Fusion p450 enzyme mutants and methods of increasing catalytic activity towards vd3, methods of whole cell production of 25(oh)vd3

Abstract

The present application relates to the field of agricultural biotechnology, in particular to fusion P450 enzyme mutants, methods for improving the catalytic activity of VD3, and methods for producing 25(OH)VD3 by whole cells, wherein a fusion P450 enzyme VK1-CYP116B46-L21 and a self-sufficient fusion P450 enzyme mutant with improved catalytic activity for synthesizing calcidiol from vitamin D3 are constructed; using VD3 as a substrate, adding glucose, a cosolvent, and a recombinant Escherichia coli expressing the fusion P450 enzyme and glucose dehydrogenase after induction, the maximum yield of 25(OH)VD3 can reach 3.26 g / L within 24 h after the catalytic reaction; directly using whole cells as catalysts without purifying enzymes, introducing glucose dehydrogenase to regenerate cofactor NAD(P)H using glucose as a substrate, avoiding the addition of expensive cofactors, reducing production costs, and being conducive to industrial production.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a method for fusion with a P450 enzyme mutant and improving its catalytic activity for VD3, and a method for whole-cell generation of 25(OH)VD3. Background Technology

[0002] Vitamin D3 (VD3), commonly known as cholecalciferol, plays a crucial role in many biological processes. Compared to VD3, 25-hydroxyvitamin D3 (25(OH)VD3) exhibits greater water solubility and higher bioavailability.

[0003] Currently, common methods for producing 25(OH)VD3 include chemical synthesis and biotransformation. Compared to chemical synthesis, C25 hydroxylase catalysis using VD3 as a substrate requires only one reaction step, exhibits high catalytic efficiency and excellent selectivity, and operates under mild conditions with minimal environmental impact. P450, a member of the heme oxidase superfamily known for its regioselective and stereoselective transformations, holds great potential for 25(OH)VD3 synthesis. However, the industrial application of P450 faces challenges such as the need for additional redox chaperones, limited catalytic activity, low coupling efficiency, and insufficient stability, resulting in high production costs.

[0004] In the catalytic cycle of P450, substrate hydroxylation depends on the efficiency of electron transfer from the redox chaperone to heme iron. This typically requires the addition of additional redox chaperone proteins to perform the electron transfer function, increasing the cost of P450 in industrial applications. Furthermore, electron transfer is the rate-limiting step in the P450 reaction. When this process is not coordinated with the substrate's catalytic efficiency, it can trigger uncoupling reactions, leading to decreased P450 coupling efficiency, reactive oxygen species accumulation, and reduced enzyme activity. Self-sufficient fusion P450 enzymes integrate heme catalytic and redox enzyme domains on a single polypeptide chain, with the domains linked by linkers, simplifying and facilitating electron transfer. Compared to multi-component P450 enzymes, they generally exhibit higher catalytic efficiency. Self-sufficient fusion P450 enzymes rely on their own conformational motion to achieve efficient interdomain electron transfer. The electron transfer efficiency depends on the efficiency of electron transfer from the reductase domain to the heme center and the ability of the heme domain to utilize transferred electrons for efficient catalysis. A comprehensive strategy that considers both electron transfer pathways and substrate specificity can effectively improve electron transfer efficiency. Therefore, developing a self-sufficient fusion P450 enzyme with C-25 hydroxylation activity for VD3, and improving its catalytic activity for VD3 through modification of electron transfer pathway and substrate specificity, has significant potential for industrial application. Summary of the Invention

[0005] The purpose of this invention is to provide a mutant fused with the P450 enzyme.

[0006] Another object of the present invention is to provide a method for improving the catalytic activity of fused P450 enzymes for VD3.

[0007] Another object of the present invention is to provide a method for producing 25-hydroxyvitamin D3 using a whole-cell catalyst, thereby solving the problem in the prior art where the low catalytic activity of P450 enzymes for VD3 leads to low yield of 25(OH)VD3, making it difficult to use in industrial production.

