Hydroxysteroid dehydrogenase mutant and application thereof in preparation of steroid drugs

Abstract

The invention discloses a hydroxysteroid dehydrogenase mutant and application thereof in preparation of steroid drugs. The mutant is obtained by performing saturated mutation and combinatorial mutation on the 89th site, the 185th site or the 190th site of an amino acid sequence as shown in SEQ ID NO.1. The invention further discloses a preparation method of the hydroxysteroid dehydrogenase mutant. The activity of the hydroxysteroid dehydrogenase mutant is improved, and the stability is improved. And a DMSO-water phase system is adopted to further improve the substrate conversion rate, so that the production cost can be reduced, and the production efficiency can be improved.

Description

(I) Technical field

[0001] The invention belongs to the technical field of enzyme gene engineering and enzyme engineering, and specifically relates 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 after their chemical structure, and refer to drugs that contain the "cyclopentaphenanthrene" parent nucleus structure in their molecular structure. In clinical practice, steroid hormone drugs are the second largest class of drugs in terms of dosage after antibiotics, and are an indispensable class of exogenous hormones. In clinical use, steroid drugs are mainly divided into three categories: adrenal cortical hormones, sex hormones, and anabolic hormones. Steroid drugs occupy an important position in the chemical drug system. Currently, there are more than 400 kinds of steroid drugs produced worldwide, and the market demand for steroid drugs has been at the forefront for many years.

[0003] The microbial transformation production of steroidal compounds refers to the use of enzymes produced by microorganisms in the metabolic process that specifically act on steroidal substrates and have specific catalytic functions to modify the structure of steroidal substrates to obtain physiologically active steroidal drug intermediates. After necessary chemical modifications, the process of steroidal drug production is completed. Compared with chemical synthesis, microbial transformation has good specificity, mild reaction conditions, fewer steps, shorter cycles, fewer side reactions, and is environmentally friendly. It has high stereoselectivity and regioselectivity, and can achieve reactions that are difficult or impossible to carry out in traditional chemical synthesis. In addition, microbial resources are abundant and diverse, and combined transformation reactions may occur in cells, so it shows greater advantages in obtaining products with novel structures and diverse forms. At present, microbial transformation has become an important way to produce new steroidal drugs, steroidal active pharmaceutical ingredients and their key intermediates.

[0004] The 3α(or 20β)-hydroxysteroid dehydrogenase (3α(or 20β)-HSD) from Pimelobacter simplex contains a 765bp DNA sequence and encodes 254 amino acids, but the catalytic activity of the wild-type 3α-hydroxysteroid dehydrogenase is currently low. The catalytic performance (catalytic activity, stability) of 3α(or 20β)-HSD can be modified by protein engineering methods such as molecular docking, semi-rational design modification, and molecular dynamics simulation to improve its catalytic activity and reaction stability, which will have high industrial application value. (III) Summary of the invention

[0005] The present invention aims to provide a hydroxysteroid dehydrogenase mutant, a coding gene, an engineering bacterium and the use thereof in the preparation of steroid drugs, so as to solve the problems of low activity and poor stability of the existing hydroxysteroid dehydrogenase.

[0006] The technical solution adopted by the present invention is:

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

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

[0009] The present invention also relates to the hydroxysteroid dehydrogenase mutant encoding gene, a recombinant expression vector containing the encoding gene, and a recombinant genetic engineering bacterium constructed by the recombinant expression vector. The basic plasmid used to construct the expression vector includes pET-28a(+), and the host bacteria used to construct the recombinant genetic engineering bacterium include Escherichia coli BL21(DE3).

[0010] The present invention also provides an application of the hydroxysteroid dehydrogenase mutant in catalyzing the dehydrogenation of steroid compounds to prepare steroid drugs. The steroid compounds include 11α, 17α-dihydroxyprogesterone.

