A process for the preparation of 25-hydroxyvitamin D3 by conversion using immobilized hydroxylase

Abstract

The present application relates to the technical field of bioengineering, and particularly relates to a method for preparing 25-hydroxyvitamin D3 by using immobilized hydroxylase conversion. The present application uses vitamin D3 as a substrate, and prepares 25-hydroxyvitamin D3 by using immobilized hydroxylase conversion, and includes preparation of a conversion system. The present application has the advantages of high feeding concentration, high substrate conversion rate and repeated use of immobilized hydroxylase. The present application has simple operation process, low production cost and high industrialization prospect.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a method for preparing 25-hydroxyvitamin D3 by using immobilized hydroxylase. Background Technology

[0002] Vitamin D3 is an important fat-soluble vitamin in living organisms, primarily serving as a precursor for calcium and phosphorus regulation. In vivo, 7-dehydrocholesterol undergoes ring-opening at position 9 of the cyclopentane-polyhydrophenanthrene structure to form the vitamin D3 structure. Vitamin D3 in vivo lacks biological activity and does not circulate in the bloodstream for extended periods; instead, it is rapidly absorbed and stored by adipose tissue or transported to the liver for further synthesis and metabolism (Sun et al., 2015). In the human body, vitamin D3 undergoes hydroxylation to transform into its active forms, 25-hydroxyvitamin D3 and 1α-hydroxyvitamin D3. The discovery of 25-hydroxyvitamin D3 is considered one of the most significant research achievements in the field of vitamins at the end of the 20th century.

[0003] With the discovery that vitamin D3, as an active hormone in metabolic synthesis, possesses broader biological activity and medicinal value, a surge of interest in chemical synthesis has emerged internationally. Currently, the mainstream chemical synthesis method, proposed by Method.F, involves a series of 20 steps, including chemical modification, substitution, and hydrogenation, to synthesize 1α,25-hydroxyvitamin D3 from cholesterol, a structurally similar compound to vitamin D3. However, the final yield is very low, only about 1% (Zhu GD, Okamura WH et al., 1995). Chemical synthesis itself is inefficient, polluting, and suffers from poor stereoselectivity in the hydroxylation of the sterol nucleus. Given the increasing demand for active vitamin D3 in the pharmaceutical market, there is an urgent need for a more efficient method to replace chemical synthesis.

[0004] Microbial transformation utilizes strains primarily from the genera *Streptomyces*, *Rhodococcus*, *Mycobacterium*, *Nocardia*, and *Saccharomyces*. Microbial transformation is low-cost, causes minimal environmental pollution, exhibits high stereoselectivity, and can significantly shorten chemical synthesis reaction steps, thus it is considered to have broad application prospects in complex reaction processes.

[0005] To date, the industrial production of active pharmaceutical ingredients by hydroxylation of sterols at 11α, 11β, and 16α has been widely adopted (Mahato SB et al., 1997), providing a very good precedent for the industrialization of vitamin D3 hydroxylase conversion to produce 25-hydroxyvitamin D3.

[0006] However, the extremely low water solubility of vitamin D3 is a major limiting factor restricting the rate of vitamin D3 hydroxylation. Existing microbial fermentation methods also have the following shortcomings: ① Low feed concentration. Due to the low tolerance of bacterial cells to high concentrations of organic solvents and surfactants, the feed concentration is limited. ② Complex product extraction and purification processes, resulting in low final product yield. In the fermentation method, the product 25-hydroxyvitamin D3 is mainly adsorbed by the bacterial cells, requiring repeated whole-cell extraction to extract the product. However, a large number of impurities abundant in the bacterial cells, such as pigments, lipids, and proteins, are also extracted simultaneously, necessitating a relatively complex extraction and purification process to obtain high-purity 25-hydroxyvitamin D3. Summary of the Invention

[0007] This invention provides a method for preparing 25-hydroxyvitamin D3 using immobilized hydroxylase, which solves the problems of low feed concentration, large amount of fermentation wastewater, and low final product yield in existing fermentation methods for preparing 25-hydroxyvitamin D3.

[0008] This invention provides a conversion system for the preparation of 25-hydroxyvitamin D3 by immobilized hydroxylase. The raw materials of the conversion system include the following components: glucose 0.5-1.5 g / L, emulsifier 30.0-50.0 g / L, vitamin D3 25-50 g / L, and dispersant 0.5-1.5 g / L.

[0009] The pH value of the conversion system is 7.0-7.5. Using the conversion system of this invention, the feed concentration and conversion rate can be increased, the conversion time shortened, and water consumption and wastewater volume reduced.

