Method for chiral catalytic synthesis of 3-hydroxy-5-hydrogen cholesterol compound by using 3 alpha hydroxysteroid dehydrogenase and isopropanol dehydrogenase

Abstract

The invention provides a method for chiral catalytic synthesis of a 3-hydroxy-5-hydrogen cholesterol compound by using 3 alpha hydroxysteroid dehydrogenase and isopropanol dehydrogenase. The 3 alpha hydroxysteroid dehydrogenase Ct3 alpha-HSD and isopropanol dehydrogenase IPADH-M3 react with a substrate, isopropanol, a coenzyme and a solvent to generate the 3-hydroxy-5-hydrogen cholesterol compound. According to the method, the isopropyl alcohol dehydrogenase is directly utilized to generate the NADH through the isopropyl alcohol and the coenzyme, the pH does not need to be monitored in the whole reaction process, and glucose is not added into a reaction system, so that the reaction process is more economical, simpler and easier to control, and industrial production is facilitated; the isopropanol is used for replacing tert-butyl alcohol, so that the use amount is less, the toxicity is lower, the melting point is low, and the use is more convenient and environment-friendly.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry, and particularly relates to a method for chiral catalytic synthesis of 3-hydroxy-5-hydrocholesterol compounds by using 3α-hydroxysteroid dehydrogenase and isopropanol dehydrogenase. Background Art

[0002] 3-Hydroxy-5-hydrocholesterol compounds have important biological and pharmacological activities and have wide application values in fields such as medicine. For example, brexanolone (3α-hydroxy-5α-pregnan-20-one) is a naturally occurring positive modulator of the γ-aminobutyric acid A receptor and is a progesterone with anesthetic, hypnotic, and sedative effects, and is suitable for the treatment of postpartum depression in adults. Alfaxalone (3α-hydroxy-5α-pregnane-11,20-dione) is also a positive modulator of the γ-aminobutyric acid A receptor and is the raw drug of veterinary anesthetic drugs, and is used as an inducer, injection anesthetic, and sedative in animals. Lithocholic acid (3α-hydroxy-5β-cholestane-24-acid) is a secondary bile acid, which has the effects of inhibiting tumor growth, selectively killing breast cancer cells, and selectively inhibiting the activity of mammalian DNA polymerase, etc., and is also the raw material for the synthesis of lithocholic acid adenosine. Adenosine derivatives are a class of compounds obtained by chemically substituting or modifying the sugar group of natural adenosine, and have physiological activities such as anti-cancer, anti-viral, anti-tumor, sedative, and hypnotic effects. The synthesis methods of 3-hydroxy-5-hydrocholesterol compounds have been widely studied and concerned. At present, it is difficult to achieve efficient synthesis by conventional chemical methods because it is difficult to stereoselectively construct the two chiral carbons of 3-hydroxy (α or β configuration) and 5-hydrogen (α or β configuration) in the steroid skeleton. Utilizing the advantages of high regioselectivity, high stereoselectivity, and mild reaction conditions of enzymes for biocatalysis is a new approach for chiral drug synthesis. Compared with chemical synthesis methods, enzyme catalysis is more economical, efficient, convenient, and environmentally friendly. Based on data mining, screening, and exploration of the molecular mechanism of enzyme activity and stereoselectivity by bioinformatics and artificial intelligence, the innovation of high-performance and precisely regulated carbonyl reductase catalysts provides important assistance for the transformation and upgrading and sustainable development of China's chiral drug manufacturing industry.

[0003] Chunling Zeng et al. reported in 2024 that glucose dehydrogenase BsGDH (derived from Bacillus subtilis) and 3α-hydroxysteroid dehydrogenase Ct3α-HSD (derived from Comamonas testosteroni) expressed by Escherichia coli were used in glucose, β-NAD +, in the tert-butanol system, it can effectively reduce 3-oxo-5-hydrocholesterol to generate 3α-hydroxy-5-hydrocholesterol. Since glucose dehydrogenase BsGDH produces phosphoglucose when using glucose and coenzyme to generate NADH, which causes the pH of the reaction system to decrease, the enzyme activity of 3α-hydroxysteroid dehydrogenase Ct3α-HSD decreases, and the reaction efficiency decreases. Therefore, it is necessary to monitor and adjust the pH during the whole reaction process. In addition, the amount of tert-butanol used is large, exceeding 50% of the system, its toxicity is high, and its melting point is high (23-26 °C), which is easy to crystallize in winter and is inconvenient to use. Summary of the Invention

[0004] In view of this, the present invention provides a method for chiral catalytic synthesis of 3-hydroxy-5-hydrocholesterol compounds using 3α-hydroxysteroid dehydrogenase and isopropanol dehydrogenase. React 3α-hydroxysteroid dehydrogenase Ct3α-HSD and isopropanol dehydrogenase IPADH-M3 with substrates, isopropanol, coenzymes and solvents to generate 3-hydroxy-5-hydrocholesterol compounds. During the reaction process of this method, no acidic substances are produced, the pH of the whole reaction process remains almost unchanged, and industrial production is simpler and easier to control. In addition, glucose is not added to the reaction system, which is more economical. Using isopropanol instead of tert-butanol, the dosage is less, the toxicity is lower, and the melting point is low, making it more convenient to use.