[0008] According to the present invention, a self-sufficient fusion P450 enzyme mutant catalyzing the synthesis of calcidiol from vitamin D3 with enhanced activity, wherein the fusion P450 enzyme is P450 enzyme VK1-CYP116B46-L21, comprising the heme catalytic domain of P450 enzyme Vdh-K1 derived from the autotrophic pseudonocardia autotrophica, and the redox domain of CYP116B46 derived from the thermophilic bacterium Tepidiphilus thermophiles, forming a wild-type fusion P450 enzyme VK1-CYP116B46-L21, the amino acid sequence of which is shown in SEQ ID NO:1. Using the wild-type P450 enzyme VK1-CYP116B46-L21 shown in SEQ ID NO:1 as a template, a mutation was made at position 180, and the lysine K at position 180 was mutated to phenylalanine F, resulting in the P450 enzyme mutant K180F, the amino acid sequence of which is shown in SEQ ID NO:2.

[0009] Furthermore, the two-point mutants are A177M / K180F (SEQ ID NO:3), P83A / K180F (SEQ ID NO:4), K180F / F346K (SEQ ID NO:5), and K180F / R354M (SEQ ID NO:6).

[0010] Furthermore, the three-point mutant is P83A / A177M / K180F, with the amino acid sequences shown in SEQ ID NO:7, P83A / K180F / F346K (SEQ ID NO:8), P83A / K180F / R354M (SEQ ID NO:9), A177M / K180F / F346K (SEQ ID NO:10), A177M / K180F / R354M (SEQ ID NO:11), and K180F / F346K / R354M (SEQ ID NO:12).

[0011] Furthermore, the four-point mutants are P83A / A177M / K180F / F346K (SEQ ID NO:13), A177M / K180F / F346K / R354M (SEQ ID NO:14), P83A / K180F / F346K / R354M (SEQ ID NO:15), and P83A / A177M / K180F / R354M (SEQ ID NO:16).

[0012] Furthermore, the five-point mutant is P83A / A177M / K180F / F346K / R354M, with the amino acid sequence shown in SEQ ID NO:17.

[0013] According to a specific embodiment of the present invention, a co-expression plasmid comprising the above-described fusion P450 enzyme mutant encoding gene and glucose dehydrogenase gene is provided.

[0014] The present invention provides recombinant cells comprising a co-expression plasmid containing the above-mentioned fusion of P450 enzyme or mutant and glucose dehydrogenase encoding gene.

[0015] In an embodiment of the present invention, the recombinant cells are recombinant Escherichia coli. Escherichia coli BL21(DE3).

[0016] The present invention provides a whole-cell catalyst containing the recombinant cells.

[0017] According to the present invention, the method for catalytically preparing 25(OH)VD3 involves adding the whole-cell catalyst to a VD3 solution to catalytically prepare 25(OH)VD3.

[0018] The method for catalytically preparing 25(OH)VD3 according to the present invention comprises the following steps:

[0019] The recombinant cells were cultured in LB medium at 37°C. OD 600 When the concentration is 0.6-1.0, add β-d-1-thiogalactopyranoside (IPTG) and 0.1-1.0 mM 5-aminoacetylalanine salt (5-ALA) to a final concentration of 0.1-1.0 mM, and induce at 16-37 ℃ for 5-20 h. Collect wet cells to obtain wet cells; inoculate the wet cells into VD3 solution and react at pH 6.5-8 and 20-40 ℃ for 2-24 h.

[0020] In some embodiments of the present invention, the VD3 solution is prepared from VD3, purified water, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, cosolvent, and co-substrate.

[0021] In some embodiments of the present invention, the cosolvent is hydroxypropyl-β-cyclodextrin and isopropanol / or ethanol.

[0022] In some embodiments of the present invention, the initial concentration of VD3 is 2-10 mM.

[0023] In some embodiments of the present invention, the concentration of the hydroxypropyl-β-cyclodextrin is 20-100 g / L.

[0024] In some embodiments of the present invention, the concentration of isopropanol / or ethanol is 5%-20% (v / v).

[0025] In some embodiments of the present invention, the co-substrate is glucose.

[0026] In some embodiments of the present invention, the concentration of the co-substrate is 25 g / L.