[0010] Preferably, the emulsifier is castor oil polyoxyethylene ether or fatty alcohol polyoxyethylene ether.

[0011] Preferably, the dispersant is polyethylene glycol, more preferably polyethylene glycol 200 or polyethylene glycol 400. Using the dispersant and emulsifier described in this invention can effectively improve the hydroxylation reaction rate.

[0012] Preferably, the conversion system also includes a buffer solution.

[0013] Further preferred is that the conversion system includes phosphate buffer and MgSO4.

[0014] More preferably, the phosphate buffer comprises Na₂HPO₄ and KH₂PO₄. Even more preferably, it comprises 1.40-1.49 g / L Na₂HPO₄ and 0.22-0.26 g / L KH₂PO₄.

[0015] Further preferred concentrations include MgSO4 at 0.05-0.015 g / L.

[0016] Preferably, the conversion system comprises 1.4 g / L Na2HPO4, 0.24 g / L KH2PO4, and 0.05-0.015 g / L MgSO4.

[0017] Preferably, the conversion system further includes raw material B, which is a mixture of alcohol and water.

[0018] More preferably, the raw material B contains 70-95 wt% alcohols, with the remainder being water.

[0019] This invention also provides a method for preparing 25-hydroxyvitamin D3 using an immobilized hydroxylase conversion system. The method of this invention, using a specific immobilized enzyme catalysis, can reduce the impact on the bacterial cells and post-processing of the product after cell disruption. The production process is simple, yields high-purity 25-hydroxyvitamin D3, and the immobilized hydroxylase can be reused multiple times.

[0020] According to the method for preparing 25-hydroxyvitamin D3 by conversion using immobilized hydroxylase according to the present invention, the ratio of immobilized hydroxylase to conversion system is 1g:(50-150)mL.

[0021] According to the method for preparing 25-hydroxyvitamin D3 by conversion using immobilized hydroxylase as described in this invention, the conversion process involves controlling the reaction temperature at 30-35℃, the rotation speed at 100-220 r / min, and the conversion time at 12-16 h.

[0022] According to the method for preparing 25-hydroxyvitamin D3 by conversion using immobilized hydroxylase as described in this invention, a cofactor of vitamin D3 hydroxylase is added during the conversion process;

[0023] The cofactor is one or a mixture of NADPH, NADH and vitamin C.

[0024] According to the method for preparing 25-hydroxyvitamin D3 by conversion using immobilized hydroxylase as described in this invention, the immobilized hydroxylase is prepared using genetically engineered bacteria that produce vitamin D3 hydroxylase.

[0025] Preferably, the genetically engineered bacteria is one of yeast, Escherichia coli, or autotrophic apomyelitis bacteria.

[0026] More preferably, the genetically engineered bacteria is yeast.

[0027] The method for preparing 25-hydroxyvitamin D3 using immobilized hydroxylase according to the present invention further includes a hydroxylase induction step before preparing the immobilized hydroxylase.

[0028] According to the method for preparing 25-hydroxyvitamin D3 using immobilized hydroxylase according to the present invention, the method for preparing the immobilized hydroxylase includes the following steps:

[0029] (1) The bacterial suspension of the vitamin D3 hydroxylase-producing genetically engineered bacteria was disrupted by cell disruption, and the supernatant was obtained by centrifugation or filtration.

[0030] (2) Add sodium alginate and glutaraldehyde to the supernatant obtained in step 1) to obtain a mixture, and then add calcium chloride solution to form gel beads;

[0031] The sodium alginate has a mass fraction of 100-200 g / L.

[0032] According to the method for preparing 25-hydroxyvitamin D3 using immobilized hydroxylase as described in this invention, after the conversion is completed, the immobilized hydroxylase is recovered by filtration, then extracted with an organic solvent, the filtrate is centrifuged and the phases are separated, the organic phase is collected, and then decolorized, distilled, and purified to obtain 25-hydroxyvitamin D3. The vitamin D3 hydroxylase described herein can be reused multiple times.

[0033] Preferably, the present invention provides a method for preparing 25-hydroxyvitamin D3 using immobilized hydroxylase, which uses vitamin D3 as a substrate and prepares 25-hydroxyvitamin D3 by conversion with immobilized hydroxylase, comprising the following steps:

[0034] Step 1): Prepare the culture medium;

[0035] Step 2): Culture engineered yeast (Saccharomyces cerevisiae) that produces hydroxylase in a culture medium;

[0036] Step 3): Hydroxylase induction;

[0037] Step 4): Prepare immobilized hydroxylase;

[0038] Step 5): Prepare the conversion system;

[0039] Step 6): Substrate conversion;

[0040] Step 7): Product extraction and purification.

