A P450 enzyme mutant, a single plasmid three-enzyme co-expression system, and its application in calcifediol synthesis

Abstract

The present invention discloses a P450 enzyme mutant, a single-plasmid three-enzyme co-expression system, and their application in calcifediol synthesis. The P450 enzyme mutants include: using the wild-type P450 enzyme CYP109E1 shown in SEQ ID NO. 1 as a template, and respectively mutating the T at position 78 or the G at position 81 to form the mutants T78A, T78V, T78L, and T78I, G81V, and G81M; and mutating both the T at position 78 and the G at position 81 to form the mutant T78L / G81M. The present invention also constructs a single-plasmid three-enzyme co-expression system of the P450 enzyme mutant and a redox partner, thereby improving the electron transfer efficiency in the P450 enzyme hydroxylation reaction. This system can synthesize calcifediol with higher efficiency under mild conditions, solving the problems of low catalytic efficiency and activity in the synthesis of calcifediol catalyzed by P450 enzymes, and has important industrial application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biocatalysis, and specifically relates to a P450 enzyme mutant with enhanced activity for catalyzing the synthesis of calcifediol from vitamin D3, a single-plasmid three-enzyme co-expression system constructed thereby, and its application in the synthesis of calcifediol. Background Art

[0002] Calcifediol is the active form of vitamin D3 produced in vivo through cytochrome P450-mediated hydroxylation at the 25-position and is the primary storage form of vitamin D3 in the body. Activated vitamin D3 maintains calcium homeostasis by binding to vitamin D receptors, regulating calcium and phosphorus absorption in the intestine, phosphate reabsorption in the kidneys, and calcium and phosphate release in the bones. This valuable contribution to the prevention or treatment of various chronic diseases, such as cancer, cardiovascular disease, and immune disorders, is made. Calcifediol can also be further hydroxylated to form calcitriol, which can be used to treat conditions such as rickets and hypothyroidism, and to prevent and treat liver cirrhosis.

[0003] The main methods for synthesizing calcifediol include chemical synthesis and biocatalysis. Chemical synthesis converts cholesterol into calcifediol through approximately 20 steps, but this synthesis method is cumbersome and environmentally hazardous, with a low final yield, making it difficult to apply on a large-scale industrial scale. Biocatalysis, on the other hand, offers the advantages of high regioselectivity, simple reaction steps, and minimal environmental pollution. The hydroxylation reaction catalyzed by P450 enzymes can introduce one of the oxygen atoms of an oxygen molecule into an inactive C-H bond under relatively mild conditions, while the other is reduced to water. This reaction exhibits both regio- and stereoselectivity, making P450 enzymes the most promising enzymes for synthesizing calcifediol. However, most P450 enzymes are currently difficult to express in vitro and exhibit extremely low catalytic activity.

[0004] Cytochrome P450 enzymes are a superfamily of B-type heme-containing proteins found in diverse organisms throughout nature. They possess a versatility unmatched by chemical catalysts, catalyzing over 20 reactions, including toxin metabolism and hormone synthesis. The P450 catalytic cycle requires the participation of redox chaperone proteins. Based on the interaction patterns between the redox chaperone and the P450 enzyme, P450 enzyme catalytic systems can be divided into three main types: the first type is a three-component system, consisting of the P450 enzyme, ferredoxin reductase (FdR), and ferredoxin (Fdx), which is commonly found in prokaryotes. The second type is a two-component system, commonly found in eukaryotic P450 enzymes, whose redox partner is the cytochrome P450 reductase (CPR). The third type is a one-component system, in which the N-terminal P450 enzyme and the C-terminal FAD / FMN redox chaperone domain are naturally fused, often referred to as "self-sufficient" P450 enzymes. Since prokaryotic P450 enzymes can achieve low-level soluble expression in vitro and are more suitable for synthetic applications, developing a three-enzyme system with C-25 hydroxylation activity on vitamin D3 and improving the catalytic activity of CYP109E1 on the non-natural substrate vitamin D3 through molecular modification has significant industrial application potential. Summary of the Invention

