Hydroxysteroid dehydrogenase mutants and their applications in the preparation of steroidal drugs

Abstract

This invention discloses a hydroxysteroid dehydrogenase mutant and its application in the preparation of steroidal drugs. The mutant is obtained by saturation mutation and combination mutation at positions 89, 185, or 190 of the amino acid sequence shown in SEQ ID NO. 1. The hydroxysteroid dehydrogenase mutant of this invention exhibits improved activity and stability. Using a DMSO-aqueous phase system further improves substrate conversion, reducing production costs and increasing production efficiency.

Description

(I) Technical Field

[0001] This invention belongs to the field of enzyme gene engineering and enzyme engineering technology, specifically relating to a hydroxysteroid dehydrogenase mutant and its application in the preparation of steroid drugs. (II) Background Technology

[0002] Steroid drugs are a class of drugs named according to their chemical structure, referring to drugs whose molecular structure contains the "cyclopentane-polyhydrophenanthrene" core structure. Clinically, steroid hormones are the second most used class of drugs after antibiotics, and are an indispensable type of exogenous hormone. In clinical use, steroid drugs are mainly divided into three categories: adrenocortical hormones, sex hormones, and anabolic steroids. Steroid drugs occupy an important position in the chemical drug system; currently, more than 400 steroid drugs are produced globally, and the market demand for steroid drugs has ranked among the top for many years.

[0003] Microbial transformation of steroidal compounds refers to the use of enzymes produced by microorganisms during metabolism. These enzymes specifically target steroid substrates and possess unique catalytic functions, modifying the structure of these substrates to obtain physiologically active steroidal drug intermediates. Following necessary chemical modifications, the steroidal drug production process is complete. Compared to chemical synthesis, microbial transformation offers advantages such as high specificity, mild reaction conditions, fewer steps, shorter cycles, fewer side reactions, and environmental friendliness. It exhibits high stereoselectivity and regioselectivity, enabling reactions that are difficult or impossible to perform using traditional chemical synthesis. Furthermore, microbial resources are abundant and diverse, and intracellular combinatorial transformation reactions are possible, thus demonstrating a greater advantage in obtaining novel and diverse products. Currently, microbial transformation has become an important route for producing new steroidal drugs, active steroidal drug components, and their key intermediates.

[0004] 3α-hydroxysteroid dehydrogenase (3α(or 20β)-HSD), derived from Pimelobacter simplex, contains a 765 bp DNA sequence encoding 254 amino acids. However, the wild-type 3α-hydroxysteroid dehydrogenase currently exhibits low catalytic activity. Modifying the catalytic performance (catalytic activity and stability) of 3α(or 20β)-HSD through protein engineering techniques such as molecular docking, semi-rational design, and molecular dynamics simulations will enhance its catalytic activity and reaction stability, potentially leading to significant industrial applications. (III) Summary of the Invention

[0005] The purpose of this invention is to provide a hydroxysteroid dehydrogenase mutant, its encoding gene, an engineered bacterium, and its application in the preparation of steroid drugs, thereby solving the problems of low activity and poor stability of existing hydroxysteroid dehydrogenases.

[0006] The technical solution adopted in this invention is:

[0007] The present invention provides a hydroxysteroid dehydrogenase mutant, which is obtained by single or multiple mutations at position 89, position 185 or position 190 of the amino acid sequence shown in SEQ ID NO.1.

[0008] Furthermore, the mutant is the amino acid sequence shown in SEQ ID NO.1 that has been mutated to one of the following: (1) isoleucine at position 89 is mutated to valine (I89V, amino acid sequence as shown in SEQ ID NO.3); (2) valine at position 185 is mutated to glycine (V185G, amino acid sequence as shown in SEQ ID NO.4); (3) threonine at position 190 is mutated to proline (T190P (denoted as MK1), amino acid sequence as shown in SEQ ID NO.5); (4) threonine at position 190 is mutated to proline and isoleucine at position 89 is mutated to valine (I89V / T190P (denoted as MK2)); (5) threonine at position 190 is mutated to proline and isoleucine at position 89 is mutated to valine and valine at position 185 is mutated to glycine (I89V / V185G / T190P (denoted as MK3), amino acid sequence as shown in SEQ ID NO.6).