[0041] Preferably, step 1) includes the preparation of slant culture medium, primary seed culture medium and secondary fermentation culture medium.

[0042] The composition and content of the slant culture medium are as follows: glucose 5.0 g / L, peptone 15.0 g / L, KH2PO4 1.0 g / L, sodium chloride 5.0 g / L, raw material A 80 ppm and agar 20.0 g / L, pH 6.0-6.5.

[0043] The primary seed culture medium consists of the following components and contents: glucose 12.0 g / L, (NH4)2SO4 1.0 g / L, yeast extract 10 g / L, KH2PO4 1.0 g / L, NaCl 2.0 g / L, and raw material A 100 ppm; the pH of the culture medium is 6.0-6.5.

[0044] The secondary fermentation medium has the following components and contents: glucose 60.0 g / L, yeast extract 25.0 g / L, KH2PO4 1.0 g / L, (NH4)2SO4 5.0 g / L, K2HPO4 1.0 g / L and MgSO4 1.0 g / L; the pH of the medium is 6.0-6.5.

[0045] Preferably, step 2) includes slant culture, primary fermentation, and secondary fermentation. First, a single colony of the engineered bacteria producing hydroxylase is inoculated into a slant culture medium and cultured for 12 hours to obtain a fresh slant culture. The fresh slant culture is then inoculated into a primary seed culture medium and cultured for 12-16 hours. Then, the primary seed culture is inoculated into the secondary fermentation culture medium at an inoculation rate of 0.5-10% by volume, and fermentation takes 24-36 hours to obtain a secondary fermentation culture broth. The culture conditions for the engineered bacteria producing hydroxylase are a temperature of 28-32℃.

[0046] Preferably, step 3) specifically involves adding vitamin D3 with a mass fraction of 1.0-3.0 g / L to the secondary culture medium obtained in step 2), continuing to culture for 6-12 hours, stopping fermentation, filtering out the bacterial cells, and repeatedly washing the bacterial cells with purified water until the washing solution is colorless, and then washing three times with 0.01 M PBS buffer at pH 7.5.

[0047] Preferably, vitamin D3 with a mass fraction of 1.0 g / L is added to the secondary culture medium obtained in step 2).

[0048] Preferably, step 4) specifically involves: resuspending the bacterial cells obtained in step 3) in 0.01M PBS buffer at pH 7.5, with a ratio of bacterial cells to 0.01M PBS buffer at pH 7.5 of (1.0-2.0) g: 1 mL. After stirring evenly, the bacterial suspension is homogenized using a high-pressure homogenizer, then filtered. The supernatant obtained is the crude hydroxylase solution. An embedding agent is added to the crude hydroxylase solution, and after mixing evenly, glutaraldehyde is added. The mixture is stirred for 30 min and then allowed to stand at 4°C for 3 h to obtain a mixed solution. Then, a 2% (w / w) calcium chloride solution is added dropwise to form gel beads. The mixture is stirred and fixed for 1.0-3.0 h, filtered, and washed twice with a 0.9% (w / w) sodium chloride solution to obtain immobilized hydroxylase. The embedding agent is sodium alginate, with a mass fraction of 100-200 g / L, and the volume ratio of sodium alginate aqueous solution to crude hydroxylase solution is 1:1.

[0049] More preferably, the high-pressure homogenizer is set to a rotation speed of 10,000-18,000 rpm / min, a processing time of 15-20 min, and a homogenization process temperature of 4-15℃.

[0050] Preferably, the components and contents of the conversion system in step 5) are: glucose 1.0 g / L, emulsifier 30.0-50.0 g / L, vitamin D3 25-50 g / L, dispersant 1.0 g / L, Na2HPO4 1.4 g / L, KH2PO4 0.24 g / L and MgSO4 0.1 g / L; the pH value of the conversion system is 7.0-7.5.

[0051] Preferably, step 6) specifically involves adding the immobilized hydroxylase obtained in step 4) to the transformation system obtained in step 5), wherein the ratio of immobilized hydroxylase to transformation system is 1g:(50-150)mL, the cofactor of vitamin D3 hydroxylase is 1.0g / L, the reaction temperature is controlled at 30-35℃, the rotation speed is 100-220r / min, and the transformation time is 12-16h.

[0052] The cofactor of the vitamin D3 hydroxylase is one or a mixture of two of NADPH, NADH and vitamin C.

