Biosynthesis method of pyridoxal phosphate or pyridoxine phosphate

Abstract

The invention relates to a biosynthesis method of pyridoxal phosphate or pyridoxine phosphate, and belongs to the technical field of biosynthesis. In order to solve the existing problem of serious environmental pollution, the invention provides a biosynthesis method of pyridoxal phosphate or pyridoxine phosphate, which comprises the following steps: in the presence of polyphosphate, magnesium salt, ATP (adenosine triphosphate), pyridoxal kinase and polyphosphate kinase, performing phosphorylation reaction on pyridoxal or pyridoxine and acid salts thereof, thereby obtaining the pyridoxal phosphate or pyridoxine phosphate. The temperature of the phosphorylation reaction is controlled to be 30-38 DEG C, after the reaction is finished, protein separation is conducted on reaction liquid, mother liquor is separated, the compound pyridoxal phosphate or pyridoxine phosphate shown in the formula I is obtained, and the mother liquor is recycled and reused. According to the method, a large amount of ammonium acetophosphate does not need to be additionally added, operation is simpler, mother liquor can be recycled, a large amount of waste water is prevented from being discharged, environment friendliness is better achieved, and the yield is high.

Description

Technical Field

[0001] This invention relates to a method for the biosynthesis of pyridoxal phosphate or pyridoxine phosphate, belonging to the field of biosynthesis technology. Background Technology

[0002] Pyridoxal-5-phosphate (PLP) is a derivative of vitamin B6 and the main activated form of vitamin B6 involved in various metabolic reactions of proteins, fats, and carbohydrates. Pyridoxal-5-phosphate is also a coenzyme for the synthesis of β-carbonylamides, which have wide applications in drug bioactivity research, liquid crystal materials, and coatings, and have a large market demand.

[0003] Currently, the preparation of pyridoxal phosphate mainly relies on chemical synthesis, which involves reacting pyridoxamine dihydrochloride with phosphoric acid to generate the intermediate pyridoxamine phosphate, which is then oxidized with manganese dioxide to produce the target product. However, this method involves hazardous chemicals and suffers from high production costs, significant environmental pollution, and low recovery rates. In contrast, biological methods offer advantages such as lower risk, less pollution, higher reaction concentrations, and higher conversion efficiency. Existing biological methods for preparing PLP mainly fall into two categories. One major approach involves the one-step phosphorylation of pyridoxal or its hydrochloride by a specific active enzyme, directly catalyzing the formation of PLP. The other approach uses pyridoxine hydrochloride (PN) as a substrate, catalyzing phosphorylation with a specific active enzyme to synthesize pyridoxine phosphate, which is then further oxidized enzymatically or chemically to prepare pyridoxal phosphate. Therefore, improving the efficiency of phosphorylation is crucial for increasing the yield of PLP.

[0004] However, a significant factor affecting the biological preparation of PLP and PNP is the ATP recycling process in the reaction system. Currently, the most efficient ATP regeneration enzymes are generally acetate kinases, while polyphosphokinases generally have low ATP regeneration efficiency. However, using acetate kinase requires the addition of ammonium acetate phosphate, which has extremely poor stability in aqueous solution. Therefore, large amounts of ammonium acetate phosphate need to be continuously added during the reaction, leading to high production costs, difficult process control, and cumbersome operation. Furthermore, the addition of large amounts of ammonium acetate phosphate results in a large amount of acetate in the reaction solution, which firstly leads to lower product quality and lower mother liquor product extraction rates, and also makes it difficult to recover unreacted pyridoxal, ATP, etc., thus increasing raw material costs. Secondly, the mother liquor cannot be recycled, resulting in a large volume of wastewater with high salt content and a variety of reaction residues, leading to high treatment costs and other problems. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a method for the biosynthesis of pyridoxal phosphate or pyridoxine phosphate, which solves the problem of achieving high product yield and reducing wastewater discharge.