[0009] This invention also relates to the gene encoding the hydroxysteroid dehydrogenase mutant, a recombinant expression vector containing the gene, and a recombinant genetically engineered bacterium constructed from the recombinant expression vector. The basic plasmid used to construct the expression vector includes pET-28a(+), and the host bacterium used to construct the recombinant genetically engineered bacterium includes *Escherichia coli* BL21(DE3).

[0010] This invention also provides the application of the aforementioned hydroxysteroid dehydrogenase mutant in the catalytic dehydrogenation of steroid compounds to prepare steroidal drugs. The steroidal compound includes 11α,17α-dihydroxyprogesterone.

[0011] Furthermore, the application involves using wet bacterial cells obtained through fermentation culture of recombinant genetically engineered bacteria containing a hydroxysteroid dehydrogenase mutant encoding gene, or crude enzyme solution extracted by ultrasonic disruption of wet bacterial cells, as a catalyst; 11α,17α-dihydroxyprogesterone as a substrate; an organic solvent as a co-solvent; and a buffer solution with pH 6-9 as the reaction medium to form a transformation system. The reaction is carried out at 25-40℃ and 100-300 rpm (preferably 37℃ and 180 rpm) to obtain (11α)-11,17-dihydroxypregn-1,4-diene-3,20-dione. The organic solvent includes dimethyl sulfoxide, methanol, dimethylformamide, and ethanol, preferably dimethyl sulfoxide.

[0012] Furthermore, in the transformation system, the concentration of wet bacterial cells is 20-70 g / L (preferably 50 g / L); the concentration of the substrate is 60-100 g / L (preferably 80 g / L); and the volume concentration of the organic solvent is 3-6% (preferably 4%).

[0013] Furthermore, the reaction medium is a 50 mM Tris-HCl buffer solution with a pH of 8.0.

[0014] Further, the wet bacterial cells were prepared as follows: Recombinant genetically engineered bacteria containing the gene encoding a hydroxysteroid dehydrogenase mutant were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 180 rpm for 12 h. Then, the inoculum was transferred at a volume concentration of 3% to fresh LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 180 rpm until OD... 600 When the concentration reaches 0.6-0.8, add IPTG to a final concentration of 1 mM, induce culture at 18℃ and 180 rpm for 16 h, and collect wet cells after centrifugation at 4℃ and 8000 rpm for 10 min.

[0015] Furthermore, the crude enzyme solution is prepared as follows: wet bacterial cells are washed once with 0.9% physiological saline, centrifuged and the supernatant is discarded. The bacterial cells are resuspended in pH 8.0, 50mM Tris-HCl buffer, placed on ice for 30 min, and ultrasonically disrupted at 0℃ and 60W for 20 min with a 3s working interval and a 7s interval. The cells are then centrifuged at 4℃ and 8000rpm for 30 min, and the supernatant is collected to obtain the crude enzyme solution. The volume of the buffer solution used for resuspension is 10mL / 1.2g based on the weight of the wet bacterial cells.

[0016] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0017] 1. The hydroxysteroid dehydrogenase mutants of this invention exhibit enhanced activity. The hydroxysteroid dehydrogenase mutants obtained by screening in this invention show higher activity against steroid compounds such as 11α,17α-dihydroxyprogesterone, with the crude enzyme solution of mutant MK3 showing a 6.9-fold increase in activity compared to the original strain.

[0018] 2. The stability of the hydroxysteroid dehydrogenase mutant of this invention is improved. The optimal temperature is 37℃, and more than 50% of the residual activity is retained after incubation at temperatures below 40℃ for 0.5h; the optimal pH is 8.0, and relatively high activity is maintained even in low-acid to slightly alkaline environments; compared with the original mutant strain, the obtained superior mutant strain exhibits good stability over a wider pH range, and retains more than 80% of the activity within this range.

[0019] 3. This invention employs a DMSO-aqueous phase system to further improve substrate conversion. Since the substrate 11α,17α-dihydroxyprogesterone is poorly soluble in water, it reduces the production efficiency of the biocatalytic preparation of (11α)-11,17-dihydroxypregn-1,4-diene-3,20-dione. This invention uses a DMSO-aqueous phase (Tris-HCl buffer) system to increase substrate solubility and further improve biocatalytic efficiency. In the single-phase aqueous system (Tris-HCl buffer) without DMSO, the initial enzyme-producing engineered bacteria achieved a 20 mM substrate conversion rate of only 75.3%. However, under the same conditions, the mutant strain achieved a conversion rate of 90.1% in the Tris-HCl buffer system with added DMSO, representing a 1.2-fold increase in conversion rate.