[0005] The technical solution of the present invention is realized as follows:

[0006] In the first aspect, the present invention provides a method for chiral catalytic synthesis of 3-hydroxy-5-hydrocholesterol compounds using 3α-hydroxysteroid dehydrogenase and isopropanol dehydrogenase. React 3α-hydroxysteroid dehydrogenase Ct3α-HSD and isopropanol dehydrogenase IPADH-M3 with substrates, isopropanol, coenzymes and solvents to generate 3-hydroxy-5-hydrocholesterol compounds.

[0007] On the basis of the above technical solutions, further, the 3-hydroxy-5-hydrocholesterol compound is one of brexanolone, alfaxalone and ethyl lithocholate.

[0008] On the basis of the above technical solutions, further, the 3α-hydroxysteroid dehydrogenase Ct3α-HSD is expressed by the Pichia pastoris expression system, and the nucleotide sequence of the 3α-hydroxysteroid dehydrogenase is as shown in SEQ ID NO.1, and the corresponding amino acid sequence is as shown in SEQ ID NO.2.

[0009] SEQ ID NO.1: ATGTCTATCATCGTTATCTCTGGTTGTGCTACTGGTATTGGTGCTGCTACTAGAAAAGTTTTGGAAGCTGCTGGTCATCAAATTGTTGGTATTGATATTAGAGACGCTGAAGTTATTGCTGATTTGTCTACTGCTGAAGGTAGAAAGCAAGCTATTGCTGATGTTTTGGCTAAATGTTCTAAGGGTATGGATGGTTTGGTTTTGTGTGCTGGTTTGGGTCCTCAAACTAAGGTTTTGGGTAACGTTGTTTCTGTTAATTACTTCGGTGCTACTGAATTGATGGATGCTTTCTTGCCTGCTTTGAAAAAGGGTCATCAACCAGCTGCTGTTGTTATTTCTTCTGTTGCTTCTGCTCATTTGGCTTTTGATAAAAACCCTTTGGCTTTGGCTTTGGAAGCTGGTGAAGAAGCTAAGGCTAGAGCTATTGTTGAACATGCTGGTGAACAAGGTGGTAATTTGGCTTATGCTGGTTCTAAGAACGCTTTGACTGTTGCTGTTAGAAAGAGAGCTGCTGCTTGGGGTGAAGCTGGTGTTAGATTGAATACTATTGCTCCTGGTGCTACTGAGACTCCATTGTTGCAAGCTGGTTTGCAAGATCCAAGATATGGTGAATCTATTGCTAAATTCGTTCCACCAATGGGTAGAAGAGCTGAACCATCTGAAATGGCTTCTGTTATTGCTTTCTTGATGTCTCCAGCTGCTTCTTACGTTCATGGTGCTCAAATTGTTATTGATGGTGGTATTGATGCTGTTATGAGACCAACTCAATTCTAG。

[0010] SEQ ID NO.2: MSIIVISGCATGIGAATRKVLEAAGHQIVGIDIRDAEVIADL STAEGRKQAIADVLAKCSKGMDGLVLCAGLGPQTKVLGNVVSVNYFGATELMDAFLPALKKGHQPAAVVISSVASAHLAFDKNPLALALEAGEEAKARAIVEHAGEQGGNLAYAGSKNALTVAVRKRAAAWGEAGVRLNTIAPGATETPLLQAGLQDPRYGESIAKFVPPMGRRAEPSEMASVIAFLMSPAASYVHGAQIVIDGGIDAVMRPTQF。

[0011] On the basis of the above technical solution, further, the isopropanol dehydrogenase IPADH-M3 is expressed by the Pichia pastoris expression system, the nucleotide sequence of the isopropanol dehydrogenase is shown in SEQ ID NO.3, and the corresponding amino acid sequence is shown in SEQ ID NO.4.