[0027] The beneficial effects of this invention are:

[0028] This invention constructs a fusion P450 enzyme VK1-CYP116B46-L21 and mutants with different VK1-CYP116B46-L21 electron transport pathways and substrate binding pockets. After screening, a self-sufficient fusion P450 enzyme mutant with enhanced activity catalyzing the synthesis of calcidiol from vitamin D3 was obtained. Using VD3 as a substrate, with the addition of glucose, a solubilizer, and recombinant E. coli expressing both the fusion P450 enzyme and glucose dehydrogenase, the induced wet cells achieved a maximum yield of 3.26 g / L of 25(OH)VD3 within 24 h after catalytic reaction. Directly using whole cells as a catalyst eliminates the need for enzyme purification. The introduction of glucose dehydrogenase and the regeneration of the cofactor NAD(P)H using glucose as a substrate avoids the addition of expensive cofactors, reduces production costs, and is beneficial for industrial production. Attached Figure Description

[0029] Figure 1 The wild-type VK1-CYP116B46-L21 and its mutants showed the ability to generate 25(OH)VD3;

[0030] Figure 2 This shows the yield of 25(OH)VD3 in the whole-cell conversion reaction of substrate VD3. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0032] Experimental materials and reagents:

[0033] 1. Strains and vectors: The expression host is *Escherichia coli*. Escherichia coli BL21(DE3), the expression plasmid vector is pETDuet1;

[0034] 2. Enzymes: endonucleases, ligases;

[0035] 3. Culture medium:

[0036] LB medium (g / L): 10 g peptone, 5 g yeast extract, 10 g sodium chloride;

[0037] TB medium (g / L): peptone 12 g, yeast extract 24 g, glycerol 5 g, dipotassium hydrogen phosphate trihydrate 16.37 g, potassium dihydrogen phosphate 2.31 g.

[0038] The primers used in the following examples are: VK1-F (SEQ ID NO:18); VK1-CYP116B46-R (SEQ ID NO:19); VK1-CYP116B46-F (SEQ ID NO:20); CYP116B46-R (SEQ ID NO:21); VK1-RBS-GDH-F (SEQ ID NO:22); GDH-R (SEQ ID NO:23); P83A-F (SEQ ID NO:24); P83A-R (SEQ ID NO:25); A177M-F (SEQ ID NO:26); A177M-R (SEQ ID NO:27); K180F-F (SEQ ID NO:28); K180F-R (SEQ ID NO:29); F346K-F (SEQ ID NO:30); F346K-R (SEQ ID NO:31); R354M-F (SEQ ID NO:30); NO:32); R354M-R (SEQ ID NO:33); K180F / A177M-F (SEQ ID NO:34); K180F / A177M-R (SEQ ID NO:35).

[0039] Unless otherwise specified in the following examples, the molecular biology experimental methods were performed in accordance with the specific methods listed in J. Sambrook's "Molecular Cloning: A Laboratory Manual" (3rd Edition), or according to the kit and product instructions.

[0040] Example 1: Preparation of recombinant strain BL21 (pETDuet1-VK1-CYP116B46-L21-GDH)

[0041] The plasmid pETDuet1 was double-digested with restriction endonucleases BamHI and Hind III; the amplification of *Pseudomonas autotrophus* was performed using primers VK1-F and VK1-CYP116B46-R with homologous ends. Pseudonocardia autotrophica The P450 enzyme Vdh-K1 gene sequence was obtained; primers VK1-CYP116B46-F and CYP116B46-R with homologous ends were used to amplify the gene from thermophilic bacteria ( ). Tepidiphilus thermophiles The gene sequence of the redox domain of the P450 enzyme CYP116B46 was obtained. The PCR product and enzyme digestion product were recovered and purified separately, and recombinant ligation was performed using a recombinase kit. The recombinant product was then transformed into *E. coli* Trans1-T1 competent cells and plated on LB agar (containing 100 μg / mL Ampicillin) for selection. After successful sequencing, the plasmid pETDuet1-VK1-CYP116B46-L21 was extracted.

[0042] The plasmid pETDuet1-VK1-CYP116B46-L21 was double-digested with restriction endonucleases Hind III and NotI. Primers with homologous ends VK1-RBS-GDH-F and GDH-R were used to amplify the plasmid derived from *Priscilla megaterium*. Priestia megaterium The glucose dehydrogenase GDH of (Gene ID: 64145183) was extracted. The PCR product and enzyme digestion product were separately recovered and purified, and recombinant ligation was performed using a recombinase kit. The recombinant product was then transformed into *E. coli* Trans1-T1 competent cells and plated on LB agar (containing 100 μg / mL Ampicillin) for selection. After successful sequencing, the plasmid pETDuet1-VK1-CYP116B46-L21-GDH was extracted and transformed into *E. coli* BL21(DE3) to obtain the recombinant strain BL21.