[0011] Further, the application is: using wet cells obtained by fermentation culture of recombinant genetically engineered bacteria containing a gene encoding a hydroxysteroid dehydrogenase mutant or a crude enzyme solution extracted by ultrasonic crushing of wet cells as a catalyst, using 11α,17α-dihydroxyprogesterone as a substrate, an organic solvent as a cosolvent, and a pH 6-9 buffer as a reaction medium to form a conversion system, reacting at 25-40° C. and 100-300 rpm (preferably 37° C. and 180 rpm) to obtain (11α)-11,17-dihydroxypregnane-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 wet bacteria are added at a concentration of 20-70 g / L (preferably 50 g / L); the substrate is added at a concentration of 60-100 g / L (preferably 80 g / L); and the organic solvent is added at a concentration of 3-6% (preferably 4%) by volume.

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

[0014] Further, the wet bacteria were prepared as follows: the recombinant genetically engineered bacteria containing the hydroxysteroid dehydrogenase mutant encoding gene were inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C, 180 rpm for 12 h, and then transferred to fresh LB liquid medium containing 50 μg / mL kanamycin at a volume concentration of 3%, and cultured at 37°C, 180 rpm until the OD 600 When it reached 0.6-0.8, IPTG was added to a final concentration of 1 mM, and the culture was induced at 18°C ​​and 180 rpm for 16 h. The culture solution was centrifuged at 4°C and 8000 rpm for 10 min to collect the wet bacteria.

[0015] Furthermore, the crude enzyme solution is prepared as follows: the wet bacteria are washed once with 0.9% physiological saline, centrifuged and the supernatant is discarded, pH 8.0, 50mM Tris-HCl buffer is added to resuspend the bacteria, ice bathed for 30 minutes, ultrasonically disrupted at 0°C and 60W for 20 minutes, working for 3s and 7s intervals, centrifuged at 4°C and 8000rpm for 30 minutes, and the supernatant is collected to obtain a crude enzyme solution; the volume of the buffer used for resuspension is 10mL / 1.2g based on the weight of the wet bacteria.

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

[0017] 1. The activity of the hydroxysteroid dehydrogenase mutant of the present invention is improved. The hydroxysteroid dehydrogenase mutant screened and obtained by the present invention exhibits higher activity to steroid compounds such as 11α,17α-dihydroxyprogesterone, among which the activity of the crude enzyme solution of mutant MK3 is 6.9 times higher than that of the original strain.

[0018] 2. The stability of the hydroxysteroid dehydrogenase mutant of the present invention is improved. The optimum temperature is 37°C, and more than 50% of the residual activity is retained when the temperature is kept below 40°C for 0.5h; the optimum pH is 8.0, and relatively high activity can be maintained in a low acid and alkaline environment; compared with before mutation, the obtained excellent mutant strain shows good stability in a wider pH range, and retains more than 80% of the activity within this range.

[0019] 3. The present invention uses a DMSO-aqueous phase system to further improve the substrate conversion rate. Since the substrate 11α, 17α-dihydroxyprogesterone is poorly soluble in water, the production efficiency of the biocatalytic preparation of (11α)-11,17-dihydroxypregnant-1,4-diene-3,20-dione is reduced. The present invention uses a DMSO-aqueous phase (Tris-HCl buffer) system to increase the solubility of the substrate and further improve the biocatalytic efficiency. In a single aqueous phase system (Tris-HCl buffer) without DMSO, the initial enzyme-producing engineered bacteria catalyzed a 20mM substrate conversion rate of only 75.3%, while under the same other conditions, the mutant strain had a conversion rate of 90.1% in the Tris-HCl buffer system with DMSO added, and the conversion rate was increased by 1.2 times.

[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 application, can reduce production costs and improve production efficiency. (IV) Description of the drawings

[0021] Figure 1 Schematic diagram of the catalytic dehydrogenation of substrate 11α,17α-dihydroxyprogesterone to prepare (11α)-11,17-dihydroxypregnane-1,4-diene-3,20-dione.

[0022] Figure 2 The docking structure of 3α-HSD and substrate Distribution diagram of amino acid sites within the range.

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

[0024] Figure 4 It is the relative enzyme activity of the crude enzyme solution of the original strain and the mutant.

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

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

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

[0028] Figure 8 is the transformation rate of the original strain and mutant MK3. (V) Specific implementation methods

[0029] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

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

[0031] LB solid culture medium composition: peptone 10.0 g / L, yeast powder 5.0 g / L, NaCl 10.0 g / L, pH 7.0, agar 20 g / L.