[0005] The purpose of the present invention is to provide a P450 enzyme mutant, a single plasmid three-enzyme co-expression system and its application in the synthesis of calcifediol, so as to solve the problem in the prior art that the P450 enzyme has low catalytic activity towards its non-natural substrate vitamin D3, resulting in low calcifediol production and difficulty in industrial production.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] According to a first aspect of the present invention, a P450 enzyme mutant with enhanced activity for catalyzing the synthesis of calcifediol from vitamin D3 is provided, comprising a wild-type P450 enzyme CYP109E1 (derived from Bacillus megaterium DSM319) shown in SEQ ID NO.1 as a template, wherein the amino acid residue 78 or the amino acid residue 81 undergoes the following mutation: a P450 enzyme mutant T78A in which the threonine T at position 78 is mutated to alanine A, the amino acid sequence of which is shown in SEQ ID NO.2; a P450 enzyme mutant T78V in which the threonine T at position 78 is mutated to valine V, the amino acid sequence of which is shown in SEQ ID NO.3; a P450 enzyme mutant T78L in which the threonine T at position 78 is mutated to leucine L, the amino acid sequence of which is shown in SEQ ID NO.4; and a P450 enzyme mutant T78I in which the threonine T at position 78 is mutated to isoleucine I, the amino acid sequence of which is shown in SEQ ID NO. NO.5; a P450 enzyme mutant G81V in which the glycine G at position 81 is mutated to valine V, and the amino acid sequence is shown in SEQ ID NO.6; and a P450 enzyme mutant G81M in which the glycine G at position 81 is mutated to methionine M, and the amino acid sequence is shown in SEQ ID NO.7; and a P450 enzyme mutant T78L / G81M in which the glycine G at position 81 is mutated to methionine M using the mutant P450 enzyme CYP109E1-T78L shown in SEQ ID NO.4 as a template.

[0008] According to the second aspect of the present invention, a gene encoding the P450 enzyme mutant is provided.

[0009] According to the third aspect of the present invention, a recombinant vector comprising the encoding gene is provided.

[0010] According to a fourth aspect of the present invention, a method for constructing a single-plasmid three-enzyme co-expression system is provided, comprising the following steps: 1) using the wild-type P450 enzyme CYP109E1 shown in SEQ ID NO.1 as a template, respectively mutating the T at position 78 or the G at position 81 and mutating the T at position 78 and the G at position 81 to form P450 enzyme mutants T78A, T78V, T78L, T78I, G81V, G81M and T78L / G81M, inserting them into the expression plasmid pET28a(+) to obtain various mutant plasmids pET28a(+)-CYP109E1; 2) amplifying the ferredoxin reductase P from Pseudomonas aeruginosa, respectively. The gene sequences of dR and ferredoxin Pdx are inserted into the expression plasmid pET28a(+) to obtain plasmid pET28a(+)-PdR and plasmid pET28a(+)-Pdx, respectively; 3) the plasmid pET28a(+)-PdR, the plasmid pET28a(+)-Pdx, and any one of the mutant plasmids pET28a(+)-CYP109E1 are respectively cut with an endonuclease, and the resultant enzymes are sequentially connected and transformed into the host Escherichia coli BL21(DE3) to construct a single-plasmid three-enzyme co-expression system.

[0011] According to the construction method of the single-plasmid three-enzyme co-expression system provided by the present invention, the P450 enzyme mutant T78A in which the threonine T at position 78 is mutated to alanine A, has an amino acid sequence as shown in SEQ ID NO.2; the P450 enzyme mutant T78V in which the threonine T at position 78 is mutated to valine V, has an amino acid sequence as shown in SEQ ID NO.3; the P450 enzyme mutant T78L in which the threonine T at position 78 is mutated to leucine L, has an amino acid sequence as shown in SEQ ID NO.4; the P450 enzyme mutant T78I in which the threonine T at position 78 is mutated to isoleucine I, has an amino acid sequence as shown in SEQ ID NO.5; the P450 enzyme mutant G81V in which the glycine G at position 81 is mutated to valine V, has an amino acid sequence as shown in SEQ ID NO.6; the P450 enzyme mutant G81M in which the glycine G at position 81 is mutated to methionine M, has an amino acid sequence as shown in SEQ ID NO.7; the P450 enzyme mutant T78L / G81M in which the threonine T at position 78 is mutated to leucine L and the glycine G at position 81 is mutated to methionine M, has an amino acid sequence as shown in SEQ ID NO.8.