[0043] Example 2: Construction and screening of mutants

[0044] Constructing mutants

[0045] Three potential hotspots, P83, A177, and K180, located at the substrate-binding pocket entrance and potentially affecting substrate-enzyme binding, were identified, along with potential electron transport sites F346 and R354. Single-point saturation mutagenesis and iterative saturation mutagenesis were performed on these five amino acid residues to construct a mutant library. Based on VK1-CYP116B46-L21, mutation primers were designed according to the mutant, and the plasmid pETDuet1-VK1-CYP116B46-L21-GDH was used as a template for mutation. PCR products were digested with DpnI to remove the template. The digested PCR products were transformed into *E. coli* Tans1-T1 competent cells and sequenced for verification. Then, *E. coli* BL21(DE3) was transformed to obtain recombinant *E. coli* strain BL21. The names of each recombinant strain, the included plasmids, enzymes (wild-type WT or mutant), and amino acid sequences are shown below.

[0046] BL21 1, pETDuet1-VK1-CYP116B46-L21-GDH, WT, SEQ ID NO: 1;

[0047] BL21 2, pETDuet1-VK1-CYP116B46-L21-P83A-GDH, P83A;

[0048] BL21 3, pETDuet1-VK1-CYP116B46-L21-A177M-GDH, A177M;

[0049] BL21 4, pETDuet1-VK1-CYP116B46-L21-K180F-GDH, K180F, SEQ ID NO: 2;

[0050] BL21 5, pETDuet1-VK1-CYP116B46-L21-F346K-GDH, F346K;

[0051] BL21 6, pETDuet1-VK1-CYP116B46-L21-R354M-GDH, R354M;

[0052] BL21 7, pETDuet1-VK1-CYP116B46-L21-P83A-K180F-GDH, P83A / K180F, SEQ IDNO: 4;

[0053] BL21 8, pETDuet1-VK1-CYP116B46-L21-A177M-K180F-GDH, A177M / K180F, SEQ IDNO: 3;

[0054] BL21 9, pETDuet1-VK1-CYP116B46-L21-K180F-F346K-GDH, K180F / F346K, SEQ ID NO:5;

[0055] BL21 10, pETDuet1-VK1-CYP116B46-L21-K180F-R354M-GDH, K180F / R354M, SEQ ID NO:6;

[0056] BL21 11,pETDuet1-VK1-CYP116B46-L21-P83A-A177M-GDH,P83A / A177M;

[0057] BL21 12,pETDuet1-VK1-CYP116B46-L21-P83A-F346K-GDH,P83A / F346K;

[0058] BL21 13,pETDuet1-VK1-CYP116B46-L21-P83A-R354M-GDH,P83A / R354M;

[0059] BL21 14,pETDuet1-VK1-CYP116B46-L21-A177M-F346K-GDH,A177M / F346K;

[0060] BL21 15,pETDuet1-VK1-CYP116B46-L21-A177M-R354M-GDH,A177M / R354M;

[0061] BL21 16,pETDuet1-VK1-CYP116B46-L21-F346K-R354M-GDH,F346K / R354M;

[0062] BL21 17,pETDuet1-VK1-CYP116B46-L21-P83A-A177M-K180F-GDH,P83A / A177M / K180F,SEQ ID NO:7;

[0063] BL21 18,pETDuet1-VK1-CYP116B46-L21-P83A-A177M-F346K-GDH,P83A / A177M / F346K;

[0064] BL21 19,pETDuet1-VK1-CYP116B46-L21-P83A-A177M-R354M-GDH,P83A / A177M / R354M;

[0065] BL21 20,pETDuet1-VK1-CYP116B46-L21-P83A-K180F-F346K-GDH,P83A / K180F / F346K,SEQ ID NO:8;

[0066] BL21 21,pETDuet1-VK1-CYP116B46-L21-P83A-K180F-R354M-GDH,P83A / K180F / R354M,SEQ ID NO:9;

[0067] BL21 22,pETDuet1-VK1-CYP116B46-L21-P83A-F346K-R354M-GDH,P83A / F346K / R354M;

