High-concentration 7-dehydrocholesterol enzymatic hydroxylation method based on organic-water two-phase system

Abstract

The invention discloses a high-concentration 7-dehydrocholesterol enzymatic hydroxylation method based on an organic-water two-phase system, which comprises the following steps: loading high-concentration 7-DHC (7-dehydrocholesterol) through an organic phase, maintaining peroxygenase activity through a water phase, and catalyzing 25-site hydroxylation of 7-DHC by taking hydrogen peroxide as an oxygen source to generate 25-hydroxy-7-dehydrocholesterol. According to the method, the problem of low solubility caused by hydrophobicity of 7-DHC is solved, the substrate concentration is greatly improved, the conversion rate and the product selectivity are improved, the method is suitable for industrial production of calcifediol, the productivity can be improved, and the cost can be reduced.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically relating to a high-concentration 7-dehydrocholesterol enzyme-catalyzed hydroxylation method based on an organic-water two-phase system. Background Technology

[0002] Calcidiol, as the main active form of vitamin D3 in vivo, plays a crucial role in regulating calcium and phosphorus metabolism and promoting bone development. Currently, its biosynthetic pathway employs a two-step method, as shown in CN115181758A (publication date: 2022.10.14) and CN114075556A (publication date: 2022.02.22): First, vitamin D3 is prepared through photochemical isomerization and thermal conversion of 7-dehydrocholesterol (7-DHC), followed by a 25-position hydroxylation reaction catalyzed by an oxidase (P450 enzyme, peroxyseroase, or peroxidase, etc.). However, this process faces significant technical bottlenecks: the vitamin D3 synthesis stage generates various structural analogs such as pre-vitamin D3, paclitaxel, and photosterol; the products and impurities have similar physicochemical properties, leading to difficulties in separation and purification; traditional chromatographic separation methods are inefficient, costly, and have limited yields; the overall process cost is high, hindering industrial production.

[0003] In existing technologies, 25-hydroxy-7-dehydrocholesterol (25-OH-DHC) is obtained by hydroxylation at position 25 using 7-DHC as a substrate under the catalysis of peroxygenase. This is then directionally converted to calcidiol via UV irradiation and thermal conversion. This innovative process offers significant advantages, including high enzyme selectivity, low photoinduced impurities (<5%), and high photoconversion rate (>80%, compared to only 10-20% in traditional processes), greatly improving production efficiency and product purity, and providing an innovative solution for the industrial production of calcidiol. However, due to the extremely strong hydrophobicity (logP≈8.5) of the substrate 7-DHC as a typical sterol compound, its solubility in aqueous systems is extremely low (<0.1 μg / mL). Even in the presence of acetone as a co-solvent, the working concentration of the substrate in existing enzyme catalytic systems is still limited to below 4 g / L, and the single-pass conversion rate is only about 60% (Xu C, Huang Y, Li H, et al. Aphotoenzymatic pathway for gramscale synthesis of 25-hydroxyvitamin D3. ChemSusChem, 2025, 18(2): e202401196). This technical bottleneck severely restricts the reaction efficiency, resulting in: (1) insufficient production capacity per unit volume; (2) rising downstream purification costs; and (3) difficulties in industrial scale-up.

[0004] Therefore, there is an urgent need in the field for a system and method that can improve conversion efficiency and is suitable for the industrial production of calcidiol. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a two-phase catalytic system that innovatively adopts an aqueous-organic phase synergistic mechanism: a high concentration of 7-DHC is loaded in the organic phase, while the substrate is continuously transferred to the aqueous phase by optimizing the interfacial mass transfer kinetics; the aqueous phase maintains the optimal microenvironment for the enzyme and avoids protein denaturation caused by organic solvents.

[0006] On one hand, the present invention provides a method for high-concentration 7-dehydrocholesterol enzyme-catalyzed hydroxylation based on an organic-aqueous two-phase system, comprising the following steps:

[0007] S1. Dissolve 7-dehydrocholesterol in an organic solvent to obtain 7-dehydrocholesterol organic mother liquor;

[0008] S2. Mix the organic mother liquor from S1 with the aqueous buffer solution and sonicate to form a homogeneous organic-aqueous two-phase system.

[0009] S3. Add peroxygenase and hydrogen peroxide to the two-phase system and carry out an enzymatic hydroxylation reaction under stirring to obtain 25-hydroxy-7-dehydrocholesterol.

[0010] Specifically, the organic solvent in step S1 is selected from any two or more of methanol, ethanol, acetonitrile, acetone, chloroform, ethyl acetate, diethyl ether, tetrahydrofuran, tert-butanol, methyl tert-butyl ether, and dichloromethane.

[0011] Specifically, the organic solvent accounts for 30-65% of the reaction volume.

[0012] Specifically, the aqueous buffer in step S2 is a phosphate buffer or a citrate buffer, the concentration of which is 30-120 mM and the pH range is 4-9, preferably 5-7.