[0020] 4. Compared with the original genetically engineered bacteria, the hydroxysteroid dehydrogenase mutant of the present invention has higher catalytic activity, is more suitable for industrial applications, and can reduce production costs and improve production efficiency. (iv) Description of the attached drawings

[0021] Figure 1 A schematic diagram of the preparation of (11α)-11,17-dihydroxypregn-1,4-diene-3,20-dione by catalytic dehydrogenation of substrate 11α,17α-dihydroxyprogesterone.

[0022] Figure 2 In the docking structure of 3α-HSD and substrate A diagram showing the distribution of amino acid sites within the range.

[0023] Figure 3 This is a bar graph showing the substrate conversion rate of 3α-HSD alanine.

[0024] Figure 4 The relative enzyme activities of the crude enzyme solutions of the original strain and the mutant are given.

[0025] Figure 5 pH dependence (A) and pH stability (B) of mutant MK3.

[0026] Figure 6 The temperature dependence (A, B) and thermal stability (C) of mutant MK3.

[0027] Figure 7 The effect of different substrate concentrations on the conversion rate of 11α,17α-dihydroxyprogesterone was investigated.

[0028] Figure 8 The conversion rate between the original strain and the mutant MK3 is represented. (V) Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0030] LB liquid medium: peptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, pH 7.0.

[0031] The composition of LB solid medium is: peptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, pH 7.0, and agar 20 g / L.

[0032] Example 1: Establishment of a mutant library of 3α-hydroxysteroid dehydrogenase

[0033] 1. Screening of wild-type 3α-hydroxysteroid dehydrogenase and construction of original strains

[0034] The KstD sequence derived from *Pimelobacter simplex*, which exhibits high enzyme activity and good solubility, was identified from the literature. This sequence was then used as a gene probe to search public genome databases for potential dehydrogenases with some homology to the probe sequence and catalytic function at the C1,2 position of the steroid nucleus. After comparing 30 sequences, four genes were screened and heterologously expressed. Finally, one 3α-hydroxysteroid dehydrogenase, namely the 3α(or 20β)HSD gene sequence (nucleotide sequence as shown in SEQ ID NO.2, amino acid sequence as shown in SEQ ID NO.1), was selected and sent to a company for synthesis. The synthesized gene was tagged with a His-tag at its end.

[0035] The target DNA fragment was digested with restriction endonucleases BamHI and HindIII at 37℃ for 3 h, purified by agarose gel electrophoresis, and recovered using an agarose gel DNA recovery kit. The target fragment was ligated with plasmid pET28a, which had also been digested with BamHI and HindIII, overnight at 4℃ to obtain the recombinant expression plasmid. This recombinant expression plasmid was transformed into E. coli DH5α competent cells under the following conditions: heat shock at 45℃ for 90 seconds, ice bath for 5 min, and recovery at 37℃ for 1 h. The cells were plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37℃. Single colonies were picked for PCR verification of positive clones. Positive clones were inoculated onto LB resistant plates containing 50 μg / mL kanamycin and cultured overnight at 37°C. After plasmid amplification, the plasmid was extracted and re-transformed into E. coli BL21(DE3) competent cells. The transformation solution was plated onto LB plates containing 50 μg / mL kanamycin and cultured upside down overnight at 37°C to obtain the engineered strain E. coli BL21(DE3)-pET-28a(+)-3α-HSD containing the pET-28a(+)-3α-HSD plasmid, which was used as the original strain.

[0036] 2. Mutation site screening

[0037] A 3α-HSD (SEQ ID NO.1) protein model was predicted using AlphaFold3, and molecular docking simulations of 3α-HSD and its substrate 11α,17α-dihydroxyprogesterone (DP) were performed using AutoDock. A structural model of the 3α-HSD-DP complex was constructed using AutoDock 4.2 molecular docking software, and key amino acid residues binding the substrate DP were analyzed and identified in the catalytic pocket and substrate channel regions (see [link to software description]). Figure 2 The docking process followed the AutoDock user guide. DP was selected as the flexible ligand, and 3α-HSD protein as the rigid acceptor. The docking region was chosen at the substrate binding site within the substrate inlet / outlet bag. After docking with substrate DP, preliminary analysis was performed, and the lowest-energy docking conformation was selected as the optimal conformation. The results were output as a 3α-HSD-DP.PDB file. Pymol was used to visualize the 3α-HSD-DP.PDB file, and the optimized results were output as PNG format graphics.

