Preparation method of pregnenolone

Abstract

The invention provides a preparation method of pregnenolone, and relates to the technical field of biological pharmacy and biochemical engineering. Cheap and easily available steroid progesterone is used as a raw material, 3 # site esterification is carried out through a chemical method, then a catalytic reaction of hydrolase and ketoreductase is carried out, and finally a target product is obtained with the yield as high as 91%. The process has no loss except conversion, and the intermediate is relatively stable, so that the reaction product is relatively high in yield and good in quality, and is suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the technical fields of biopharmaceuticals and biochemical engineering, and in particular to a preparation method of pregnenolone. Background Art

[0002] Pregnenolone is an important endogenous steroid hormone in humans and an inactive precursor neurosteroid compound. It is highly effective in treating bipolar disorder, gliomas, schizophrenia, and has analgesic and anesthetic effects, anti-epileptic and anxiolytic properties, memory enhancement, and anti-inflammatory properties. Although most research on neurosteroid activity remains at the preclinical stage, and concentration levels in the human body exhibit significant gender and individual differences, finding efficient production methods remains crucial.

[0003] Currently, the main methods for preparing pregnenolone include biofermentation and chemical synthesis. However, these methods generally suffer from drawbacks such as lengthy reaction steps, harsh reaction conditions, and environmental concerns. For example, Chinese patent CN115786152B discloses a method for preparing pregnenolone from cholesterol. This method involves a three-step, sequential catalytic degradation of the cholesterol side chain by cholesterol side-chain cleavage enzyme (P450scc), involving hydroxylation at C22, followed by hydroxylation at C20 to produce 20R and 22R hydroxycholesterol, followed by further cleavage between C20 and C22 to produce pregnenolone. However, this method suffers from a long reaction process and low P450scc catalytic efficiency. For example, reference DOI: 10.3184 / 174751917X14955339414758 reports a chemical method for synthesizing pregnenolone. Its core mechanism involves desilication and simultaneous olefin isomerization over a novel catalyst, ultimately yielding pregnenolone. The single-step conversion is 97% and the reaction time is 3 hours. However, due to the inability to control chirality, the method requires the introduction of chirality at the tertiary position into the raw materials, making it suitable only for research and not for production. Therefore, a highly efficient, mild, and suitable for large-scale industrial production of pregnenolone is urgently needed. Hydrolases, a class of enzymes that catalyze the reaction of substrates with water to achieve hydrolysis, are commonly used in fields such as chiral drug synthesis and prodrug hydrolysis, but their direct application in the preparation of pregnenolone has not been reported. Summary of the Invention

[0004] In view of this, the present invention proposes a method for preparing pregnenolone to solve the technical problems of low reaction efficiency, harsh conditions and unsuitability for large-scale production in the preparation of pregnenolone.

[0005] The technical solution of the present invention is achieved as follows: The present invention provides a method for preparing pregnenolone. The preparation process includes a hydrolase catalysis step, wherein the amino acid sequence of the hydrolase is shown in SEQ ID NO: 1: SPVAVEKRQIFGPGDNDLRDGPCKDITFIFARGSTEPGLMGITVGPDTCDELNEFRGRVACQGVGPRYEASLAGNFLPRGTTQAAIDEAAELFNLAHTKCPNTQIVGGGYSQGAAVMHGAIPGLSNAVKDQIKGVVLYGDTRNEQDGGRIPNFPTDKTNIICNPGDLVCDGTLILTAAHFTYGTRVRGAVDWLEDRLS.

[0006] The structural formula of pregnenolone of the present invention is as follows:

[0007] Based on the above scheme, preferably, the preparation method comprises the following steps: S1, preparing progesterone into progesterone ester; S2, adding the above-mentioned hydrolase and ketoreductase to the progesterone ester in step S1, performing enzymatic catalysis to prepare pregnenolone, and then purifying it.

[0008] Reaction formula of the present invention is as follows:

[0009] Based on the above scheme, preferably, in step S2, the mass volume ratio of the progesterone ester: hydrolase: ketoreductase is (4-5) g: (3-6) mL: (3.5-18.5) mL.