[0006] The objective of this invention is achieved through the following technical solution: a method for the biosynthesis of pyridoxal phosphate or pyridoxine phosphate, characterized in that the method comprises the following steps:

[0007] A. In the presence of polyphosphate, magnesium salt, ATP, pyridoxal kinase and polyphosphate kinase, a compound of formula II or its acid salt is subjected to a phosphorylation reaction, wherein the temperature of the phosphorylation reaction is controlled at 30°C to 38°C.

[0008]

[0009] In the compound of formula II, R is a -CH2OH group or a -HC=O group;

[0010] B. After the phosphorylation reaction is completed, the reaction solution is subjected to protein separation, and the mother liquor is separated to obtain compound I, pyridoxal phosphate or pyridoxine phosphate. The mother liquor is then recycled.

[0011]

[0012] The R group in the compound of formula I corresponds to the R group in the compound of formula II.

[0013] This invention utilizes polyphosphokinase to regenerate ATP and combines it with pyridoxal kinase to catalyze the production of pyridoxal phosphate or pyridoxine phosphate from the substrate. By leveraging the synergistic effect of polyphosphokinase and pyridoxal kinase and controlling the reaction system temperature at 30℃~38℃, it effectively achieves efficient ATP regeneration. More importantly, by employing the synergistic effect of these two kinases, it eliminates the need for adding large amounts of ammonium acetylated phosphate, thus preventing the formation of acetate in the reaction solution after the reaction. This significantly improves product utilization and simplifies the operation. The mother liquor collected after product extraction can be recycled, effectively reducing wastewater generation and avoiding large-scale wastewater discharge, thus promoting environmental friendliness. Furthermore, the product yield is high. Compared with the use of acetate kinase in the existing technology, the cost in the preparation of pyridoxal phosphate or pyridoxine phosphate can be reduced by half; it is not only applicable to the pyridoxal hydrochloride-catalyzed synthesis of pyridoxal phosphate system, but also has a high applicability in the phosphorylation of pyridoxine to prepare pyridoxine phosphate (PNP) system, reducing PNP production cost and improving PNP production efficiency.

[0014] When R in the above compound of formula II is a -CH2OH group, the substrate is pyridoxine or its hydrochloride, and its structural formula is shown below:

[0015]

[0016] When R is a -HC=O group in the above-mentioned compound II, the substrate is pyridoxal or its hydrochloride salt, and its structural formula is shown below:

[0017]

[0018] In the above-described biosynthesis method for pyridoxal phosphate or pyridoxine phosphate, preferably, the phosphorylation reaction in step A is carried out at a pH of 6-8. This better ensures the activity of polyphosphokinase and pyridoxal kinase in the reaction system, making the reaction more efficient and better guaranteeing the yield and quality of the product.

[0019] In the above-described biosynthesis method of pyridoxal phosphate or pyridoxine phosphate, preferably, the polyphosphate in step A is sodium hexametaphosphate or potassium hexametaphosphate; and the magnesium salt is magnesium chloride or magnesium sulfate. Using sodium hexametaphosphate or potassium hexametaphosphate enables efficient ATP production with high reaction conversion, and the magnesium salt synergistically promotes the reaction.

[0020] In the aforementioned biosynthesis methods of pyridoxal phosphate or pyridoxine phosphate, preferably, the mother liquor described in step B is first dephosphorized using a struvite precipitation method before recycling. The main chemical component of struvite is MgNH4PO4·6H2O. The struvite precipitation method refers to adding MgNH4PO4·6H2O to a solution containing ammonia nitrogen and phosphate. 2+ This method involves generating a sparingly soluble magnesium ammonium phosphate precipitate, thereby removing phosphorus from the solution. In the biosynthesis of pyridoxal phosphate or pyridoxine phosphate, removing phosphorus from the crystallization mother liquor allows for the efficient recovery of unused raw materials and residual products, reducing production costs. Simultaneously, the precipitate serves as an excellent slow-release fertilizer, enabling the recycling of phosphorus resources.

