WO2018023205A1

WO2018023205A1 - Purification method for nicotinamide mononucleotide

Info

Publication number: WO2018023205A1
Application number: PCT/CN2016/092455
Authority: WO
Inventors: 傅荣昭; 张琦; 张冬民
Original assignee: 邦泰生物工程(深圳)有限公司
Priority date: 2016-07-30
Filing date: 2016-07-30
Publication date: 2018-02-08
Also published as: CN108026132B; CN108026132A

Abstract

A purification method for nicotinamide mononucleotide, relating to a method for purifying an NMN crude product obtained via a biocatalysis method. The purpose of the present method is to solve the problems in existing NMN purification methods of low yield and low purified product purity. The present method comprises the following steps: A, placing a nicotinamide mononucleotide crude product obtained via a biocatalysis method in an anion exchange resin column, eluting with water, and collecting an eluate; B, performing nanofiltration concentration treatment on the eluate of step A, and collecting a concentrate; C, placing the concentrate of step B in a chelating resin column, eluting with water, and collecting an eluate; D, performing concentration and drying treatment on the eluate of step C, and obtaining a purified nicotinamide mononucleotide product. The present method is universally applicable for the purification of NMN crude products obtained via biocatalysis methods.

Description

Method for purifying nicotinamide mononucleotide

Technical field

[0001] The present invention relates to the technical field of a method for preparing a nicotinamide mononucleotide, and more particularly to a method for purifying a crude product of a nicotinamide mononucleotide prepared by a biocatalytic method.

Background technique

[0002] Nicotinamide mononucleotide (NMN) is a biochemical substance present in biological cells and is a synthetic substrate of coenzyme I. It is adenosine nucleotide adenosine transferase. After being turned into, it becomes a coenzyme I (NAD). The level of NMN in the body and the activity of nicotinamide nucleotide adenosyltransferase (NAMPT) directly affect the concentration of NAD. The same NMN is directly involved in adenosine transfer in vivo, which is an important synthetic substrate and function regulation in vivo. substance. In terms of therapeutic applications, NMN can be used for anti-aging, treatment of chronic diseases, etc. Peer studies have shown that NMN also regulates insulin secretion and has an effect on mRNA expression levels. Therefore, NMN has broad application prospects in medical treatment, and peers also have broad market prospects in chemical industry as a reaction substrate.

[0003] At present, the preparation method of nicotinamide mononucleotide mainly includes the following three types: 1. yeast fermentation method; 2. chemical synthesis method; ₃ , biocatalysis method. Among them, the chemical synthesis method has the disadvantages of high cost and the production of chiral compounds; and the NMN produced by the yeast fermentation method contains certain organic solvent residues; the biocatalytic method does not contain the solvent residue, and there is no chiral problem and is prepared. NMN is the same as the same type in the body and has become the most green and environmentally friendly NMN preparation method.

[0004] The existing biocatalytic method for preparing NMN is generally based on nicotinamide and 5'-phosphoribosyl-ruthenium-pyrophosphate (PR PP), in Nicotinamide phosphoribosyltransferase (abbreviated as Preparation of NMN under the catalysis of Nampt). The crude NMN prepared by the method is generally purified by ion exchange resin, but because NMN and various analogs have similar charge and polarity, it is difficult to separate and purify, and it is often impossible to completely remove the analog impurities therein. . Existing methods of purifying ion exchange resins often only obtain products with a purity of about 60%, and the yield is only about 40%, and the production efficiency is low, which is not suitable for large-scale production.

technical problem [0005] The present invention aims to provide a method for purifying NMN having high yield and high product purity, in view of the problems of low yield and low purity of purified product of the prior art NMN purification method mentioned in the above background art. .