[0038] Select the substrate activity pocket Alanine was scanned for 19 amino acids within the range (L17, N86, G88, I89, S90, N111, K136, I137, S138, M146, Y152, P182, G183, M184, V185, T187, M189, T190). The substrate conversion rate was tested using the method in Example 2, and the results are shown in [Figure 2]. Figure 3 I89, V185, and T190 were selected for subsequent site-directed saturation mutagenesis.

[0039] 3. Location-based saturation mutation

[0040] (1) Plasmid extraction:

[0041] Plasmids were extracted from the original strain constructed in step 1 using the AxyPrep Plasmid DNA Mini Kit:

[0042] 1) Take 1-4 mL of bacterial culture that has been cultured overnight in LB medium (if using rich medium, the volume of bacterial culture should be halved or less) and add it to a centrifuge tube. Centrifuge at 12,000 rpm for 1 min and discard the supernatant.

[0043] 2) Add 250 μL of Buffer S1 to suspend the bacterial precipitate. The suspension should be uniform and there should be no small bacterial clumps (make sure RNase A has been added to Buffer S1).

[0044] 3) Add 250 μL of Buffer S2, gently and thoroughly mix by turning the container up and down 4 to 6 times to ensure complete lysis of the bacteria until a clear solution is formed (this step should not exceed 5 minutes, and avoid vigorous shaking, otherwise it will lead to contamination of genomic DNA).

[0045] 4) Place the preparation tube into a 2mL centrifuge tube (provided in the kit) and centrifuge at 12,000×g for 1 min.

[0046] 5) Take the supernatant from step 4) and transfer it to the preparation tube (place it in a 2 mL centrifuge tube (provided in the kit), centrifuge at 12,000×g for 1 min, and discard the filtrate.

[0047] 6) Place the preparation tube back into the centrifuge tube, add 500 μl of Buffer W1, centrifuge at 12,000 × g for 1 min, and discard the filtrate.

[0048] 7) Place the preparation tube back into the centrifuge tube, add 700 μl of Buffer W2, centrifuge at 12,000 × g for 1 min, and discard the filtrate; wash again with 700 μl of Buffer W2 in the same manner. Discard the filtrate. (Confirm that anhydrous ethanol has been added to the Buffer W2 concentrate according to the volume specified on the reagent bottle).

[0049] 8) Place the preparation tube back into a 2mL centrifuge tube and centrifuge at 12,000×g for 1 min.

[0050] 9) Transfer the preparation tube to a new 1.5 mL centrifuge tube (provided in the kit). Add 60-80 μL of Eluent or deionized water to the center of the membrane in the preparation tube and let it stand at room temperature for 1 min. Centrifuge at 12,000 × g for 1 min (heating the Eluent or deionized water to 65℃ will improve the elution efficiency). The liquid at the bottom of the centrifuge tube is the plasmid. Finally, take 1-2 μL and run it on an agarose gel at 120V for 12 min. Observe whether there are bright bands that match the size of the plasmid. Label the centrifuge tube containing the plasmid (pET-28a(+)-3α-HSD) and store it in a -20℃ freezer for use as a PCR template or during transformation.

[0051] (2) Primer design

[0052] Using a semi-rational design approach, degenerate primers Ile89(I)-F, ILe89(I)-R, Val185(V)-F, Val185(V)-R, and Thr190(T)-F, Thr190(T)-R (as shown in Table 1) were designed based on SEQ ID NO.1 and the screened mutation sites. Using pET-28a(+)-3α-HSD plasmid as a template, plasmid PCR was performed using the PrimeSTAR MaxPremix(2X) enzyme from Takara's mutation kit. A mutant library was constructed using site-directed saturation mutagenesis technology, and mutants that enhance the catalytic activity of 3α-hydroxysteroid dehydrogenase on the substrate 11α,17α-dihydroxyprogesterone were further screened.

[0053] Table 1: Primer sequences

[0054]

[0055] Note: The underlined parts are mutation sites. In Table 1, M represents C / A, N represents A / C / T / G, and K represents T / G.

[0056] (3) PCR amplification

[0057] The PCR amplification system (20 μL) consisted of: 0.5–20 ng template, 10 μL PrimeSTAR Max Premix (2X), 0.5 μL each of F and R mutant primers, and ddH2O to make up to 20 μL.