[0010] Based on the above scheme, preferably, in step S1, the prepared raw materials include progesterone, acetic anhydride and p-toluenesulfonic acid.

[0011] Based on the above scheme, preferably, the progesterone ester is progesterone acetate.

[0012] Based on the above scheme, it is further preferred that, under the condition of 20-30°C, 200 g of progesterone and 400 mL of acetic anhydride are added in sequence to a 3 L three-necked flask equipped with a thermometer and a stirrer, stirring is started and the air is replaced with nitrogen, and then 2 g of p-toluenesulfonic acid is added, and the temperature is raised to 30±2°C and stirred for 10-12 hours, until TLC shows that the raw materials are completely reacted.

[0013] Based on the above scheme, preferably, in step S2, the raw materials for enzyme catalysis further contain nicotinamide adenine dinucleotide, glucose and glucose dehydrogenase.

[0014] Based on the above scheme, preferably, in step S2, the enzyme-catalyzed solvent comprises tert-butanol and PBS buffer.

[0015] Based on the above scheme, preferably, in step S2, the pH of the enzyme catalysis is 6.0-7.0.

[0016] Based on the above scheme, preferably, in step S2, the purified raw material contains ethyl acetate or methanol.

[0017] Based on the above scheme, a further preferred method is to weigh 24 g of the substrate progesterone acetate and place it in a three-necked flask. 18.8 g of D(+)-anhydrous glucose, 200 mL of tert-butanol, and 200 mL of 0.2 M PBS solution are added, and the mixture is stirred in a 30°C water bath for 1 hour. Nicotinamide adenine dinucleotide (NAD) 0.6 g, hydrolase 18.48 mL, reductase 22.08 mL, and glucose dehydrogenase (GDH) 7.92 mL are also added. The pH is stabilized at 6.5, and the reaction is continued for 18 hours. TLC indicates complete conversion, thereby obtaining pregnenolone.

[0018] Based on the above scheme, preferably, the nucleotide sequence of the hydrolase is shown in SEQ ID NO: 2:.

[0019] The preparation method of pregnenolone of the present invention has the following beneficial effects compared with the prior art: (1) Different from other hydrolases, the hydrolase of the present invention can specifically recognize progesterone acetate and hydrolyze its ester group to generate the corresponding intermediate, providing a new route for the efficient synthesis of pregnenolone; (2) The enol intermediate formed by the progesterone esterification under the action of the hydrolase is very stable. With the participation of the ketoreductase in the reaction, it can selectively asymmetric reduce the keto carbonyl at position 3 to obtain a new β-chiral product. This intermediate is rapidly reduced to pregnenolone. As the intermediate decreases, the hydrolase reaction activity returns to its highest speed, further accelerating the formation of the intermediate. Under the alternating reaction of the two main enzymes, the content of the intermediate in the system is very low, making the greatest use of the relative stability of the intermediate, reducing the formation of oxidative impurities, and thus improving the yield and purity of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 Schematic diagram of the recombinant plasmid Cu-Mc-pPICZaA of the present invention; Figure 2 1 is an SDS-PAGE image of the hydrolase solution of the present invention, wherein 1, 2, 3, and 4 are expression supernatant samples after induction for 1, 2, 3, and 4 days, respectively, and M is a protein marker band; Figure 3 is a high-performance liquid chromatography chromatogram of the progesterone ester compound of the present invention; Figure 4 is the H NMR spectrum of the progesterone ester of the present invention; Figure 5 is a high-phase liquid chromatogram of the hydrolysis intermediate of the present invention; Figure 6 is a high-phase liquid chromatogram of pregnenolone of the present invention; Figure 7 It is the nuclear magnetic hydrogen spectrum of pregnenolone of the present invention. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0023] All the reagents used in the present invention can be purchased from the market by conventional means. The purchase sources and product numbers of the main reagents are shown in Table 1.