[0068] BL21 23,pETDuet1-VK1-CYP116B46-L21-A177M-K180F-F346K-GDH,A177M / K180F / F346K,SEQ ID NO:10;

[0069] BL21 24,pETDuet1-VK1-CYP116B46-L21-A177M-K180F-R354M-GDH,A177M / K180F / R354M,SEQ ID NO:11;

[0070] BL21 25,pETDuet1-VK1-CYP116B46-L21-A177M-F346K-R354M-GDH,A177M / F346K / R354M;

[0071] BL21 26,pETDuet1-VK1-CYP116B46-L21-K180F-F346K-R354M-GDH,K180F / F346K / R354M,SEQ ID NO:12;

[0072] BL21 27,pETDuet1-VK1-CYP116B46-L21-A177M-K180F-F346K-R354M-GDH,A177M / K180F / F346K / R354M,SEQ ID NO:14;

[0073] BL21 28, pETDuet1-VK1-CYP116B46-L21-P83A-K180F-F346K-R354M-GDH, P83A / K180F / F346K / R354M, SEQ ID NO: 15;

[0074] BL21 29, pETDuet1-VK1-CYP116B46-L21-P83A-A177M-F346K-R354M-GDH, P83A / A177M / F346K / R354M;

[0075] BL21 30, pETDuet1-VK1-CYP116B46-L21-P83A-A177M-K180F-R354M-GDH, P83A / A177M / K180F / R354M, SEQ ID NO: 16;

[0076] BL21 31, pETDuet1-VK1-CYP116B46-L21-P83A-A177M-K180F-F346K-GDH, P83A / A177M / K180F / F346K, SEQ ID NO: 13;

[0077] BL21 32, pETDuet1-VK1-CYP116B46-L21-P83A-A177M-K180F-F346K-R354M-GDH, P83A / A177M / K180F / F346K / R354M, SEQ ID NO: 17.

[0078] (2) Mutant screening:

[0079] The 32 mutant strains obtained in step (1) were inoculated into LB medium and cultured at 200 rpm and 37 ℃ for about 10 h. Then, they were inoculated into TB medium at an inoculation rate of 1% of the medium volume and cultured at 200 rpm and 37 ℃ until OD. 600 =Approximately 0.8, add IPTG to a final concentration of 1.0 mM and 5-ALA of 1.0 mM, induce at 200 rpm and 25 ℃ for 18 h, centrifuge at 6000 rpm for 10 min to collect the bacterial cells, and then proceed with the substrate VD3.

[0080] Whole-cell transformation reaction was used to screen for mutants with improved transformation efficiency.

[0081] Whole-cell transformation reaction system: VD3 substrate (final concentration 1 mM) and recombinant E. coli cells after induction (final cell concentration OD) were added to potassium phosphate buffer (50 mM, pH 7.4) supplemented with 50 g / L hydroxypropyl-β-cyclodextrin and 10% (v / v) ethanol. 600 = 10), and finally add glucose (final concentration 25 g / L). React at 25°C and 200 rpm for 1 h. After the reaction is complete, add an equal volume of ethyl acetate to terminate the reaction and extract. Vortex the mixture at high speed for 10 min, then centrifuge at high speed (12000 rpm, 10 min). Collect the upper organic phase, filter it through a 0.22 μm organic filter membrane, and perform HPLC analysis.

[0082] The concentrations of VD3 and 25(OH)VD3 in the reaction solution were analyzed by HPLC: an Agilent C18 column (5 μm × 4.6 mm × 250 mm) was used, with methanol / acetonitrile (30:70, v / v) as the mobile phase; the detection wavelength was set to 264 nm; the flow rate was maintained at 1.0 mL / min; the injection volume was 10 μL; the column temperature was 30 °C; and the detection time was 15 min. The catalytic activity of the constructed mutants was evaluated by the conversion of substrate VD3. The mutants with higher 25(OH)VD3 production capacity than the wild type were obtained, such as... Figure 1 As shown in the figure, the activity of all mutants is improved to varying degrees. Among them, the five-point mutant VK1-CYP116B46 L21P83A / A177M / K180F / F346K / R354M has the highest 25(OH)VD3 production, which is 8.8 times that of the wild type.