[0012] The ferredoxin reductase PdR and ferredoxin Pdx are both derived from Pseudomonas putida. The amino acid sequence of the ferredoxin reductase PdR is shown in SEQ ID NO.9, and the amino acid sequence of the ferredoxin Pdx is shown in SEQ ID NO.10.

[0013] According to a fifth aspect of the present invention, a single-plasmid three-enzyme co-expression system constructed by the construction method is provided.

[0014] Preferably, the single-plasmid three-enzyme co-expression system uses the P450 enzyme double-point combined mutant T78L / G81M.

[0015] According to a sixth aspect of the present invention, there is provided a single-plasmid three-enzyme co-expression system for use in the synthesis of calcifediol.

[0016] According to a preferred embodiment of the present invention, the application includes the following steps: S1: culturing the single-plasmid three-enzyme co-expression system in TB culture medium, inducing expression, and obtaining a crude enzyme solution; S2: mixing the solubilizer hydroxypropyl-β-cyclodextrin and Tris-HCl buffer in a mass-to-volume ratio of 45%, adding the substrate vitamin D3 after vortexing, vortexing thoroughly until clear, preparing a 4mM mother liquor, adding the substrate vitamin D3 to a final concentration of 0.3-0.5mM, then adding the crude enzyme solution and a certain amount of cofactor NAD(P)H, placing the mixture in a constant temperature shaker at 30°C and 200-250rpm for 20-30h to carry out the conversion reaction of the substrate vitamin D3 to achieve the synthesis of calcifediol.

[0017] The present invention first docks vitamin D3 into the P450 enzyme CYP109E1 (PDB ID: 5L92, resolution: ), through analysis of the binding pocket of vitamin D3 for P450 enzymes, combined with molecular dynamics simulations, the amino acid residues 78 and 81 were identified as key to the substrate specificity of the P450 enzyme CYP109E1. After saturation mutagenesis of these residues, the activity of each mutant in catalyzing the synthesis of calcifediol was measured. To analyze the synergistic effects between them, mutants with significantly improved activity were combined, and the optimal double-point mutant CYP109E1-T78L / G81M was identified, in which amino acid residues 78 and 81 were simultaneously mutated. Through these protein engineering strategies, multiple mutants with enhanced P450 enzyme activity were obtained, and the acquisition of these mutants has great potential for industrial production.

[0018] Therefore, the present invention uses the wild-type P450 enzyme shown in SEQ ID NO.1 as a template, analyzes and determines the amino acid sites that play a key role in activity, and obtains the following multiple P450 enzyme mutants, including: the T at position 78 is mutated to A, V, L and I to form mutants T78A, T78V, T78L and T78I; the G at position 81 is mutated to V and M to form mutants G81V and G81M; the mutant T78L / G81M is formed by the combined mutation of positions 78 and 81.

[0019] Furthermore, the present invention applies the aforementioned P450 enzyme mutants to the synthesis of calcifediol. The wild-type P450 enzyme CYP109E1, constructed using a single-plasmid, three-enzyme co-expression system, only achieved a 2.8% conversion rate for 0.4 mM vitamin D3 within 24 hours. However, through modification of the binding pocket and a saturation mutagenesis strategy, multiple P450 enzyme mutants with enhanced catalytic performance were obtained. The two mutants with the greatest improvements, CYP109E1-T78L and CYP109E1-G81M, saw their 12-hour conversion rates at 0.4 mM vitamin D3 increased from 2.8% of the wild-type CYP109E1 to 16.9% and 17.1%, respectively. The double-site combined mutant CYP109E1-T78L / G81M achieved a 12-hour conversion rate of 36.5% at 0.4 mM vitamin D3.

[0020] In summary, the present invention has the following beneficial effects: Through semi-rational design, the present invention molecularly modifies the P450 enzyme CYP109E1, obtaining multiple P450 enzyme mutants with enhanced catalytic activity and improving the conversion rate of vitamin D3. Furthermore, by constructing a single-plasmid three-enzyme co-expression system comprising the P450 enzyme mutants and a redox partner, the present invention improves the electron transfer efficiency in the P450 enzyme hydroxylation reaction, enabling the efficient synthesis of calcifediol under mild conditions, without the production of other hydroxylation byproducts. Therefore, the present invention has significant application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the SDS-PAGE protein gel electrophoresis diagram of the single-plasmid three-enzyme co-expression system of the wild-type P450 enzyme CYP109E1 and the redox chaperones PdR and Pdx, where M is a protein molecular weight standard reagent and A is the supernatant of the cell lysis fluid of the single-plasmid three-enzyme co-expression system. DETAILED DESCRIPTION

[0022] The present invention will be further described below with reference to specific examples. It should be understood that the following examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Unless otherwise specified, the techniques used in the examples are conventional in the art, or in accordance with the experimental methods recommended by the kit and instrument manufacturers.