[0053] This invention provides a method for preparing 25-hydroxyvitamin D3, which utilizes hydroxylase immobilization technology and non-aqueous phase reaction system technology to achieve hydroxylation reaction of vitamin D3 under high concentration conditions, and can effectively solve the problems existing in the field.

[0054] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0055] (1) High feed concentration. Due to the high proportion of dispersant in the reaction system, both the substrate and product can be well distributed and dissolved in the reaction system, which can effectively improve the hydroxylation reaction rate. The feed concentration of vitamin D3 in a single enzyme reaction can reach 50 g / L, the molar conversion rate can reach more than 92%, and the conversion time can be shortened to less than 16 h.

[0056] (2) Immobilized enzymes can be recycled multiple times. Immobilized hydroxylases can be repeatedly recycled for hydroxylation reactions more than 3 times, which greatly reduces production costs.

[0057] (3) The extraction and purification process is relatively simple. Since the present invention uses enzyme catalysis and the reaction system has simple and clear components, and the product can be dissolved in the organic phase, the subsequent separation and purification process of the product is simplified to a certain extent. Detailed Implementation

[0058] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0059] HPLC detection conditions: Thermo 3000U HPLC; Thermo C18 column (250mm×4.6mm, 5μm) column; mobile phase: water / methanol / (10 / 90); flow rate: 1.0mL / min; temperature: room temperature; injection volume: 10ul; detection wavelength: 264nm.

[0060] The preparation method of raw material A in this embodiment of the invention is as follows: 10.0g of glucose, 14.0g of Na2HPO4, 2.4g of KH2PO4, 1.0g of MgSO4, 10.0g of dispersant and 400g of emulsifier are dissolved in 9L of sterile water, and the pH is adjusted to 7.5 with 15% phosphoric acid to obtain phase A.

[0061] In this embodiment of the invention, the preparation method of phase B is as follows: 400g of vitamin D3 is dissolved in 1000mL of raw material B (a mixture of isoamyl alcohol and water, containing 90w% isoamyl alcohol) to obtain phase B.

[0062] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0063] Example 1

[0064] The hydroxylase-producing engineered yeast (Saccharomyces cerevisiae) was cultured on slant agar to obtain the production strain required for fermentation.

[0065] I. Preparation of engineered yeast (Saccharomyces cerevisiae) slant culture and seed culture.

[0066] (1) Slant culture

[0067] Slant culture medium: glucose 5.0 g / L, peptone 15.0 g / L, KH2PO4 1.0 g / L, sodium chloride 5.0 g / L, raw material A (in this example, the dispersant in raw material A is polyethylene glycol 200 and the emulsifier is fatty alcohol polyoxyethylene ether) 80 ppm, agar 20.0 g / L, pH 6.5, sterilized by moist heat at 121℃ for 30 min.

[0068] Under aseptic conditions, a certain amount of bacterial culture preserved in the glycerol tube was evenly spread onto an agar slant culture medium and incubated at 30°C for 12 hours.

[0069] (2) Seed culture

[0070] The primary culture medium consists of the following components by weight (g): glucose 12.0 g / L, (NH4)2SO4 1.0 g / L, yeast extract 10 g / L, KH2PO4 1.0 g / L, NaCl 2.0 g / L, raw material A 100 ppm, pH 6.0, autoclaved at 121℃ for 30 min, and cooled before use.

[0071] The strain cultured in step (1) was used to pick up one loop of bacterial cells under aseptic conditions and put them into a seed culture medium containing 30 mL. Three bottles were inoculated in the same way and cultured at 28°C and 200 r / min for 12 h to obtain the seed liquid.

[0072] Secondary fermentation culture and induction

[0073] The secondary culture medium consists of the following components by weight (g): glucose 60.0 g / L, yeast extract 25.0 g / L, KH2PO4 1.0 g / L, (NH4)2SO4 5.0 g / L, K2HPO4 1.0 g / L, MgSO4 1.0 g / L, pH 6.5. It is autoclaved at 121℃ for 30 min and then cooled before use.

[0074] Secondary fermentation was performed in 500mL shake flasks with a volume of 50mL. The seed culture was transferred to the secondary fermentation medium at a 10% inoculation rate. The temperature was 28℃ and the rotation speed was 220 rpm. After 16 hours of cultivation, vitamin D3 at 1g / L was added, and the culture was continued for another 8 hours. Fermentation was then stopped, and the bacterial cells were collected by filtration. The cells were repeatedly washed with purified water until the washing solution was colorless, and then washed three times with 0.01M PBS buffer (pH 7.5).