[0024] Table 1 Purchase sources and product numbers of reagents

[0025] Example 1 Construction of yeast strain carrying recombinant plasmid Cu-Mc-pPICZaA and preparation of hydrolytic enzyme solution Hydrolases Malbranchea cinnamomeaThe Cu-Mc-pPICZaA hydrolase from the source has an amino acid sequence as shown in SEQ ID NO: 1, and the corresponding nucleotide sequence encoding the amino acid is shown in SEQ ID NO: 2. It is synthesized into the pPICZaA vector to obtain the recombinant plasmid Cu-Mc-pPICZaA, as shown in FIG. Figure 1 shown.

[0026] The recombinant plasmid Cu-Mc-pPICZaA was transformed into competent cells of Escherichia coli cloning strain TOP10, and the transformed transformants were subjected to plasmid extraction. The extracted plasmid was linearized with the restriction endonuclease SacI, recovered by gel electroporation, and transformed into Pichia pastoris X33 competent cells. Finally, it was spread on YPD plates containing 200 μg / mL bleomycin and cultured at 28°C for 72 h. The positive clones were selected for fermentation in a fermentor.

[0027] The specific steps are as follows: An appropriate amount of Pichia pastoris engineered bacteria on the plate was inoculated into 250 mL of liquid YPD culture medium and cultured in a constant temperature shaker at 28°C and 220 rpm for 24 h to activate the bacteria.

[0028] The seed solution was added to the fermentor at a 5% (v / v) inoculum. The temperature during the glycerol growth and glycerol feeding phases was set at 30°C. The dissolved oxygen (DO) in the fermentor was maintained between 20% and 50% by controlling the stirring speed at 200-800 rpm, the aeration volume at 10-20 L / min, and the feed rate. The pH was approximately 5.0. When the DO continued to rise, the glycerol in the culture medium was depleted by the cells. At this time, glycerol feed medium was added. When the wet weight of the cells reached approximately 200 g / L, glycerol addition was stopped and the cells were starved for approximately 1 hour. When the DO in the fermentor reached its maximum value again, methanol feed medium was added to induce protein expression. The temperature during the induction phase was set at 28°C. Ammonia was automatically added to maintain the culture pH at approximately 6.0. After 4 days of induction, the fermentation supernatant was collected to obtain the hydrolase solution (fermentation broth protein concentration: 0.45 g / L-0.70 g / L). The SDS-PAGE electrophoresis of the hydrolase is shown in the figure. Figure 2 As shown in the figure, 1, 2, 3, and 4 are expression supernatant samples after induction for 1, 2, 3, and 4 days, respectively, and M is a protein marker band.

[0029] The specific components of the fermentation tank culture 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, and PTM1 stock solution 4.4 mL / L.

[0030] The composition of PTM1 stock solution is: 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, dissolved in ultrapure water and diluted to 1 L.

[0031] The composition of the glycerol feed medium is: 50% (m / v) glycerol, 12 mL / L PTM1.

[0032] The composition of methanol feed medium is as follows: 6 L of anhydrous methanol is added to 72 mL of PTM1 stock solution.

[0033] The feeding rate of methanol feed medium was maintained at 7.2~7.8 mL / h / L starting fermentation broth.

[0034] Example 2 Preparation and Detection of Progesterone Acetate Preparation of progesterone acetate: To a 3 L three-necked flask equipped with a thermometer and a stirrer at room temperature (20-30°C), add 200 g of progesterone and 400 mL of acetic anhydride, sequentially. Stirring is initiated and the air is replaced with nitrogen. Then, 2 g of p-toluenesulfonic acid is added. The temperature is raised to 30 ± 2°C and stirred for 10-12 hours (the reaction may involve dissolution followed by solid precipitation) until TLC indicates complete reaction of the starting material (developing solvent: PE / EA = 3 / 1, vanillin for color development). Sampling method: Add an appropriate amount of the reaction solution to 0.5 mL of water, extract with 0.5 mL of ethyl acetate, and collect the organic phase for testing. Cool the reaction solution to 0-5°C while rapidly stirring. Add 2000 mL of ice water, previously cooled to 0-5°C, to the reaction system. Maintain stirring at 0-5°C for 0.5-1 hour, filter until almost no mother liquor drips. Slurry the filter cake with 500 mL of water at room temperature (20-30°C) for 0.5 hour, filter, and wash with water until neutral and drain. Spread the material on a plate and air-dry at 45-50°C for 12 hours to obtain the esterified product as a white powdery solid with a weight yield of 108-113% (molar yield of 95-100%) and a purity of ≥98%.