[0058] PCR amplification conditions: 94℃ pre-denaturation for 2 min; [98℃ denaturation for 10 s, 55℃ / 57℃ annealing for 15 s, 72℃ extension for 1 min] 35 cycles; 72℃ final extension for 10 min; PCR amplification products stored at 4℃.

[0059] After verification by gel electrophoresis, the PCR products were digested with the restriction enzyme Dpn I at 37°C for 2 hours and then transformed into E. coli BL21(DE3) competent cells. The cells were then evenly spread on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C until single colonies grew. Single colonies were picked and sent to Qingke Biotechnology Co., Ltd. for sequencing to obtain a single mutant library.

[0060] (4) Screening for dominant mutants

[0061] Single-clonal transformants were picked and inoculated into 800 μL of LB medium containing kanamycin (50 μg / mL) in 96-well plates and incubated at 37°C and 180 rpm for 12 h until the bacterial OD of the culture was measured. 600Once the concentration reached 0.6–0.8, IPTG was added to a final concentration of 0.1 mmol / L for induction expression (18℃, 16 h). After cultivation, the 96-well deep-well plates were centrifuged at 5000 rpm and 4℃ for 10 min to collect the bacterial cells. After removing the supernatant, the bacterial cells in each well were resuspended in 800 μL of 2 mg / mL lysozyme solution (0.1 mM, pH 8.0 Tris-HCl buffer) and incubated at 37℃ for 2 h to lyse the bacterial cells. Subsequently, the 96-well deep-well plates were centrifuged at 5000 rpm and 4℃ for 10 min to obtain the supernatant.

[0062] A reaction system of 100 μL was prepared by adding 20 mM of substrate 11α,17α-dihydroxyprogesterone (substrate first dissolved in methanol), 5% methanol (v / v), 5 mM 2,6-dichlorobenzylindolephenol (DCPIP), 15 mM phenazine methyl sulfate (PMS), and 5 μL of cell-free crude extract (supernatant) to 50 mM Tris-HCl (pH 8.0). The reaction system was compared with the original genetically engineered bacteria reaction system, which showed a rapid color change from deep blue to light yellow, to screen for wells with faster color change times. The enzyme activity was then tested using the method described in Example 2. Figure 3 Transformants with high activity were selected and sent to a sequencing company for sequencing. The dominant single mutants 3α-HSD-I89V, amino acid sequence as shown in SEQ ID NO.3; 3α-HSD-V185G, amino acid sequence as shown in SEQ ID NO.4; and 3α-HSD-T190P (denoted as MK1), amino acid sequence as shown in SEQ ID NO.5 were obtained.

[0063] Based on the dominant single mutants, combined mutations were performed, and the enzyme activities of different mutants were compared to screen for the dominant mutants 3α-HSD-I89V / T190P (denoted as MK2) and 3α-HSD-I89V / V185G / T190P (denoted as MK3).

[0064] The relative enzyme activities of the original strain, MK1, MK2, and MK3 are as follows: Figure 4 With the enzyme activity of the original strain as 100%, the activity of mutant MK3 was 6.9 times higher than that of the original strain pET28a(+)-3α-HSD.

[0065] Example 2: Detection of enzyme activity and substrate conversion rate of 3α-hydroxysteroid dehydrogenase and its mutants

[0066] 1. Enzyme activity detection

[0067] The principle of detecting 3α-HSD and mutant enzyme activity using the DCPIP method: 3α-HSD and its mutants catalyze the dehydrogenation of 11α,17α-dihydroxyprogesterone to (11α)-11,17-dihydroxypregn-1,4-diene-3,20-dione. The 2H removed at the C1-2 position of the substrate binds to PMS (phenazine dimethyl sulfate) to form PMSH2, which is then immediately transferred to DCPIP to form DCPIPH2, thus reducing DCPIP. PMS and DCPIP act as electron acceptors. DCPIP has a characteristic absorption peak at 600 nm. By measuring the change in absorbance, the reduction rate of DCPIP can be determined, thereby characterizing the activity of 3α-HSD and its mutants.

[0068] Enzyme activity definition: The amount of enzyme required to reduce 1 nmol of DCPIP per minute at 37℃ and pH=8.0 is defined as 1 unit.

[0069] (U).