[0083] Example 3: Gram-scale preparation of 25(OH)VD3

[0084] The recombinant strain constructed in Example 2 E. coli BL21 32 / pETDuet1-VK1-CYP116B46-L21-P83A-A177M-K180F-F346K-R354M-GDH was inoculated into LB medium and cultured at 200 rpm and 37 ℃ for about 10 h. Then, it was inoculated into TB medium at 1% of the medium volume and cultured at 200 rpm and 37 ℃ until OD... 600 = 0.8, add IPTG and 5-ALA to a final concentration of 1.0 mM, induce at 200 rpm and 25 ℃ for 18 h, centrifuge at 6000 rpm for 10 min to collect the cells, and carry out whole-cell transformation reaction of substrate VD3.

[0085] The VD3 concentration in the entire reaction system was 10 mM during the whole-cell transformation reaction, and the reaction time was 24 h. All other operations were the same as in Example 2. After the reaction was completed, an equal volume of ethyl acetate was added to terminate the reaction and extract the mixture. The mixture was then vortexed at high speed for 10 min, centrifuged at 12000 rpm for 10 min, and the upper organic phase was collected. This phase was filtered through a 0.22 μm organic filter membrane and analyzed by HPLC. The yield was as follows: Figure 2 As shown, VK1-CYP116B46-L21 P83A / A177M / K180F / F346K / R354M achieved 82% substrate conversion at 24 h, with a yield of 8.16 mM (approximately 3.26 g / L).

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention; that is, all simple and equivalent changes and modifications made based on the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. Mutant of a fusion P450 enzyme with increased activity for the synthesis of 25(OH)VD3 from catalytic vitamin D3, characterized in that, The amino acid sequence of the fusion P450 enzyme mutant is shown in any one of SEQ ID NO:2~SEQ ID NO:

17.

2. A fused P450 enzyme gene characterized in that, The fusion P450 enzyme gene encodes a protein with an amino acid sequence shown in any one of SEQ ID NO:2~SEQ ID NO:

17.

3. A recombinant expression vector comprising the fusion P450 enzyme gene of claim 2.

4. The recombinant expression vector of claim 3, wherein, The recombinant expression vector comprises the fusion P450 enzyme gene of claim 2 and a glucose dehydrogenase gene.

5. A recombinant strain comprising the fusion P450 enzyme gene of claim 2 and a glucose dehydrogenase gene.

6. Use of the recombinant strain of claim 5 as a whole-cell catalyst for catalyzing the synthesis of 25(OH)VD3 from vitamin D3.

7. A method of improving the catalysis of vitamin D3 synthesis to 25(OH)VD3 by a fused P450 enzyme, characterized in that, The method comprises the following steps: The fusion P450 enzyme with an amino acid sequence shown in SEQ ID NO:1 is subjected to any one of the following mutations: K180F; P83A / A177M / K180F, P83A / K180F / F346K, P83A / K180F / R354M, A177M / K180F / F346K, A177M / K180F / R354M or K180F / F346K / R354M; P83A / A177M / K180F / F346K, A177M / K180F / F346K / R354M, P83A / K180F / F346K / R354M or P83A / A177M / K180F / R354M; or P83A / A177M / K180F / F346K, A177M / K180F / F346K / R354M, P83A / K180F / F346K / R354M or P83A / A177M / K180F / R354M; or P83A / A177M / K180F / F346K, A177M / K180F / F346K / R354M, P83A / K180F / F346K / R354M or P83A / A177M / K180F / R354M; or 8. A method of catalytically producing 25(OH)VD3, characterized in that, P83A / A177M / K180F / F346K, A177M / K180F / F346K / R354M, P83A / K180F / F346K / R354M or P83A / A177M / K180F / R354M; or The method comprises the following steps:

9. The method of catalytically producing 25(OH)VD3 according to claim 8, wherein, The recombinant strain comprising the fusion P450 enzyme gene of claim 2 and a glucose dehydrogenase gene is added to a solution containing VD3 and glucose to catalyze the preparation of 25(OH)VD3, wherein the solution containing VD3 and glucose contains a cosolvent, and the cosolvent is hydroxypropyl-β-cyclodextrin and ethanol.

10. The method of catalytically producing 25(OH)VD3 according to claim 8, wherein, After the addition of the recombinant strain, the reaction is carried out at pH 6.5~8 and 20-40℃ for 2-24h. The initial concentration of VD3 is 2-10mM, and the concentration of glucose is 25g / L.

Images