[0035] The prepared progesterone acetate was detected using a high performance liquid chromatography analysis method, and the specific detection conditions were as follows: Liquid phase: Agilent, chromatographic column: ZORBAX ODS (4.6×250 mm, 5-Micron), elution conditions: acetonitrile: sodium dihydrogen phosphate aqueous solution (0.78 g / L, phosphoric acid adjusted to pH 3): methanol volume ratio = 30:37:40, flow rate: 0.8 ml / min, injection volume: 20 μL, detector: differential index detector (RID), column oven temperature: 35°C; the HPLC chromatogram of progesterone acetate is shown below. Figure 3 The separated progesterone acetate fraction was measured on a conventional nuclear magnetic resonance spectrometer, and the nuclear magnetic hydrogen spectrum was as shown in FIG. Figure 4 shown.

[0036] Example 3 Detection of double bond transfer intermediates Preparation of the double bond transfer intermediate: Weigh 20 g of the substrate progesterone ester into a 500 mL three-necked flask. Add 200 mL of tert-butyl alcohol and 200 mL of 0.2 M PBS solution (120 mL of 0.2 M sodium dihydrogen phosphate solution + 80 mL of 0.2 M sodium hydrogen phosphate solution). Stir in a 30°C water bath for 1 hour. Add 18.48 mL of hydrolase, stabilize the pH at 6.5, and react for 3 hours. The results showed that TLC showed a conversion of 60%-70% (developing solvent: PE / EA = 4 / 1, vanillin color development; chromatographic sampling method: take 100 μl of the reaction solution, add 900 μl of acetonitrile, mix well and then detect); The prepared double bond transfer intermediate was detected by high performance liquid chromatography analysis. The specific detection conditions were the same as those in Example 2. The high performance liquid chromatography of the double bond transfer intermediate was as follows: Figure 5 shown.

[0037] Example 4 Preparation, Purification and Detection of Pregnenolone Weigh 24 g of the substrate, lutein acetate, into a 500 mL three-necked flask. Add 18.8 g of D(+)-anhydrous glucose, 200 mL of tert-butanol, and 200 mL of 0.2 M PBS (120 mL of 0.2 M sodium dihydrogen phosphate solution + 80 mL of 0.2 M sodium dihydrogen phosphate solution). Stir in a 30°C water bath for 1 h. Add 0.6 g of NAD (nicotinamide adenine dinucleotide), 18.48 mL of hydrolase, 22.08 mL of reductase, and 7.92 mL of GDH enzyme. Maintain the pH at 6.5. React for 18 h until complete conversion is indicated by TLC (developing solvent: PE / EA = 4 / 1, vanillin as colorimetric visualization; chromatographic sampling: aspirate 100 μl of the reaction solution, add 900 μl of acetonitrile, mix well, and analyze. After the reaction is complete, tert-butanol is removed by rotary evaporation, and the remaining aqueous phase is filtered to collect the filter cake. 10 times the amount of ethyl acetate is added, stirred and refluxed at 80°C, and the filtrate is filtered and collected. The filter cake is again stirred and refluxed with 4 times the amount of ethyl acetate at 80°C, and the filtrate is filtered and collected. The two filtrates are combined, the ethyl acetate is dried in a rotary evaporation oven at 60°C overnight, and 21.74 g of crude extract is obtained; the crude extract is slurried with 3 times the amount of ethyl acetate to obtain 21.84 g, with a purity of 99% and a yield of 91%. The product is a yellow powder.

[0038] The prepared pregnenolone was detected by high performance liquid chromatography. The detection conditions of high performance liquid chromatography were the same as those in Example 2. The high performance liquid chromatogram of pregnenolone was as follows: Figure 6 As shown, the separated pregnenolone fraction was measured on a conventional nuclear magnetic resonance spectrometer, and the nuclear magnetic hydrogen spectrum was as follows Figure 7 shown.

