A 3-sterone-Δ 1 -Dehydrogenase mutants and their applications
By mutating specific amino acid sites of 3-sterone-Δ1-dehydrogenase, a 3-sterone-Δ1-dehydrogenase mutant with high catalytic activity was constructed, solving the problems of long reaction cycle and low conversion rate in the existing technology. This enabled a highly efficient and environmentally friendly biodehydrogenation process for methylprednisolone, which is suitable for industrial application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
- Filing Date
- 2026-03-24
- Publication Date
- 2026-07-24
AI Technical Summary
Existing technologies have long reaction cycles and low conversion rates in the biodehydrogenation process of methylprednisolone. Furthermore, existing 3-sterone-Δ1-dehydrogenases have low catalytic activity for steroid substrates with methyl substitution at the C6 position, making it difficult to meet the needs of industrial production.
By mutating specific amino acid sites of 3-sterone-Δ1-dehydrogenase, a mutant of 3-sterone-Δ1-dehydrogenase with high catalytic activity was constructed. The enzyme was then expressed in host cells using a recombinant expression vector, achieving efficient conversion of high-concentration substrates.
The mutant significantly improved catalytic efficiency and shortened the reaction cycle under high substrate concentrations, avoiding the environmental hazards of chemical methods and the low efficiency of microbial fermentation methods. It is suitable for industrial production and reduces production costs.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of enzyme gene engineering and enzyme engineering technology, specifically relating to a 3-sterone-Δ 1 - Dehydrogenase mutants and engineered bacteria and their applications, capable of dehydrogenating steroid compounds at the C1,2 positions. Background Technology
[0002] Steroid drugs possess strong pharmacological effects such as anti-infection, anti-allergy, antiviral, and anti-shock, and have now become the second largest class of drugs after antibiotics. Steroid hormones are classified as follows: adrenocortical hormones, including methylprednisolone, hydrocortisone, and prednisone, which can treat Addison's disease, and have anti-inflammatory, anti-allergic, and anti-shock effects; anabolic steroids, whose main physiological function is to inhibit protein catabolism and promote protein synthesis, mainly used to treat diseases caused by increased protein production and insufficient protein synthesis; and sex hormones, including estrogens, androgens, and progestins.
[0003] Methylprednisolone (11β,17α,21-trihydroxy-6α-methylpregn-1,4-diene-3,20-dione) and its derivatives are potent adrenocortical steroids with significant immunosuppressive and anti-inflammatory activities, widely used in organ transplantation and the clinical treatment of immune stress. This drug is a synthetic glucocorticoid with typical glucocorticoid-like effects, such as anti-inflammation and immunosuppression. Compared to hydrocortisone, a representative glucocorticoid, its anti-inflammatory efficacy is approximately four times that of the latter. In the structure of steroid drugs, the introduction of a double bond at the C1,2 position of the A ring can significantly enhance anti-inflammatory activity and is an important modification reaction in the industrial production of corticosteroids. For example, dehydrocortisone acetate, produced by dehydrogenation at the C1,2 position, has an anti-inflammatory effect 3-4 times greater than that of the parent compound. Currently, selenium dioxide is commonly used for C1,2 dehydrogenation in chemical synthesis. However, this method involves significant side reactions and can easily leave harmful selenium compounds in the product. Therefore, biological dehydrogenation methods, with their advantages of being green, environmentally friendly, and highly selective, are gradually becoming a key technology for synthesizing anti-inflammatory steroid hormones.
[0004] In recent years, research on the biodehydrogenation of methylprednisolone has progressed relatively slowly. Patent CN101760496A discloses a method for biodehydrogenation of methylprednisolone using Arthrobacterium, but this process has a long reaction cycle (over 48 hours) and a conversion rate of less than 80%, limiting its industrial application. Existing literature indicates that steroid C1,2-position dehydrogenase (3-ketosteroid-Δ...) 1KstD enzymes exhibit low catalytic activity towards steroidal substrates with a methyl substituent at the C6 position. Furthermore, due to poor substrate solubility, it is difficult to increase substrate concentrations in practical applications, thus failing to meet the demands of industrial production. Therefore, identifying and utilizing novel KstD enzyme genes through genomics, bioinformatics, and protein engineering, and then improving their conversion efficiency for high-concentration substrates or shortening reaction time through molecular modification, is of significant research and application value. Summary of the Invention
[0005] To address the above problems, this invention provides a 3-sterone-Δ catalyst with significantly improved catalytic performance. 1 - The dehydrogenase mutant enables the C1,2 position dehydrogenation reaction of the intermediate with a methyl substitution at C6 on the steroid nucleus and the conversion of high-concentration substrates, laying the foundation for its further industrial application.
