Fusion P450 enzyme, mutant thereof and method for producing 25-hydroxyvitamin D3 by using whole-cell catalyst

Abstract

The invention relates to the technical field of agricultural biology, in particular to a method for producing 25-hydroxyvitamin D3 by fusing P450 enzyme, a mutant thereof and a whole-cell catalyst. The fusion P450 enzyme VK1-CYP116B46 comprises a Vdh-K1 redox structure domain of the P450 enzyme and a redox structure domain of the CYP116B46, and the Vdh-K1 redox structure domain and the CYP116B46 redox structure domain are connected through a linker. And carrying out linker screening on the VK1-CYP116B46, so as to obtain the mutant. Whole cells are directly adopted as a catalyst, enzyme purification is not needed, glucose dehydrogenase is introduced, glucose serves as a substrate to regenerate the cofactor NAD (P) H, expensive cofactors are prevented from being added, the production cost is reduced, and industrial production is facilitated; compared with a multi-component P450 enzyme adopted in the prior art, the conversion rate of VD3 is greatly improved by fusing P450 enzyme conversion.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and specifically relates to a method for producing 25-hydroxyvitamin D3 by fusing P450 enzyme, its mutants and whole cell catalysts. Background Art

[0002] Vitamin D3 (VD3), commonly known as cholecalciferol, plays a key role in many biological processes. Compared with VD3, 25-hydroxyvitamin D3 (25(OH)VD3) exhibits stronger water solubility, and 25(OH)VD3 can treat VD3 deficiency more effectively than supplementing VD3 alone.

[0003] Currently, the common methods for producing 25(OH)VD3 are chemical synthesis method and biotransformation method. Compared with the chemical synthesis route, the C25 hydroxylase-catalyzed reaction using VD3 as a substrate only requires one reaction step, has high catalytic efficiency and excellent selectivity, mild reaction conditions, and little environmental pollution. P450 is a member of the heme oxidase superfamily known for its specific region and stereoselective transformation, and has great potential in the synthesis of 25(OH)VD3. However, the industrial application of P450 faces challenges such as the need for additional addition of redox partners, limited catalytic activity, low coupling efficiency, and insufficient stability, resulting in high production costs.

[0004] In the catalytic cycle of P450, the hydroxylation of the substrate depends on the efficiency of electron transfer from the redox partner to the heme iron. Usually, an additional redox partner protein needs to be added to perform the function of electron transfer, resulting in an increase in the cost of P450 in industrial applications. In addition, the rate-limiting step in the P450 reaction is electron transfer. When this process is not coordinated with the catalytic efficiency of the substrate, it may trigger a decoupling reaction, resulting in a decrease in the coupling efficiency of P450, an accumulation of reactive oxygen species, and a decrease in enzyme activity. The self-sufficient fusion P450 enzyme integrates the heme catalytic and redox enzyme domains on a single polypeptide chain, and the domains are connected by a linker, which simplifies and promotes electron transfer. Compared with multi-component P450, it usually exhibits higher catalytic efficiency. The self-sufficient fusion P450 enzyme relies on its own conformational movement to achieve effective inter-domain electron transfer. The linker region located between the heme and reductase domains does not directly participate in ET, but can regulate the interaction between domains, and thus regulate the conformational movement to affect the inter-domain electron transfer efficiency. Therefore, developing a self-sufficient fusion P450 enzyme with C-25 hydroxylation activity towards VD3 and improving its catalytic activity towards VD3 by modifying the linker between domains has great industrial application potential. Summary of the Invention

[0005] The object of the present invention is to solve the problem in the prior art that the low catalytic activity of P450 enzyme on VD3 leads to low yield of 25(OH)VD3, which is difficult to be used in industrial production.