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

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

[0034] We found KstD sequences from Pimelobacter simplex with high enzyme activity and good solubility in the literature, and then used them as gene probes to search in public genome databases, and explored potential dehydrogenases that have certain homology with the probe sequence and have catalytic function on the C1,2 position of the steroid nucleus. After comparing 30 sequences, we screened 4 genes and expressed them heterologously. Finally, we screened out a 3α-hydroxysteroid dehydrogenase, namely 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) and sent it to the company for synthesis, and the end of the synthetic gene has a His-tag label.

[0035] The target DNA fragment was double-digested with restriction endonucleases BamHI and HindIII at 37°C for 3h, purified by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit. The target fragment was ligated with the plasmid pET28a, which was also double-digested with BamHI and HindIII, at 4°C overnight under the action of T4 ligase to obtain a recombinant expression plasmid. The above recombinant expression plasmid was transformed into E.coli DH5α competent cells under the transformation conditions of 45°C heat shock for 90 seconds, ice bath for 5min, and recovery at 37°C for 1h. Spread on LB resistance plates containing 50μg / mL kanamycin, cultured at 37°C overnight, and pick monoclonal colonies for PCR verification of positive clones. The positive clones were inoculated onto LB resistance plates containing 50 μg / mL kanamycin and cultured at 37°C overnight. After plasmid amplification, the plasmid was extracted and re-transformed into E. coli BL21 (DE3) competent cells. The transformation solution was spread onto LB plates containing 50 μg / mL kanamycin and cultured upside down at 37°C overnight to obtain the engineered bacteria E. coli BL21 (DE3)-pET-28a (+) -3α-HSD containing the pET-28a (+) -3α-HSD plasmid as the original strain.

[0036] 2. Screening of mutation sites

[0037] The protein model of 3α-HSD (SEQ ID NO.1) was predicted by AlphaFold3, and the molecular docking simulation of 3α-HSD and substrate 11α,17α-dihydroxyprogesterone (DP) was performed using AutoDock. The structural model of the 3α-HSD-DP complex was constructed using AutoDock 4.2 molecular docking software, and the key amino acid residues that bind to the substrate DP were analyzed and identified in the catalytic pocket and substrate channel regions (see Figure 2 ). The docking process refers to the AutoDock user guide. DP is selected as a flexible ligand, 3α-HSD protein is selected as a rigid receptor, and the docking region is selected at the substrate binding site within the substrate inlet and outlet pockets. After docking the substrate DP, a preliminary analysis is performed, and the docking conformation with the lowest energy is selected as the optimal conformation, and the result is output as a 3α-HSD-DP.PDB file. Pymol is used to visualize the 3α-HSD-DP.PDB file, and the optimization results are output as png format graphics.

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

[0039] 3. Site-directed saturation mutagenesis

[0040] (1) Plasmid extraction:

[0041] Use the AxyPrep Plasmid DNA Mini Kit to extract the plasmid from the original strain constructed in step 1:

[0042] 1) Take 1-4 mL of bacterial culture cultured overnight in LB medium (if rich medium is used, 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 Buffer S1 to suspend the bacterial pellet. The suspension should be uniform and no small bacterial clumps should be left (make sure RNase A has been added to Buffer S1).

[0044] 3) Add 250 μL Buffer S2, gently and thoroughly invert up and down 4 to 6 times to mix evenly to fully lyse the bacteria until a transparent solution is formed (this step should not exceed 5 minutes, and avoid violent shaking, otherwise it will lead to genomic DNA contamination).

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

[0046] 5) The supernatant from step 4) was aspirated and transferred to a preparation tube (placed in a 2 mL centrifuge tube (provided in the kit)), centrifuged at 12,000×g for 1 min, and the filtrate was discarded.

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

[0048] 7) Return the preparation tube to the centrifuge tube, add 700μl Buffer W2, centrifuge at 12,000×g for 1 min, and discard the filtrate; wash once again with 700μL Buffer W2 in the same way. Discard the filtrate. (Confirm that anhydrous ethanol has been added to the Buffer W2concentrate according to the specified volume on the reagent bottle).

