WO2020056713A1 - Method for preparing high-purity nad - Google Patents

Abstract

A method for preparing high-purity NAD, comprising: adjusting a pH value to 6.0-8.0, removing insoluble substances, heating to 35 ± 1°C, adding ethanol, cooling to 6 ± 0.5°C, crystallizing, filtering, heating a filtrate to 25-30°C, adjusting the pH value to 1.5-3.0, adding an NAD seed crystal, cooling to 12 ± 0.5°C, crystallizing, adding ethanol again after crystallinity reaching 40%, cooling to 3 ± 0.5°C, crystallizing, filtering after the crystallinity being above 90%, and drying a filter cake to obtain a high-purity NAD product.

Description

Method for preparing high-purity NAD Technical field

The invention relates to the technical field of purification of nucleotide coenzymes, and in particular to a method for purifying a crude solution of NAD.

Background technique

NAD is the abbreviated form of Nicotinamide Adenine Dinucleotide. It is a physiological substance that exists in all living cells, including human cells, and has no side effects on the body. This substance is Cofactors of many enzymes that catalyze oxidation-reduction reactions are therefore called coenzymes I.

NAD participates in a variety of physiological activities such as cell material metabolism, energy synthesis, and cell DNA repair in the body. It is a control marker in the energy production chain in mitochondria and plays an important role in the body's immune capacity. Today, NAD is widely used in chemical catalytic reactions, API production, health care product manufacturing, and cosmetics industries. With the in-depth study of NAD in the treatment and prevention of diseases, the importance of NAD as a health care product has become increasingly prominent. Since then, the market demand has increased year by year.

At present, the preparation methods of NAD are mainly divided into chemical synthesis methods and biocatalytic methods. Among them, biocatalytic methods include biological fermentation methods and enzyme catalytic methods. Compared with chemical synthesis methods, it has gradually become environmentally friendly and harmless. Mainstream direction. The crude product solution (enzyme reaction solution or biological fermentation broth) prepared by biocatalytic method contains a large amount of impurities in addition to NAD. The purity of NAD is only 20-40%, so it needs to be further separated and purified to obtain a comparative Pure NAD products.

Chinese invention patent CN105131065B, a method for preparing an oxidized coenzyme I, and Chinese invention patent CN105481923B, a method for preparing nicotinamide adenine dinucleotide, both disclose a method for separating and purifying NAD, which mainly uses resin columns. Isolate and purify. This separation and purification method using a resin column is cumbersome, time-consuming, and uses a large amount of solvents, which is not conducive to large-scale industrial production. In the later stage, the resin needs to be activated and a large amount of solvents such as acids, alkalis, and salts will be used. Environmental pollution is relatively large and costly.

technical problem

The purpose of the present invention is to solve the technical problems of tedious operation, time-consuming, high cost, and large pollution in the existing method for separating and purifying NAD, and to provide a high-purity preparation with simple and fast operation, low cost, and low pollution. NAD method is used to purify the crude NAD solution.

Technical solutions

To achieve the above object, the present invention provides a method for preparing high-purity NAD, including: 1) adjusting the pH value of the crude NAD solution to 6.0 to 8.0, and then removing insoluble matters; 2) increasing the temperature to 35 ± 1 ° C, And add ethanol to the final concentration of ethanol in the solution is 20% to 40% by volume, stir until clear; 3) cool to 6 ± 0.5 ℃, maintain the pH value of 6.0 to 8.0 to crystallize, after the crystals precipitate, filter and collect The filtrate; 4) The temperature of the filtrate in step 3) was raised to 25-30 ° C, and then the pH was adjusted to 1.5-3.0, and stirred until clear; 5) The NAD seed was added, and then the temperature was lowered to 12 ± 0.5 ° C, and the pH was maintained. 1.5 ～ 3.0, when the crystallization rate of NAD in the solution reaches 40%, add ethanol until the final concentration of ethanol in the solution is 10% -30% by volume, and mix well; 6) Reduce the temperature to 3 ± 0.5 ℃ and maintain the pH Crystallize from 1.5 to 3.0. When the crystallization rate of NAD in the solution is more than 90%, filter, collect the filter cake, and obtain a high-purity NAD product after drying.

In the method of the present invention, the crude solution of NAD in step 1) refers to a solution containing a large amount of impurities in addition to NAD, such as a solution obtained by dissolving NAD products purchased from the market with insufficient purity in water, or The crude product solution obtained by NAD was prepared by biocatalysis.