[0070] Crude enzyme solution: The original strain and engineered bacteria (including MK1, MK2, and MK3) screened in Example 1, taken from a -80℃ freezer, were streaked onto LB agar plates containing 50 μg / mL kanamycin (Kan) and incubated at 37℃. After colony growth, a single colony was picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL Kan, and incubated at 37℃ and 180 rpm for 12 h. The culture was then transferred to 100 mL of LB liquid medium containing 50 μg / mL Kan at a volume concentration of 3%, and incubated at 37℃ and 200 rpm for 2–3 h until OD (oxidative stress) was observed. 600 When the concentration reaches 0.6–0.8, the culture flask is cooled, and IPTG is added to a final concentration of 1 mM. The culture is then induced at 18°C ​​and 180 rpm for 16 h. After centrifugation at 8000 rpm and 4°C for 10 min, the resting wet bacterial cells are collected. 1.2 g of the collected wet bacterial cells are washed once with 0.9% physiological saline, centrifuged, and the supernatant is discarded. Finally, 10 mL of 50 mM Tris-HCl (pH 8.0) buffer is added to resuspend the cells. The cells are incubated on ice for 30 min, then sonicated at 0°C (60 W, total time 20 min, sonication on time 3 s, sonication off time 7 s). After centrifugation at 4°C and 8000 rpm for 30 min, the supernatant, containing the target protein, is collected and stored at 4°C.

[0071] Enzyme activity assay reaction system: PMS solution was prepared with DMSO at 15 mM, DCPIP solution was prepared with DMSO at 5 mM, and substrate DP solution was prepared with DMSO at 20 mM. The enzyme activity assay reaction system was constructed as shown in Table 2.

[0072] Table 2 Reaction system for enzyme activity assay

[0073]

[0074] The above enzyme activity assay reaction system was incubated at 37°C for 20 min. The absorbance at a wavelength of 600 nm was detected by a spectrophotometer. The concentration of DCPIP in the crude enzyme solution was calculated based on the standard curve of DCPIP standard concentration and absorbance, and then the corresponding enzyme activity was calculated.

[0075] 2. Substrate conversion rate detection:

[0076] After 96 h of conversion reaction, the sample was collected in a 1.5 mL EP tube, and the substrate and product were extracted with ethyl acetate. During extraction, the EP tube was first vortexed for 3 min to ensure complete extraction of the substrate and product into the ethyl acetate. After extraction, the sample was centrifuged at 8000 rpm for 4 min to clarify the ethyl acetate phase. 200 μL of the supernatant was transferred to a 1.5 mL EP tube and evaporated to dryness in a fume hood. The evaporated liquid was then reconstituted with 500 μL of acetonitrile. The reconstituted liquid was filtered through a 0.45 μm microporous organic membrane, and 200 μL of the filtrate was collected in the inner liner of a liquid chromatography vial. The peak areas of the substrate and product were quantitatively analyzed using high-performance liquid chromatography (HPLC), and the substrate conversion rate was calculated based on the standard curve of substrate peak area versus concentration.

[0077] HPLC conditions: Agilent 1100 Series LC (G1314 Pump, G1322 ADEGASSER G1314 VWD detector, 10 μL LAN injector, HP Chem Station); C18 (5 μm, 250 mm x 4.6 mm) column; mobile phase: acetonitrile:methanol:water (25:15:60, v / v / v); flow rate: 1 mL / min; column temperature: 30 °C; detector: UV detector, wavelength: 240 nm; injection volume: 10 μL. Substrate elution time: 10.473 min; product elution time: 12.232 min.

[0078] Example 3: Optimal reaction pH of mutant MK3

[0079] Optimal pH: The buffer solution in the enzyme activity assay reaction system of Example 2 was changed to a buffer solution with a concentration of 50mM, namely MES (pH 6.0, 6.5, 7.0), PBS (pH 6.5, 7.0, 7.5, 8.0, 8.5), HEPES (pH 7.0, 7.5, 8.0), Tris–HCl (pH 7.0, 7.5, 8.0, 8.5, 9.0) and Gly–NaOH (pH 8.0, 8.5, 9.0).

[0080] The crude enzyme solution was selected from mutant MK3. Enzyme activity was detected using the method described in Example 2. Activity in pH 7.5 Tris-HCl buffer was defined as 100%. The enzyme maintained relatively high activity under low-acid to slightly alkaline conditions; under strongly acidic conditions, the relative activity was below 40%. The highest enzyme activity was observed at a reaction pH of pH 8.0 in Tris-HCl buffer, representing a 0.5-fold increase compared to the original strain (see Example 2). Figure 5 (A)

[0081] pH stability: After incubating the crude enzyme solution of mutant MK3 in the above-mentioned pH 7.0–9.0 buffer at 37°C for 20 min, the relative enzyme activity was detected using the method in Example 2 (see Example 2). Figure 5 The strain B) exhibited good stability within a pH range of 7.0–9.0, retaining over 80% of its activity within this range. The original strain, however, showed good pH stability within a pH range of 7.0–8.5.