[0006] In a first aspect, the present invention provides a 3-sterone-Δ 1 -Dehydrogenase mutant, which includes mutations at one or more sites corresponding to wild-type 3-sterone-Δ 1 -Dehydrogenase SEQ ID NO.: 1 contains amino acids 1 to 507 at positions 52, 114, 129, 132, 168, 353, and 419, and is associated with wild-type 3-sterone-Δ 1 The amino acid sequence of the dehydrogenase, SEQ ID NO. 1, has at least 90% homology.
[0007] The mutant has one of the following sequences:
[0008] In another preferred embodiment, the alanine (A) at position 52 is mutated to cysteine (C), leucine (L), or methionine (M), preferably methionine (M);
[0009] In another preferred embodiment, the tyrosine (Y) at position 114 is mutated to phenylalanine (F), histidine (H), or lysine (K), preferably phenylalanine (F);
[0010] In another preferred embodiment, the glutamic acid at position 129 (E) is mutated to lysine (K), serine (S), or glutamine (Q), preferably lysine (K);
[0011] In another preferred embodiment, the histidine (H) at position 132 is mutated to alanine (A), glycine (G), isoleucine (I), leucine (L), serine (S), threonine (T) and valine (V), preferably serine (S);
[0012] In another preferred embodiment, the leucine (L) at position 168 is mutated to cysteine (C), glutamic acid (E), isoleucine (I), and valine (V), preferably isoleucine (I);
[0013] In another preferred embodiment, the phenylalanine (F) at position 353 is mutated to isoleucine (I), asparagine (N), serine (S) and valine (V), preferably valine (V);
[0014] In another preferred embodiment, the methionine (M) at position 419 is mutated to histidine (H), asparagine (N), glutamine (Q) and tryptophan (W), preferably tryptophan (W);
[0015] In another preferred embodiment, the alanine (A) at position 52 is mutated to methionine (M), and the histidine (H) at position 132 is mutated to serine (S).
[0016] In another preferred embodiment, the tyrosine (Y) at position 114 is mutated to phenylalanine (F), and the histidine (H) at position 132 is mutated to serine (S).
[0017] In another preferred embodiment, the histidine (H) at position 132 is mutated to serine (S), and the phenylalanine (F) at position 353 is mutated to asparagine (N);
[0018] In another preferred embodiment, the histidine (H) at position 132 is mutated to serine (S), and the methionine (M) at position 419 is mutated to alanine (A).
[0019] In another preferred embodiment, the phenylalanine (F) at position 353 is mutated to asparagine (N), and the methionine (M) at position 419 is mutated to tryptophan (W).
[0020] In another preferred embodiment, the alanine at position 52 (A) is mutated to methionine (M), the histidine at position 132 (H) is mutated to serine (S), and the phenylalanine at position 353 (F) is mutated to asparagine (N).
[0021] In another preferred embodiment, the histidine (H) at position 132 is mutated to serine (S), the phenylalanine (F) at position 353 is mutated to asparagine (N), and the methionine (M) at position 419 is mutated to alanine (A).
[0022] Secondly, the present invention provides 3-sterone-Δ 1 The invention relates to the coding gene of a 3-dehydrogenase mutant and a recombinant expression vector containing said coding gene. The recombinant expression vector can be used to express the 3-sterone-Δ-dehydrogenase mutant of the present invention using conventional methods in the art. 1The nucleotide sequence of the dehydrogenase gene is linked to various commercially available empty vectors for construction. These commercially available empty vectors can be various plasmid vectors conventional in the art, as long as the recombinant expression vector can replicate normally in the corresponding expression host and express the corresponding 3-sterone-Δ 1 - A dehydrogenase mutant is acceptable. The preferred plasmid vector varies depending on the expression host. Those skilled in the art will understand how to select an appropriate vector, promoter, enhancer, and host cell. For *E. coli*, the preferred plasmid vector is pET-21a(+); for *Pichia pastoris*, the preferred plasmid vector is pPICZαA.