[0075] III. Preparation of Immobilized Hydroxylase

[0076] The obtained bacterial cells were resuspended in 0.01M PBS buffer at pH 7.5, with a ratio of 1.0g:1mL. After thorough mixing, the bacterial suspension was homogenized using a high-speed homogenizer at 10,000 rpm for 15 minutes, maintaining the system temperature at 4-15°C using a low-temperature circulator. The mixture was then filtered, and the supernatant was obtained as the crude hydroxylase solution. An encapsulating agent, sodium alginate (100g / L), was added to the crude hydroxylase solution at a volume ratio of 1:1. After thorough mixing, glutaraldehyde was added, and the mixture was stirred for 30 minutes and then allowed to stand at 4°C for 3 hours to obtain the final solution. Then, add dropwise a 2% calcium chloride solution to form gel beads; stir and fix for 1.0 h, filter, and wash twice with a 0.9% sodium chloride solution to obtain immobilized hydroxylase.

[0077] IV. Formulation and Conversion System

[0078] The weight composition of the 1L conversion system includes (g): glucose 1.0, emulsifier 30.0, vitamin D3 25.0, dispersant 1.0, Na2HPO4 1.4, KH2PO4 0.24, MgSO4 0.1, raw material B 100, and pH value 7.0.

[0079] The specific preparation method is as follows: Dissolve 1.0g of glucose, 1.4g of Na2HPO4, 0.24g of KH2PO4, 0.1g of MgSO4, 1.0g of dispersant polyethylene glycol 200, and 30g of emulsifier fatty alcohol polyoxyethylene ether in 900ml of sterile water, and adjust the pH to 7.0 with 15% phosphoric acid to obtain phase A; dissolve 25g of vitamin D3 in 100mL of raw material B to obtain phase B; under stirring, slowly add phase B to phase A and stir evenly to obtain the conversion system.

[0080] V. Transformation

[0081] Add coenzyme NADPH to the transformation system obtained in step (iv) above at a mass fraction of 1.0 g / L, control the reaction temperature at 30℃, the rotation speed at 180 r / min, and stir for 15 min. Then add the immobilized enzyme obtained in step (iii) above into the transformation system. The mass-volume ratio of immobilized enzyme to the reaction transformation system is 1 g: 150 mL. The transformation time is 12 h. The molar conversion rate is 92.5% as detected by HPLC.

[0082] VI. Product Extraction and Refining

[0083] After the conversion was completed, the immobilized enzyme was removed by filtration. Ethyl acetate was added to the filtrate, and the mixture was stirred for 1 hour. After standing and separating into layers, the upper organic phase was taken and subjected to activated carbon decolorization and vacuum distillation to obtain crude 25-hydroxyvitamin D3.

[0084] The crude product was then purified with a mixture of ethanol and water in a certain proportion to finally obtain 19.6g of refined 25-hydroxyvitamin D3, with a yield of 81.5% and a 25-hydroxyvitamin D3 content of 98.0%.

[0085] Example 2

[0086] The hydroxylase-producing engineered yeast (Saccharomyces cerevisiae) was cultured on slant agar to obtain the production strain required for fermentation.

[0087] I. Preparation of engineered yeast (Saccharomyces cerevisiae) slant culture and seed culture.

[0088] (1) Slant culture

[0089] Slant culture medium: glucose 5.0 g / L, peptone 15.0 g / L, KH2PO4 1.0 g / L, sodium chloride 5.0 g / L, raw material A (in this example, the dispersant in raw material A is polyethylene glycol 200 and the emulsifier is castor oil polyoxyethylene ether) 80 ppm, agar 20.0 g / L, pH 6.0, sterilized by moist heat at 121℃ for 30 min.

[0090] Under aseptic conditions, a certain amount of bacterial culture preserved in the glycerol tube was evenly spread onto an agar slant culture medium and incubated at 30°C for 12 hours.

[0091] (2) Seed culture

[0092] The primary culture medium consists of the following components by weight (g): glucose 12.0 g / L, (NH4)2SO4 1.0 g / L, yeast extract 10 g / L, KH2PO4 1.0 g / L, NaCl 2.0 g / L, raw material A 100 ppm, pH 6.0, autoclaved at 121℃ for 30 min, and cooled before use.

[0093] The strain cultured in step (1) was used to pick up 3 loops of bacterial cells under aseptic conditions and put them into 100 mL of seed culture medium. Three bottles were inoculated in the same way and cultured at 30°C and 220 r / min for 16 h to obtain seed liquid.

