpm in a shaker. The next day, transfer 10 μL of the overnight culture to a new 96-well plate and add 500 μL of TB medium containing 50 μg / mL kanamycin. Store the remaining culture. Inoculate the 96-well plate at 37°C with shaking until the OD600 reaches 0.6-0.8. Add 0.1 mM IPTG to induce expression. Incubate at 20°C and 200 rpm for 18 hours. This plate is designated a "replica plate."

[0054] (2) Cell collection:

[0055] Collect cells by centrifugation: After induction, place the replicate plate in a centrifuge and centrifuge at 3000 rpm at 4°C for 15 minutes, then discard the supernatant.

[0056] Bacterial suspension: Resuspend the cells in 500 μL of 50 mM Tris-HCl (pH 8.0) buffer, mix well, and centrifuge again at 4°C, 3000 rpm for 10 minutes. Discard the supernatant.

[0057] Re-suspension: Resuspend the cells in 200 μL 50 mM Tris-HCl (pH 8.0) buffer for later use.

[0058] Freeze-thaw treatment: Place the 96-well plate in a -80°C freezer for 2 hours. After thawing, add 0.5 mg / mL lysozyme and incubate for 30 minutes to obtain crude enzyme solution.

[0059] (3) Enzyme activity assay: 2,6-dichlorophenolindoxyl (DCPIP) and benzazine methanesulfonic acid (PMS) were used as components of the electron transport system at 40°C and 600 nm (ε600 = 18.7 × 10 3 cm -1 M -1The assay was performed under 40°C (40°F) conditions. A 200 μL reaction mixture consisted of 50 mM Tris-HCl buffer (pH 8.0), 1.5 mM PMS, 0.12 mM DCPIP, an appropriate volume of crude enzyme solution, 2% methanol by volume, and 500 μM substrates AD, which were pre-dissolved in methanol. Enzyme activity is expressed as units (U), where 1 U is defined as the reduction of 1 μmol of DCPIP per minute at 40°C and pH 8.0.

[0060] (4) For samples whose absorbance changes faster than that of the wild type, record their corresponding positions and calculate the enzyme activity.

[0061] (5) Sequencing confirmation: The mutation position was verified by sequencing. A series of mutants were screened, including F93S / A168V, G167D / Q405R, M103T / D238G, E355G / I426V, etc. Finally, the enzyme activity of the preferred mutant obtained by screening reached 67.7U / mg wet bacteria, which was significantly improved compared with the wild-type enzyme activity of 54.3U / mg wet bacteria. The preferred mutant was obtained by mutating the 167th glycine of KstD2 shown in SEQ ID NO.1 to aspartic acid and the 405th glutamine to arginine, and was named KstD2 G167D / Q405R The amino acid sequence is shown in SEQ ID NO. 3. The method of Example 1 was used to construct the engineering bacteria E. coli BL21 (DE3)-pET-28a (+)-KstD2 G167D / Q405R .

[0062] 3. Induced expression of steroid dehydrogenase mutant engineered bacteria

[0063] Store E. coli BL21(DE3)-pET-28a(+)-KstD2 G167D / Q405R The glycerol tube was inoculated into 20 mL of TB medium containing 50 μg / mL kanamycin and cultured at 37°C and 180 rpm for 12 h as seed solution. The seed solution was inoculated into 100 mL of TB medium containing 50 μg / mL kanamycin at a volume concentration of 3% and cultured at 37°C and 180 rpm in a shaking incubator until the OD 600 When the pH reached 0.6-0.8, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.1 mM, and the cells were induced on a shaker at 20°C and 180 rpm for 16 hours. After induction, the cells were centrifuged at 8000 rpm for 10 minutes, and the pellet was washed once with 0.9% (w / v) saline to remove residual culture medium and other impurities. The wet cells were collected, and the enzyme activity was 67.7 U / mg.