[0021] In the above-mentioned biosynthesis method of pyridoxal phosphate or pyridoxine phosphate, as another preferred embodiment, the mother liquor in step B is treated with ammonia and magnesium salts for dephosphorization before recycling. The presence of ammonia and magnesium salts causes residual phosphorus in the mother liquor to form magnesium ammonium phosphate precipitate, which is then removed by filtration, thus achieving effective dephosphorization. Furthermore, recycling the filtrate after filtration allows for a recovery rate of over 90% for residual pyridoxal phosphate and unreacted substrate, and a total ATP recovery rate of over 55%. The dephosphorized filtrate can be directly fed with the corresponding raw materials and substrates, avoiding the characteristic of unrecoverable mother liquor, improving raw material utilization, and effectively reducing wastewater discharge. As a further preferred embodiment, the molar ratio of residual phosphorus in the mother liquor to ammonia is 1:1 to 1.1; the molar ratio of residual phosphorus in the mother liquor to magnesium salt is 1:1.2 to 1.3.

[0022] In the above-described biosynthesis method of pyridoxal phosphate or pyridoxine phosphate, preferably, the protein separation in step B is performed by flocculation, ultrafiltration, or acidification. This better ensures the quality of the product and achieves high purity.

[0023] In the above-mentioned biosynthetic methods of pyridoxal phosphate or pyridoxine phosphate, preferably, the molar ratio of the compound of formula II or its acid salt: polyphosphate: magnesium salt: ATP is 1:0.5-0.8:0.5-0.8:0.03-0.05.

[0024] In the above-described biosynthesis method for pyridoxal phosphate or pyridoxine phosphate, preferably, the phosphorylation reaction in step A is carried out in the presence of water. An aqueous system is more conducive to maintaining enzyme activity and allows the reaction to proceed more completely.

[0025] The specific reaction equation for the biosynthesis method of pyridoxal phosphate or pyridoxine phosphate of the present invention (taking hexametaphosphate as an example of polyphosphate) can be expressed by the following method:

[0026]

[0027] In summary, compared with the prior art, the present invention has the following advantages:

[0028] 1. This invention eliminates the need for adding large amounts of ammonium acetylated phosphate, preventing the formation of acetate in the reaction solution and effectively improving product utilization. The operation is also simpler, and the mother liquor collected after product extraction can be recycled. This recyclability effectively reduces wastewater generation and avoids large-scale wastewater discharge, making it more environmentally friendly and achieving a high yield. Compared to the use of acetate kinase in existing technologies, the cost in the preparation of pyridoxal phosphate or pyridoxine phosphate can be reduced by half.

[0029] 2. The present invention utilizes the presence of ammonia and magnesium salts to precipitate residual phosphorus in the mother liquor into magnesium ammonium phosphate, which is then removed by filtration, thereby achieving effective phosphorus removal. Furthermore, the filtration and subsequent recycling process can achieve a recovery rate of over 90% for residual pyridoxal phosphate and unreacted substrates, and a total ATP recovery rate of over 55%, avoiding the drawback of unrecoverable filtrate. Attached Figure Description

[0030] Figure 1 This is a liquid phase detection spectrum of the reaction substrate and product for the synthesis of pyridoxal phosphate in this invention.

[0031] Figure 2 This is a liquid phase detection spectrum of the reaction substrate and product for the synthesis of pyridoxine phosphate in this invention.

[0032] The above Figure 1 In the reaction, the retention time of the substrate pyridoxal (PL) was 3.351 min, and the retention time of the product pyridoxal phosphate (PLP) was 4.573 min.

[0033] Figure 2 In the reaction, the retention time of the substrate pyridoxine (PN) was 3.180 min, and the retention time of the product pyridoxine phosphate (PNP) was 3.603 min. Detailed Implementation

[0034] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the present invention is not limited to these embodiments.