[0094] Secondary fermentation culture and induction

[0095] The secondary culture medium consists of the following components by weight (g): glucose 60.0 g / L, yeast extract 25.0 g / L, KH2PO4 1.0 g / L, (NH4)2SO4 5.0 g / L, K2HPO4 1.0 g / L, MgSO4 1.0 g / L, pH 6.5. It is autoclaved at 121℃ for 30 min and then cooled before use.

[0096] Secondary fermentation was carried out in a 50L fermenter. The seed culture was transferred to the secondary fermentation medium at an inoculation rate of 0.5%. The temperature was 30℃, and the dissolved oxygen should not be lower than 30. Sugar and ammonia were added appropriately. When the culture time was 24h, vitamin D3 was added at a rate of 2g / L. The culture was continued for another 12h. The fermentation broth was milky white in appearance and had a strong yeast odor. Fermentation was stopped, the cells were filtered and the cells were washed repeatedly with purified water until the washing solution was colorless. Then, the cells were washed three times with 0.01M PBS buffer at pH 7.5.

[0097] III. Preparation of Immobilized Hydroxylase

[0098] The obtained bacterial cells were resuspended in 0.01M PBS buffer at pH 7.5, with a ratio of 2.0 g to 1 mL. After thorough mixing, the bacterial suspension was homogenized using a high-speed homogenizer at 18,000 rpm for 25 minutes, maintaining the system temperature at 4-15°C using a low-temperature circulator. The mixture was then filtered, and the supernatant was obtained as the crude hydroxylase solution. An encapsulating agent, sodium alginate (200 g / L), was added to the crude hydroxylase solution at a volume ratio of 1:1. After thorough mixing, glutaraldehyde was added, and the mixture was stirred for 30 minutes and then allowed to stand at 4°C for 3 hours to obtain the final solution. Then, add dropwise a 2% calcium chloride solution to form gel beads; stir and fix for 1.0 h, filter, and wash twice with a 0.9% sodium chloride solution to obtain immobilized hydroxylase.

[0099] IV. Formulation and Conversion System

[0100] The 10L conversion system comprises (g) the following components by weight: glucose 10.0, emulsifier 400.0, vitamin D3 400.0, dispersant 10.0, Na2HPO4 14.0, KH2PO4 2.4, MgSO4 1.0, raw material B 1000, and pH 7.0.

[0101] The specific preparation method is as follows: Dissolve 10.0g of glucose, 14.0g of Na2HPO4, 2.4g of KH2PO4, 1.0g of MgSO4, 10.0g of dispersant polyethylene glycol 200, and 400g of emulsifier castor oil polyoxyethylene ether in 9L of sterile water, and adjust the pH to 7.5 with 15% phosphoric acid to obtain phase A; dissolve 400g of vitamin D3 in 1000mL of raw material B to obtain phase B; under stirring, slowly add phase B to phase A and stir evenly to obtain the conversion system.

[0102] V. Transformation

[0103] Add the coenzyme NADH and vitamin mixture to the transformation system obtained in step (iv) above at a mass fraction of 1.0 g / L. Control the reaction temperature at 35℃, the rotation speed at 200 r / min, and stir for 15 min. Then add the immobilized enzyme obtained in step (iii) above to the transformation system. The mass-volume ratio of immobilized enzyme to the reaction transformation system is 1 g: 100 mL. The transformation time is 16 h. The molar conversion rate is 93% as detected by HPLC.

[0104] VI. Product Extraction and Refining

[0105] After the conversion was completed, the immobilized enzyme was removed by filtration. Butyl acetate was added to the filtrate, stirred for 1 hour, allowed to stand and separate into layers, and the upper organic phase was taken. After vacuum distillation, activated carbon decolorization, crystallization and other steps, crude 25-hydroxyvitamin D3 was obtained.

[0106] The crude product was then purified with a mixture of ethanol and water in a certain proportion to finally obtain 321.7g of refined 25-hydroxyvitamin D3, with a yield of 83% and a 25-hydroxyvitamin D3 content of 98.5%.

[0107] Example 3

[0108] The hydroxylase-producing engineered yeast (Saccharomyces cerevisiae) was cultured on slant agar to obtain the production strain required for fermentation.

[0109] I. Preparation of engineered yeast (Saccharomyces cerevisiae) slant culture and seed culture.

[0110] (1) Slant culture

[0111] Slant culture medium: glucose 5.0 g / L, peptone 15.0 g / L, KH2PO4 1.0 g / L, sodium chloride 5.0 g / L, raw material A (in this example, the emulsifier is fatty alcohol polyoxyethylene ether, and the dispersant is polyethylene glycol 400) 80 ppm, agar 20.0 g / L, pH 6.0, sterilized by moist heat at 121℃ for 30 min.