[0012] SEQ ID NO.3: ATGAGATTCAAGGATAAGGTTGTTATCGTTACTGGTG GTGCTTCTGGTATTGGTGAAGCTACTGCTAGAGCTTTTATTAGAGAAGGTGCTAAGGTTGTTATTGCTGATTTCTCTGATCATGGTCAACAATTGGCTGATGAATTGGCTGGTGCTCATGAACAAGCTTTGTTTATTAAAACTGACGTTGCTGATACTAGAGCTGTTCAAGCTTTGATTGCTAGAACTGTTGAAAATTATGGTAGATTGGATATCATGTTCGCTAACGCTGGTATTGCTGCTGATGCTCCAATTGATGAATTGGATGAAGCTGCTTGGCAAAAAACTATTGATATTAATCTGACCGGTGTTTATTTGTGTGATAAGTATGCTATTGACCAAATGAGATCCCAAGGTGGTGGTGTTATTGTTAATTGTGGTTCTATTCATTCCCATGTTGGTAAGTCTGGTGTTACTGCTTATGCTGCTGCTAAAGGTGGTGTTAAGTTGTTGACTCAAACTTTGGCTATTGATTATGGTCCTCAAAATATTAGAGTCAACGCTGTTTGTCCAGGTTACATTGATACTCCTTTGTTGAAAACTATCCCAGATGATAAGTACCAAGCTTTGGTTGCTTTGCATCCAATGGGTAGATTGGGTAGAGCTGAAGAAGTTGCTAACGCTGTTTTGTTTTTGGCTTCTGATGAAGCTTCTTTCGTTAACGGTGCTTCTTTGTTGGTTGATGGTGGTTATACTGCTCAATAA。

[0013] SEQ ID NO.4: MRFKDKVVIVTGGASGIGEATARAFIREGAKVVIADFSDHGQQLADELAGAHEQALFIKTDVADTRAVQALIARTVENYGRLDIMFANAGIAADAPIDELDEAAWQKTIDINLTGVYLCDKYAIDQMRSQGGGVIVNCGSIHSHVGKSGVTAYAAAKGGVKLLTQTLAIDYGPQNIRVNAVCPGYIDTPLLKTIPDDKYQALVALHPMGRLGRAEEVANAVLFLASDEASFVNGASLLVDGGYTAQ。

[0014] The isopropanol dehydrogenase IPADH-M3 and 3α-hydroxysteroid dehydrogenase Ct3α-HSD expressed by Pichia pastoris can avoid the risks of phage contamination and bacterial endotoxin during the microbial fermentation stage, ensuring safe use in the food and pharmaceutical fields.

[0015] On the basis of the above technical solution, further, when the 3-hydroxy-5-hydrocholesterol compound is brexanolone, the substrate is 5α-dihydroprogesterone; the reaction system is: 80 - 120 g / L substrate, 0.2 - 0.5 g / L coenzyme, and 10% - 20% isopropanol of the total volume of the reaction system. 100 - 150 U of 3α-hydroxysteroid dehydrogenase and 280 - 330 U of isopropanol dehydrogenase are added per gram of substrate; the reaction conditions are to react at 28 - 33 °C for 2 - 4 h.

[0016] On the basis of the above technical solution, further, when the 3-hydroxy-5-hydrocholesterol compound is alfaxalone, the substrate is 5α-androstane-3,11,20-trione; the reaction system is: 40 - 80 g / L substrate, 0.2 - 0.5 g / L coenzyme, and 10% - 20% isopropanol of the total volume of the reaction system. 220 - 270 U of 3α-hydroxysteroid dehydrogenase and 280 - 330 U of isopropanol dehydrogenase are added per gram of substrate; the reaction conditions are to react at 28 - 33 °C for 5 - 8 h.

[0017] On the basis of the above technical solution, further, when the 3-hydroxy-5-hydrocholesterol compound is ethyl lithocholate, the substrate is ethyl 5β-cholestane-3-one-24-oate; the reaction system is: 80 - 120 g / L substrate, 0.2 - 0.5 g / L coenzyme, and 10% - 20% isopropanol of the total volume of the reaction system. 100 - 150 U of 3α-hydroxysteroid dehydrogenase and 280 - 330 U of isopropanol dehydrogenase are added per gram of substrate; the reaction conditions are to react at 28 - 33 °C for 2 - 4 h.

[0018] On the basis of the above technical solutions, further, the coenzyme is β-NAD + .

[0019] On the basis of the above technical solutions, further, the solvent is a sodium phosphate buffer solution with a concentration of 0.15 - 0.3 M and a pH of 6.3 - 6.7.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The method for chiral catalytic synthesis of 3-hydroxy-5-hydrocholesterol compounds using 3α-hydroxysteroid dehydrogenase and isopropanol dehydrogenase provided by the present invention directly uses isopropanol dehydrogenase to generate NADH from isopropanol and coenzyme. The whole reaction process does not require pH monitoring, and glucose is not added to the reaction system, making the reaction process more economical, simple, easy to control, and suitable for industrial production.