[0023] Thirdly, the present invention provides a solution comprising the 3-sterone-Δ of the present invention. 1 - Recombinant bacteria containing a dehydrogenase mutant gene or its recombinant expression vector. The recombinant expression transformant can be prepared by transforming the above-mentioned recombinant expression vector into the corresponding host cell using conventional techniques in the art. The host cell is a conventional host cell in the art, provided that the recombinant expression vector can stably replicate spontaneously and that the encoded 3-sterone-Δ 1 - The dehydrogenase gene must be effectively expressed. The host cells are preferably Escherichia coli and Pichia pastoris, more preferably Escherichia coli BL21(DE3) or Pichia pastoris X33.
[0024] Fourthly, the present invention provides the aforementioned 3-sterone-Δ 1 The use of the dehydrogenase mutant, the nucleic acid molecule, the recombinant expression vector, or the host cell in the preparation of steroidal drugs, preferably in the catalytic dehydrogenation reaction of an intermediate with a methyl substitution at the C6 position on the steroid nucleus.
[0025] Specifically, the intermediate with a methyl substitution at the C6 position on the steroid nucleus is selected from one of 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione and 11β,17α,21-trihydroxy-6α-methylpregn-4-ene-3,20-dione.
[0026] Fifthly, the present invention provides a method for preparing C1,2-dehydrosteroidal compounds, using an intermediate with a methyl substitution at the C6 position of the steroidal core as a substrate, and adding the 3-sterone-Δ 1 A dehydrogenase mutant, or the host cell or the cell culture thereof, is subjected to a catalytic reaction at pH 8.0 and a temperature of 30°C to obtain a dehydrogenated compound. Preferably, the catalytic reaction system further contains an electron acceptor (PMS) and a co-solvent. Preferably, the co-solvent is selected from at least one of methanol, ethanol, isopropanol, Tween 80, etc.
[0027] Compared with the prior art, the present invention has the following beneficial effects:
[0028] This invention provides a 3-sterone-Δ 1 -Dehydrogenases and their mutants exhibit significantly improved catalytic performance, demonstrating high catalytic activity for intermediates with methyl substitution at the C6 position of the steroid nucleus, thus solving the problem of existing 3-sterone-Δ 1 -Dehydrogenases suffer from low activity and poor tolerance to these substrates. The mutant can efficiently transform the target substrate at a high substrate concentration of 100 g / L, shortening the transformation cycle and meeting the needs of industrial production. The reaction does not require large amounts of organic solvents, avoiding the environmental hazards of chemical methods and the inefficiency of microbial fermentation methods; the process is simple and environmentally friendly. Furthermore, the mutant construction and preparation methods are mature and easy to scale up, providing strong technical support for the green and efficient synthesis of steroidal drug intermediates, significantly reducing production costs, and showing good prospects for industrial application. Attached Figure Description
[0029] Figure 1 The figure shown is an HPLC chromatogram of 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione transformed by the AeKstD mutant M13 recombinant bacteria in Example 3.
[0030] Where a is a standard of the substrate 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione, and b is a transformation sample of 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione by the mutant M13 recombinant bacteria.
[0031] Figure 2 The figure shown is an HPLC chromatogram of 11β,17α,21-trihydroxy-6α-methylpregn-4-ene-3,20-dione transformed by the AeKstD mutant M13 recombinant bacteria in Example 3.
[0032] Where a is a standard of substrate 11β,17α,21-trihydroxy-6α-methylpregn-4-ene-3,20-dione, and b is a transformed sample of 11β,17α,21-trihydroxy-6α-methylpregn-4-ene-3,20-dione by mutant M13. Detailed Implementation
[0033] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention.
[0034] Example 1: Preparation of recombinant expression plasmids and recombinant expression transformants
[0035] 1.1 3-Sterone-Δ 1Synthesis of -dehydrogenase (AeKstD) gene
[0036] Using bioinformatics databases such as NCBI, KEGG, Uniprot, Brenda, and Foldseek, and combined with various software such as Mega, Discovery Studio, and Cytoscape, a comprehensive analysis of the enzyme protein was conducted from multiple levels, including sequence, three-dimensional structure, evolutionary relationship, and protein-protein interaction. Ultimately, a possible 3-sterone-Δ from *Altererythrobacter estronivorus* was identified. 1 -Dehydrogenase, the gene was synthesized by codon optimization based on the protein sequence (amino acid sequence as shown in SEQ ID NO: 1) (optimized nucleotide sequence as shown in SEQ ID No: 2), and constructed into the pET21a expression vector, with gene insertion sites at NdeI and HindIII.