Problem solution

Technical solution

[0006] In order to achieve the above object, the present invention provides a method for purifying a nicotinamide mononucleotide, characterized in that

: The method comprises the following steps:

[0007] A, the biocatalytic preparation of the crude nicotinamide mononucleotide product on the anion exchange resin column, eluted with water, collecting the eluate;

[0008] B, the eluent of step A is subjected to nanofiltration concentration treatment, and the concentrated liquid is collected;

[0009] C, step B of the concentrated liquid chelating resin column, eluted with water, collecting the eluent;

[0010] D, the eluent of step C is concentrated and dried to obtain a purified nicotinamide single nucleotide product.

[0011] The NMN purification method provided by the present invention is applicable to a NMN crude product obtained by preparing a NMN by a biocatalytic method, and the biocatalytic method specifically refers to a method for converting a substrate into a NMN by using a biological enzyme, wherein The biological enzyme is a combination of nicotinamide phosphoribosyltransferase or nicotinamide phosphoribosyltransferase and one or more other enzymes, wherein the substrate may be PRPP and nicotinamide, or may be converted to PRPP or nicotinamide. Precursor substance.

[0012] The purpose of the step B in the above purification method of NMN is to concentrate the eluent to shorten the subsequent loading of the mash, and to improve the efficiency; on the other hand, remove a part of the salt to make the eluent Impurities are more easily adsorbed on the chelating resin.

[0013] The concentration treatment in step D of the above-described purification method of NMN may be carried out by any suitable concentration method known in the art, such as nanofiltration concentration, reverse osmosis concentration, etc.; the drying treatment may be any suitable one known in the art. Drying methods, such as freeze drying, spray drying, vacuum drying, and the like.

[0014] Preferably, the anion exchange resin is a weakly basic anion exchange resin, including D201, D354 and A

510 and so on.

More preferably, the weakly basic anion exchange resin is an anion exchange resin containing a tertiary amine group, including

D301, D311 and LX-67, etc.

[0016] Preferably, in step A and step C, the whole process is detected by a nucleic acid protein detector, and the detection wavelength is 260. Nm, the eluent is collected in a manner that starts from the beginning of the reading of the nucleic acid protein detector, and stops collecting after the reading starts to fall.

[0017] Preferably, the nanofiltration membrane used in the nanofiltration concentration treatment in the method has a molecular weight cut off of 100-300.

Preferably, the method further comprises: adjusting the pH of the crude nicotinamide mononucleotide to 5.5-6.5 prior to step A. The benefits of this are: On the one hand, the stability of the product in the pH range is relatively good, and on the other hand the pH range matches the acid resistance of the container.

[0019] Preferably,

The method further comprises: adjusting the pH of the eluate of step C to 2.0-2.4 prior to step D. Since the pH of the eluent eluted from the chelate resin column is low, and the nanofiltration equipment used in the subsequent nanofiltration concentration treatment has an acid resistance of 2.0 or more, the pH of the eluent needs to be protected. The value is adjusted to 2.0-2.4. If the subsequent concentrating equipment has a higher acid resistance, it is not necessary to adjust its pH.

[0020] Preferably, in the step D, the concentration treatment process adopts a nanofiltration concentration device with a molecular weight cutoff of 100-300, and the mass percentage of sodium ions washed into the product by multiple times with pure water is less than 1%.

[0021] In order to recycle the anion exchange resin to reduce the production cost, preferably, the method further comprises the step of regenerating the anion exchange resin, wherein the regeneration liquid used has a pH of 1.0 and a content of 1.0 mol/L. Sodium chloride solution.

[0022] In order to recycle the chelating resin to reduce the production cost, preferably, the method further comprises the step of regenerating the chelating resin, and the regenerant used is a 1.0 mol/L hydrochloric acid solution.

Advantageous effects of the invention

Beneficial effect

Compared with the prior art, the purification method of the nicotinamide mononucleotide provided by the invention has the advantages of high yield and high purity of the purified product, and the yield of the purification method can be up to 60% as confirmed by industrial practice. Above, the purity of the purified NMN is as high as 97% or more. Moreover, the method does not use toxic and harmful organic solvents, green environmental protection, simple and easy process, and low production cost, so that the purified product is highly competitive in the market. This method has general applicability to the purification of crude NMN obtained by biocatalytic preparation of NMN.