[0006] The self-sufficient fusion P450 enzyme VK1-CYP116B46 of the present invention comprises a P450 enzyme Vdh-K1 heme catalytic domain derived from Pseudonocardia autotrophica autotrophic Pseudonocardia, and a redox domain of a P450 enzyme derived from a thermophilic bacterium Tepidiphilus thermophiles CYP116B46. The Vdh-K1 heme catalytic domain and the CYP116B46 redox domain of the fusion P450 enzyme VK1-CYP116B46 are connected by a linker. Among them, the amino acid sequence of Vdh-K1 is shown as SEQ ID NO:1, the amino acid sequence of the redox domain of CYP116B46 is shown as SEQ ID NO:2, the linker is the natural linker between domains of CYP116B46, and its amino acid sequence is AQNPERRDPDILRLRQPVRIGPPRAKDV (SEQ ID NO:3), or a modified linker, and the amino acid sequence of the modified linker is shown as any one of SEQ ID NO:4 to SEQ ID NO:14: The yields of L6, L7, L10, L12, L16, L18, L19, L20, L21, L22, and L23 are higher than those of the wild type.

[0007] L6: RDPDILRLRQPVRIGPPRAKDV (SEQ ID NO:4); L7: DPDILRLRQPVRIGPPRAKDV (SEQ ID NO:5); L10: ILRLRQPVRIGPPRAKDV (SEQ ID NO:6); L12: RLRQPVRIGPPRAKDV (SEQ ID NO:7); L16: PVRIGPPRAKDV (SEQ ID NO:8); L18: RIGPPRAKDV (SEQ ID NO:9); L19: IGPPRAKDV (SEQ ID NO:10); L20: GPPRAKDV (SEQ ID NO:11); L21: PPRAKDV (SEQ ID NO:12); L22: PRAKDV (SEQ ID NO:13); L23: RAKDV (SEQ ID NO:14).

[0008] SEQ ID NO:1 is the amino acid sequence of Vdh-K1: MALTTTGTEQHDLFSGTFWQNPHPAYAALRAEDPVRKLALPDGPVWLLTRYADVREAFVDPRLSKDWRHRLPEDQRADMPATPTPMMILMDPPDHTRLRKLVGRSFTVRRMNELEPRITEIADGLLAGLPTDGPVDLMREYAFQIPVQVICELLGLPAEDRDDFSAWSSVLVDDSPADDKNAAMGKLHGYLSDLLERKRTEPDDALLSSLLAVSDMDGDRLSQEELVAMAMLLLIAGHETTVNLIGNGVLALLTHPDQRKLLAEDPSLISSAVEEFLRFDSPVSQAPIRFTAEDVTYSGVTIPAGEMVMLGLAAANRDADWMPEPDRLDITRDASGGVFFGHGIHFCLGAQLARLEGRVAIGRLFADRPELALAVGLDELVYRRSTLVRGLSRMPVTMGPRSA.

[0009] SEQ ID NO:2 is the amino acid sequence of the redox domain of CYP116B46: VRTMEVAAVERPSEDIVVLHLTRPDRRPLPRWSPGAHIDIECGEPDRSRQYSLCSDPENRDAWRVAVQRDPASRGGSRWIHEEVRPGMLLRVRGPRNSFRLDEHAPRYLFLAGGIGITPIMTMAARAKELGTDYELHYSVRSRTSLIFVDELRQIHGDRLHVYVSEEGVRNDLAALIRRASAGTQIYACGPQRMLDTLERLIENRPEVTLRVEHFFGEPSHLDPAKERPFQVVLRNSGLTVEVPADKTLLEVLRAYNIEVQSDCEEGLCGTCEVSVVEGEVDHRDSVLTRAERRENRRMMCCCSRAKTERLVLDLKLAAA.

[0010] The present invention provides the coding gene of the above-mentioned fusion P450 enzyme.

[0011] The present invention provides a co-expression vector carrying the coding gene of the above-mentioned fusion P450 enzyme and the coding gene of glucose dehydrogenase.

[0012] According to a specific embodiment of the present invention, the glucose dehydrogenase is from Priestia megaterium ( Priestia megaterium ), and its amino acid sequence is shown in SEQ ID NO:15.