[0112] Under aseptic conditions, a certain amount of bacterial culture preserved in the glycerol tube was evenly spread onto an agar slant culture medium and incubated at 30°C for 12 hours.

[0113] (2) Seed culture

[0114] The primary culture medium consists of the following components by weight (g): glucose 12.0 g / L, (NH4)2SO4 1.0 g / L, yeast extract 10 g / L, KH2PO4 1.0 g / L, NaCl 2.0 g / L, raw material A 100 ppm, pH 6.0, autoclaved at 121℃ for 30 min, and cooled before use.

[0115] Two fresh slant cultures from step (1) were washed with sterile water under sterile conditions and then inoculated into a 30L seed tank. The culture was then incubated at 32°C for 12 hours to obtain the seed culture.

[0116] Secondary fermentation culture and induction

[0117] The secondary culture medium consists of the following components by weight (g): glucose 60.0 g / L, yeast extract 25.0 g / L, KH2PO4 1.0 g / L, (NH4)2SO4 5.0 g / L, K2HPO4 1.0 g / L, MgSO4 1.0 g / L, pH 6.5. It is autoclaved at 121℃ for 30 min and then cooled before use.

[0118] Secondary fermentation was carried out in a 5000L fermenter. The seed culture was transferred to the secondary fermentation medium at an inoculation rate of 0.5%. The temperature was 32℃, and the dissolved oxygen should not be lower than 30. Sugar and ammonia were added appropriately. When the culture time was 24h, vitamin D3 was added at a rate of 3g / L. The culture was continued for another 12h. The fermentation broth was milky white in appearance and had a strong yeast odor. Fermentation was stopped, the cells were filtered and the cells were repeatedly washed with purified water until the washing solution was colorless. Then, the cells were washed three times with 0.01M PBS buffer at pH 7.5.

[0119] III. Preparation of Immobilized Hydroxylase

[0120] The obtained bacterial cells were resuspended in 0.01M PBS buffer at pH 7.5, with a ratio of 2.0 g to 1 mL. After thorough mixing, the bacterial suspension was homogenized using a high-speed homogenizer at 18,000 rpm for 25 minutes, maintaining the system temperature at 4-15°C using a low-temperature circulator. The mixture was then filtered, and the supernatant was obtained as the crude hydroxylase solution. An encapsulating agent, sodium alginate (200 g / L), was added to the crude hydroxylase solution at a volume ratio of 1:1. After thorough mixing, glutaraldehyde was added, and the mixture was stirred for 30 minutes and then allowed to stand at 4°C for 3 hours to obtain the final solution. Then, add dropwise a 2% calcium chloride solution to form gel beads; stir and fix for 1.0 h, filter, and wash twice with a 0.9% sodium chloride solution to obtain immobilized hydroxylase.

[0121] IV. Formulation and Conversion System

[0122] The weight composition (kg) of the 1000L conversion system includes: glucose 1.0, emulsifier fatty alcohol polyoxyethylene ether 50.0, vitamin D3 50.0, dispersant polyethylene glycol 400 1.0, Na2HPO4 1.40, KH2PO4 0.24, MgSO4 0.1, raw material B 100, and pH value 7.0.

[0123] The specific preparation method is as follows: Dissolve glucose, Na2HPO4, KH2PO4, MgSO4, dispersant and emulsifier in 900L of sterile water, adjust the pH to 7.5 with 15% phosphoric acid to obtain phase A; dissolve vitamin D3 in 100L of raw material B to obtain phase B; under stirring, slowly add phase B to phase A and stir evenly to obtain the conversion system.

[0124] V. Transformation

[0125] Add the coenzyme NADPH mixture to the transformation system obtained in step (iv) above at a mass fraction of 1.0 g / L. Control the reaction temperature at 35℃, the rotation speed at 100 r / min, and stir for 15 min. Then add the immobilized enzyme obtained in step (iii) above into the transformation system. The mass-volume ratio of immobilized enzyme to the reaction transformation system is 1 g: 50 mL. The transformation time is 16 h. The molar conversion rate is 92.8% as detected by HPLC.

[0126] VI. Product Extraction and Refining

[0127] After the conversion was completed, the immobilized enzyme was removed by filtration. Butyl acetate was added to the filtrate, stirred for 1 hour, allowed to stand and separate into layers, and the upper organic phase was taken. After vacuum distillation, activated carbon decolorization, crystallization and other steps, crude 25-hydroxyvitamin D3 was obtained.