[0037] 1.2 Transformation of recombinant plasmids
[0038] Competent Escherichia coli cells were prepared using the calcium chloride method.
[0039] (1) Take 10 μL of recombinant plasmid into 50 μL of Escherichia coli BL21(DE3) competent cells and incubate on ice for 30 min.
[0040] (2) Heat shock in a 42℃ water bath for 45 s, then quickly place on ice for 1-2 min.
[0041] (3) Add 600 μL of fresh LB liquid medium and incubate at 37°C with shaking for 45–60 min.
[0042] (4) Spread 200 μL of bacterial culture onto the surface of LB solid medium containing ampicillin and incubate at 37℃ for 12-16 h until single colonies appear. Pick positive clones to obtain the recombinant expression transformant E. coli BL21(DE3) / pET21a-AeKstD.
[0043] Example 2: 3-Sterone-Δ 1 Construction and screening of dehydrogenase (AeKstD) mutants
[0044] 1.1 Construction of AeKstD mutant
[0045] 3-Sterone-Δ was predicted using Alphafold 3.0. 1The structure of the dehydrogenase (AeKstD) was selected, and non-conserved residues in its substrate binding pocket and substrate channel amino acids were saturated and mutated. Mutation primers were designed using the degenerate codon NNK. PCR was performed using pET21a-AeKstD as a template and high-fidelity polymerase FastPfu. The PCR reaction conditions were as follows: Round 1: In a PCR reaction system with a total volume of 50 μL, 10-50 ng of template, 10 μL of 5×buffer, 4 μL of dNTP (2.5 mM), 1 μL (10 μM) of each of the two mutant primers, and sterile distilled water was added to a final volume of 50 μL. The PCR reaction program was as follows: (1) Denaturation at 98℃ for 10 sec, (2) Annealing at 58℃ for 20 sec, (3) Extension at 72℃ for 40 sec. Steps (1) to (3) were performed for a total of 30 cycles. Round 2: In a PCR reaction system with a total volume of 50 μL, add 10–50 ng of template, 10 μL of 5× buffer, 4 μL of dNTP (2.5 mM), 1 μL of MgSO4 (50 mM), 2 μL of mutant primer (from Round 1), and add sterile distilled water to a final volume of 50 μL. PCR reaction procedure: (1) denaturation at 98℃ for 10 sec, (2) annealing at 58℃ for 20 sec, (3) extension at 72℃ for 4 min. Perform steps (1) to (3) for a total of 30 cycles. Store the product at 4℃. After verification by agarose gel electrophoresis, the PCR product was digested with the limiting enzyme DpnI at 37℃ for 2 h. The digested product was then transferred into E. coli BL21(DE3) competent cells and plated on a plate containing ampicillin antibiotic. The cells were then incubated at 37℃ for approximately 12 h.
[0046] 1.2 High-throughput screening
[0047] Single clones were selected and induced to express on a small scale in 96-well plates. Cells were collected by centrifugation, lysed, and the expressed proteins were screened for activity. Mutants with high activity were selected and sequenced.
[0048] Enzyme activity assay system: Total reaction volume 0.2 mL, 50 mM Tris-HCl pH 8.0, 1.5 mM methyl phenazine sulfate (PMS), 0.12 mM 2,6-dichlorophenolindophenol (DCPIP), appropriate concentration of substrate, after adding an appropriate amount of enzyme solution, start detection at 30℃, and measure the change in absorbance at 600 nm.
[0049] 1.3 Combinatorial mutations of AeKstD
[0050] Based on the results of saturation mutagenesis, combinatorial mutagenesis was constructed. The resulting single-clone colonies were picked and cultured in Erlenmeyer flasks containing 30 mL of LB medium. The activity of the expressed protein was then measured. The enzyme activity assay method is shown above.