[0035] Example 1

[0036] I. Expression and preparation of pyridoxal kinase

[0037] The pyridoxal kinase mutant bacterial culture was inoculated into LB medium and cultured at 37°C and 200 rpm for 12 hours to obtain the primary seed culture. The pyridoxal kinase mutant can be the pyridoxal kinase mutant disclosed in the previously filed patent application with patent number CN202411985002.0 and publication number CN119709686A, entitled "A Pyridoxal Kinase Mutant, Recombinant Expression Vector and Microbial Cells and Their Applications"; other existing pyridoxal kinases can also be used for catalysis.

[0038] The primary seed culture was inoculated into TB medium at an inoculation rate of 2.5% (V / V) and cultured on a shaker at 37°C and 200 rpm for 4 h. IPTG was then added to a final concentration of 0.5 mM and cultured on a shaker at 25°C and 150 rpm for 12 h to induce the expression of the target protein. After the culture was completed, the fermentation broth was centrifuged at 8000 rpm / min at 4°C to collect the wet cells, which were then frozen and stored at -20°C.

[0039] Weigh the above wet bacterial cells, add 3 times the weight of purified water, stir and mix well, sonicate to disrupt the cells, centrifuge, collect the supernatant, and obtain the enzyme solution of the pyridoxal kinase mutant.

[0040] The culture medium formulations for pyridoxal kinase and PPK expression are as follows:

[0041] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, sterilized at 121℃ for 20 min.

[0042] TB medium: potassium dihydrogen phosphate 2.31 g / L, dipotassium hydrogen phosphate trihydrate 16.44 g / L, yeast extract 24 g / L, peptone 12 g / L, glycerol 4 g / L.

[0043] II. Expression and Enzyme Solution Preparation of Polyphosphoric Acid Kinase

[0044] The polyphosphate kinase mutant bacterial culture was inoculated into LB medium and cultured on a shaker at 37°C and 200 rpm for 12 hours to obtain the primary seed culture.

[0045] The primary seed culture was inoculated into TB medium at an inoculation rate of 2.5% (V / V) and cultured on a shaker at 37°C and 200 rpm for 4 h. IPTG was then added to a final concentration of 0.5 mM and cultured on a shaker at 25°C and 150 rpm for 12 h to induce the expression of the target protein. After the culture was completed, the fermentation broth was centrifuged at 8000 rpm / min at 4°C to collect the wet cells, which were then frozen and stored at -20°C.

[0046] Weigh the above wet bacterial cells, add 3 times the weight of purified water, stir and mix well, sonicate to break up the cells, centrifuge, collect the supernatant, and obtain the polyphosphoric acid kinase solution.

[0047] The culture medium formulations involved in polyphosphate kinase expression are as follows:

[0048] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, sterilized at 121℃ for 20 min.

[0049] TB medium: potassium dihydrogen phosphate 2.31 g / L, dipotassium hydrogen phosphate trihydrate 16.44 g / L, yeast extract 24 g / L, peptone 12 g / L, glycerol 4 g / L.

[0050] Example 2

[0051] Methods for determining the enzyme activity of polyphosphate kinases and pyridoxal kinases

[0052] Polyphosphate kinase (PPK2) activity assay system: 1 mL of reaction solution contained 10 mM ADP, 15 mM sodium hexametaphosphate, 30 mM magnesium chloride, pH 7.0. 0.1 mL of diluted PPK2 was added. The reaction was carried out at 35℃ for 5 min. After sampling, the concentration of ATP in the reaction solution was determined by HPLC. The PPK2 enzyme activity was determined to be 3-4 U / mL.

[0053] The pyridoxal kinase activity assay system consisted of 3 mL of reaction solution containing 50 g / L PN, 30 g / L ATP, 2 g / L magnesium sulfate heptahydrate, and 0.2 mL pyridoxal kinase. The solution was set at pH 5.0 and reacted at 35℃ for 10 min. After sampling, the concentration change of PNP was determined by HPLC. The measured pyridoxal kinase activity was 15-20 U / mL.

[0054] Example 3

[0055] The synthesis of pyridoxal phosphate and treatment of the extract (mother liquor) were carried out in a 200 mL system.