[0022] (2) The method for chiral catalytic synthesis of 3-hydroxy-5-hydrocholesterol compounds using 3α-hydroxysteroid dehydrogenase and isopropanol dehydrogenase provided by the present invention uses isopropanol instead of tert-butanol, with less dosage, lower toxicity, and a low melting point (-89.5 °C), making it more convenient and environmentally friendly to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0024] Figure 1 Schematic diagram of recombinant plasmid Ct3α-HSD-pPICZαA;

[0025] Figure 2 SDS-PAGE electrophoresis diagram of the supernatant of the shake flask expression of the yeast strain of recombinant plasmid Ct3α-HSD-pPICZαA;

[0026] Figure 3 Schematic diagram of recombinant plasmid IPADH-M3--pPICZαA;

[0027] Figure 4 SDS-PAGE electrophoresis diagram of the supernatant of the shake flask expression of the yeast strain of recombinant plasmid IPADH-M3--pPICZαA;

[0028] Figure 5TLC chromatogram of the reaction catalyzed by 3α-hydroxysteroid dehydrogenase Ct3α-HSD in combination with isopropanol dehydrogenase IPADH-M3 using 5α-dihydroprogesterone as a substrate to produce brexanolone;

[0029] Figure 6 High-performance liquid chromatography (HPLC) chromatogram of the substrate 5α-dihydroprogesterone;

[0030] Figure 7 HPLC chromatogram of the reaction product of the reaction catalyzed by 3α-hydroxysteroid dehydrogenase Ct3α-HSD in combination with isopropanol dehydrogenase IPADH-M3 using 5α-dihydroprogesterone as a substrate to produce brexanolone;

[0031] Figure 8 TLC chromatogram of the reaction catalyzed by 3α-hydroxysteroid dehydrogenase Ct3α-HSD in combination with isopropanol dehydrogenase IPADH-M3 using 5α-androstane-3,11,20-trione as a substrate to produce alfaxalone;

[0032] Figure 9 HPLC chromatogram of the substrate 5α-androstane-3,11,20-trione;

[0033] Figure 10 HPLC chromatogram of the reaction product of the reaction catalyzed by 3α-hydroxysteroid dehydrogenase Ct3α-HSD in combination with isopropanol dehydrogenase IPADH-M3 using 5α-androstane-3,11,20-trione as a substrate to produce alfaxalone;

[0034] Figure 11 TLC chromatogram of the reaction catalyzed by 3α-hydroxysteroid dehydrogenase Ct3α-HSD in combination with isopropanol dehydrogenase IPADH-M3 using ethyl 5β-cholestane-3-one-24-carboxylate as a substrate to produce ethyl lithocholate;

[0035] Figure 12 HPLC chromatogram of the substrate ethyl 5β-cholestane-3-one-24-carboxylate;

[0036] Figure 13 HPLC chromatogram of the reaction product of the reaction catalyzed by 3α-hydroxysteroid dehydrogenase Ct3α-HSD in combination with isopropanol dehydrogenase IPADH-M3 using ethyl 5β-cholestane-3-one-24-carboxylate as a substrate to produce ethyl lithocholate. Detailed implementation manners

[0037] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0038] In the following specific embodiments, the experimental materials used, unless otherwise specified, are purchased through conventional channels. The specific experiments refer to the conventional methods and conditions in the art or follow the instructions of the kit product manual.

[0039] In the following specific embodiments, 5α-dihydroprogesterone, 5α-androstane-3,11,20-trione, ethyl 5β-cholestane-3-one-24-carboxylate, etc. are all produced by our company, and the concentration of isopropanol is 95%.

[0040] In the following specific embodiments, the components of the fermenter medium are: H3PO4 9.8 mL / L, CaSO4 0.93 g / L, MgSO4 14.9 g / L, K2SO4 18.2 g / L, (NH4)2SO4 5 g / L, glycerol 40 g / L, KOH 4.13 g / L, PTM1 stock solution 4.4 mL / L.

[0041] The components of the PTM1 stock solution are: MnSO4 2.68 g, CuSO4·5H2O 6 g, NaI 0.08 g, H3BO3 0.02 g, Na2MoO4·2H2O 0.2 g, FeSO4·7H2O 65 g, CoCl2·6H2O 0.5 g, ZnCl2·6H2O 35.88 g, biotin 0.2 g, concentrated sulfuric acid 5 mL, and the volume is fixed to 1 L after dissolving with ultrapure water.

[0042] The components of the glycerol feeding medium are: glycerol 50% (m / v), 12 mL / L PTM1.

[0043] The components of the methanol feeding medium are: 6 L of anhydrous methanol added with 72 mL of PTM1 stock solution.

[0044] The feeding rate of the methanol feeding medium is maintained at 7.2 - 7.8 mL / h / L of the initial fermentation broth.

[0045] The temperature during the cell glycerol growth and glycerol feeding stage is set at 30°C. By controlling the stirring speed at 200 - 800 rpm, the aeration rate at 10 - 20 L / min, and the feeding rate, the dissolved oxygen (DO) in the fermenter is maintained within the range of 20% - 50%, and the pH is about 5.0.