[0051] Through screening, mutants that enhanced the activity of the target steroid substrate were obtained. The mutation sites and activities of these mutants are listed in Table 1.
[0052] Table 1. Relative activities of wild type and mutants against 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione
[0053] .
[0054] Example 3: 3-Sterone-Δ 1 Transformation of 3-ketosteroid compounds by the dehydrogenase AeKstD and its mutant recombinant bacteria
[0055] Seed culture: 3-sterone-Δ was picked up with an inoculation loop 1 - The dehydrogenase AeKstD and its mutant recombinant bacteria were inoculated into LB medium containing ampicillin and cultured overnight at 37 °C and 200 rpm.
[0056] Fermentation induction culture: The overnight cultured seed culture was transferred to the fermentation medium at an inoculum rate of 1%, and cultured at 37 ℃ and 200 rpm until OD was reached. 600nm Add 0.1 mM IPTG to a concentration of approximately 0.6-1.0 and incubate at 25°C and 200 rpm for 10-12 hours to obtain the fermentation broth. Centrifuge to collect the bacterial cells (6000 rpm) and wash the cells twice with sodium chloride solution (0.9%, w / v). Use the bacterial cells as a biocatalyst.
[0057] 3.1 3-Sterone-Δ 1 - Transformation of 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione by recombinant bacteria and mutants of dehydrogenase AeKstD
[0058] The bacterial cell weight was resuspended in 100 mL Tris-HCl buffer (pH 8.0, 50 mM) to obtain a reaction solution with a cell concentration of 10 g / L. 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione was added to the reaction solution at a concentration of 40 g / L, followed by 0.3 g / L phenazine sulfate. The reaction was carried out at 30 °C with stirring for 6 h. The mixture was extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The conversion rate was determined by HPLC. Table 2 shows that, compared to the wild type, mutants M4, M10, and M13 significantly improved the conversion rate of 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione.
[0059] Table 2 Comparison of transformation of 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione between wild type and mutant.
[0060] .
[0061] 3.2 Transformation of 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione by the mutant M4 recombinant strain
[0062] The bacterial cell weight was resuspended in 100 mL Tris-HCl buffer (pH 8.0, 50 mM) to obtain a reaction solution with a cell concentration of 10 g / L. 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione was added to the above reaction solution at a concentration of 60 g / L, followed by 0.5 g / L methyl phenazine sulfate and 5% (v / v) methanol. The mixture was stirred at 30 °C for 24 h. After the reaction was complete, the mixture was extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. HPLC analysis showed that the conversion rate was 97.2%.
[0063] 3.3 Transformation of 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione by the mutant M10 recombinant strain
[0064] The bacterial cell resuspended in 100 mL Tris-HCl buffer (pH 8.0, 50 mM) to obtain a reaction solution with a cell concentration of 10 g / L. 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione was added to the above reaction solution at a concentration of 80 g / L, followed by 1.0 g / L methyl phenazine sulfate and 5% (v / v) ethanol. The mixture was stirred at 30 °C for 48 h. After the reaction was complete, the mixture was extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. HPLC analysis showed that the conversion rate was 96.5%.
[0065] 3.4 Transformation of 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione by the mutant M13 recombinant strain
[0066] The bacterial cell weight was resuspended in 100 mL Tris-HCl buffer (pH 8.0, 50 mM) to obtain a reaction solution with a cell concentration of 10 g / L. 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione was added to the above reaction solution at a concentration of 100 g / L, followed by 1.5 g / L methyl phenazine sulfate and 10% (v / v) isopropanol. The mixture was stirred at 30 °C for 48 h. After the reaction was complete, the mixture was extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. HPLC analysis showed that the conversion rate was 95.2%. Figure 1 In the HPLC chromatograms shown, a is the standard of the substrate 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione, and b is the transformed sample of 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione by the mutant M13 recombinant bacteria.