[0082] Example 4: Optimal reaction temperature of mutant MK3

[0083] Optimal Temperature: In Example 2, the crude enzyme solution for the enzyme activity detection reaction system was selected from the mutant MK3 crude enzyme solution, and the buffer solution was changed to Tris-HCl buffer (50mM, pH 8.0). Enzyme activity was detected at different temperatures (25, 30, 34, 40, 45, 50, 55℃) using the method of Example 2. The results are shown in [Figure 2]. Figure 6 In step A, enzyme activity was then measured at 33, 35, 37, 39, 41, and 43°C. The results are shown in [the table below]. Figure 6 B. The activity at 35°C is defined as 100%. Figure 6 Figures A and B show that the optimal temperature for the activity of mutant MK3 is 37℃, and the activity begins to decline sharply when the temperature exceeds 40℃.

[0084] Temperature stability: The crude enzyme solution of mutant MK3 was incubated in Tris-HCl buffer (50 mM, pH 8.0) at different temperatures (25, 30, 35, 40, 45, 50 °C) for 30 minutes, and then cooled on ice for 10 minutes. Enzyme activity was then detected using the method in Example 2. Mutant MK3 showed better thermostability after incubation at 25–40 °C for 0.5 hours, retaining more than 50% of its residual activity before reaching 40 °C. Figure 6 (C). Under the same conditions, the original strain showed good thermal stability at 25-35℃.

[0085] Example 5: Effect of cosolvent on C1,2-position dehydrogenation reaction

[0086] The total volume of the reaction system was 5 mL: the substrate 11α,17α-dihydroxyprogesterone concentration was 80 g / L, the co-solvent volume concentration was 4%, the final concentration of the mutant MK3 wet cells prepared by the method in Example 2 was 50 g / L, and 50 mM Tris-HCl buffer (pH 8.0) was added to make up to 5 mL. DMSO, methanol, ethanol, isopropanol, and acetone were used as co-solvents, and the reaction was carried out at 37 °C and 180 rpm for 96 h. After the reaction was completed, the reaction solution was centrifuged (8000 rpm, 4 °C, 10 min). The supernatant was extracted three times with an equal volume of ethyl acetate, and the ethyl acetate phases were combined. 200 μL of the upper extract was transferred to a 1.5 mL EP tube and evaporated to dryness in a fume hood. After evaporation, it was reconstituted with 500 μL of acetonitrile. The substrate conversion rate was detected by the liquid chromatography method described in Example 2, and the results are shown in Table 3.

[0087] Table 3. Effects of different cosolvents on the C1,2-position dehydrogenation reaction.

[0088]

[0089] Example 6: Transformation Validation of 3α-HSD Engineered Bacteria

[0090] 1. Wet bacteria

[0091] The original strain and the engineered mutant MK3 screened in Example 1 were picked from the slant and inoculated into LB liquid medium containing 50 μg / mL kanamycin. The cultures were incubated at 37°C with shaking at 160 rpm for 12 h. The culture was then transferred at a volume concentration of 3% to 250 mL Erlenmeyer flasks containing 50 mL of fresh LB liquid medium containing 50 μg / mL kanamycin. Initial OD... 600 Value 0.2, 37℃, shake at 180 rpm for 1.5-2 h until OD 600 When the concentration reaches 0.6-0.8, add IPTG to a final concentration of 1 mM, and induce culture at 18°C ​​and 180 rpm for 16 h. After centrifugation at 4°C and 8000 rpm for 10 min, collect the wet cells.

[0092] 2. Transformation reaction

[0093] (1) Substrate concentration

[0094] The 50 mL transformation system in a 250 mL reaction flask consisted of: 50 g / L of wet bacterial cells prepared in step 1, substrate 11α,17α-dihydroxyprogesterone (60, 70, 80, 90, 100 g / L), 4% DMSO, and 50 mM Tris-HCl buffer solution (pH 8.0) to a final volume of 50 mL. The biotransformation reaction was carried out at 37 °C and 180 rpm for 96 h. A 500 μL sample was taken and extracted with twice the volume of ethyl acetate. The substrate conversion rate was determined using the method described in Example 2. Under the same conditions, the original strain of wet bacterial cells served as a control.