[0046] Example 1: Construction of Yeast Strain with Recombinant Plasmid Ct3α-HSD-pPICZαA and Screening of Flask Expression

[0047] 3α-Hydroxysteroid dehydrogenase is 3α-hydroxysteroid dehydrogenase from Comamonas testosteroni, Ct3α-HSD, which has the nucleotide sequence shown in SEQ ID NO.1, and its corresponding amino acid sequence is SEQ ID NO.2. It was synthesized onto the pPICZ(alpha)A vector by Wuhan Kingcare Bioengineering Co., Ltd. to obtain the recombinant plasmid Ct3α-HSD-pPICZαA (as Figure 1 shown).

[0048] The recombinant plasmid Ct3α-HSD-pPICZαA was transformed into the competent cells of Escherichia coli cloning strain TOP10. The transformed transformants were subjected to plasmid extraction. After the extracted plasmid was linearized by digestion with the restriction endonuclease SacI and recovered by gel extraction, it was electrotransformed into the competent cells of Pichia pastoris X33, and finally spread on a YPD plate containing 200 μg / mL bleomycin and cultured at 28 °C for 72 h.

[0049] Four recombinants were picked and inoculated into Erlenmeyer flasks containing 15 mL of BMGY medium respectively, and cultured at 28 °C and 220 rpm for 24 h. Then it was changed to BMMY medium, and 1% volume of methanol was added every 24 h for induction. After 2 days of induction, the supernatant was collected by centrifugation for SDS-PAGE detection, and the results are as Figure 2 shown.

[0050] Figure 2 In which M represents the protein marker band, and 1, 2, 3, 4 represent the supernatants of flask expression of 4 different recombinants Ct3α-HSD-pPICZαAX33. It can be seen that all 4 single colonies have expression, and the expression levels of strains 2 and 4 are slightly higher. Strains 2 and 4 expressing strains were preserved, and strain 2 was selected for fermentation tank fermentation culture later.

[0051] Example 2: Preparation of 3α-Hydroxysteroid Dehydrogenase Enzyme Solution

[0052] The recombinant Pichia pastoris Ct3α-HSD-pPICZαAX33 was placed in a 15 L fermenter for conventional yeast fermentation, and the specific steps are as follows:

[0053] An appropriate amount of Pichia pastoris engineering bacteria on the plate was inoculated into a medium containing 250 mL of liquid YPD and cultured in a constant temperature shaker at 28 °C and 220 rpm for 24 h to activate the bacteria.

[0054] The seed culture was added to the fermenter at an inoculum size of 5% (v / v). The temperature during the glycerol growth and glycerol feeding stages of the cells was set at 30 °C. By controlling the stirring speed, aeration rate, and feeding rate, the dissolved oxygen (DO) in the fermenter was maintained within the range of 20% - 50%, and the pH was approximately 5.0. When the DO continued to rise, it indicated that the glycerol in the medium had been exhausted by the cells. At this time, glycerol feeding medium was added. When the wet weight of the cell strain reached about 200 g / L, the addition of glycerol was stopped and the cells were starved for about 1 h. When the DO in the fermenter rose to the maximum again, methanol feeding medium was added to induce protein expression. The temperature during the induction stage was set at 28 °C, and ammonia water was automatically added to maintain the pH of the medium at approximately 6.0. After 5 days of induction, the fermentation broth was taken out of the tank, and the fermentation supernatant was collected. After filtration through a 0.2-μm membrane, protein concentration was carried out using a 10-kDa membrane package, and protease activity was measured.

[0055] The detection protocol for Ct3α-HSD enzyme activity was as follows: Take 1.9 mL of sodium phosphate buffer with a pH of 6.5 and a concentration of 0.2 M, 0.3 mL of 200 mM COBE (ethyl 4-chloroacetoacetate), and 0.5 mL of 3α-hydroxysteroid dehydrogenase enzyme dilution solution in a quartz cuvette, mix well, place it in a constant-temperature spectrophotometer at 37 °C, zero the absorbance at 340 nm, add 0.3 mL of 0.15 mM NADH, mix well, and start reading. Record the change in absorbance within 3 min to obtain the initial velocity, and calculate the enzyme activity according to the enzyme activity formula.

[0056] Definition of enzyme activity: Under the conditions of 37 °C and pH 6.5, the amount of enzyme required to consume micromoles of NADH per minute is 1 enzyme activity unit (U).

[0057] E (U / mL) = ΔA × V1 × n ÷ (6.22 × d × V2)

[0058] Where: △A is the difference in absorbance values per minute within the initial velocity range, min -1 , △A = 60*K (K: slope of the absorbance change curve); V1 is the total reaction volume, 3 mL; n is the enzyme solution dilution factor; 6.22 is the extinction coefficient of NADH, mmol / (L·cm); d is the diameter of the cuvette, 1 cm; V2 is the volume of enzyme solution added, 0.5 mL.