[0023] Example 1 Strain Construction

[0024] Sequences for the P450 enzyme CYP109E1 from Bacillus megaterium DSM319 and the redox chaperones PdR and Pdx from Pseudomonas putida were cloned and inserted into the expression plasmid pET28a(+) to generate pET28a(+)-CYP109E1, pET28a(+)-PdR, and pET28a(+)-Pdx. The amino acid sequence of CYP109E1 is shown in SEQ ID NO. 1, the amino acid sequence of PdR is shown in SEQ ID NO. 9, and the amino acid sequence of Pdx is shown in SEQ ID NO. 10. After sequencing, the three plasmids were transformed into the expression host Escherichia coli BL21(DE3) for subsequent recombinant enzyme expression.

[0025] Example 2 Selection of mutation sites

[0026] Select the target protein crystal structure (PDB ID: 5L92, resolution: ) as the receptor protein and perform molecular docking with the substrate ligand. After docking, the results are sorted by binding energy and analyzed, and the ideal docked complex is saved.

[0027] Through docking structure analysis, two potential hotspots T78 and G81 located in the substrate binding pocket that may affect the binding of substrate and enzyme were screened out. Subsequently, these two amino acid residues were subjected to saturation mutation to construct a mutant library, and the optimal mutants at the two sites were jointly mutated.

[0028] Example 3 Construction of CYP109E1 mutants

[0029] Escherichia coli carrying the pET28a(+)-CYP109E1 recombinant plasmid was cultured in an LB liquid medium test tube for 10-12 hours, and the plasmid was extracted as a template for subsequent mutant construction. The primers used for mutation (SEQ ID NO. 11-24) are shown in Table 1.

[0030] Table 1 Mutation primer information

[0031]

[0032]

[0033] The PCR reaction system is shown in Table 2.

[0034] Table 2 Point mutation PCR reaction system

[0035]

[0036] PCR reaction conditions: pre-denaturation at 95°C for 3 min; denaturation at 98°C for 5 s, annealing at 58°C for 15 s, and extension at 72°C for 6 min, repeated 30 times; final extension at 72°C for 5 min; and storage at 4°C.

[0037] After PCR amplification is complete and positive results are confirmed by agarose gel electrophoresis, 0.5 μL of DpnI enzyme is added to specifically remove the methylated template DNA and placed in a 37°C metal bath for 3 hours. Afterwards, transformation, bacterial selection, and sequencing can be performed.

[0038] Example 4 Construction of a single plasmid three-enzyme co-expression system

[0039] The plasmid pRSFDuet-1 was digested with NdeI and XhoI, and pET28a(+)-Pdx was PCR amplified using the primer pairs RSP-Pdx-MCS2-F and RSP-Pdx-MCS2-R (listed in Table 3). The digested product and the resulting PCR product were then ligated using a seamless cloning kit and transformed into E. coli BL21(DE3) cells to obtain the pRSFDuet-1-Fdx recombinant strain. The plasmid was further cultured and extracted, and the plasmid was digested with NdeI. The pET28a(+)-PdR was PCR amplified using the primer pairs RSP-PdR-F and RSP-PdR-R. The digested product and the resulting PCR product were then ligated again and transformed into E. coli BL21(DE3) cells to obtain the pRSFDuet-1-PdR / Pdx recombinant strain. Continue to culture and extract the plasmid, cut it with BamHI and HindIII, PCR amplify pET28a(+)-CYP109E1 with primers RSP-E1-F and RSP-E1-R, repeat the above ligation and transformation steps, and finally obtain the single-plasmid recombinant strain pRSFDuet-1-CYP109E1 / PdR / Pdx.