[0064] Example 3: Engineering bacteria expressing fusion of steroid dehydrogenase mutant and catalase

[0065] The katA gene (GenBank: KT963080) from Bacillus pumilus was codon-optimized based on the codon preference of Escherichia coli and synthesized (the amino acid sequence is shown in SEQ ID NO. 4, and the nucleotide sequence is shown in SEQ ID NO. 5).

[0066] To ensure KstD2 G167D / Q405R The two proteins do not interfere with each other during fusion expression. To reduce the structural interference between the two, a flexible connecting peptide (Gly4Ser, abbreviated as G4S) was inserted between the two amino acid sequences to artificially synthesize the recombinant plasmid pET-28a(+)-katA-L-KstD2. G167D / Q405R (Plasmid map as Figure 2 (as shown in a).

[0067] The recombinant plasmid pET-28a(+)-katA-L-KstD2 G167D / Q405R Introduced into host E. coli BL21 (DE3) to obtain strain E. coli BL21 (DE3)-pET-28a (+) -katA-L-KstD2 G167D / Q405R , the corresponding wet cells were prepared using the method of Example 3.

[0068] The recombinant strain E. coli BL21 (DE3)-pET-28a (+)-katA-L-KstD2 G167D / Q405R Activate and culture, obtain the culture medium and extract the plasmid to obtain the plasmid pET-28a(+)-katA-L-KstD2 G167D / Q405R . Using plasmid pET-28a(+)-katA-L-KstD2 G167D / Q405R As a template, PCR amplification of KstD2 was performed using primers F1 and R1 in Table 2 and PCR amplicon in Table 4. G167D / Q405R The L-katA fragment was amplified using primers F2 and R2.

[0069] Table 3. Primers used in PCR

[0070]

[0071] Table 4. PCR amplification system (50 μL):

[0072] Reaction components Dosage PrimeSTAR Max Premix (2X) 25 μL template 2μL Upstream primer 1 μL Downstream primer 1 μL <![CDATA[ddH2O]]> 21 μL

[0073] The PCR amplification program was as follows: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 1 min, 30 cycles, final extension at 72°C for 10 min, and storage at 4°C.

[0074] After PCR amplification, the product was detected and analyzed by agarose gel electrophoresis, and the target band was purified and recovered using a gel recovery kit.

[0075] Plasmid pET-28a(+) was linearized by double enzyme digestion with BamHI and HindIII. The digestion products were analyzed by agarose gel electrophoresis and recovered.

[0076] The recovered PCR product and the enzyme-digested product were seamlessly cloned and connected, and the connected product was introduced into E. coli DH5α competent cells and cultured overnight.

[0077] The clones obtained were selected for sequencing verification, and the successful clones were cultured and plasmids were extracted to obtain the recombinant plasmid pET-28a(+)-KstD2 G167D / Q405R -L-katA (plasmid map as Figure 2 The plasmid was introduced into E. coli BL21 (DE3) competent cells to obtain strain E. coli BL21 (DE3)-pET-28a (+)-KstD2 G167D / Q405R -L-katA.

[0078] Example 4: Preparation of ADD by Biotransformation

[0079] 1. KstD2 G167D / Q405R

[0080] E. coli BL21 (DE3)-pET-28a (+)-KstD2 prepared by the method of Example 2 G167D / Q405R The wet cells were resuspended in 50mM Tris-HCl buffer at pH 8.0 to a concentration of 40g / L. Substrate AD was added at a final concentration of 40g / L and reacted at 35°C and 180rpm for 14 hours. After the reaction, the reaction solution was extracted three times with equal volumes of ethyl acetate. The ethyl acetate layer was evaporated to remove the ethyl acetate and then re-dissolved with methanol. The product was analyzed by liquid chromatography. The results are as follows: Figure 4 As shown in Group 1, the conversion rate of AD reached 76.8%.