Embodiments of the invention The present invention will be further described in detail below with reference to the specific embodiments. The following examples are illustrative of the invention, and the invention is not limited to the following examples.

Embodiment 1

[0026] Treatment target: Bangtai Bioengineering (Shenzhen) Co., Ltd. uses the enzyme-catalyzed method (prepared with PRPP and nicotinamide as substrate, catalyzed by nicotinamide phosphoribosyltransferase to prepare NMN) to prepare four batches of NMN crude product solution. The content and purity of NMN in the four batches of NMN crude product solution were determined by high performance liquid chromatography as shown in Table 1.

[0027] The purification process of the above four batches of NMN crude product solution is as follows:

[0028] 1, one-time adjustment of PH value: Because the above four batches of NMN crude product solution has a higher pH value, so first adjust its p H to about 6.0 with hydrochloric acid;

[0029] 2. One-time nanofiltration concentration: The nanofiltration product solution is nanofiltration concentrated to about 1/6 of the original volume by a nanofiltration concentration device to shorten the loading time, and the molecular weight of the nanofiltration membrane used is 200. ;

[0030] 3. One-time loading: Two ion columns (r=300 nm, h=2700 nm, 190L) packed with an anion exchange resin D301 containing a tertiary amino group are connected in series, and all the valves connected in series are adjusted; Step 2 The concentration of the concentrate is loaded, slowly adjusting the loading speed to 200-300 IJh (1-1.5BV); observe the change of flow rate; the whole process is detected by nucleic acid protein detector, the detection wavelength is 260nm, and the reading of the nucleic acid protein detector is recorded. And observe changes in readings;

[0031] 4, one rinse: After the sample is completed, adjust the valve to rinse the ion column with pure water, and adjust the flow rate of the rinse to make it at 200-300IJh (1-1.5BV);

[0032] 5. One-time purification product collection: After the reading of the nucleic acid protein detector starts to rise, the product is collected, and the reading of the nucleic acid protein detector begins to decrease, the collection is stopped, and the sample is sent for detection;

[0033] 6, secondary nanofiltration concentration: the primary purification product collected in step 5 is subjected to nanofiltration concentration treatment, nanofiltration to about 1/2 of the original volume, the nanofiltration membrane used has a molecular weight cutoff of 200;

[0034] 7. Secondary loading: Two ion columns (r=300nm, h=2700nm, 190L) packed with chelating resin are connected in parallel, and all the valves connected in parallel are adjusted; the concentrated liquid in step 6 is loaded. Slowly adjust the loading speed to 100-200IJh (0.5-1.0BV); observe the change of flow rate; use the nucleic acid protein detector to detect the whole wavelength, the detection wavelength is 260nm, record the reading of the nucleic acid protein detector and observe the reading change;

[0035] 8. After the second rinsing, after the sample is completed, the regulating valve rinses the ion column with pure water, and adjusts the rinsing flow. Speed, make it at 100-200IJh (0.5-1.0BV);

[0036] 9. The second purified product is collected, and the reading of the nucleic acid protein detector starts to rise, and the product is collected, and the reading of the nucleic acid protein detector is decreased to 1.0, the collection is stopped, and the sample is sent for detection;

[0037] 10, the second adjustment of the pH value: the secondary purification product collected in step 9 with sodium hydroxide to adjust the pH to 2.0-2.4;

[0038] 11, three nanofiltration concentration: the pH of the step 10 product is subjected to nanofiltration concentration treatment, and washed with pure water multiple times until the mass percentage of sodium ions in the product is less than 1%, used The nanofiltration membrane has a molecular weight cut off of 200;

[0039] 12. Drying: The concentrated liquid of step 11 is lyophilized to obtain a purified NMN finished product.

[0040] The content and purity of the four batches of the purified NMN product were measured by high performance liquid chromatography, and the yield was calculated. The results are shown in Table 1.