[0040] The mutants sequenced correctly in Example 3 were cultured and plasmids were extracted to construct a single-plasmid three-enzyme co-expression system for the mutants. After obtaining the pRSFDuet-1-PdR / Pdx plasmid, it was cut with BamHI and HindIII, and the mutant plasmid pET28a(+)-CYP109E1 was PCR amplified using primer pairs RSP-E1-F and RSP-E1-R, respectively. The above ligation and transformation steps were repeated to finally obtain a single-plasmid recombinant strain of pRSFDuet-1-mutant CYP109E1 / PdR / Pdx. The primers used in the co-expression system (SEQ ID NOs. 25-30) are shown in Table 3.

[0041] Table 3 Primers required for co-expression system construction

[0042]

[0043]

[0044] Example 5 Induction and expression of a single plasmid three-enzyme co-expression system

[0045] The recombinant cells were spread on a solid plate and cultured in a 37°C constant temperature incubator for 12 hours. A single colony was picked and placed in a 5 mL LB medium test tube. A 1 / 1000 concentration of kanamycin solution was added and the test tube was placed in a 37°C constant temperature shaker for 12 hours. 500 μL of the bacterial solution was transferred into 50 mL of TB liquid medium and a 1 / 1000 concentration of kanamycin solution was also added. The culture was continued for 2-2.5 hours until the cell OD value reached 0. 600 Reach between 0.6-0.8. Add the inducer IPTG at a concentration of 1 part per ten thousand (final concentration of 0.1 mM) to the cultured bacterial solution for induction, and then place the shaking flask in a constant temperature incubator at 20°C and 200 rpm for incubation for 18 hours. After these bacterial solutions are balanced, centrifuge (4°C, 8000 rpm, 10 min), discard the supernatant, add 15 mL of normal saline and wash twice, take the washed bacteria and add 15 mL of Tris-HCl buffer (pH 7.4) at a mass ratio of 1:12, shake and resuspend thoroughly, and then use an ultrasonic cell disruptor to disrupt them, take the suspended bacterial solution after disruption and centrifuge (4°C, 8000 rpm, 10 min), pour out the supernatant on ice, and obtain the crude enzyme solution. Among them, the SDS-PAGE protein gel electrophoresis diagram of the single plasmid three-enzyme co-expression system of the wild-type P450 enzyme CYP109E1 is shown as follows. Figure 1 shown.

[0046] Example 6 Screening of mutants

[0047] The crude enzyme solution prepared in Example 5 was used as a catalyst to carry out a conversion reaction of the substrate vitamin D3 to screen out mutants with improved conversion rates.

[0048] The reaction system consisted of a 45% mass (g) to volume (mL) mixture of solubilizer hydroxypropyl-β-cyclodextrin and Tris-HCl buffer (20mM pH 7.4), vortexed, and then added to the substrate vitamin D3. The solution was thoroughly vortexed until clear to create a 4mM stock solution. 100 μL of the substrate vitamin D3 stock solution (final concentration 0.4mM) was added to each reaction tube, followed by the addition of the three-enzyme co-expression wet bacteria. The reaction was initiated by the addition of a certain amount of the cofactor NAD(P)H (final concentration 0.4mM), and then placed in a 30°C, 250 rpm constant temperature shaker for 24 hours. The reaction series is shown in Table 4.

[0049] Table 4 Reaction system

[0050]

[0051] Extraction method: After the reaction is completed, 750 μL of ethyl acetate is added to terminate the reaction and extract. After the mixture is vortexed at high speed for 10 minutes, it is placed in a high-speed centrifuge for centrifugation (14000 rpm, 10 minutes), the upper organic phase is collected, and the above steps are repeated once. The organic phases are combined and placed in a fume hood to evaporate overnight. 200 μL of chromatographic grade methanol is added, and the mixture is vigorously shaken for 5 minutes. The mixture is further centrifuged in a high-speed centrifuge (14000 rpm, 10 minutes). 150 μL of the supernatant is filtered through a 0.22 μm organic filter membrane and loaded into HPLC for detection.

[0052] The concentrations of vitamin D3 and calcifediol in the reaction solution were analyzed by HPLC: Agilent C18 column (5 μm × 4.6 mm × 250 mm), the mobile phase was methanol and water, the gradient elution conditions were as shown in Table 5, ultraviolet detection wavelength: 265 nm, column temperature: 30 ° C, flow rate: 1.0 mL / min, injection volume was 10 μL, and detection time was 25 min.