[0013] Specifically, the peroxygenase is derived from Agrocybe aegerita, Marasmius rotula, Psathyrella aberdarensis, or Coprinellus radians, with corresponding GenBank accession numbers of CBJ94532.1, JQ654436.1, MH880928, or FM872459.1, respectively.

[0014] Specifically, the peroxygenase can be any one or more of crude enzyme powder, crude enzyme solution, or purified enzyme. Preferably, the peroxygenase does not require the provision of additional cofactors for catalysis. The crude enzyme powder, crude enzyme solution, or purified enzyme is prepared using conventional methods, such as by introducing recombinant bacteria with a peroxygenase encoding gene.

[0015] Specifically, the concentration of 7-dehydrocholesterol in the substrate ranges from 2 to 15 g / L, and the concentration of the peroxygenase is 600 nM to 4 μM. Preferably, the concentration of 7-dehydrocholesterol in the substrate ranges from 8 to 12 g / L, and the concentration of the peroxygenase is 600 nM to 3 μM. The injection rate of hydrogen peroxide is 15 to 300 mM / h.

[0016] Specifically, in step S2, the ultrasonic treatment time is 5-20 minutes, and in step S3, the stirring conditions are 28-50℃ constant temperature and 300-800 rpm stirring speed for 2-12 hours.

[0017] On the one hand, the present invention provides the application of the above method in the preparation of 25-hydroxy-7-dehydrocholesterol.

[0018] On the other hand, the present invention also provides the application of the above method in the preparation of calcidiol.

[0019] Beneficial effects: This invention optimizes the reaction system for the synthesis of 25-OH-DHC from a single aqueous phase to a two-phase system of aqueous and organic phases by peroxygenase catalysis, significantly increasing the substrate loading to over 8 g / L. The method features mild reaction conditions, high single-batch product yield, and a breakthrough conversion efficiency of 94.8%. After the reaction, the product is directly dissolved in the organic phase, reducing the cumbersome downstream processing in the aqueous phase. Furthermore, the organic phase can be recycled 5-8 times, significantly improving the process economy. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the technical route of the present invention;

[0021] Figure 2 This is an HPLC chromatogram of the peroxygenase catalyzing the conversion of substrate 7-DHC to 25-OH-DHC, as described in Example 1. Detailed Implementation

[0022] Detailed Description of Embodiments. The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description unfolds. However, these embodiments are merely illustrative and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] In the embodiments of this invention, the method for determining the substrate 7-DHC and the product 25-OH-DHC is high performance liquid chromatography (HPLC). The chromatographic conditions are: Hypersil C18 (5µm, 250*4.6mm); UV detection wavelength: 285nm; flow rate: 1mL / min; injection volume: 10mL; mobile phase: water-methanol (10%:90%, v / v%).

[0026] This invention utilizes peroxygenase to catalyze the 25-hydroxylation reaction of high concentrations of 7-DHC, and the technical route is as follows: Figure 1 As shown, the enzymatic system includes a 7-DHC mother liquor dissolved in an organic phase, using peroxygenases from different sources as enzyme catalysts and hydrogen peroxide as an oxygen source to promote the enzymatic hydroxylation reaction of 7-dehydrocholesterol. The substrate conversion rate and product selectivity in the organic-water two-phase system were detected.

[0027] Example 1

[0028] In a 20 mL standard reaction system, 80 mg 7-DHC (8 g / L), 5 mL acetone, and 5 mL chloroform were added sequentially, and the mixture was sonicated for 5 min to ensure complete dissolution. Then, 10 mL phosphate buffer (100 mM, pH 6.0) was added, and sonication was continued for 10 min to form a homogeneous two-phase system. Finally, 600 nM crude enzyme powder AaeUPO (a peroxyenzyme from Agrocybeaegerita, its amino acid sequence is available in GenBank: CBJ94532.1) was added, and the reaction was carried out at a constant temperature of 35 °C with magnetic stirring at 500 rpm for 5 h, while hydrogen peroxide was continuously injected at a rate of 50 mM / h. After the reaction was complete, the organic phase was separated and rotary evaporated under reduced pressure at 40 °C. HPLC analysis showed that the substrate conversion rate reached 94.8%, and the product selectivity was 97.1%. The results are as follows: Figure 2 As shown.

[0029] Example 2

[0030] In a 20 mL standard reaction system, 100 mg of 7-DHC (10 g / L), 7 mL of acetone, and 3 mL of ethyl acetate were added sequentially, and the mixture was sonicated for 5 min to ensure complete dissolution. Then, 8 mL of phosphate buffer (100 mM, pH 6.0) was added, and sonication was continued for 10 min to form a homogeneous two-phase system. Finally, 2 mL (final concentration 1 μM) of crude enzyme solution MroUPO (peroxygenase MroUPO derived from Marasmius rotula, its amino acid sequence GenBank: JQ654436.1) was added, and the reaction was carried out at a constant temperature of 40 °C with magnetic stirring at 500 rpm for 8 h, while hydrogen peroxide was continuously injected at a rate of 30 mM / h. After the reaction was complete, the organic phase was separated and rotary evaporated under reduced pressure at 40 °C. HPLC analysis showed that the substrate conversion rate reached 90.1% and the product selectivity was 92.1%.