[0067] 3.5 3-Sterone-Δ 1 - Transformation of 11β,17α,21-trihydroxy-6α-methylpregn-4-ene-3,20-dione by dehydrogenase AeKstD and its mutant recombinant bacteria
[0068] The bacterial cell weight was resuspended in 100 mL Tris-HCl buffer (pH 8.0, 50 mM) to obtain a reaction solution with a cell concentration of 10 g / L. 11β,17α,21-trihydroxy-6α-methylpregn-4-ene-3,20-dione was added to this reaction solution at a concentration of 20 g / L (0.5% w / v) Tween 80, followed by 0.3 g / L methyl phenazine sulfate. The reaction was stirred at 30 °C for 6 h. After extraction with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The conversion rate was determined by HPLC. Table 3 shows that, compared to the wild type, mutants M4, M10, and M13 significantly improved the conversion rate of 11β,17α,21-trihydroxy-6α-methylpregn-4-ene-3,20-dione.
[0069] Table 3. Comparison of transformation of 11β,17α,21-trihydroxy-6α-methylpregn-4-ene-3,20-dione between wild type and mutant.
[0070] .
[0071] 3.6 Transformation of 11β,17α,21-trihydroxy-6α-methylpregn-4-ene-3,20-dione by the M13 mutant recombinant strain
[0072] The bacterial cell weight was resuspended in 100 mL Tris-HCl buffer (pH 8.0, 50 mM) to obtain a reaction solution with a cell concentration of 10 g / L. 11β,17α,21-trihydroxy-6α-methylpregn-4-ene-3,20-dione was added to the above reaction solution at a concentration of 40 g / L, followed by 0.8 g / L phenazine sulfate and 10% (v / v) isopropanol. The mixture was stirred at 30 °C for 24 h. After the reaction was complete, the mixture was extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. HPLC analysis showed that the conversion rate was 95.0%. Figure 2 In the HPLC chromatograms shown, a is the standard of the substrate 11β,17α,21-trihydroxy-6α-methylpregn-4-ene-3,20-dione, and b is the transformed sample of 11β,17α,21-trihydroxy-6α-methylpregn-4-ene-3,20-dione by mutant M13.
Claims
1. A 3-sterone-Δ catalyst with significantly enhanced catalytic performance 1 -Dehydrogenase mutant, characterized by Corresponding to wild-type 3-sterone-Δ 1 The amino acid sequence of the dehydrogenase SEQ ID NO. : 1 contains only the following mutations: H132S; A52M and H132S; Y114F and H132S; H132S and F353N; H132S and M419A; A52M, H132S and F353N; H132S, F353N and M419A.
2. The 3-sterone-Δ as described in claim 1 1 - The gene encoding the dehydrogenase mutant.
3. A recombinant expression vector containing the encoding gene as described in claim 2.
4. The recombinant expression vector as described in claim 3, characterized in that, It is either pET-21a(+) plasmid or pPICZαA.
5. Recombinant bacteria containing the coding gene as described in claim 2 or the recombinant expression vector as described in claim 3 or 4.
6. The recombinant bacteria as described in claim 5, characterized in that, It is either Escherichia coli or Pichia pastoris.
7. The 3-sterone-Δ as described in claim 1 1 - The application of a dehydrogenase mutant, or the encoding gene as described in claim 2, or the recombinant expression vector as described in claim 3 or 4, or the recombinant bacteria as described in claim 5 or 6 in catalyzing the C1,2 dehydrogenation reaction of an intermediate with a methyl substitution at the C6 position on the steroid nucleus; wherein the intermediate with a methyl substitution at the C6 position on the steroid nucleus is selected from 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione and 11β,17α,21-trihydroxy-6α-methylpregn-4-ene-3,20-dione.
8. A method for preparing C1,2 dehydrosteroidal compounds, characterized in that, Using an intermediate with a methyl substitution at the C6 position of the steroid nucleus as a substrate, the 3-sterone-Δ as described in claim 1 is added. 1 - A dehydrogenase mutant or a cell or cell culture of the recombinant bacteria as described in claim 5 or 6 is subjected to a catalytic reaction to obtain a dehydrogenated compound; The intermediate with a methyl substitution at the C6 position on the steroid nucleus is selected from one of 11β,17α-dihydroxy-6α-methylpregn-4-ene-3,20-dione and 11β,17α,21-trihydroxy-6α-methylpregn-4-ene-3,20-dione.
9. The method as described in claim 8, characterized in that, The reaction conditions are pH 7.5-8.5 and temperature 25-35℃; The catalytic reaction system also contains an electron acceptor and a co-solvent, wherein the co-solvent is selected from at least one of methanol, ethanol, isopropanol, and Tween 80.