[0128] The crude product was then purified with a mixture of ethanol and water in a certain proportion to finally obtain 41 kg of refined 25-hydroxyvitamin D3, with a yield of 85% and a 25-hydroxyvitamin D3 content of 98.5%.

[0129] The method for preparing 25-hydroxyvitamin D3 using immobilized hydroxylase has the following advantages:

[0130] This helps to improve the molar conversion rate of vitamin D3, shorten the conversion cycle, and increase the total product yield. As described in the examples, at a feed concentration of 50 g / L, the substrate molar conversion rate can reach over 90%, the conversion time is within 16 hours, and the total product yield is over 80%.

[0131] This helps reduce production costs. The immobilized hydroxylase can be reused more than three times. The organic solvent in the reaction system can be recovered and recycled, which helps reduce the production cost of the product.

[0132] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing 25-hydroxyvitamin D3 using an immobilized hydroxylase, characterized in that, The conversion reaction is carried out in a conversion system using an immobilized hydroxylase. The raw materials of the conversion system include the following components: glucose 0.5-1.5 g / L, emulsifier 30.0-50.0 g / L, vitamin D3 25-50 g / L, and dispersant 0.5-1.5 g / L. The emulsifier is castor oil polyoxyethylene ether or fatty alcohol polyoxyethylene ether; The dispersant is polyethylene glycol; The pH value of the conversion system is 7.0-7.5; The method for preparing the immobilized hydroxylase includes the following steps: (1) The bacterial suspension of the vitamin D3 hydroxylase-producing genetically engineered bacteria was disrupted by cell disruption, and the supernatant was obtained by centrifugation or filtration; (2) Add sodium alginate and glutaraldehyde to the supernatant obtained in step 1) to obtain a mixture, and then add calcium chloride solution to form gel beads; The amount of sodium alginate used is 100-200 g / L.

2. The method for preparing 25-hydroxyvitamin D3 using immobilized hydroxylase according to claim 1, characterized in that, The ratio of immobilized hydroxylase to the transformation system was 1 g: (50-150) mL.

3. The method for preparing 25-hydroxyvitamin D3 using immobilized hydroxylase according to claim 1, characterized in that, The dispersant is polyethylene glycol 200 or polyethylene glycol 400.

4. The method for preparing 25-hydroxyvitamin D3 using immobilized hydroxylase according to claim 1, characterized in that, The raw materials for the conversion system also include a buffer solution, which includes phosphate buffer and MgSO4.

5. The method for preparing 25-hydroxyvitamin D3 using immobilized hydroxylase according to claim 4, characterized in that, The phosphate buffer solution includes Na2HPO4 and KH2PO4.

6. The method for preparing 25-hydroxyvitamin D3 using immobilized hydroxylase according to claim 4, characterized in that, The phosphate buffer solution comprises 1.40-1.49 g / L Na₂HPO₄ and 0.22-0.26 g / L KH₂PO₄.

7. The method for preparing 25-hydroxyvitamin D3 using immobilized hydroxylase according to claim 4 or 6, characterized in that, The buffer solution contains 0.015-0.05 g / L MgSO4.

8. The method for preparing 25-hydroxyvitamin D3 using immobilized hydroxylase according to any one of claims 1-2, characterized in that, A cofactor for vitamin D3 hydroxylase is added during the conversion process; The cofactor is one or a mixture of NADPH, NADH and vitamin C.

9. The method for preparing 25-hydroxyvitamin D3 using immobilized hydroxylase according to any one of claims 1-2, characterized in that, During the conversion process: control the reaction temperature at 30-35℃, the rotation speed at 100-220r / min, and the conversion time at 12-16h.

10. The method for preparing 25-hydroxyvitamin D3 using immobilized hydroxylase according to claim 1, characterized in that, The genetically engineered bacteria are one of yeast, Escherichia coli, or autotrophic amylopectin bacteria.

11. The method for preparing 25-hydroxyvitamin D3 using immobilized hydroxylase according to claim 1, characterized in that, The genetically engineered bacteria is yeast.

12. The method for preparing 25-hydroxyvitamin D3 using immobilized hydroxylase according to any one of claims 1-2, characterized in that, The preparation of the immobilized hydroxylase also includes a hydroxylase induction step.

13. The method for preparing 25-hydroxyvitamin D3 using immobilized hydroxylase according to any one of claims 1-2, characterized in that, After the conversion was completed, the immobilized hydroxylase was recovered by filtration, and then extracted with organic solvent. The filtrate was centrifuged and the organic phase was collected. After decolorization, distillation and purification, 25-hydroxyvitamin D3 was obtained.