Preparation method and application of pyridoxine phosphate
By using an enzyme composition of pyridoxine kinase and polyphosphate kinase and an adenine nucleoside triphosphate recycling system, the problems of low yield in enzyme-catalyzed synthesis of pyridoxine phosphate and environmental pollution in chemical synthesis methods have been solved, achieving efficient, green and environmentally friendly production of pyridoxine phosphate.
Patent Information
- Application Number
- CN202511728563.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-24
- Publication Date
- 2026-01-16
AI Technical Summary
Existing technologies for enzyme-catalyzed synthesis of pyridoxine phosphate suffer from limitations in enzyme composition and low yield, while chemical synthesis methods are characterized by high cost, severe environmental pollution, and low output.
An enzyme composition of pyridoxine kinase and polyphosphokinase is used, combined with an adenine nucleoside triphosphate recycling system. Pyridoxine kinase catalyzes the phosphorylation of the 5' hydroxyl group of pyridoxine to generate pyridoxine phosphate, and polyphosphokinase is used to regenerate ATP, thereby realizing the recycling of coenzyme factors.
This method achieves efficient and environmentally friendly synthesis of pyridoxine phosphate, reduces production costs, increases yield to 99.6%, simplifies the process, and reduces impurity generation.
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Figure CN121344113A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for preparing pyridoxine phosphate and its application. Background Technology
[0002] Pyridoxine phosphate (PNP), the active form of vitamin B6, is a water-soluble compound that participates in amino acid metabolism, neurotransmitter synthesis, and heme production as a coenzyme for over 140 enzymes, possessing core functions such as neuroprotection and immune regulation. Its medicinal applications are wide-ranging, covering the treatment of vitamin B6 deficiency, diabetic neuropathy, epilepsy, and hyperemesis gravidarum, with particularly significant efficacy in neurological disorders. Recent research has confirmed its ability to improve cognitive impairment in Alzheimer's disease and its potential to inhibit tumor angiogenesis. In conclusion, pyridoxine phosphate has wide applications in many fields and enjoys a large market demand.
[0003] The production of pyridoxine phosphate mainly relies on chemical synthesis or extraction from natural organisms, but these methods suffer from high costs, severe environmental pollution, and low yields. With the development of synthetic biology and metabolic engineering, in vitro biosynthesis technology has provided a new pathway for the efficient and green production of pyridoxine phosphate. Compared to existing industrial chemical synthesis processes, the enzymatic synthesis of pyridoxine phosphate has been achieved through the study of recombinantly expressed pyridoxine kinase and its multi-enzyme cascade. However, current enzymatic synthesis of pyridoxine phosphate suffers from limitations such as limited enzyme compositions and low yields. Summary of the Invention
[0004] Purpose of the invention: The purpose of this invention is to provide a method and application for preparing pyridoxine phosphate using an enzyme composition of pyridoxine kinase and polyphosphate kinase.
[0005] Technical solution: The method for preparing pyridoxine phosphate includes a reaction system comprising the substrate pyridoxine, an enzyme composition, and a coenzyme factor. The enzyme composition comprises pyridoxine kinase and polyphosphate kinase, and the coenzyme factor comprises a metal cofactor, ATP, and a phosphate donor.
[0006] Reaction principle: Simultaneously, while pyridoxine kinase catalyzes the phosphorylation of the 5'-hydroxyl group of pyridoxine (PN) to generate pyridoxine phosphate (PNP), an adenine nucleoside triphosphate (ATP) recycling system is introduced. Utilizing the catalytic action of pyridoxine kinase, ATP contributes a phosphate group and corresponding chemical energy to generate ADP. Furthermore, polyphosphate kinases regenerate ADP back into ATP using polyphosphates, thus achieving the recycling of the coenzyme factor ATP.
[0007] Preferably, the amino acid sequence of the pyridoxine kinase is shown in SEQ ID NO:1, and the amino acid sequence of the polyphosphate kinase is shown in SEQ ID NO:2.
[0008] The pH of the reaction is preferably 7 to 7.5, with 7.5 being the most preferred.
[0009] The preferred metal cofactor is MgCl2. 2+ The preferred concentration is 25-75 mM, with 25 mM being the most preferred.
[0010] The phosphate donor is sodium hexametaphosphate (polyP6). The concentration of sodium hexametaphosphate is preferably 60-200 mM, and most preferably 100 mM.