[0059] When n = 600, K = 0.00638,

[0060] E = 60 × 0.00638 × 3 × 600 ÷ (6.22 × 1 × 0.5) = 221.56 U / mL.

[0061] Example 3: Construction of yeast strain with recombinant plasmid IPADH-M3--pPICZαA and screening of flask expression

[0062] Isopropanol dehydrogenase IPADH-M3 is an isopropanol dehydrogenase derived from Brucella suis via. IPADH-M3 has the nucleotide sequence shown in SEQ ID NO.3, and its corresponding amino acid sequence is SEQ ID NO.4. It was synthesized by Wuhan Kingcare Bioengineering Co., Ltd. onto the pPICZ(alpha)A vector to obtain the recombinant plasmid IPADH-M3-pPICZαA (as Figure 3 shown).

[0063] The recombinant plasmid IPADH-M3-pPICZαA was transformed into competent cells of Escherichia coli cloning strain TOP10. The transformants were subjected to plasmid extraction. The extracted plasmid was linearized by digestion with the restriction enzyme SacI and then recovered by gel electrophoresis. It was electrotransformed into competent cells of Pichia pastoris X33. Finally, it was spread on a YPD plate containing 200 μg / mL bleomycin and cultured at 28 °C for 72 h. Eight recombinants were picked and inoculated into Erlenmeyer flasks containing 15 mL of BMGY medium and cultured at 28 °C and 220 rpm for 24 h. Then it was changed to BMMY medium, and 1% (v / v) methanol was added every 24 h for induction. After 2 days of induction, the supernatant was obtained by centrifugation and subjected to SDS-PAGE detection. The results are as Figure 4 shown.

[0064] Figure 4 In the figure, M represents the protein marker band, and 1, 2, 3, 4, 5, 6, 7, 8 represent the culture supernatants of 8 different recombinants IPADH-M3--pPICZαA X33 in shake flasks. It can be seen that recombinants 1, 3, and 4 have expression, and the expression level of strain 3 is slightly higher. Strains 1, 3, and 4 with expression were preserved, and strain 3 was selected for fermentation culture in a fermenter later.

[0065] Example 4: Preparation of isopropanol dehydrogenase IPADH-M3 enzyme solution

[0066] The recombinant Pichia pastoris IPADH-M3-pPICZαA X33 was placed in a 15 L fermenter for conventional yeast fermentation. The specific steps are as follows:

[0067] An appropriate amount of Pichia pastoris engineering bacteria on the plate was inoculated into a medium containing 250 mL of liquid YPD and cultured in a constant temperature shaker at 28 °C and 220 rpm for 24 h to activate the bacteria.

[0068] The temperature during the growth of the bacterial cells in glycerol and the glycerol feeding stage was set at 30 °C. By controlling the stirring speed, aeration rate, and feeding rate, the dissolved oxygen (DO) in the tank was maintained within the range of 20% - 50%, and the pH was approximately 5.0. When the DO continued to rise, it indicated that the glycerol in the culture medium had been depleted by the cells. At this time, glycerol feeding medium was added. When the wet weight of the cell strain reached about 200 g / L, the addition of glycerol was stopped and the cells were starved for about 1 h. When the DO in the tank rose to the maximum value again, methanol feeding medium was added to induce protein expression. The temperature during the induction stage was set at 28 °C, and ammonia water was automatically added to maintain the pH of the culture medium at approximately 6.0. After 5 days of induction, the fermentation broth was taken out of the tank, and the fermentation supernatant was collected. After filtration through a 0.2 μm membrane, protein concentration was carried out using a 10 kDa membrane package, and protease activity was measured.

[0069] The detection protocol for IPADH - M3 enzyme activity was as follows: Take 2.7 mL of potassium phosphate buffer with a concentration of 0.1 M and a pH of 8.0, 0.1 mL of 10 mg / mL β - NAD + and 0.1 mL of isopropanol in a quartz cuvette, mix well, place it in a constant - temperature spectrophotometer at 37 °C, zero the absorbance at 340 nm, add 0.1 mL of IPADH - M3 enzyme dilution, mix well, and start reading. Record the change in absorbance within 3 min to obtain the initial velocity, and calculate the enzyme activity according to the enzyme activity formula.

[0070] Definition of enzyme activity: Under the conditions of 37 °C and a pH of 8.0, the amount of enzyme required to catalyze the production of micromoles of NADH per minute is defined as 1 enzyme activity unit (U).

[0071] E (U / mL) = ΔA × V1 × n ÷ (6.22 × d × V2)

[0072] Where: △A is the difference in absorbance values per minute within the initial velocity range, min -1 , △A = 60 * K (K: slope of the absorbance change curve); V1 is the total reaction volume, 3 mL; n is the enzyme solution dilution factor; 6.22 is the extinction coefficient of NADH, mmol / (L·cm); d is the diameter of the cuvette, 1 cm; V2 is the volume of the enzyme solution added, 0.01 mL.