[0081] High-performance liquid chromatography (HPLC) detection conditions were as follows: Shimadzu LC-20A, equipped with a Welch Ultimate XB-C18 column (4.5×250 mm, 5 μm), with a mobile phase of methanol:water (75:25) at a flow rate of 0.8 mL / min and a detection wavelength of 254 nm.

[0082] 2.katA-L-KstD2 G167D / Q405R

[0083] The wet cells in step 1 were replaced with E. coli BL21(DE3)-pET-28a(+)-katA-L-KstD2 prepared by the method of Example 3. G167D / Q405R The other operations are the same as the wet bacteria. The results are as follows Figure 4 As shown in Group 2, the conversion rate of AD reached 82.6%.

[0084] 3. KstD2 G167D / Q405R -L-katA

[0085] The wet cells in step 1 were replaced with E. coli BL21(DE3)-pET-28a(+)KstD2 prepared by the method of Example 3. G167D / Q405R -L-katA wet cells, other operations are the same, the results are as follows Figure 4 As shown in Group 3, the conversion rate of AD reached 90.4%.

[0086] 4. KstD2 under optimized conditions G167D / Q405R -L-katA biotransformation to produce ADD

[0087] E. coli BL21 (DE3)-pET-28a (+) KstD2 prepared by the method of Example 3 G167D / Q405R The wet cells of -L-katA were resuspended in 50mM Tris-HCl buffer at pH 8.0 to a concentration of 40g / L. Substrate AD and cosolvent HP-β-CD were added at a final concentration of 40g / L (maintaining a molar ratio of 1:1 with substrate AD). The reaction was continued at 40°C and 180rpm for 14 hours. Other operations were the same as in step 1. The results were as shown in FIG. Figure 4 As shown in Group 4, the conversion rate of AD reached 98.6%.

[0088] The above embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Any equivalent substitution or modification made by those skilled in the art based on the present invention is within the protection scope of the present invention.

Claims

1. A steroid dehydrogenase mutant, characterized in that The steroid dehydrogenase mutant is obtained by mutating the glycine at position 167 of the amino acid sequence shown in SEQ ID NO.1 to aspartic acid and the glutamine at position 405 to arginine.

2. A recombinant genetically engineered bacterium containing a gene encoding the steroid dehydrogenase mutant according to claim 1.

3. Use of the steroid dehydrogenase mutant according to claim 1 in the dehydrogenation of androst-4-ene-3,17-dione to produce androst-1,4-diene-3,17-dione.

4. The use according to claim 3, characterized in that The application method comprises the following steps: using wet bacteria cultured through fermentation of an engineered bacterium expressing a fusion of a steroid dehydrogenase mutant and a catalase as a catalyst, using androst-4-ene-3,17-dione as a substrate, and using a buffer solution with a pH of 5-10 as a reaction medium to form a conversion system, reacting at 25-55° C. and 180 rpm, separating and purifying the reaction solution to obtain androst-1,4-diene-3,17-dione.

5. The use according to claim 4, characterized in that In the transformation system, the substrate is added at a concentration of 20-60 g / L; the wet bacteria are added at a concentration of 20-60 g / L.

6. The use according to claim 4, characterized in that A cosolvent is added to the conversion system, wherein the cosolvent is hydroxypropyl-β-cyclodextrin, and the molar ratio of the added amount to the substrate is 1:

1.

7. The use according to claim 4, characterized in that The amino acid sequence of the catalase is shown in SEQ ID NO.

4.

8. The use according to claim 4, characterized in that The engineering bacteria construction method comprises: connecting a steroid dehydrogenase mutant and a catalase via a connecting peptide to form a fusion protein, inserting the fusion protein into a plasmid pET-28a(+) to construct a recombinant expression vector, and introducing the fusion protein into a host bacteria; the amino acid sequence of the connecting peptide is GGGGS.

9. The use according to claim 8, characterized in that The connection order of the fusion protein is: steroid dehydrogenase mutant-connector peptide-catalase or catalase-connector peptide-steroid dehydrogenase mutant.

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