[0013] SEQ ID NO:15 MYKDLEGKVVVITGSSTGLGKAMAIRFATEKAKVVVNYRSKEEEANSVLEEIKKVGGEAIAVKGDVTVESDVINLVQSAIKEFGKLDVMINNAGMENPVSSHEMSLSDWNKVIDTNLTGAFLGSREAIKYFVENDIKGTVINMSSVHEKIPWPLFVHYAASKGGMKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPEQRADVESMIPMGYIGEPEEIAAVAAWLASSEASYVTGITLFADGGMTQYPSFQAGRG。

[0014] According to a specific embodiment of the present invention, the method for constructing the co-expression plasmid is as follows: VK1-CYP116B46 or its mutant and the glucose dehydrogenase gene are ligated to a plasmid to obtain a co-expression plasmid.

[0015] The present invention provides a recombinant cell containing the above-mentioned co-expression plasmid encoding the fusion P450 enzyme or its mutant and the glucose dehydrogenase gene.

[0016] In one embodiment, the recombinant cell is recombinant Escherichia coli Escherichia coli BL21(DE3).

[0017] The present invention provides a whole-cell catalyst, and the whole-cell catalyst contains the above-mentioned recombinant cell.

[0018] The present invention provides a method for preparing 25(OH)VD3 by whole-cell catalysis. The method is to add the whole-cell catalyst to a VD3 solution for catalytic preparation of 25(OH)VD3.

[0019] According to the method for preparing 25(OH)VD3 by whole-cell catalysis of the present invention, the steps of the method are as follows: The recombinant cells are cultured in LB medium at 37°C, OD 600When it is 0.6 - 1.0, add β - d - 1 - thiopyranosyl galactoside (IPTG) with a final concentration of 0.1 - 1.0 mM and 5 - aminolevulinate (5 - ALA) with a final concentration of 0.1 - 1.0 mM, induce at 16 - 37 °C for 5 - 20 h, and collect the wet bacterial cells; inoculate the wet bacterial cells into the VD3 solution, and react at pH 6.5 - 8 and 20 - 40 °C 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, co - solvent, and co - substrate.

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

[0022] In some embodiments of the present invention, the initial concentration of VD3 is 2 - 8 mM.

[0023] In some embodiments of the present invention, the concentration of 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] Advantages of the present invention: The present invention constructs the fusion P450 enzyme VK1 - CYP116B46, and constructs different VK1 - CYP116B46 linker mutants. After screening, mutants with improved conversion rates are obtained; Using VD3 as the substrate, adding glucose, co - solvent, and the wet bacterial cells of recombinant Escherichia coli co - expressing the fusion P450 enzyme and glucose dehydrogenase after induction, the highest yield of 25(OH)VD3 within 24 h after the catalytic reaction can reach 4.89 mM (1.96 g / L); Directly using whole cells as catalysts does not require enzyme purification. Introducing glucose dehydrogenase to regenerate the co - factor NAD(P)H with glucose as the substrate avoids the addition of expensive co - factors, reduces production costs, and is conducive to industrial production. Brief Description of the Drawings

[0028] Figure 1 Showing the 25(OH)VD3 production ability of VK1 - CYP116B46 containing wild - type and mutant linkers; Figure 2 Show the yield of 25(OH)VD3 in the whole-cell conversion reaction of substrate VD3. Detailed implementation manners

[0029] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] Test materials and reagents: 1. Strains and vectors: The expression host is Escherichia coli Escherichia coli BL21(DE3), and the expression plasmid vector is pETDuet1.

[0031] 2. Enzymes: Restriction enzymes, ligases.

[0032] 3. Media: LB medium (g / L): Tryptone 10 g, Yeast extract 5 g, Sodium chloride 10 g; TB medium (g / L): Tryptone 12 g, Yeast extract 24 g, Glycerol 5 g, Dipotassium hydrogen phosphate trihydrate 16.37 g, Potassium dihydrogen phosphate 2.31 g.