[0011] The concentration of ATP is preferably 15-25 mM, and most preferably 25 mM.
[0012] Preferably, the preparation steps of the pyridoxine kinase or polyphosphate kinase include: constructing a recombinant vector containing the gene of the target protein; preparing recombinant microorganisms; and expressing the target protein.
[0013] The application refers to the application of the preparation method in the field of biopharmaceuticals.
[0014] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. By introducing an adenine nucleoside triphosphate recycling system, a process for the in vitro biosynthesis of pyridoxine phosphate with highly stereoselectivity through a dual-enzyme cascade is obtained, thereby reducing the amount of ATP added and significantly reducing production costs; 2. It avoids the problems of complex steps and high-phosphorus wastewater generation in chemical synthesis methods, and realizes a simple, efficient, green and environmentally friendly synthesis of pyridoxine phosphate; 3. This method has the advantages of mild reaction conditions, low impurity generation, easy product purification, and high conversion efficiency, with a yield of up to 99.6%. Attached Figure Description
[0015] Figure 1 High performance liquid chromatogram of pyridoxine standard (retention time: 6.906 min);
[0016] Figure 2 High performance liquid chromatogram of pyridoxine phosphate standard (retention time: 5.504 min);
[0017] Figure 3 High-performance liquid chromatography (HPLC) chromatograms of the in vitro biosynthesis of pyridoxine phosphate using the dual-enzyme cascade method of the present invention (retention times: pyridoxine 7.118 min, pyridoxine phosphate 5.503 min).
[0018] Figure 4 This is a schematic diagram illustrating the effect of pH on the conversion rate of pyridoxine phosphate.
[0019] Figure 5 A schematic diagram showing the effect of MgCl2 concentration on the conversion rate of pyridoxine phosphate;
[0020] Figure 6 A schematic diagram showing the effect of PolyP6 concentration on pyridoxine phosphate conversion;
[0021] Figure 7 This is a schematic diagram illustrating the effect of ATP concentration on the conversion rate of pyridoxine phosphate. Detailed Implementation
[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings.
[0023] Example 1
[0024] This embodiment provides a method for preparing pyridoxine phosphate. The reaction system includes the substrate pyridoxine, an enzyme composition, and a coenzyme factor. The enzyme composition is pyridoxine kinase and polyphosphate kinase, and the coenzyme factor includes the metal cofactor MgCl2, ATP, and the phosphate donor PolyP6. The preparation method of the pyridoxine kinase and polyphosphate kinase is as follows.
[0025] (1) Vector construction and cloning: The nucleotide sequences encoding pyridoxine kinase and polyphosphokinase (SEQ ID NO: 1-2) were ligated to vector pET28a using a seamless cloning method to obtain pyridoxine kinase-pET28a and polyphosphokinase-pET29a, respectively. The above-mentioned ligation vectors were operated as follows: 10 μl of ligation product was added to 100 μl of E. coli BL21(DE3) competent cells in an ice bath, followed by an ice bath for 30 min, heat shock at 42℃ for 60 s, and then an ice bath for 5 min. 300 μl of antibiotic-free LB medium at 37℃ was added to the tube, and the cells were incubated at 37℃ and 200 rpm for 1 h. Then, the cells were plated on solid LB plates with kanamycin, incubated at 37℃, and single colonies were picked.
[0026] Verification: Selected single colonies were added to a 20 μl PCR Mix containing T7 universal primers for PCR amplification. The PCR reaction conditions were: 95℃ for 15 min, 94℃ for 15 s for denaturation, 55℃ for 15 s for annealing, and 72℃ for 1 min for 30 cycles, followed by a final extension at 72℃ for 5 min. Electrophoresis was performed after PCR amplification to confirm positive clones, yielding *E. coli* strains containing the target enzyme sequences: pyridoxine kinase-*E. coli* and polyphosphate kinase-*E. coli*.
[0027] (2) Enzyme expression and extraction: The previously obtained *E. coli* were added to LB medium supplemented with kanamycin and cultured at 37°C until OD600 = 0.7. Then, IPTG was added to a final concentration of 0.5 mM. *E. coli* containing the pyridoxine kinase sequence was induced to express for 16 h at 25°C, and *E. coli* containing the polyphosphate kinase sequence was induced to express for 16 h at 16°C. The bacterial culture was then centrifuged at 5000 rpm for 10 min to collect the bacterial cells. After discarding the supernatant, 5 mL of pH 7.2, 20 mM PBS solution was added per gram of bacterial cells to resuspend the bacterial cells. The resuspended bacterial cells were then disrupted by a high-pressure cell disruptor to obtain an enzyme-containing lysate. After centrifugation at 12500 rpm for 1 h, the supernatant was extracted to obtain a crude enzyme solution containing pyridoxine kinase and polyphosphate kinase, which is the enzyme composition described above. It was stored at 4°C for later use.