Table 1

[]

Embodiment 2

[0043] The regeneration process of the anion exchange resin is as follows:

[0044] 1. Rinsing: flush out the by-products remaining in the resin with a large amount of pure water until the reading of the nucleic acid protein detector drops below 0.5;

[0045] 2. Regeneration: Sodium chloride regeneration liquid with a pH of 1.0 and 1.0 mol/L, 400-500 L, rotary regenerative valve

, reclaiming the regenerative pump, regenerating the anion exchange resin with a regeneration rate of 200-300 IJh (1-1.5 BV); [0046] 3. Washing: After the regeneration liquid is regenerated, rinse the ion column with 2-3 column volumes of pure water. Then, the anion exchange resin column can be used for the next purification treatment, and the adsorptivity is good. Embodiment 3

[0048] Regeneration of Chelating Resin

[0049] 1. Rinsing: flush out the by-products remaining in the resin with a large amount of pure water until the reading of the nucleic acid protein detector drops below 0.5;

[0050] 2, regeneration: preparation of 1.0mol / L hydrochloric acid regeneration liquid 400-500L, rotary regeneration valve, snoring regeneration pump, with 20

0-300L/h U-1.5BV) regeneration rate regeneration chelating resin;

3. Water washing: After the regeneration liquid is regenerated, the ion column is washed with 2-3 column volumes of pure water, and the chelating resin column can be used for the next purification treatment, and the adsorption property is good.

Claims

Claim

[Claim 1] A method for purifying a nicotinamide mononucleotide, comprising: the method comprising the steps of:

A. The crude nicotinamide mononucleotide prepared by the biocatalytic method is applied to an anion exchange resin column, and eluted with water to collect the eluate;

B, the eluent of step A is subjected to nanofiltration concentration treatment, and the concentrated liquid is collected;

C. The concentrated liquid of step B is chelating the resin column, eluting with water, and collecting the eluent;

D. The eluent of step C is concentrated and dried to obtain a purified nicotinamide mononucleotide product.

[Claim 2] The method for purifying a nicotinamide mononucleotide according to claim 1, wherein the anion exchange resin is a weakly basic anion exchange resin.

[Claim 3] The method for purifying a nicotinamide mononucleotide according to claim 2, wherein the weakly basic anion exchange resin is an anion exchange resin containing a tertiary amino group.

[Claim 4] The method for purifying nicotinamide mononucleotide according to claim 1, wherein: in step A and step C, the whole process is detected by a nucleic acid protein detector, and the detection wavelength is 260 η m, the washing The method of collecting the liquid is collected from the beginning of the reading of the nucleic acid protein detector, and the collection is stopped after the reading starts to decrease.

[Claim 5] The method for purifying a nicotinamide mononucleotide according to claim 1, wherein the nanofiltration membrane for nanofiltration concentration treatment in the method has a molecular weight cut off of 100 to 300.

[Claim 6] The method for purifying a nicotinamide mononucleotide according to claim 1, wherein the method further comprises: before step A, the pH of the crude nicotinamide mononucleotide product The value is adjusted to 5.5-6.5.

[Claim 7] The method for purifying nicotinamide mononucleotide according to claim 1, wherein the method further comprises: adjusting the pH of the eluate of step C to 2.0 before step D. -2.4

[Claim 8] The method for purifying nicotinamide mononucleotide according to claim 7, wherein: in step D, the concentration treatment process adopts a nanofiltration concentration device with a molecular weight cutoff of 100-300, and uses pure water. The mass percentage of sodium ions washed into the product is less than 1%.

[Claim 9] The method for purifying a nicotinamide mononucleotide according to claim 1, wherein the method further comprises the step of regenerating the anion exchange resin, and the regenerant used is p

H is a sodium chloride solution having a 1.0 content of 1.0 mol/L.

The method for purifying a nicotinamide mononucleotide according to claim 1, wherein the method further comprises the step of regenerating the chelate resin, wherein the regenerant used is 1.0 mol/ L hydrochloric acid solution.