[0033] For the molecular biology experimental methods not specifically described in the following examples, they are all carried out according to the specific methods listed in "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook, or according to the kit and product instructions. Example 1: Preparation of recombinant strain BL21(pETDuet1-VK1-CYP116B46-GDH)

[0034] Use restriction enzymes BamHI and Hind III to double-digest plasmid pETDuet1; use primers VK1-F and VK1-CYP116B46-R with homologous ends to amplify the P450 enzyme Vdh-K1 gene sequence from Pseudonocardia autotrophica ( Pseudonocardia autotrophica )), and use primers VK1-CYP116B46-F and CYP116B46-R with homologous ends to amplify the gene sequence of the redox domain of the P450 enzyme CYP116B46 from Thermophilic bacteria ( Tepidiphilus thermophiles )); Recover and purify the PCR products and the enzyme-digested products respectively, perform recombinant ligation through the kit recombinase, and then transform the recombinant product into Escherichia coli Trans1-T1 competent cells, and coat them on LB (containing 100 μg / mL Ampicillin) for screening. After the sequencing is correct, extract the plasmid to obtain plasmid pETDuet1-VK1-CYP116B46.

[0035] Table 1 Primer Primer sequence VK1-F (SEQ ID NO:16) CCATCATCACCACAGCCAGGATCCGATGGCGCTGACCACCAC VK1-CYP116B46-R (SEQ ID NO:17) CGTTCCGGATTCTGCGCTGCGCTACGCGGACCCA VK1-CYP116B46-F (SEQ ID NO:18) TGGGTCCGCGTAGCGCAGCGCAGAATCCGGAACG CYP116B46-R (SEQ ID NO:19) AGCATTATGCGGCCGCAAGCTTCAGGTCCAGAACCAGACG VK1-RBS-GDH-F (SEQ ID NO:20) AAGCTTGAGCTCGGTACCCGGGGATCCAAGGAGATATACATGTACACCGATCTGAAAGA GDH-R (SEQ ID NO:21) GACTTAAGCATTATGCGGCCGCACCACGACCTGCCTGAAA 。

[0036] The plasmid pETDuet1-VK1-CYP116B46 was double digested with the restriction endonucleases Hind III and NotI, and the glucose dehydrogenase GDH derived from Priestia megaterium ( Priestia megaterium )(Gene ID: 64145183) was amplified using the primers VK1-RBS-GDH-F and GDH-R with homologous ends; the PCR product and the digested product were respectively recovered and purified, recombinantly ligated by a kit recombinase, and then the recombinant product was transformed into Escherichia coli Trans1-T1 competent cells and spread on LB (containing 100 μg / mL Ampicillin) for screening. After correct sequencing, the plasmid was extracted to obtain the plasmid pETDuet1-VK1-CYP116B46-GDH, which was transformed into Escherichia coli BL21(DE3) to obtain the recombinant strain BL21(pETDuet1 - VK1-CYP116B46-GDH). Example 2: Construction and screening of mutants

[0037] Construction of mutants Based on VK1-CYP116B46, mutant primers were designed. Using the plasmid pETDuet1-VK1-CYP116B46-GDH as a template, the linker was mutated. The amino acid sequences of the linker before and after mutation are shown in Table 2. The PCR product was digested with DpnⅠ to remove the template. The digested PCR product was transformed into Escherichia coli Tans1-T1 competent cells, and its sequencing was verified. Then it was transformed into Escherichia coli BL21(DE3) to obtain the recombinant Escherichia coli strain BL21(pETDuet1 -VK1-CYP116B46 L5G-GDH, pETDuet1-VK1-CYP116B46 L1-GDH, pETDuet1-VK1-CYP116B46 L2-GDH, pETDuet1-VK1-CYP116B46 L3-GDH, pETDuet1-VK1-CYP116B46 L4-GDH, pETDuet1-VK1-CYP116B46 L5-GDH, pETDuet1-VK1-CYP116B46 L6-GDH, pETDuet1-VK1-CYP116B46 L7-GDH, pETDuet1-VK1-CYP116B46 L8-GDH, pETDuet1-VK1-CYP116B46 L9-GDH, pETDuet1-VK1-CYP116B46 L10-GDH, pETDuet1-VK1-CYP116B46 L11-GDH, pETDuet1-VK1-CYP116B46 L12-GDH, pETDuet1-VK1-CYP116B46 L13-GDH, pETDuet1-VK1-CYP116B46 L14-GDH, pETDuet1-VK1-CYP116B46 L15-GDH, pETDuet1-VK1-CYP116B46 L16-GDH, pETDuet1-VK1-CYP116B46 L17-GDH, pETDuet1-VK1-CYP116B46 L18-GDH, pETDuet1-VK1-CYP116B46 L19-GDH, pETDuet1-VK1-CYP116B46 L20-GDH, pETDuet1-VK1-CYP116B46 L21-GDH, pETDuet1-VK1-CYP116B46 L22-GDH, pETDuet1-VK1-CYP116B46 L23-GDH, pETDuet1-VK1-CYP116B46 L24-GDH, pETDuet1-VK1-CYP116B46 L25-GDH, pETDuet1-VK1-CYP116B46 L26-GDH, pETDuet1-VK1-CYP116B46 L27-GDH, pETDuet1-VK1-CYP116B46 L28-GDH).