Preferably, the concentration of the crude NAD solution is controlled to 100-300 mmol / L to ensure that enough crystals can be precipitated in the later stage, thereby ensuring a sufficiently high yield.

Biocatalysis refers to the process of transforming substances using biological enzymes or biological organisms (cells, organelles, tissues, etc.) as catalysts (referred to as enzyme catalysis and biological fermentation respectively). This reaction process is also called biotransformation ). The biological organisms commonly used in biocatalysis are mainly microorganisms. The essence is to use enzymes in the cells of microorganisms to catalyze and promote the process of biological transformation. The biocatalytic method has mild action conditions (basically completed in normal temperature, neutral, water and other environments); unique and efficient substrate selectivity (because the enzyme in the catalytic process has specific characteristics, that is, an enzyme only Can catalyze a specific substrate to react, but one substrate may be catalyzed by multiple enzymes); it has unique advantages for the synthesis of chiral active pharmaceutical ingredients.

The so-called crude product solution obtained by NAD prepared by the biocatalytic method in the present invention specifically refers to an enzyme reaction solution obtained by using a biological enzyme to catalyze the conversion of a substrate into NAD, or to remove a biological organism by fermentation and induction of expression using a biological organism containing a biological enzyme After the fermentation broth containing NAD. The so-called biological enzyme refers to an enzyme that can specifically catalyze the conversion of a substrate into NAD, and the so-called substrate refers to a precursor substance that can be converted into NAD. For example, the substrate can be NMN and ATP, or a precursor substance that can be converted into NMN or ATP. The corresponding biological enzyme is nicotinamide mononucleotide adenosyltransferase, or nicotinamide mononucleoside. Acid adenosine transferase is used in combination with one or more other enzymes.

The effect of adjusting the pH value to 6.0 to 8.0 in step 1) of the method of the present invention is that on the one hand, impurities (mainly high-valent cations) contained in the solution are precipitated in the form of precipitation; on the other hand, it provides necessary for subsequent crystallization pH conditions. Preferably, the pH is adjusted using sodium hydroxide.

Preferably, in the step 1) of the method of the present invention, the insoluble matter is removed by performing a microfiltration treatment. Microfiltration, also known as microfiltration, uses a microporous membrane as a filter medium. Under the pressure of 0.1 to 0.3 MPa, it traps particles and bacteria between 0.1 and 1 micron, but allows macromolecular organic substances and inorganic salts to pass through. . The microfiltration membrane used in the method of the present invention is preferably a hollow fiber membrane having a pore diameter of 0.1 to 0.5 μm.

The purpose of step 2) of the method of the present invention is to raise the temperature to 35 ± 1 ° C. The purpose is to prevent the formation of a gelatinous precipitate in the solution after adding ethanol, thereby adsorbing some NAD, and increasing the difficulty of filtration.

The effect of adding the ethanol solution in step 2) of the method of the present invention is that ethanol is a poor solution for crystallization of the substrate and by-products, which is beneficial to speeding up and increasing the conversion rate of the crystallization of the substrates and by-products.

In step 3) of the method of the present invention, the reason why the pH value is maintained at 6.0 to 8.0 is that crystals cannot be precipitated when the NAD is above 6.0, and the suitable pH value of the crystals of impurities (mainly catalyzed substrates, by-products and heteroproteins) is 6.0 ～ 8.0; cooling down to 6 ± 0.5 ℃ is conducive to the maximum crystallization of impurities.

When the purity of NAD in the solution is greater than 80%, the efficiency of crystallization of NAD is the best, and the purity of the obtained product is the highest. Therefore, in step 3) of the method of the present invention, preferably, the crystals are precipitated until the purity of the NAD in the solution is above 80%, and then filtering is performed.

The filtration in the method of the present invention refers to a process of separating solids and liquids in a solution by a physical method. Common filtration methods are applicable to the present invention, including atmospheric pressure filtration, reduced pressure filtration, centrifugal filtration, and the like. Preferably, in step 3) of the method of the present invention, filtration is performed using a 500-mesh filtration device.

In step 4) of the method of the present invention, adjusting the pH value to 1.5 to 3.0 is beneficial to the precipitation of NAD. Preferably, hydrochloric acid is used to adjust the pH value; raising the temperature to 25 to 30 ° C can prevent NAD from forming an oily precipitate during the pH adjustment process. Transition state, making it difficult to precipitate crystals.