[0095] The results are as follows Figure 7 As shown, increasing the substrate concentration leads to a longer reaction time. When the final substrate concentration is less than or equal to 80 g / L, the conversion rate is 90.1% after 96 h. When the substrate concentration is greater than 80 g / L, the substrate conversion rate decreases.

[0096] (2) Reaction time

[0097] Step (1) The substrate concentration was changed to 80 g / L, and the other operations were the same. Samples were taken at 24 h, 48 h, 72 h, 96 h and 120 h, respectively. Each sample was 500 μL. Two volumes of ethyl acetate were added for extraction. The substrate conversion rate was detected using the method in Example 2.

[0098] See the results Figure 8 As shown, the transformation efficiency of the mutant strain was significantly higher than that of the original strain in every 24-hour sampling. In the first 48 hours, the transformation efficiency of the mutant strain MK3 was 4.12 times higher than that of the original strain. When the transformation was completed after 96 hours, the substrate transformation efficiency of the original strain was only 28.4%, while under the same conditions, the substrate transformation efficiency of the mutant strain MK3 was 90.1%, which was 3.17 times higher than that of the original strain.

[0099] The above description of the embodiments is intended to enable those skilled in the art to understand and use the invention. Those skilled in the art can readily make various modifications to these embodiments and apply the general principles of this description to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A hydroxysteroid dehydrogenase mutant, characterized in that, The mutant is formed by mutating threonine at position 190 to proline, isoleucine at position 89 to valine, and valine at position 185 to glycine in the amino acid sequence shown in SEQ ID NO.

1. The amino acid sequence of the mutant is shown in SEQ ID NO.

6.

2. The use of the hydroxysteroid dehydrogenase mutant of claim 1 in the preparation of steroid drugs by catalyzing the dehydrogenation of steroid compounds.

3. The application as described in claim 2, characterized in that, The steroidal compound includes 11α,17α-dihydroxyprogesterone.

4. The application as described in claim 3, characterized in that, The application is as follows: using wet bacterial cells obtained by fermentation culture of recombinant genetically engineered bacteria containing the gene encoding a hydroxysteroid dehydrogenase mutant, or crude enzyme solution extracted by ultrasonic disruption of wet bacterial cells, as a catalyst, 11α,17α-dihydroxyprogesterone as a substrate, an organic solvent as a co-solvent, and a buffer solution with pH 6-9 as a reaction medium to form a transformation system, and reacting at 25-40℃ and 100-300rpm to obtain (11α)-11,17-dihydroxypregn-1,4-diene-3,20-dione.

5. The application as described in claim 4, characterized in that, The organic solvents include dimethyl sulfoxide, methanol, dimethylformamide, and ethanol.

6. The application as described in claim 4, characterized in that, The reaction medium is a 50 mM Tris-HCl buffer solution with a pH of 8.

0.

7. The application as described in claim 4, characterized in that, In the transformation system, the concentration of wet bacterial cells is 20-70 g / L; the concentration of the substrate is 60-100 g / L; and the volumetric concentration of the organic solvent is 3-6%.

8. The application as described in claim 4, characterized in that, The wet bacterial cells were prepared as follows: Recombinant genetically engineered bacteria containing the gene encoding a hydroxysteroid dehydrogenase mutant were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 180 rpm for 12 h. The inoculum was then transferred at a volume concentration of 3% to fresh LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 180 rpm until OD (digestive end-expiratory) reached. 600 When the concentration reaches 0.6-0.8, add IPTG to a final concentration of 1 mM, induce culture at 18℃ and 180 rpm for 16 h, and collect wet cells after centrifugation at 4℃ and 8000 rpm for 10 min.

9. The application as described in claim 4, characterized in that, The crude enzyme solution was prepared as follows: the wet bacterial cells were washed once with 0.9% physiological saline, centrifuged and the supernatant was discarded. The bacterial cells were resuspended in pH 8.0, 50mM Tris-HCl buffer, placed on ice for 30 min, and then sonicated at 0℃ and 60W for 20 min with a 3-second working interval of 7 seconds. The mixture was then centrifuged at 4℃ and 8000rpm for 30 minutes, and the supernatant was collected to obtain the crude enzyme solution.

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