[0038] Table 2 Amino acid sequences before and after linker mutation VK1-CYP116B46 Linker WT AQNPERRDPDILRLRQPVRIGPPRAKDV 5G GGGGGAQNPERRDPDILRLRQPVRIGPPRAKDV L1 QNPERRDPDILRLRQPVRIGPPRAKDV L2 NPERRDPDILRLRQPVRIGPPRAKDV L3 PERRDPDILRLRQPVRIGPPRAKDV L4 ERRDPDILRLRQPVRIGPPRAKDV L5 RRDPDILRLRQPVRIGPPRAKDV L6 RDPDILRLRQPVRIGPPRAKDV L7 DPDILRLRQPVRIGPPRAKDV L8 PDILRLRQPVRIGPPRAKDV L9 DILRLRQPVRIGPPRAKDV L10 ILRLRQPVRIGPPRAKDV L11 LRLRQPVRIGPPRAKDV L12 RLRQPVRIGPPRAKDV L13 LRQPVRIGPPRAKDV L14 RQPVRIGPPRAKDV L15 QPVRIGPPRAKDV L16 PVRIGPPRAKDV L17 VRIGPPRAKDV L18 RIGPPRAKDV L19 IGPPRAKDV L20 GPPRAKDV L21 PPRAKDV L22 PRAKDV L23 RAKDV L24 AKDV L25 KDV L26 DV L27 V L28 - 。

[0039] (2)Mutant screening: The 29 mutant strains obtained in step (1) were respectively inoculated into LB medium and cultured at 200 rpm and 37 °C for about 10 h. Then, they were inoculated into TB medium at an inoculation amount of 1% of the medium volume and cultured at 200 rpm and 37 °C until 600 OD 600 = about 0.8. IPTG with a final concentration of 1.0 mM and 5-ALA with a final concentration of 1.0 mM were added, and induction was carried out at 200 rpm and 25 °C for 18 h. The cells were collected by centrifugation at 6000 rpm for 10 min to obtain the cells, and whole-cell transformation reaction of substrate VD3 was performed to screen out mutants with improved conversion rate.

[0040] Whole-cell transformation reaction system: VD3 substrate (final concentration of 1 mM) and the induced cells of recombinant Escherichia coli (final cell concentration OD 600 = 20) were added to potassium phosphate buffer (50 mM, pH 7.4) containing 50 g / L hydroxypropyl-β-cyclodextrin and 10% (v / v) ethanol. Finally, glucose (final concentration of 25 g / L) was added, and the reaction was carried out at 25 °C and 200 rpm for 2 h. After the reaction was completed, an equal volume of ethyl acetate was added to terminate the reaction and perform extraction. The mixture was vortexed at high speed for 10 min and then centrifuged in a high-speed centrifuge (12000 rpm, 10 min). The upper organic phase was collected, filtered through a 0.22 μm organic filter membrane, and loaded onto HPLC for detection.