Preferably, in step 5) of the method of the present invention, the amount of NAD seed added is 0.5% to 1.5% of the mass of NAD contained in the filtrate of step 3).

Because directly lowering the temperature from 25-30 ° C to 3 ± 0.5 ° C or adding an excessive amount of ethanol solution at one time will cause NAD to form an oily transition state, thereby delaying the crystallization time, reducing the crystal conversion rate and even causing crystallization failure. In steps 5) and 6) of the method of the present invention, the crystallization process of NAD is divided into two stages, which are: the first stage is cooled to 12 ± 0.5 ° C, and ethanol is added when the crystallization rate of NAD reaches more than 40% The final concentration of ethanol in the solution is 10% -30% by volume; in the second stage, the temperature is reduced to 3 ± 0.5 ° C. This can effectively avoid the occurrence of the oily transition state of NAD, thereby improving the crystallization efficiency and crystal conversion rate.

Preferably, before the filter cake of step 6) is dried, the filter cake is washed with a 30% ± 2% ethanol aqueous solution with a volume fraction of 10 to 15 ° C.

Preferably, the filter cake of step 6) is dried by a vacuum drying method.

When the crude NAD solution is a crude product solution prepared by biocatalytic NAD, in order to improve the efficiency of separation and purification and increase the yield of NAD crystals, preferably, the method of the present invention further includes: before step 1), The crude product solution was concentrated to remove a large amount of water. Concentration in the present invention refers to a process in which the concentration of a solution is increased by reducing the solvent using a physical method, and includes a vacuum distillation method, an ultrafiltration method, a dialysis method, an adsorption method, and a freeze-drying method.

Preferably, the above-mentioned concentration treatment is concentrated by nanofiltration. Nanofiltration is a pressure-driven membrane separation process between reverse osmosis and ultrafiltration. Nanofiltration membranes are used as filter media. The pore size of nanofiltration membranes is about several nanometers, allowing solvent molecules or certain relative molecular masses. Smaller solutes or low-priced ions penetrate to achieve separation and concentration effects. Preferably, the molecular weight cut-off of the nanofiltration membrane used in the present invention is 200-400 Daltons.

If the concentration of the crude product solution after the concentration treatment is too low, the yield of subsequent NAD crystallization will be reduced, while if the concentration is too high, the pressure of the nanofiltration membrane will be increased, and the life of the nanofiltration membrane will be shortened. Preferably, the crude product solution is concentrated to a concentration of NAD in the solution of 100-300 mmol / L.

For enzyme-catalyzed reactions, iron, nickel, magnesium, and other activators for activating enzymes need to be used during the reaction. These activators will form a large amount of inorganic salt ion precipitation during the nanofiltration concentration process, thereby blocking the nanofiltration. Membrane, so, preferably, prior to nanofiltration concentration, the crude product solution needs to be pretreated as follows: the crude product solution is adjusted to a pH value of 3.0 to 5.0, and then subjected to a microfiltration treatment to collect the microfiltrate.

Preferably, the microfiltration membrane used in the microfiltration process is a hollow fiber membrane having a pore diameter of 0.1 to 0.5 μm.

Beneficial effect

Compared with the prior art, the NAD purification method provided by the present invention has the advantages of high yield and high purity of the purified product. It has been confirmed by industrial practice that the purification method can be prepared from a crude product solution of NAD prepared by a biocatalytic method. A pure NAD product with a purity of more than 99% was obtained, and the yield was more than 85%. The purification method is simple and fast to operate, does not require the use of a large number of toxic and harmful reagents, has little environmental pollution, and has low purification costs, and is suitable for large-scale industrialization to separate and purify NAD products from biological enzyme reaction solutions and biological fermentation solutions.