[0073] When n = 200 and k = 0.00158,

[0074] E = 60 × 0.00158 × 3 × 200 ÷ (6.22 × 1 × 0.01) = 914.47 U / mL.

[0075] Example 5: Ct3α - HSD was combined with isopropanol dehydrogenase IPADH - M3, and 5α - dihydroprogesterone was used as a substrate to catalyze the reaction to produce brexanolone

[0076] According to the reaction system of 5α-dihydroprogesterone (substrate) at 100 g / L, coenzyme β-NAD at 0.4 g / L, sodium phosphate buffer at pH 6.5 with a concentration of 0.2 M, and isopropanol at 20% of the total volume of the reaction system, 130 U of Ct3α-HSD and 300 U of IPADH-M are added per gram of substrate for enzymatic reaction. The reaction process is continuously stirred and maintained at a temperature of 30 °C for 2.5 h. + 、pH6.5 concentration of 0.2M sodium phosphate buffer and 20% isopropanol of the total volume of the reaction system, 130U of Ct3α-HSD and 3300U of IPADH-M are added per gram of substrate for enzymatic reaction. The reaction process is continuously stirred and maintained at a temperature of 30 °C for 2.5 h.

[0077] Structural formula of 5α-dihydroprogesterone:

[0078] Structural formula of brexanolone:

[0079] Sampling for TLC chromatography (developer: petroleum ether: ethyl acetate = 2:1), vanillin coloring. When there is no substrate spot, the reaction is complete and the reaction is terminated. The results are as Figure 5 shown, Figure 6 and Figure 7 respectively represent the results of HPLC analysis of the substrate and the reaction product. It shows that Ct3α-HSD catalyzes 5α-dihydroprogesterone by combining with isopropanol dehydrogenase IPADH-M3, with a conversion rate > 99% and de > 99%. The reaction product is relatively single, facilitating the separation and purification of the subsequent product.

[0080] Example 6: Ct3α-HSD catalyzes the reaction to generate alfaxalone using 5α-androstane-3,11,20-trione as a substrate by combining with isopropanol dehydrogenase IPADH-M3

[0081] According to the reaction system of 5α-androstane-3,11,20-trione (substrate) at 60 g / L, coenzyme β-NAD at 0.3 g / L, + 、pH6.5 concentration of 0.2M sodium phosphate buffer and 20% isopropanol of the total volume of the reaction system, 250U of Ct3α-HSD and 3300U of IPADH-M are added per gram of substrate for enzymatic reaction. The reaction process is continuously stirred and maintained at a temperature of 30 °C for 6 h.

[0082] Structural formula of 5α-androstane-3,11,20-trione:

[0083] Structural formula of alfaxalone:

[0084] Sampling for TLC chromatography (developer: petroleum ether: ethyl acetate = 2:1), vanillin coloring. When there is no substrate spot, the reaction is complete and the reaction is terminated. The results are as Figure 8 shown, Figure 9 and Figure 10They respectively represent the results of HPLC analysis of the substrate and the reaction product. It shows that Ct3α-HSD catalyzes 5α-androstane-3,11,20-trione in combination with isopropanol dehydrogenase IPADH-M3, with a conversion rate > 99% and de > 99%. The reaction product is relatively single, facilitating the separation and purification of the subsequent product.

[0085] Example 7: Ct3α-HSD catalyzes the reaction to produce ethyl lithocholate using ethyl 5β-cholestane-3-one-24-carboxylate as the substrate in combination with isopropanol dehydrogenase IPADH-M3

[0086] In a reaction system of 100 g / L of ethyl 5β-cholestane-3-one-24-carboxylate (substrate), 0.4 g / L of coenzyme β-NAD+, a sodium phosphate buffer solution with a pH of 6.5 and a concentration of 0.2 M, and 20% isopropanol in the total volume of the reaction system, 130 U of Ct3α-HSD and 300 U of IPADH-M3 are added per gram of substrate for the enzyme solution reaction. The reaction process is continuously stirred and maintained at a temperature of 30 °C for 2.5 h.

[0087] Sampling for TLC chromatography (developer: petroleum ether: ethyl acetate = 2:1), coloring with vanillin. When there is no substrate spot, the reaction is complete and the reaction is terminated. The results are as Figure 11 shown, Figure 12 and Figure 13 They respectively represent the results of HPLC analysis of the substrate and the reaction product. It shows that Ct3α-HSD catalyzes ethyl 5β-cholestane-3-one-24-carboxylate in combination with isopropanol dehydrogenase IPADH-M3, with a conversion rate > 99% and de > 99%. The reaction product is relatively single, facilitating the separation and purification of the subsequent product.