[0041] The concentrations of VD3 and 25(OH)VD3 in the reaction solution were analyzed by HPLC: Agilent C18 column (5 μm×4.6 mm×250 mm), mobile phase was methanol / acetonitrile (30:70, v / v); detection wavelength was set at 264 nm; flow rate was maintained at 1.0 mL / min; injection volume was 10 μL, column temperature was 30 °C, and detection duration was 15 min. The catalytic activity of the mutants constructed above was evaluated by the conversion of substrate VD3, and mutants with a higher 25(OH)VD3 production ability than the wild type were obtained as Figure 1 shown. From the data in the figure, it can be seen that the yields of linker mutants L6, L7, L10, L12, L16, L18, L19, L20, L21, L22, L23 are higher than that of the wild type, proving that modifying the linker can effectively improve the activity of the self-sufficient fusion P450 enzyme. The mutant VK1-CYP116B46 L21 produced the highest yield of 25(OH)VD3, which was 1.53 times that of the wild type. However, completely removing the linker (L28) led to the complete inactivation of P450, indicating the importance of the linker for the self-sufficient fusion P450 enzyme. Example 3: Gram-scale preparation of 25(OH)VD3

[0042] The recombinant strain constructed in Example 2 E.coli BL21 / pETDuet1 - VK1-CYP116B46 L21-GDH was inoculated into LB medium and cultured at 200 rpm and 37 °C for about 10 h. Then, it was inoculated into TB medium at an inoculation amount of 1% of the medium volume and cultured at 200 rpm and 37 °C until OD 600 = about 0.8. IPTG with a final concentration of 1.0 mM and 5-ALA with a final concentration of 1.0 mM were added, and induction was carried out at 200 rpm and 25 °C for 18 h. The cells were collected by centrifugation at 6000 rpm for 10 min to obtain the cells, and the whole-cell conversion reaction of the substrate VD3 was carried out.

[0043] During the whole-cell conversion reaction, the concentration of VD3 in the whole reaction system was 8 mM, and the reaction time was 24 h. The rest of the operations were the same as those in Example 2. After the reaction was completed, an equal volume of ethyl acetate was added to terminate the reaction and extract. After the mixture was vortexed at high speed for 10 min, it was centrifuged in a high-speed centrifuge (12000 rpm, 10 min). The upper organic phase was collected, filtered through a 0.22 μm organic filter membrane, and loaded and detected on HPLC. The yield was as Figure 2 shown. VK1-CYP116B46-L21 achieved 50% substrate conversion at 8 h, and the final yield at 24 h was 4.89 mM (about 1.96 g / L), and the conversion rate was 61%.

[0044] As described above, only the preferred embodiments of the present invention are provided, and are not intended to limit the scope of the present invention. Various changes can be made to the above embodiments of the present invention. That is, all simple, equivalent changes and modifications made according to the claims and the content of the specification of the present invention application fall within the scope of the claims of the present invention patent. Those not described in detail in the present invention are all conventional technical contents.

Claims

1. A self-sufficient fusion P450 enzyme, characterized in that The self-sufficient fusion P450 enzyme includes a Vdh-K1 heme catalytic domain and a redox domain of CYP116B46 connected by a linker, wherein the amino acid sequence of the Vdh-K1 heme catalytic domain is shown in SEQ ID NO:1, the amino acid sequence of the redox domain of CYP116B46 is shown in SEQ ID NO:2, and the linker is a natural linker between domains of CYP116B46 or a modified linker, wherein the amino acid sequence of the natural linker between domains of CYP116B46 is shown in SEQ ID NO:3, and the amino acid sequence of the modified linker is shown in any one of SEQ ID NO:4 to SEQ ID NO:

14.

2. Fusion P450 enzyme gene, characterized in that, Encoding the self-sufficient fusion P450 enzyme of claim 1.

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

4. The recombinant expression vector according to claim 3, characterized in that The recombinant expression vector also includes a gene encoding glucose dehydrogenase.

5. A recombinant expression strain comprising the recombinant expression vector according to claim 3 or 4.

6. Use of the self-sufficient fusion P450 enzyme according to claim 1 for preparing 25(OH)VD3.

7. A method for preparing 25(OH)VD3 by whole-cell catalysis, characterized in that: The method comprises adding the recombinant expression strain according to claim 5 into a VD3 solution to catalyze and ferment the preparation of 25(OH)VD3.

8. The method for preparing 25(OH)VD3 by whole-cell catalysis according to claim 7, characterized in that: The VD3 solution contains glucose.

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