[0028] Preparation of pyridoxine phosphate: The enzyme composition prepared above was reacted with pyridoxine (320 mM), ATP (25 mM), MgCl2 solution (25 mM), polyP6 solution (100 mM), and PBS at 37°C and pH 7.5 for 24 h. After the reaction, the efficiency of pyridoxine phosphate preparation was detected by high performance liquid chromatography (see [link to relevant documentation]). Figure 3 ).
[0029] Example 2
[0030] This embodiment uses the enzyme composition prepared in Example 1 to investigate the optimal reaction pH for the preparation of pyridoxine phosphate. The reaction pH was set to 6.0, 7.0, and 7.5, respectively, and other reaction conditions were the same as in Example 1.
[0031] The results are as follows Figure 4 As shown: at pH 6.0, the amount of PNP generated is extremely low; as the pH increases to 7.0~7.5, the amount of PNP generated increases; at pH 7.5, the catalytic effect is the best, and the conversion rate of PNP is 99.5%.
[0032] Example 3
[0033] This embodiment uses the enzyme composition prepared in Example 1 to investigate the optimal MgCl2 concentration for the preparation of pyridoxine phosphate. MgCl2 was set separately. 2+ The concentrations were 10, 20, 25, 50, and 75 mM, and other reaction conditions were the same as in Example 1.
[0034] The results are as follows Figure 5 As shown: the conversion rate of PNP increases with the conversion rate of Mg. 2+ The concentration increases with increasing Mg 2+ The yield reached its maximum of 72% at a concentration of 25 mM.
[0035] Example 4
[0036] This embodiment uses the enzyme composition prepared in Example 1 to investigate the optimal PolyP6 concentration for the preparation of pyridoxine phosphate. The PolyP6 concentration was set at 0, 60, 75, 100, 200, 300, and 350 mM, respectively, and other reaction conditions were the same as in Example 1.
[0037] The results are as follows Figure 6 As shown, almost no PNPs are produced when PolyP6 is not present. The conversion rate increases with the increase of PolyP6 concentration. When the PolyP6 concentration is 100 mM and the reaction time is 6 h, the conversion rate reaches the maximum of 99.6%.
[0038] Example 5
[0039] This embodiment uses the enzyme composition prepared in Example 1 to investigate the optimal ATP concentration for the preparation of pyridoxine phosphate. ATP concentrations of 10, 15, 20, and 25 mM were set, with other reaction conditions remaining the same as in Example 1.
[0040] The results are as follows Figure 7 As shown, the conversion rate of PNP reaches 71.1% when the ATP solution is at 25 mM.
Claims
1. A method for preparing pyridoxine phosphate, wherein the reaction system comprises a substrate pyridoxine, an enzyme composition, and a coenzyme factor, characterized in that, The enzyme composition is pyridoxine kinase and polyphosphate kinase, and the coenzyme factor includes metal cofactor, ATP and phosphate donor.
2. The production method according to claim 1, characterized by, The amino acid sequence of the pyridoxine kinase is shown as SEQ ID NO: 1, and the amino acid sequence of the polyphosphate kinase is shown as SEQ ID NO:
2.
3. The production method according to claim 1, characterized by, The pH of the reaction is 7-7.
5.
4. The preparation method according to claim 1, characterized in that, The metal cofactor is MgCl2.
5. The production method according to claim 4, characterized by, Mg 2+ at a concentration of 25-75 mM.
6. The method of claim 1, wherein, The phosphate donor is sodium hexametaphosphate.
7. The production method according to claim 6, characterized by, The concentration of the sodium hexametaphosphate is 60-200 mM.
8. The method of claim 1, wherein, The concentration of the ATP is 15-25 mM.
9. The method of claim 1, wherein, The preparation step of the pyridoxine kinase or polyphosphate kinase includes constructing a recombinant vector containing a target protein gene, preparing a recombinant microorganism, and expressing the target protein.
10. Use of the preparation method according to claim 1 in the field of biopharmaceuticals.