[0088] Structural formula of ethyl 5β-cholestane-3-one-24-carboxylate:

[0089]

[0090] Structural formula of ethyl lithocholate:

[0091]

[0092] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for chiral catalytic synthesis of 3-hydroxy-5-hydrocholesterol compounds using 3α-hydroxysteroid dehydrogenase and isopropanol dehydrogenase, characterized in that, React 3α-hydroxysteroid dehydrogenase Ct3α-HSD and isopropanol dehydrogenase IPADH-M3 with a substrate, isopropanol, coenzyme, and solvent to produce 3-hydroxy-5-hydrocholestane compounds.

2. The method for chiral catalytic synthesis of 3-hydroxy-5-hydrocholesterol compounds using 3α-hydroxysteroid dehydrogenase and isopropanol dehydrogenase according to claim 1, characterized in that, The 3-hydroxy-5-hydrocholestane compound is one of brexanolone, alfaxalone, and ethyl lithocholate.

3. The method for chiral catalytic synthesis of 3-hydroxy-5-hydrocholesterol compounds using 3α-hydroxysteroid dehydrogenase and isopropanol dehydrogenase according to claim 1, characterized in that, The 3α-hydroxysteroid dehydrogenase Ct3α-HSD is expressed by the Pichia pastoris expression system. The nucleotide sequence of the 3α-hydroxysteroid dehydrogenase is shown in SEQ ID NO.1, and the corresponding amino acid sequence is shown in SEQ ID NO.

2.

4. The method for chiral catalytic synthesis of 3-hydroxy-5-hydro cholesteroid compounds using 3α-hydroxysteroid dehydrogenase and isopropanol dehydrogenase according to claim 1, characterized in that, The isopropanol dehydrogenase IPADH-M3 is expressed by the Pichia pastoris expression system. The nucleotide sequence of the isopropanol dehydrogenase is shown in SEQ ID NO.3, and the corresponding amino acid sequence is shown in SEQ ID NO.

4.

5. The method for chiral catalytic synthesis of 3-hydroxy-5-hydrocholesterol compounds by using 3α-hydroxysteroid dehydrogenase and isopropanol dehydrogenase according to claim 2, characterized in that, When the 3-hydroxy-5-hydrocholestane compound is brexanolone, the substrate is 5α-dihydroprogesterone; the reaction system is: 80 - 120 g / L substrate, 0.2 - 0.5 g / L coenzyme, and 10% - 20% isopropanol of the total volume of the reaction system. Add 100 - 150 U of 3α-hydroxysteroid dehydrogenase and 280 - 330 U of isopropanol dehydrogenase per gram of substrate; the reaction conditions are to react at 28 - 33 °C for 2 - 4 h.

6. The method for chiral catalytic synthesis of 3-hydroxy-5-hydrocholesterol compounds using 3α-hydroxysteroid dehydrogenase and isopropanol dehydrogenase according to claim 2, characterized in that, When the 3-hydroxy-5-hydrocholestane compound is alfaxalone, the substrate is 5α-androstane-3,11,20-trione; the reaction system is: 40 - 80 g / L substrate, 0.2 - 0.5 g / L coenzyme, and 10% - 20% isopropanol of the total volume of the reaction system. Add 220 - 270 U of 3α-hydroxysteroid dehydrogenase and 280 - 330 U of isopropanol dehydrogenase per gram of substrate; the reaction conditions are to react at 28 - 33 °C for 5 - 8 h.

7. The method for chiral catalytic synthesis of 3-hydroxy-5-hydro cholestane compounds using 3α-hydroxysteroid dehydrogenase and isopropanol dehydrogenase according to claim 2, wherein When the 3-hydroxy-5-hydrocholestane compound is ethyl lithocholate, the substrate is ethyl 5β-cholestan-3-one-24-oate; the reaction system is: 80 - 120 g / L substrate, 0.2 - 0.5 g / L coenzyme, and 10% - 20% isopropanol of the total volume of the reaction system. Add 100 - 150 U of 3α-hydroxysteroid dehydrogenase and 280 - 330 U of isopropanol dehydrogenase per gram of substrate; the reaction conditions are to react at 28 - 33 °C for 2 - 4 h.

8. The method for chiral catalytic synthesis of 3-hydroxy-5-hydrocholesterol compounds using 3α-hydroxysteroid dehydrogenase and isopropanol dehydrogenase according to claim 1, characterized in that, The coenzyme is β-NAD + .

9. The method for chiral catalytic synthesis of 3-hydroxy-5-hydrocholesterol compounds by using 3α-hydroxysteroid dehydrogenase and isopropanol dehydrogenase according to claim 1, wherein The solvent is a sodium phosphate buffer solution with a concentration of 0.15 - 0.3 M and a pH of 6.3 - 6.7.

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