[0039] Example 5 Preparation and purification of pregnenolone Weigh 20 g of the substrate, progesterone acetate, into a 500 mL three-necked flask. Add 18.8 g of D(+)-anhydrous glucose, 200 mL of tert-butyl alcohol, and 200 mL of 0.2 M PBS (120 mL of 0.2 M sodium dihydrogen phosphate solution + 80 mL of 0.2 M sodium dihydrogen phosphate solution). Stir in a 30°C water bath for 1 h. Add 0.6 g of NAD (nicotinamide adenine dinucleotide), 24 mL of hydrolase, 73.2 mL of reductase, and 7.92 mL of GDH enzyme. Maintain the pH at 6.5 and allow the reaction to continue for 18 h. TLC indicates complete conversion (developing solvent: PE / EA = 4 / 1, vanillin as colorimetric visualization; chromatographic sampling: aspirate 100 μl of the reaction solution, add 900 μl of acetonitrile, mix well, and analyze).

[0040] After the reaction is complete, tert-butanol is removed by rotary evaporation, and the remaining aqueous phase is filtered to collect the filter cake. 10 times the amount of ethyl acetate is added, stirred and refluxed at 80°C, and the filtrate is filtered and collected. The filter cake is again stirred and refluxed with 4 times the amount of ethyl acetate at 80°C, and the filtrate is filtered and collected. The two filtrates are combined, the ethyl acetate is dried by rotary evaporation, and the product is dried in an oven at 60°C overnight to obtain 19.1 g of crude extract; the crude product is recrystallized from 16 times methanol to obtain 15.8 g, with a purity of 99.83% and a yield of 79%. The product is a white powder.

[0041] Example 6 Weigh 22.5 g of the substrate progesterone acetate into a 500 mL three-necked flask, add 18.8 g of D(+)-anhydrous glucose, 200 mL of tert-butyl alcohol, and 200 mL of 0.2 M PBS solution (120 mL of 0.2 M sodium dihydrogen phosphate solution + 80 mL of 0.2 M sodium dihydrogen phosphate solution), and stir in a 30°C water bath for 1 h. Add 0.6 g of NAD (nicotinamide adenine dinucleotide), 22.5 mL of hydrolase, 55 mL of reductase, and 7.92 mL of GDH enzyme. Maintain the pH at 6.5 and react for 18 h until complete conversion is indicated by TLC (developing solvent: PE / EA = 4 / 1, vanillin as colorimetric visualization; chromatographic sampling method: aspirate 100 μl of the reaction solution, add 900 μl of acetonitrile, mix well, and analyze. After the reaction is complete, tert-butanol is removed by rotary evaporation, and the remaining aqueous phase is filtered to collect the filter cake. 10 times the amount of ethyl acetate is added, stirred and refluxed at 80°C, and the filtrate is collected by filtration. The filter cake is again stirred and refluxed with 4 times the amount of ethyl acetate at 80°C, and the filtrate is collected by filtration. The two filtrates are combined and the ethyl acetate is dried in a 60°C oven overnight to obtain 21g of crude extract; the crude extract is slurried with 3 times ethyl acetate to obtain 18.9g, with a purity of 99% and a yield of 90%. The product is a yellow powder.

[0042] Comparative Example 1 This comparative example is the same as the preparation method of the intermediate in Example 3, except that the hydrolase is replaced by another lipase Fs-Cut-pPICZaA with the same protein concentration, which is derived from Fusarium vanettenii, The amino acid sequence is as follows: LPTSNPAQELEARQLGRTTRDDLINGNSASCADVIFIYARGSTETGNLGTLGPSIASNLESAFGKDGVWIQGVGGAYRATLGDNALPRGTSSAAIREMLGLFQQANTKCPDATLIAGGYSQGAALAAASIEDLDSAIRDKIAGTVLFGYTKNLQNRGRIPNYPADRTKVFCNTGDLVCTGSLIVAAPHLAYGPDARGPAPEFLIEKVRAVRGSA. TLC showed no conversion.

[0043] Comparative Example 2 The preparation method of the intermediate in this comparative example is the same as that in Example 3, except that the hydrolase was replaced by another commercial lipase CALB with the same protein concentration. The result showed that TLC did not show any conversion.

