Steroid dehydrogenase mutant and application thereof in preparation of steroid drugs

Abstract

The invention discloses a steroid dehydrogenase mutant and application of the steroid dehydrogenase mutant in preparation of steroid drugs, the steroid dehydrogenase mutant is screened to improve enzyme activity, and the enzyme activity is improved from original 54.3 U / mg wet thallus to 67.7 U / mg wet thallus. The steroid dehydrogenase mutant and the catalase are connected through the connecting peptide to form the fusion protein, and the fusion protein is expressed for catalyzing AD to prepare ADD, so that the substrate conversion rate is increased and reaches 98.6%, the problem of poison of hydrogen peroxide to wet thallus cells in the reaction process is solved, the production cost is reduced, and the method is suitable for industrial production. Good industrial application prospects are realized.

Description

(I) Technical field

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

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

[0003] The biotransformation technology of steroid compounds is the process of converting steroid compounds into steroid derivatives with medicinal or industrial value through biocatalysis. It is an important means of structural modification of steroids and other complex natural products. The biotransformation method has significant advantages such as mild reaction conditions, few by-products, simple steps, and green environmental protection. Therefore, it is widely used in the research and production of steroid drugs.

[0004] Androstane-1,4-diene-3,17-dione (ADD) is one of the important steroidal derivatives produced during the degradation of sterols and is widely used as a precursor for various steroidal drugs such as synthetic contraceptives, estrogens and progestins. ADD can be produced by biocatalytic dehydrogenation of androstane-4-ene-3,17-dione (AD). 3-Androstane-Δ1-dehydrogenase (EC:1.3.99.4, abbreviated as KstD or KSDD) is a key enzyme that catalyzes the 1,2 dehydrogenation reaction of steroids. It achieves the synthesis of ADD by effectively introducing double bonds at the 1,2 positions of the steroid A ring. However, during the biotransformation of AD, the FAD regeneration system produces hydrogen peroxide (H 2 O 2 ), and H 2 O 2 As a strong oxidant, it has a toxic effect during whole-cell catalytic conversion and may affect the conversion efficiency.

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

[0006] The preferred mutant of 3-sterol-Δ1-dehydrogenase is obtained by using error-prone PCR technology, so that it can further improve the bioconversion efficiency and promote its application in the conversion of sterols in the field of biomedicine. (III) Summary of the invention

[0007] The purpose of the present invention is to provide a steroid dehydrogenase mutant and its application in the preparation of steroid drugs. The present invention uses 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 influence of hydrogen peroxide on bioconversion, improve the bioconversion rate, and solve the problem of the toxic effect of hydrogen peroxide generated by the FAD regeneration system on bioconversion during the whole-cell catalytic conversion process.

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

[0009] The present invention provides a steroid dehydrogenase mutant, wherein the steroid dehydrogenase mutant is a steroid dehydrogenase (KstD 2 ) obtained by single or double mutation at position 167 and position 405 of the amino acid sequence.

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

[0011] Furthermore, it is preferred that the steroid dehydrogenase mutant is a mutant in which 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, denoted as KstD 2 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 bacterium.

[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 is: using wet bacteria cultured by fermentation of an engineered bacterium that fusion-expresses 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 under the conditions of 25-55°C and 180rpm, 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 is 50 mM Tris-HCl buffer at pH 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] Further, the preferred engineering bacteria are constructed as follows: the synthetic recombinant plasmid pET-28a(+)-katA-L-KstD 2 G167D / Q405R As a template, katA fragment and KstD with connecting peptide were obtained by PCR amplification. 2 G167D / Q405R , and then seamlessly cloned with pET-28a(+) that was also digested with BamHI and HindIII to obtain the fusion protein KstD 2 G167D / Q405R -L-katA.

[0022] Further, 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 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 an OD 600When it reaches 0.6-0.8, add IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 0.1mM, place it in a shaking incubator at 20°C and 180rpm for induction for 16h. After the induction, centrifuge it at 8000rpm for 10min, and wash the precipitate 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, 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 drawings

[0026] Figure 1 This is a diagram showing 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 is the SDS-PAGE electrophoresis of the fusion expression protein, where M is the protein molecular weight standard (Marker); Lane 1 is E.coli BL21-pET28a-KstD 2 G167D / Q405R Lane 2 is E. coli BL21-pET28a-katA-L-KstD 2 G167D / Q405R Lane 3 is E. coli BL21-pET28a-KstD 2 G167D / Q405R -L-katA.