[0031] Example 3

[0032] In a 20 mL standard reaction system, 120 mg of 7-DHC (12 g / L), 6 mL of tert-butanol, and 5 mL of diethyl ether were added sequentially, and the mixture was sonicated for 5 min to ensure complete dissolution. Then, 7 mL of phosphate buffer (100 mM, pH 6.0) was added, and the mixture was sonicated for another 10 min to form a homogeneous two-phase system. Finally, 2 mL (final concentration 1.5 μM) of crude enzyme solution CraUPO (peroxygenase CraUPO from Coprinellus radians, its amino acid sequence GenBank: FM872459.1) was added, and the reaction was carried out at a constant temperature of 40 °C with magnetic stirring at 500 rpm for 10 h, while hydrogen peroxide was continuously injected at a rate of 18 mM / h. After the reaction was complete, the organic phase was separated and rotary evaporated under reduced pressure at 40 °C. HPLC analysis showed that the substrate conversion rate reached 89.9% and the product selectivity was 90.7%.

[0033] Example 4

[0034] In a 20 mL standard reaction system, 120 mg 7-DHC (12 g / L), 3 mL acetone, 3 mL tert-butanol, and 6 mL isopropanol were added sequentially, and the mixture was sonicated for 5 min to ensure complete dissolution. Then, 7 mL phosphate buffer (100 mM, pH 6.0) was added, and the mixture was sonicated for another 10 min to form a homogeneous two-phase system. Finally, 1 mL (final concentration 2 μM) of crude enzyme solution PabUPO (peroxygenase PabUPO derived from Psathyrella aberdarensis, its amino acid sequence GenBank: MH880928) was added, and the reaction was carried out at 38 °C with magnetic stirring at 500 rpm for 3 h, while hydrogen peroxide was continuously injected at a rate of 150 mM / h. After the reaction was complete, the organic phase was separated and rotary evaporated under reduced pressure at 40 °C. HPLC analysis showed that the substrate conversion rate reached 86.0%, and the product selectivity was 90.3%.

[0035] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for high-concentration 7-dehydrocholesterol enzyme-catalyzed hydroxylation based on an organic-aqueous two-phase system, characterized in that, Includes the following steps: S1. Dissolve 7-dehydrocholesterol in an organic solvent to obtain 7-dehydrocholesterol organic mother liquor; S2. Mix the organic mother liquor from S1 with the aqueous buffer solution and sonicate to form a homogeneous organic-aqueous two-phase system. S3. Add peroxygenase and hydrogen peroxide to the two-phase system and carry out an enzymatic hydroxylation reaction under stirring to obtain 25-hydroxy-7-dehydrocholesterol.

2. The method according to claim 1, characterized in that, The organic solvent in step S1 is selected from any two or more of methanol, ethanol, acetonitrile, acetone, chloroform, ethyl acetate, diethyl ether, tetrahydrofuran, tert-butanol, methyl tert-butyl ether, and dichloromethane.

3. The method according to claim 2, characterized in that, The organic solvent accounts for 30-65% of the reaction volume.

4. The method according to claim 1, characterized in that, The aqueous buffer in step S2 is a phosphate buffer or a citrate buffer, the concentration of which is 30-120 mM and the pH range is 4-9, preferably 5-7.

5. The method according to claim 1, characterized in that, The peroxygenase is derived from *Agrocybe aegerita*, *Pteris vittata*, *Pteris brittle*, or *Coprinus radiata*, and their amino acid sequences have GenBank accession numbers of CBJ94532.1, JQ654436.1, MH880928, or FM872459.1, respectively.

6. The method according to claim 5, characterized in that, The peroxygenase is any one or more of crude enzyme powder, crude enzyme solution, or purified enzyme. Preferably, the peroxygenase does not require the provision of additional cofactors for catalysis.

7. The method according to claim 1, characterized in that, The concentration of 7-dehydrocholesterol in the substrate ranges from 2 to 15 g / L, and the concentration of the peroxygenase is 500 nM to 4 μM. Preferably, the concentration of 7-dehydrocholesterol in the substrate ranges from 8 to 12 g / L, and the concentration of the peroxygenase is 600 nM to 3 μM. The injection rate of hydrogen peroxide is 15 to 300 mM / h.

8. The method according to claim 1, characterized in that, In step S2, the ultrasonic treatment time is 5-20 minutes, and in step S3, the stirring conditions are 28-50℃ constant temperature and 300-800 rpm stirring speed for 2-12 hours.

9. Use of the method according to any one of claims 1-8 in the preparation of 25-hydroxy-7-dehydrocholesterol.

10. Use of the method according to any one of claims 1-8 in the preparation of calcidiol.

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