[0053] Table 5 Gradient elution conditions

[0054]

[0055] The catalytic activity of the constructed mutant single-plasmid three-enzyme co-expression system was evaluated by converting the substrate vitamin D3. The mutants obtained with higher calcifediol production capacity than the wild type are shown in Table 6. The data in the table show that the long side chain non-polar mutations at the T78 and G81 sites significantly improved the conversion rate of calcifediol. The best single-point mutants, T78L and G81M, achieved conversion rates of 16.9% and 17.1%, respectively, at a 12-hour concentration of 0.4 mM vitamin D3. The double-point combined mutant CYP109E1-T78L / G81M achieved a conversion rate of 36.5% at a 12-hour concentration of 0.4 mM vitamin D3.

[0056] Table 6 Conversion rates of P450 enzymes and mutants in a single plasmid triple-enzyme co-expression system

[0057]

[0058] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Various modifications may be made to the above-described embodiments of the present invention. In other words, any simple, equivalent changes and modifications made in accordance with the claims and the description of the present invention fall within the scope of protection of the patent claims. Anything not fully described in this invention constitutes conventional technology.

Claims

1. A P450 enzyme mutant with enhanced activity for catalyzing the synthesis of calcifediol from vitamin D3, characterized in that: Using the wild-type P450 enzyme CYP109E1 shown in SEQ ID NO. 1 as a template, the P450 enzyme mutant was formed by the following mutation at amino acid residue 78: A P450 enzyme mutant T78A in which the threonine T at position 78 is mutated to alanine A, and the amino acid sequence is shown in SEQ ID NO. 2; or A P450 enzyme mutant T78V in which the threonine T at position 78 is mutated to valine V, and the amino acid sequence is shown in SEQ ID NO. 3; or A P450 enzyme mutant T78L in which the threonine T at position 78 is mutated to leucine L, and the amino acid sequence is shown in SEQ ID NO. 4; or The P450 enzyme mutant T78I in which the threonine T at position 78 is mutated to isoleucine I has an amino acid sequence as shown in SEQ ID NO.

5.

2. A gene encoding the P450 enzyme mutant according to claim 1.

3. A recombinant vector comprising the encoding gene according to claim 2.

4. A method for constructing a single plasmid three-enzyme co-expression system, characterized in that: The following steps are involved: 1) Providing an enzyme mutant according to claim 1, inserting the mutant into the expression plasmid pET28a(+) to obtain the mutant plasmid pET28a(+)-CYP109E1; 2) The gene sequences of ferredoxin reductase PdR and ferredoxin Pdx from Pseudomonas aeruginosa were amplified and inserted into the expression plasmid pET28a(+) to obtain plasmids pET28a(+)-PdR and pET28a(+)-Pdx, respectively; 3) Using endonucleases, the plasmid pET28a(+)-PdR, the plasmid pET28a(+)-Pdx, and any one of the mutant plasmids pET28a(+)-CYP109E1 were digested separately, and the plasmids were ligated in sequence and transformed into the host Escherichia coli BL21(DE3) to construct a single-plasmid three-enzyme co-expression system.

5. The construction method according to claim 4, wherein: The amino acid sequence of the ferredoxin reductase PdR is shown in SEQ ID NO.9, and the amino acid sequence of the ferredoxin Pdx is shown in SEQ ID NO.

10.

6. A single plasmid three-enzyme co-expression system constructed according to the construction method according to any one of claims 4 to 5.

7. Use of the single-plasmid three-enzyme co-expression system according to claim 6 in the synthesis of calcifediol.

8. The use according to claim 7, characterized in that The following steps are involved: S1: culturing the single-plasmid three-enzyme co-expression system in TB medium, inducing expression, and obtaining a crude enzyme solution; S2: Mix the solubilizer hydroxypropyl-β-cyclodextrin and Tris-HCl buffer at a mass volume ratio of 45%, add the substrate vitamin D3 after vortexing, vortex thoroughly until clear, prepare a 4 mM mother liquor, add the substrate vitamin D3 to a final concentration of 0.3~0.5 mM, then add the crude enzyme solution and the cofactor NADPH, place in a constant temperature shaker at 30°C and 200~250 rpm and shake for 20~30 hours to carry out the conversion reaction of the substrate vitamin D3, thereby achieving efficient synthesis of calcifediol.

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