Best Mode of the Invention

The method for preparing high-purity NAD provided by the present invention preferably includes the following steps:

1) Adjusting the pH value of the crude product solution prepared by biocatalytic NAD to 3.0 to 5.0, and then performing microfiltration with a hollow fiber membrane having a pore size of 0.1 to 0.5 μm, and collecting the micro filtrate;

2) Nanofiltration of the microfiltrate from step 1) with a nanofiltration membrane having a molecular weight cutoff of 200 to 400 Daltons to a concentration of NAD of 100 to 300 mmol / L;

3) The pH value of the concentrated solution in step 2) was adjusted to 6.0 to 8.0 with a sodium hydroxide solution, and then microfiltration was performed with a hollow fiber membrane having a pore size of 0.1 to 0.5 μm, and the micro filtrate was collected;

4) The micro filtrate of step 3) is heated to 35 ± 1 ° C, and ethanol is added to a final concentration of ethanol in the solution of 20% to 40% by volume, and stirred until clear;

5) The clarified solution of step 4) is cooled to 6 ± 0.5 ° C, and the pH is maintained at 6.0 to 8.0 for crystallization. When the crystals are precipitated until the purity of NAD in the solution is more than 80%, filtration is performed with a 500-mesh filtering device to collect the filtrate;

6) Raise the filtrate of step 5) to 25-30 ° C, then adjust the pH to 1.5-3.0 with hydrochloric acid, and stir until clear;

7) Add 0.5% to 1.5% of the NAD seed contained in the solution to the clarified solution in step 6), and then lower the temperature to 12 ± 0.5 ° C and maintain the pH value of 1.5 to 3.0 until the NAD crystals in the solution When the rate reaches 40%, add ethanol to the solution to a final ethanol concentration of 10% to 30% by volume, and mix well;

8) Step 7) cool the uniformly mixed solution to 3 ± 0.5 ° C and maintain the pH value of 1.5-3.0 for crystallization. When the crystallization rate of NAD in the solution is above 90%, filter and collect the filter cake;

Wash the filter cake in step 8) with a 30% ± 2% ethanol aqueous solution at a volume fraction of 10 to 15 ° C, and then vacuum-dry the washed filter cake to obtain a high-purity NAD product.

Embodiments of the invention

The following further describes the present invention in detail with reference to specific embodiments. The following embodiments explain the present invention, and the present invention is not limited to the following embodiments.

Example 1

Treatment object: Bangtai Bioengineering (Shenzhen) Co., Ltd. prepared four batches of crude product solution obtained by NAD using biological enzyme catalysis method (with NMN and ATP as substrates and catalysis with nicotinamide single nucleotide adenosyltransferase). The NAD content and purity in the four batches of crude NAD solution are shown in Table 1.

The purification process of the above four batches of crude NAD solution is as follows:

1. The pH of the four batches of the crude NAD solution was adjusted to 3.0-5.0 with hydrochloric acid, and then microfiltration was performed with a hollow fiber membrane having a pore size of 0.1-0.5 μm.

2. The solution after microfiltration was concentrated by nanofiltration with a nanofiltration membrane having a molecular weight cutoff of 200 to 400 Daltons, and concentrated to a concentration of NAD in the solution of 100 to 300 mmol / L.

3. Use sodium hydroxide to adjust the pH value of the solution concentrated in step 2 to 6.0 to 8.0. During the pH adjustment process, a large amount of precipitation will occur, mainly high-valent cation precipitation, and the hollow fiber membrane with a pore size of 0.1 to 0.5 μm is used for micro-adjustment. Filter to remove the precipitate.

4. Transfer the microfiltration solution from step 3 to the crystallization kettle, turn on the stirring device, set the jacket temperature of the crystallization kettle to 35 ° C, add ethanol to the final ethanol concentration in the solution to 20-40% by volume, and stir until The solution was clear.

5. Set the jacket temperature of the crystallization kettle to 6 ° C. After the solution temperature drops to 6 ° C, take a sample every 3h to check the purity of the NAD in the solution, monitor the pH value of the solution online, and use sodium hydroxide to adjust the pH of the solution at any time. When the purity of the NAD in the solution is greater than 80%, the solution is discharged, filtered through a 500 mesh filter cloth, the filtrate is collected, and the purity of the NAD in the obtained filtrate is measured by high-performance liquid chromatography to obtain the above four The purity of the batch NAD crude product solution after one time crystallization is shown in Table 2.

6. Transfer the filtrate filtered in step 5 to the crystallization kettle, set the jacket temperature of the crystallization kettle to 30 ° C, and when the solution temperature is greater than 25 ° C, adjust the pH of the solution to between 1.5 and 3.0 with hydrochloric acid, and stir To clarify.

7. After the solution is clarified, add NAD seeds in an amount of 0.5% to 1.5% of the NAD mass in the solution, and set the jacket temperature of the crystallization kettle to 12 ° C. After the solution temperature reaches 12 ° C, take samples every 3h to detect NAD When the crystallization rate reaches 40%, add ethanol to the solution until the final concentration of ethanol in the solution is 10-30% by volume, and adjust the pH of the solution to between 1.5 and 3.0 with hydrochloric acid and mix well. .