[0029] Figure 4 is the conversion rate of the recombinant engineered bacteria biocatalyzing AD to produce ADD in Example 4. 1 is E. coli BL21 (DE3) - pET-28a (+) - KstD 2 G167D / Q405R 2 for E. coli BL21 (DE3) -pET-28a (+) -katA-L-KstD 2 G167D / Q405R3 for E.coli BL21(DE3)-pET-28a(+)KstD 2 G167D / Q405R -L-katA conversion rate; 4 is E.coli BL21(DE3)-pET-28a(+)KstD under optimized conditions 2 G167D / Q405R -Conversion rate of L-katA. (V) Specific implementation methods

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

[0031] The culture medium composition involved in the embodiments of the present invention is as follows:

[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 culture 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 KstD from Mycolicibacterium neoaurum DMS1381 in the Gene Bank was 2 The codons of Escherichia coli were 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 synthesized by Beijing Qingke Biotechnology Co., Ltd. and inserted into the restriction sites BamHI and HindIII of plasmid pET-28a(+) to obtain the recombinant plasmid pET-28a(+)-KstD 2 .

[0037] The recombinant plasmid pET-28a(+)-KstD 2 Introduce the host E. coli BL21 (DE3) to obtain the strain E. coli BL21 (DE3)-pET-28a (+) -KstD 2 , as the starting strain.

[0038] 2. Expression of starting strain

[0039] E. coli BL21(DE3)-pET-28a(+)-KstD 2 The glycerol tube was streaked onto a plate of LB solid medium (containing 50μg / mL kanamycin) and incubated at 37℃ overnight. A single colony was picked from the plate and inoculated into 20mL LB liquid medium containing 50μg / mL kanamycin, and cultured at 37℃ and 180rpm for 12h as seed liquid. The seed liquid was inoculated into 100mL TB medium containing 50μg / mL kanamycin at a volume concentration of 3%, and cultured in a shaking incubator at 37℃ and 180rpm until OD600 reached 0.6-0.8, then IPTG (isopropyl-β-D-thiogalactoside) was added at a final concentration of 0.1mM, and induced at 20℃ and 180rpm for 16h. After induction, centrifuged at 8000rpm for 10min, and the precipitate was washed once with 0.9% (w / v) saline to remove residual culture medium components and other impurities, and the wet bacteria were collected, with an enzyme activity of 54.3U / mg.

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

[0041] 1. Error-prone PCR

[0042] Extract the E. coli BL21(DE3)-pET-28a(+)-KstD constructed in Example 1 2 Plasmid pET-28a(+)-KstD 2 Random mutations were introduced by error-prone PCR using the PCR product as a template.

[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 1μL 10×Taq Buffer 5μL Upstream primer 1μL Downstream primer 1μL template 0.5μL <![CDATA[25mM MgCl 2 ]]> 4μL <![CDATA[10mM MnCl 2 ]]> 0.5μL <![CDATA[dd H 2 The]]> 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 restriction digestion with BamHI and HindIII. The digestion products were analyzed by agarose gel electrophoresis and recovered by gel excision.

[0050] The recovered PCR product and the enzyme-cut 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) into which the mutant plasmid was introduced in step 1, single clones were randomly selected and placed in a 96-well plate containing TB medium with 50 μg / mL kanamycin, and cultured overnight at 37°C and 180 rpm in a shaker. The next day, 10 μL of the overnight culture was transferred to a new 96-well plate, and 500 μL of TB medium containing 50 μg / mL kanamycin was added, and the remaining bacterial solution was preserved. The inoculated 96-well plate was shaken and cultured at 37°C to OD600 0.6-0.8, 0.1 mM IPTG was added to induce expression, and cultured at 20°C and 200 rpm for 18 hours, named "replica plate".

[0054] (2) Cell collection:

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

[0056] Bacterial suspension: resuspend the bacteria with 500 μL 50 mM Tris-HCl (pH 8.0) buffer, mix well, centrifuge again at 4°C, 3000 rpm for 10 min, and 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 a crude enzyme solution.

[0059] (3) Enzyme activity assay: 2,6-dichlorophenolindoxyl (DCPIP) and phenazine methanesulfonic acid (PMS) were used as components of the electron transfer system at 40°C and 600 nm (ε600 = 18.7 × 10 3 cm -1 M -1) was measured under 40 °C. The reaction mixture of 200 μL included 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 substrate AD, which was first dissolved in methanol. The enzyme activity unit is expressed as 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 screened 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 KstD shown in SEQ ID NO.1 2 The 167th glycine was mutated to aspartic acid, and the 405th glutamine was mutated to arginine, and it was named KstD 2 G167D / Q405R The amino acid sequence is shown in SEQ ID NO.3. The method in Example 1 was used to construct the engineering bacteria E. coli BL21 (DE3) - pET-28a (+) - KstD 2 G167D / Q405R .