[0044] Comparative Example 3 This comparative example is the same as the preparation method of Example 4 for pregnenolone, except that the raw material is replaced by the intermediate in Example 3, no hydrolase is added, and only ketoreductase is added to carry out the enzyme reduction reaction at position 3. As a result, TLC shows no conversion.

[0045] Comparative Example 4 This comparative example is the same as the preparation method of Example 4 for pregnenolone, except that 12 mL of hydrolase and 15 mL of ketoreductase with the same protein concentration are added. The results show that the reaction is not complete after 24 hours of reaction.

[0046] Comparative Example 5 This comparative example is the same as the preparation method of pregnenolone in Example 4, except that 50 mL of hydrolase and 120 mL of ketoreductase with the same protein concentration are added. As a result, the reaction also requires 18 hours for complete reaction.

[0047] Comparative Example 6 This comparative example was prepared in the same manner as in Example 4, except that the amount of tert-butanol in the system was reduced to 100 ml, and the amount of 0.2 M PBS buffer was reduced to 100 ml. As a result, the hydrolysis reaction conversion rate was less than 50%, and the ketone reduction reaction conversion rate was less than 30%.

[0048] Examples 4-6 demonstrate that the preparation method of the present invention can produce pregnenolone in high yield and purity. When other hydrolases are used for catalysis, as in Comparative Examples 1 and 2, the substrate progesterone acetate is not recognized at all, demonstrating that the hydrolases of the present invention can specifically bind to the substrate progesterone acetate and efficiently hydrolyze it.

[0049] As in Example 3 and Comparative Example 3, when the hydrolase and ketoreductase were used separately, the enzyme reduction reaction at position 3 could not be completed because the activation energy required for the reaction increased after the intermediates were stabilized.

[0050] As shown in Example 4 and Comparative Examples 4 and 5, controlling the amount of hydrolase and ketoreductase added has a significant impact on the dual-enzyme reaction. When the enzyme dosage is too low, the complete reaction time is too long. When the enzyme dosage is too high, the reaction cost gradually increases, but the reaction speed is not improved at all.

[0051] As shown in Example 4 and Comparative Example 6, controlling the amount of 0.2M PBS buffer and tert-butanol has a significant impact on the dual-enzyme reaction. When the reaction solvent is too much, the reaction cost gradually increases, which is not conducive to production. However, when the reaction solvent is too little, the substrate solubility is too poor, and the reaction cannot proceed smoothly.

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing pregnenolone, characterized in that: The preparation process comprises a hydrolase catalysis step, and the amino acid sequence of the hydrolase is shown in SEQ ID NO:

1.

2. The method for preparing pregnenolone according to claim 1, wherein The preparation method comprises the following steps: S1, preparing progesterone into progesterone ester; S2, adding the hydrolase and ketoreductase as described in claim 1 to the progesterone ester in step S1, performing enzymatic catalysis to prepare pregnenolone, and then purifying it.

3. The method for preparing pregnenolone according to claim 2, wherein In step S2, the mass volume ratio of the progesterone ester: hydrolase: ketoreductase is (4-5) g: (3-6) mL: (3.5-18.5) mL.

4. The method for preparing pregnenolone according to claim 2, wherein In step S1, the prepared raw materials include progesterone, acetic anhydride and p-toluenesulfonic acid.

5. The method for preparing pregnenolone according to claim 2, wherein In step S1, the progesterone ester is progesterone acetate.

6. The method for preparing pregnenolone according to claim 2, wherein In step S2, the raw materials for enzyme catalysis also contain nicotinamide adenine dinucleotide, glucose and glucose dehydrogenase.

7. The method for preparing pregnenolone according to claim 2, wherein: In step S2, the enzyme-catalyzed solvent comprises tert-butanol and PBS buffer.

8. The method for preparing pregnenolone according to claim 2, wherein In step S2, the pH of the enzyme catalysis is 6.0-7.

0.

9. The method for preparing pregnenolone according to claim 2, wherein: In step S2, the purified raw material comprises ethyl acetate or methanol.

10. The method for preparing pregnenolone according to claim 1, wherein The nucleotide sequence of the hydrolase is shown in SEQ ID NO: 2.

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