8. Set the jacket temperature of the crystallization kettle to 3 ° C. After the solution reaches 3 ° C, take a sample every 4h to detect the NAD conversion in the solution, monitor the pH value of the solution online, and keep the pH of the solution at any time with hydrochloric acid. 1.5 ～ 3.0; when the conversion rate of NAD in the solution reaches more than 90%, the solution is discharged and filtered, and then the filter cake is washed 3 times with a 30% ethanol solution with a volume fraction of 10-15 ° C. High-purity NAD products are obtained after vacuum drying.

The weight and purity of the four purified NAD products were measured, and the yield was calculated. The results are shown in Table 3.

Table 1

batch	Solution volume / L	NAD content / g	NAD purity /%
1	4330	36593	27.3
2	4000	39774	25.5
3	3800	40127	29.6
4	4300	43979	26.9

Table 2

batch	Solution volume / L	Concentration (mmol / L)	NAD purity /%
1	224	244	88.3
2	253	234	95.1
3	216	279	93.0
4	430	154	90.6

table 3

batch	Product weight / g	NAD purity /%	NAD yield /%
1	32410	99.54	88.6
2	37050	99.39	93.2
3	36120	99.58	90.0
4	37900	99.62	86.2

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Claims (11)

1. A method for preparing high-purity NAD, characterized in that the method includes the following steps:

   1) The pH value of the crude NAD solution is adjusted to 6.0 to 8.0, and then the insoluble matter is removed;

   2) The temperature is raised to 35 ± 1 ° C, and ethanol is added until the final concentration of ethanol in the solution is 20% to 40% by volume, and stirred until clear;

   3) Reduce the temperature to 6 ± 0.5 ° C, maintain the pH value of 6.0-8.0 for crystallization, filter after the crystals precipitate, and collect the filtrate;

   4) The filtrate of step 3) is heated to 25-30 ° C, and then the pH is adjusted to 1.5-3.0, and stirred until clear;

   5) Add NAD seed, then cool down to 12 ± 0.5 ℃, keep the pH value 1.5 ～ 3.0, when the crystallization rate of NAD in the solution reaches more than 40%, add ethanol to the final concentration of ethanol in the solution is 10% ～ 30% volume fraction, mix well;

   6) The temperature is reduced to 3 ± 0.5 ° C, the pH value is maintained at 1.5-3.0, and when the crystallization rate of NAD in the solution is above 90%, it is filtered and the filter cake is collected.
2. The method for preparing high-purity NAD according to claim 1, wherein the concentration of the crude NAD solution is 100-300 mmol / L.
3. The method for preparing high-purity NAD according to claim 1, wherein in step 1), insoluble matter is removed by a microfiltration method.
4. The method for preparing high-purity NAD according to claim 1, characterized in that in the step 3), filtering is performed when the crystals are precipitated into the solution and the purity of NAD is above 80%.
5. The method for preparing high-purity NAD according to claim 1, wherein in the step 5), the amount of the NAD seed added is 0.5% to the mass of the NAD contained in the filtrate of the step 3). 1.5%.
6. The method for preparing high-purity NAD according to claim 1, characterized in that: before drying the filter cake in step 6), the filter is filtered with an aqueous solution of ethanol having a volume fraction of 30% ± 2% at 10 to 15 ° C. The cake was washed.
7. The method for preparing high-purity NAD according to any one of claims 1 to 6, wherein the crude solution of NAD is a crude product solution obtained by preparing NAD by a biocatalytic method.
8. The method for preparing a high-purity NAD according to claim 7, further comprising: before the step 1), concentrating the crude product solution.
9. The method for preparing high-purity NAD according to claim 7, wherein the concentration treatment is nanofiltration concentration using a nanofiltration membrane having a molecular weight cutoff of 200 to 400 Daltons.
10. The method for preparing high-purity NAD according to claim 7, further comprising: before performing the concentration treatment, adjusting the pH value of the crude product solution to 3.0 to 5.0, and then performing microfiltration treatment Collect the micro filtrate.
11. The method for preparing a high-purity NAD according to claim 3 or 10, wherein the microfiltration membrane used in the microfiltration process is a hollow fiber membrane with a pore size of 0.1-0.5 μm.