[0056] Weigh out 242 mmol / L pyridoxal hydrochloride, 130 mmol / L sodium hexametaphosphate, 130 mmol / L magnesium chloride, 10 mmol / L ATP, and 200 ml of purified water. Turn on mechanical stirring and heat the reaction system to 35°C. Adjust the pH of the system to 7.0. Add 15 ml of pyridoxal kinase (201) and 6 ml of polyphosphokinase cell-broken centrifuged supernatant enzyme solution. React for 14 h. Detect the formation process of pyridoxal phosphate using high performance liquid chromatography. The reaction was terminated when the conversion rate of pyridoxal phosphate in the system reached 80.37%. Separate the enzyme protein by ultrafiltration. Concentrate the filtrate and crystallize to extract the final product pyridoxal phosphate (PLP). HPLC analysis showed that the dry PLP content was 98.96% and the molar yield was 69.97%. The mother liquor after extraction was measured, and the results showed that the phosphorus concentration in the mother liquor was 10.2 g / L. Then, 0.5 eq magnesium chloride was added to the mother liquor and stirred to dissolve it. Then, 1 eq ammonia water was added and stirred at room temperature for 2 hours. After the process was completed, the precipitate was removed by filtration, and 120 ml of dephosphorized filtrate was obtained.

[0057] Example 4

[0058] The dephosphorization filtrate obtained in Example 3 was recycled and reused.

[0059] The mother liquor for the synthesis of pyridoxal phosphate was reused in a 200 mL system for feeding:

[0060] Take 120 ml of the dephosphorization filtrate from Example 3, add 80 ml of purified water, start stirring, heat to 35°C, and sequentially add 186 mmol / L pyridoxal hydrochloride, 105 mmol / L sodium hexametaphosphate, 130 mmol / L magnesium chloride, and 5 mmol / L ATP. Adjust the pH to 7.0, then add 15 ml of pyridoxal kinase and 6 ml of polyphosphokinase cell-disrupting centrifuged supernatant enzyme solution. React for 15 h. During the reaction, the formation process of pyridoxal phosphate is monitored by high performance liquid chromatography. The conversion rate of pyridoxal phosphate is measured to be 82.01%. The reaction is terminated, and the product pyridoxal phosphate (PLP) and mother liquor are extracted according to the method in Example 3. The PLP product content is 97.32%, and the molar yield is 70.86%. The mother liquor is dephosphorized according to the method in Example 3, and the process is repeated.

[0061] Example 5

[0062] Synthesis of pyridoxal phosphate in a 3.5L system and treatment of the extract:

[0063] Weigh out 175g of pyridoxal hydrochloride, 280g of sodium hexametaphosphate, 42g of magnesium chloride, 21g of ATP, and 3500g of purified water. Turn on mechanical stirring, heat to 35℃, adjust pH to 7.0, add 260g of pyridoxal kinase (201) and 110g of polyphosphokinase, break down the enzyme supernatant, and maintain the temperature for 15h. During the reaction, the formation process of pyridoxal phosphate was monitored by high performance liquid chromatography. According to the results, the conversion rate of pyridoxal phosphate was 80.96%. The reaction was terminated, and the enzyme protein was separated by ultrafiltration. The filtrate was concentrated and crystallized to extract the final product, pyridoxal phosphate (PLP). HPLC analysis showed that the dry PLP content was 99.35%, and the molar yield was 71.01%.

[0064] The extracted mother liquor was tested and the phosphorus concentration was found to be 11.65 g / L. 35.82 g of magnesium chloride was added and stirred to dissolve. 49.42 g of ammonia water was slowly added dropwise, stirred at room temperature for 2 hours, and filtered to obtain 1950 ml of dephosphorized filtrate.