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

[0050] 9) Move the preparation tube into a new 1.5mL centrifuge tube (provided in the kit), add 60-80μL Eluent or deionized water to the center of the preparation tube membrane, and let it stand at room temperature for 1 minute. Centrifuge at 12,000×g for 1 minute (heating Eluent or deionized water to 65°C will improve the elution efficiency). The liquid at the bottom of the centrifuge tube is the plasmid. Finally, take 1-2μL and run it for 12 minutes using agarose gel electrophoresis at 120V to observe whether there is a bright band that matches the size of the plasmid. Mark the centrifuge tube containing the plasmid (pET-28a(+)-3α-HSD) and store it in a -20°C refrigerator for use as a PCR template or transformation.

[0051] (2) Primer design

[0052] A semi-rational design method was used to design 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) according to SEQ ID NO.1 and the screened mutation sites. The pET-28a(+)-3α-HSD plasmid was used as a template and the PrimeSTAR MaxPremix (2X) enzyme in the mutation kit of Takara was used for plasmid PCR. A mutation library was constructed by site-directed saturation mutagenesis technology, and mutants that improved 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 part is the mutation site. 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) was as follows: 0.5-20 ng template, 10 μL PrimeSTAR Max Premix (2X), 0.5 μL each of F and R mutation primers, and ddH2O to make up to 20 μL.

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

[0059] After gel verification, the PCR product was digested with endonuclease Dpn I at 37°C for 2 h and transformed into E. coli BL21 (DE3) competent cells. The cells were evenly coated with LB agar plates containing 50 μg / mL kanamycin and cultured in a 37°C incubator overnight until a single clone grew. A single colony was picked and sent to Qingke Biotechnology Co., Ltd. for sequencing to obtain a single mutant library.

[0060] (4) Screening of dominant mutants

[0061] Pick a single clone transformant and inoculate it into 800 μL of LB medium containing kanamycin (50 μg / mL) in a 96-deep-well plate and culture it at 37°C and 180 rpm for 12 h. 600After reaching 0.6-0.8, IPTG with a final concentration of 0.1mmol / L was added to induce expression (18℃, 16h). After the culture was completed, the 96-well deep-well plate was centrifuged at 5000rpm and 4℃ for 10min to collect the bacteria. After removing the supernatant, the bacteria in each well were resuspended with 800μL 2mg / mL lysozyme solution (0.1mM, pH 8.0 Tris-HCl buffer solution) and incubated at 37℃ for 2h to lyse the bacterial cells. Subsequently, the 96-well deep-well plate was centrifuged at 5000rpm and 4℃ for 10min to obtain the supernatant.

[0062] 20mM substrate 11α,17α-dihydroxyprogesterone (the substrate was first dissolved in methanol), 5% methanol by volume, 5mM 2,6-dichlorophenylindolephenol (DCPIP), 15mM phenazine methylsulfate (PMS) and 5μL cell-free crude extract (supernatant) were added to 50mM Tris-HCl (pH 8.0) to form a reaction system with a total volume of 100μL. The color change time from dark blue to light yellow of the original genetically engineered bacteria reaction system was used as a control to screen the culture wells with fast color change time. The enzyme activity was then tested using the method in Example 2 ( Figure 3 ), selected transformants with higher vitality and sent them to a sequencing company for sequencing, and screened out the dominant single mutants 3α-HSD-I89V, whose amino acid sequence is shown in SEQ ID NO.3; 3α-HSD-V185G, whose amino acid sequence is shown in SEQ ID NO.4; and 3α-HSD-T190P (denoted as MK1), whose amino acid sequence is shown in SEQ ID NO.5.

[0063] Combination mutations were performed on the basis of the dominant single mutants, and by comparing the enzyme activities of different mutants, the dominant mutants 3α-HSD-I89V / T190P (denoted as MK2); 3α-HSD-I89V / V185G / T190P (denoted as MK3) were screened out.