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

[0063] Save E. coli BL21(DE3)-pET-28a(+)-KstD 2 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 in a shaking incubator at 37°C and 180 rpm until 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 placed in a shaker at 20°C and 180 rpm for induction for 16 hours. After the induction, the mixture was centrifuged at 8000 rpm for 10 minutes, and the precipitate was washed once with 0.9% (w / v) saline to remove the residual culture medium components and other impurities, and the wet cells were collected, and the enzyme activity of the wet cells 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 according to 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 KstD 2 G167D / Q405R The two proteins, katA and katA, do not interfere with each other during fusion expression. To reduce the structural interference between the two proteins, a flexible connecting peptide (Gly4Ser, referred to as G 4 S), artificially synthesized recombinant plasmid pET-28a(+)-katA-L-KstD 2 G167D / Q405R (Plasmid map as Figure 2 (shown in a).

[0067] The recombinant plasmid pET-28a(+)-katA-L-KstD 2 G167D / Q405R Introduce the host E. coli BL21 (DE3) to obtain the strain E. coli BL21 (DE3)-pET-28a (+) -katA-L-KstD 2 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-KstD 2 G167D / Q405R Activate and culture, obtain the culture fluid and extract the plasmid to obtain the plasmid pET-28a(+)-katA-L-KstD 2 G167D / Q405R . Using plasmid pET-28a(+)-katA-L-KstD 2 G167D / Q405R As a template, PCR amplification of KstD was performed using primers F1 and R1 in Table 2 and PCR amplicon in Table 4. 2 G167D / Q405R Fragment, primers F2 and R2 were used to amplify the L-katA fragment.

[0069] Table 3. Primers used for PCR

[0070]

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

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

[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 restriction digestion with BamHI and HindIII. The digestion products were analyzed by agarose gel electrophoresis and recovered by gel excision.

[0076] The recovered PCR product and the enzyme-cut 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(+)-KstD 2 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 (+)-KstD 2 G167D / Q405R -L-katA.

[0078] Example 4: Preparation of ADD by Bioconversion

[0079] 1. KstD 2 G167D / Q405R

[0080] The E. coli BL21(DE3)-pET-28a(+)-KstD prepared by the method of Example 2 was 2 G167D / Q405R The wet bacteria were resuspended in 50mM Tris-HCl buffer at pH 8.0 to a concentration of 40g / L. The 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 an equal volume 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] The high performance liquid chromatography (HPLC) detection conditions were as follows: Shimadzu LC-20A, liquid chromatograph equipped with Welch Ultimate XB-C18 column (4.5×250 mm, 5 μm), mobile phase was methanol: water (75:25), flow rate was 0.8 mL / min, and detection wavelength was 254 nm.

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

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

[0084] 3. KstD 2 G167D / Q405R -L-katA

[0085] The wet cells in step 1 were replaced with E. coli BL21(DE3)-pET-28a(+)KstD prepared by the method of Example 3. 2 G167D / Q405R -L-katA wet bacteria, 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. KstD under optimized conditions 2 G167D / Q405R -L-katA biotransformation to produce ADD

[0087] The E. coli BL21 (DE3)-pET-28a (+) KstD prepared by the method of Example 3 was 2 G167D / Q405R The wet cells of -L-katA were resuspended in 50 mM Tris-HCl buffer at pH 8.0 to a concentration of 40 g / L. The substrate AD and cosolvent HP-β-CD were added at a final concentration of 40 g / L (the molar ratio with the substrate AD was kept at 1:1), and the reaction was carried out at 40°C and 180 rpm for 14 hours. The other operations were the same as step 1. The results are 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 change made by a person 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 single mutation or double mutation at positions 167 and 405 of the steroid dehydrogenase amino acid sequence shown in SEQ ID NO.

1.

2. The steroid dehydrogenase mutant according to claim 1, 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 / or mutating the glutamine at position 405 to arginine.

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

4. 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.

5. The use according to claim 4, characterized in that The application method comprises the following steps: using wet bacteria cultured by fermentation of an engineered bacterium expressing 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, and obtaining androst-1,4-diene-3,17-dione.

6. The use according to claim 5, 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.

7. The use according to claim 5, 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.

8. The use according to claim 5, characterized in that The amino acid sequence of the catalase is shown in SEQ ID NO.

4.

9. The use according to claim 5, 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 vector into a host bacteria; the connecting peptide amino acid sequence is GGGGS.

10. The use according to claim 5, 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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