[0065] Example 6

[0066] The above-mentioned dephosphorization filtrate is recovered and reused. The specific reuse operation is as follows:

[0067] The mother liquor for the synthesis of pyridoxal phosphate in a 3.5L system was reused for feeding:

[0068] Take 1950 ml of the dephosphorized filtrate obtained in Example 5, add 1550 ml of purified water, start stirring, heat to 35°C, and then add 135 g of pyridoxal hydrochloride, 224 g of sodium hexametaphosphate, 42 g of magnesium chloride, and 10.5 g of ATP in sequence. Adjust the pH to 7.0 with 10 N sodium hydroxide, then add 260 g of pyridoxal kinase (201) and 110 g of polyphosphokinase. Centrifuge the supernatant enzyme solution and react for 16 h. HPLC was used to detect the pyridoxal phosphate formation process, and the result showed that the pyridoxal phosphate conversion rate was 79.82%. The reaction was terminated, and the enzyme protein was separated by ultrafiltration. The filtrate was concentrated and crystallized to extract the final product, pyridoxal phosphate (PLP). HPLC was used to detect the PLP content in the dry product, which was 98.64%, with a yield of 70.62%. The collected mother liquor was dephosphorized, and the mother liquor was repeatedly reused after dephosphorization according to the method in Example 5.

[0069] The results of Example 6 show that this method significantly improves the catalytic efficiency for the formation of pyridoxal phosphate. After treatment, the recovery rates of sodium hexametaphosphate, ATP, and PNP in the mother liquor are relatively high and stable. The conversion rate and yield of the reaction process show no significant abnormalities after reuse. The mother liquor, after phosphorus removal, can be reused at least four batches or more, significantly reducing costs. The reusability of the mother liquor effectively achieves the advantage of cost reduction and also reduces wastewater discharge, further contributing to environmental friendliness.

[0070] Example 7

[0071] Pyridoxine phosphate (PNP) was synthesized in a 200 mL system, and the extract (mother liquor) was treated accordingly.

[0072] Weigh out 242 mmol / L pyridoxine hydrochloride, 130 mmol / L sodium hexametaphosphate, 130 mmol / L magnesium chloride, 10 mmol / L ATP, and 200 ml purified water. Turn on mechanical stirring and heat the reaction system to 36°C. Adjust the pH of the system to 7.0. Add 15 ml of pyridoxal kinase (201) and 6 ml of polyphosphate kinase cell disruption and centrifugation supernatant enzyme solution. React for 10 h. Detect the pyridoxine phosphate formation process by high performance liquid chromatography. The reaction was terminated when the conversion rate of pyridoxine phosphate in the system reached 81.61%. Separate the enzyme protein by ultrafiltration. Concentrate the filtrate and crystallize to extract the final product pyridoxine phosphate (PNP). HPLC analysis showed that the dry PNP content was 99.02% and the molar yield was 71.3%.

[0073] The mother liquor after extraction was measured, and the results showed that the phosphorus concentration in the mother liquor was 10.7 g / L. Then, 1.2 eq of magnesium chloride was added to the mother liquor and stirred to dissolve it. Then, 1.1 eq of ammonia water was added and stirred at room temperature for 2 hours. After the stirring was completed, the precipitate was removed by filtration, and 110 ml of dephosphorized filtrate was obtained.

[0074] Example 8

[0075] The dephosphorization filtrate obtained in Example 7 was recycled and reused.

[0076] The mother liquor for synthesizing pyridoxine phosphate in a 200 mL system was reused for feeding:

[0077] Take 110 ml of the dephosphorization filtrate from Example 7, add 90 ml of purified water, start stirring, heat to 35°C, and sequentially add 195 mmol / L pyridoxine hydrochloride, 105 mmol / L sodium hexametaphosphate, 130 mmol / L magnesium chloride, and 10 mmol / L ATP. Adjust the pH to 7.0, then add 15 ml of pyridoxal kinase and 6 ml of polyphosphokinase cell-disrupting centrifuged supernatant enzyme solution. React for 12 h. During the reaction, the formation process of pyridoxine phosphate is monitored by high performance liquid chromatography. The conversion rate of pyridoxine phosphate is measured to be 80.41%. The reaction is then terminated. The product pyridoxine phosphate (PNP) and mother liquor can be extracted and treated according to the method in Example 7. The PNP product content is 98.52%, and the molar yield is 70.84%. The mother liquor is used for dephosphorization treatment according to the method in Example 7, and the process is repeated. The reusability of the mother liquor effectively reduces costs and wastewater discharge, making it more environmentally friendly.