[0064] The relative enzyme activities of the original strain, MK1, MK2, and MK3 were shown in Figure 4 Taking 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 using DCPIP method to detect the enzyme activity of 3α-HSD and mutants: 3α-HSD and mutants catalyze the dehydrogenation of 11α,17α-dihydroxyprogesterone to generate (11α)-11,17-dihydroxypregna-1,4-diene-3,20-dione. The 2H removed from the C1,2 position of the substrate will bind to PMS (phenazine dimethyl sulfate) to form PMSH2, which is then immediately transferred to DCPIP to form DCPIPH2, causing DCPIP to be reduced. PMS and DCPIP act as electron acceptors. DCPIP has a characteristic absorption peak at 600nm. By measuring the change in absorbance, the reduction rate of DCPIP can be determined, thereby characterizing the enzyme activity of 3α-HSD and mutants.

[0068] Enzyme activity definition: At 37°C and pH=8.0, the amount of enzyme required to reduce 1 nmol DCPIP per minute is 1 unit

[0069] (U).

[0070] Crude enzyme solution: Take out the original strain and the mutants (including MK1, MK2, and MK3) screened in Example 1 from the -80°C refrigerator and streak them onto LB plate medium containing 50 μg / mL kanamycin (Kan) and culture at 37°C. After the colonies grow, pick a single colony and inoculate it into 5 mL of LB liquid medium containing 50 μg / mL Kan, and culture it at 37°C, 180 rpm for 12 h. The culture solution is transferred to 100 mL of LB liquid medium containing 50 μg / mL Kan at an inoculum concentration of 3% by volume, and culture it at 37°C, 200 rpm for 2-3 h. When the OD 600 When the pH reaches 0.6-0.8, add IPTG to a final concentration of 1mM after cooling the culture flask, and induce the culture at 18°C ​​and 180rpm for 16h. Centrifuge at 8000rpm and 4°C for 10min to collect wet bacterial resting cells. 1.2g of the collected wet bacteria were washed once with 0.9% physiological saline, centrifuged and discarded the supernatant, and finally 10mL of 50mM Tris-HCl (pH8.0) buffer was added to resuspend the bacteria. Ice bath for 30min, ultrasonically disrupted at 0°C (power 60W, total working time 20min, ultrasonic on time 3s, ultrasonic off time 7s), centrifuged at 4°C and 8000rpm for 30 minutes, and the supernatant, i.e., the crude enzyme solution containing the target protein, was collected and stored in a 4°C refrigerator.

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

[0072] Table 2 Enzyme activity assay reaction system

[0073]

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

[0075] 2. Substrate conversion rate detection:

[0076] The samples after 96 hours of conversion reaction were collected in 1.5mL EP tubes, and the substrate and product were extracted with ethyl acetate. During extraction, the EP tube was first shaken with a vortex oscillator for 3 minutes to fully extract the substrate and product into ethyl acetate. After the extraction, centrifuged at 8000rpm for 4 minutes to clarify the ethyl acetate phase. 200μL of supernatant was taken into a 1.5mL EP tube and evaporated in a fume hood. After evaporation, it was re-dissolved with 500μL of acetonitrile. The re-dissolved liquid was filtered with a 0.45μm microporous organic membrane, and 200μL of filtrate was taken into the inner tube of the liquid phase bottle. The peak area of ​​the substrate and product was quantitatively analyzed by high performance liquid chromatography (HPLC), and the substrate conversion rate was calculated according to the standard curve of substrate peak area and concentration.

[0077] HPLC conditions: Agilent 1100 Series LC (G1314 Pump, G1322ADEGASSERG1314VWD detector, 10μLAN injector, HP Chem Station); C18 (5um, 250mmx4.6mm) column; mobile phase: acetonitrile: methanol: water (25:15:60, v / v / v); flow rate: 1mL / min; column temperature: 30℃; detector: UV Detector, wavelength: 240nm. Injection volume: 10μL. Substrate elution time 10.473min, product elution time 12.232min.

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

[0079] Optimum pH: The buffer in the enzyme activity assay reaction system of Example 2 was changed to buffers with a concentration of 50 mM, 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 of mutant MK3 was selected, and the enzyme activity was detected by the method of Example 2. The activity of pH 7.5 Tris-HCl buffer was defined as 100%. The enzyme maintained a relatively high activity in a low acid and alkaline environment; in a strong acid environment, the relative activity of the enzyme was less than 40%. When the reaction pH value was pH 8.0 Tris-HCl buffer, the enzyme activity was the highest, which was 0.5 times higher than that of the original strain (see Figure 5 (A).