[0078] Example 9

[0079] Synthesis of pyridoxine phosphate and treatment of the extract in a 3.5L system:

[0080] Weigh 177g of pyridoxine hydrochloride, 280g of sodium hexametaphosphate, 42g of magnesium chloride, 21g of ATP, and 3500g of purified water. Turn on mechanical stirring, heat to 35℃, adjust pH to 7.0, add 260g of pyridoxal kinase (201) and 110g of polyphosphoric acid kinase, break down the enzyme supernatant, and maintain the temperature for 11h. During the reaction, the formation of pyridoxine phosphate was monitored by high performance liquid chromatography. According to the results, the conversion rate of pyridoxine phosphate was 80.54%. The reaction was terminated, and the enzyme protein was separated by ultrafiltration. The filtrate was concentrated and crystallized to extract the final product, pyridoxine phosphate (PNP). HPLC analysis showed that the dry PNP content was 99.42% and the molar yield was 72.43%.

[0081] The mother liquor after extraction was tested, and the phosphorus concentration was found to be 11.45 g / L. 35.78 g of magnesium chloride was added and stirred to dissolve. 49.5 g of ammonia water was slowly added dropwise, stirred at room temperature for 2 hours, and filtered to obtain 1974 ml of dephosphorized filtrate.

[0082] Example 10

[0083] The above-mentioned dephosphorization filtrate is recovered and reused. The specific reuse operation is as follows:

[0084] The mother liquor for synthesizing pyridoxine phosphate in a 3.5L system was reused for feeding:

[0085] Take 1974 ml of the dephosphorized filtrate obtained in Example 10, add 1550 ml of purified water, start stirring, heat to 35°C, and then add 140 g of pyridoxine hydrochloride, 224 g of sodium hexametaphosphate, 42 g of magnesium chloride, and 10.5 g of ATP in sequence. Adjust the pH to 7.0 with 10 N sodium hydroxide, then add 260 g of pyridoxal kinase (201) and 110 g of polyphosphokinase. Centrifuge the supernatant enzyme solution and react for 10 h. Detect the pyridoxine phosphate formation process by high performance liquid chromatography. The result shows that the pyridoxine phosphate conversion rate is 79.85%. Terminate the reaction and separate the enzyme protein by ultrafiltration. Concentrate the filtrate, crystallize and extract the final product pyridoxine phosphate (PNP). HPLC detects the PNP content of the dry product, which is 98.76%, with a yield of 72.2%. Dephosphorize the collected mother liquor. The mother liquor is then repeatedly reused after dephosphorization according to the method in Example 9.

[0086] The results of Example 10 show that this method significantly improves the catalytic efficiency for the formation of pyridoxine phosphate. After treatment, the recovery rates of sodium hexametaphosphate, ATP, and PNP in the mother liquor are relatively high and stable. No significant abnormalities were observed in the conversion rate and yield of the reaction process after reuse. Furthermore, through repeated implementation, the mother liquor can be reused at least four batches, significantly reducing costs. The reusability of the mother liquor effectively reduces costs and wastewater discharge, thus contributing to environmental friendliness.

[0087] Example 11

[0088] The synthesis of pyridoxal phosphate and treatment of the extract (mother liquor) were carried out in a 200 mL system.