[0081] pH stability: After the crude enzyme solution of mutant MK3 was incubated in the above pH 7.0-9.0 buffer at 37°C for 20 min, the relative enzyme activity was detected using the method of Example 2 (see Figure 5 In B), it showed good stability in the pH range of 7.0–9.0 and retained more than 80% of its activity in this range. The original strain showed good pH stability in the pH range of 7.0 to 8.5.

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

[0083] Optimum temperature: Example 2 The crude enzyme solution of the enzyme activity detection reaction system selected the mutant MK3 crude enzyme solution, and the buffer was changed to Tris-HCl buffer (50mM, pH8.0). The enzyme activity was detected at different temperatures (25, 30, 34, 40, 45, 50, 55°C) using the method of Example 2. The results are shown in Table 2. Figure 6 A, then the enzyme activity was tested at 33, 35, 37, 39, 41, 43°C. The results are shown in Figure 6 B. The activity at 35°C was defined as 100%. Figure 6 A and B in the figure show that the optimum temperature for the activity of mutant MK3 is 37°C. When the temperature exceeds 40°C, the activity begins to drop sharply.

[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, and the enzyme activity was detected using the method of Example 2. After incubation at 25-40°C for 0.5 hours, mutant MK3 showed better thermal stability, retaining more than 50% of the residual activity before 40°C ( Figure 6 C). When the original strain was tested under the same conditions, it showed good thermal stability at 25-35℃.

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

[0086] The total volume of the reaction system is 5 mL: the concentration of substrate 11α, 17α-dihydroxyprogesterone is 80 g / L, the volume concentration of the cosolvent is 4%, the final concentration of the mutant MK3 wet bacteria prepared by the method in Example 2 is 50 g / L, and 50 mM Tris-HCl buffer (pH 8.0) buffer is added to 5 mL. DMSO, methanol, ethanol, isopropanol, and acetone are used as cosolvents, respectively, and the reaction is carried out at 37°C and 180 rpm for 96 hours. After the reaction is completed, the reaction solution is centrifuged (8000 rpm, 4°C, 10 min), the reaction supernatant is extracted 3 times with an equal volume of ethyl acetate, and the ethyl acetate phase is combined. 200 μL of the upper layer extract is drawn into a 1.5 mL EP tube, and evaporated in a fume hood. After evaporation, it is redissolved with 500 μL of acetonitrile. The liquid phase method described in Example 2 is used to detect the substrate conversion rate, and the results are shown in Table 3.

[0087] Table 3 Effect of different co-solvents on C1,2 dehydrogenation reaction

[0088]

[0089] Example 6: 3α-HSD engineering bacteria transformation verification

[0090] 1. Wet bacteria

[0091] The original strain and the engineered strain of mutant MK3 selected in Example 1 were picked from the slant and inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C with shaking at 160 rpm for 12 h. The inoculum was transferred to a 250 mL Erlenmeyer flask containing 50 mL of fresh LB liquid medium containing 50 μg / mL kanamycin at a volume concentration of 3%. The initial OD 600 The value was 0.2, and the culture was carried out at 37°C and 180 rpm with shaking for 1.5 to 2 h until the OD 600 When the pH value reached 0.6-0.8, IPTG was added to a final concentration of 1 mM, and the culture was induced on a shaker at 18°C ​​and 180 rpm for 16 h. The culture solution was centrifuged at 4°C and 8000 rpm for 10 min to collect the wet bacteria.

[0092] 2. Conversion reaction

[0093] (1) Substrate concentration

[0094] The 50 mL conversion system in a 250 mL reaction bottle was composed of: 50 g / L of wet cells prepared in step 1, substrate 11α, 17α-dihydroxyprogesterone (60, 70, 80, 90, 100 g / L), 4% DMSO by volume, and 50 mM Tris-HCl buffer solution at pH 8.0 to 50 mL; the bioconversion reaction was carried out at 37° C. and 180 rpm for 96 h, 500 μL of the sample was taken, and two volumes of ethyl acetate were added for extraction, and the substrate conversion rate was detected by the method of Example 2. Under the same conditions, the wet cells of the original strain were used as the 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% in 96 h. When the substrate concentration is greater than 80 g / L, the substrate conversion rate decreases.

[0096] (2) Response time

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

[0098] Results Figure 8 As shown in the figure, the conversion rate of the mutant strain was significantly improved compared with the original strain in every 24-hour sampling. The conversion efficiency of the mutant strain MK3 in the first 48 hours was 4.12 times higher than that of the original strain. When the conversion was 96 hours, the substrate conversion rate of the original strain was only 28.4%, while under the same conditions, the substrate conversion rate 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 to facilitate the understanding and use of the invention by those skilled in the art. Those familiar with the art can easily make various modifications to these embodiments and apply the general principles of this description to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and 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 present invention should be within the scope of protection of the present invention.

Claims

1. A hydroxysteroid dehydrogenase mutant, characterized in that The mutant is obtained by performing single mutation or multiple mutation on the 89th, 185th or 190th position of the amino acid sequence shown in SEQ ID NO.

1.

2. The mutant according to claim 1, characterized in that The mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.1 is mutated into one of the following: (1) the isoleucine at position 89 is mutated into valine; (2) the valine at position 185 is mutated into glycine; (3) the threonine at position 190 is mutated into proline; (4) the threonine at position 190 is mutated into proline and the isoleucine at position 89 is mutated into valine; (5) the threonine at position 190 is mutated into proline and the isoleucine at position 89 is mutated into valine and the valine at position 185 is mutated into glycine.

3. Use of the hydroxysteroid dehydrogenase mutant according to claim 1 in catalyzing the dehydrogenation of steroidal compounds to prepare steroidal drugs.

4. The use according to claim 3, characterized in that The steroidal compound includes 11α,17α-dihydroxyprogesterone.

5. The use according to claim 4, characterized in that The application is: using wet bacteria obtained by fermentation culture of recombinant genetic engineering bacteria containing a gene encoding a hydroxysteroid dehydrogenase mutant or a crude enzyme solution extracted by ultrasonic crushing of wet bacteria as a catalyst, using 11α,17α-dihydroxyprogesterone as a substrate, an organic solvent as a cosolvent, and a pH 6-9 buffer as a reaction medium to form a conversion system, reacting at 25-40°C and 100-300rpm to obtain (11α)-11,17-dihydroxypregnane-1,4-diene-3,20-dione.

6. The use according to claim 5, characterized in that The organic solvent includes dimethyl sulfoxide, methanol, dimethylformamide and ethanol.

7. The use according to claim 5, characterized in that The reaction medium is a 50 mM Tris-HCl buffer solution with a pH of 8.

0.

8. The use according to claim 5, characterized in that In the transformation system, the wet bacteria are added at a concentration of 20-70 g / L; the substrate is added at a concentration of 60-100 g / L; and the organic solvent is added at a concentration of 3-6% by volume.

9. The use according to claim 5, characterized in that The wet bacteria were prepared as follows: the recombinant genetic engineering bacteria containing the hydroxysteroid dehydrogenase mutant encoding gene were inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C, 180 rpm, and shaken for 12 h; the inoculum was transferred to a fresh LB liquid medium containing 50 μg / mL kanamycin at a volume concentration of 3%, and cultured at 37°C, 180 rpm, and shaken until the OD 600 When it reached 0.6-0.8, IPTG was added to a final concentration of 1 mM, and the culture was induced at 18°C ​​and 180 rpm for 16 h. The culture solution was centrifuged at 4°C and 8000 rpm for 10 min to collect the wet bacteria.

10. The use according to claim 5, characterized in that The crude enzyme solution was prepared as follows: the wet bacteria were washed once with 0.9% physiological saline, centrifuged and the supernatant was discarded, pH 8.0, 50 mM Tris-HCl buffer was added to resuspend the bacteria, ice bathed for 30 minutes, ultrasonically disrupted at 0°C and 60W for 20 minutes, working for 3 seconds and taking 7 seconds intervals, centrifuged at 4°C and 8000 rpm for 30 minutes, and the supernatant was collected to obtain the crude enzyme solution.

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