[0089] Weigh out 242 mmol / L pyridoxal hydrochloride, 130 mmol / L sodium hexametaphosphate, 130 mmol / L magnesium chloride, 10 mmol / L ATP, and 200 ml of purified water. Turn on mechanical stirring and heat the reaction system to 35°C. Adjust the pH of the system to 7.0. Add 15 ml of pyridoxal kinase and 6 ml of polyphosphokinase cell-disrupting supernatant enzyme solution. React for 15 h. Detect the formation process of pyridoxal phosphate using high performance liquid chromatography. The reaction was terminated when the conversion rate of pyridoxal phosphate in the system reached 80.14%. Separate the enzyme protein by ultrafiltration. Concentrate the filtrate and crystallize to extract the final product pyridoxal phosphate (PLP). HPLC analysis showed that the dry PLP content was 98.89% and the molar yield was 69.54%. The mother liquor after product extraction was measured, and the results showed that the phosphorus concentration in the mother liquor was 10.8 g / L. Then, 7.97 g of magnesium chloride was added to the mother liquor, stirred and dissolved, and 2.5 g of ammonia water was added dropwise for dephosphorization treatment. The mixture was stirred at room temperature for 2 hours. After the treatment, the mixture was filtered to remove the precipitate, and 130 ml of dephosphorized filtrate was obtained.

[0090] The specific embodiments described in this invention are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

[0091] Although the present invention has been described in detail and specific embodiments have been cited, it will be apparent to those skilled in the art that various changes or modifications can be made without departing from the spirit and scope of the invention.

Claims

1. A method for the biosynthesis of pyridoxal phosphate or pyridoxine phosphate, characterized in that, The method includes the following steps: A. In the presence of polyphosphate, magnesium salt, ATP, pyridoxal kinase and polyphosphate kinase, a compound of formula II or its acid salt is subjected to a phosphorylation reaction, wherein the temperature of the phosphorylation reaction is controlled at 30°C to 38°C. In the compound of formula II, R is a -CH2OH group or a -HC=O group; B. After the phosphorylation reaction is completed, the reaction solution is subjected to protein separation, and the mother liquor is separated to obtain compound I, pyridoxal phosphate or pyridoxine phosphate. The mother liquor is then recycled. The R group in the compound of formula I corresponds to the R group in the compound of formula II.

2. The method for biosynthesizing pyridoxal phosphate or pyridoxine phosphate according to claim 1, characterized in that, The phosphorylation reaction described in step A is carried out under conditions of pH 6 to 8.

3. The biosynthetic method for pyridoxal phosphate or pyridoxine phosphate according to claim 1, characterized in that, The polyphosphate mentioned in step A is sodium hexametaphosphate or potassium hexametaphosphate; the magnesium salt is magnesium chloride or magnesium sulfate.

4. The method for biosynthesizing pyridoxal phosphate or pyridoxine phosphate according to claim 1, 2, or 3, characterized in that, The mother liquor described in step B is first dephosphorized by struvite precipitation before being recycled.

5. The method for biosynthesizing pyridoxal phosphate or pyridoxine phosphate according to claim 4, characterized in that, Before recycling the mother liquor described in step B, ammonia and magnesium salts are added for dephosphorization treatment.

6. The method for biosynthesizing pyridoxal phosphate or pyridoxine phosphate according to claim 5, characterized in that, The molar ratio of residual phosphorus in the mother liquor to the ammonia water is 1:1.0 to 1.1; the molar ratio of residual phosphorus in the mother liquor to the magnesium salt is 1:1.2 to 1.

3.

7. The method for biosynthesizing pyridoxal phosphate or pyridoxine phosphate according to claim 1, 2, or 3, characterized in that, The protein separation described in step B is performed using flocculation, ultrafiltration, or acidification.

8. The method for biosynthesizing pyridoxal phosphate or pyridoxine phosphate according to claim 1, 2, or 3, characterized in that, The molar ratio of the compound of formula II or its acid salt: polyphosphate: magnesium salt: ATP is 1:0.5-0.8:0.5-0.8:0.03-0.

05.

9. The method for biosynthesizing pyridoxal phosphate or pyridoxine phosphate according to claim 1, 2, or 3, characterized in that, The phosphorylation reaction described in step A is carried out in the presence of water.

Images