阅读说明：本技术 一种基于酶法的高纯度nmn制备工艺 (High-purity NMN preparation process based on enzyme method ) 是由 马宏峰 赵伟 王梓 陈晨 于 2020-04-20 设计创作，主要内容包括：本发明涉及NMN制备技术领域,且公开了一种基于酶法的高纯度NMN制备工艺,制备工艺步骤如下：①原料预处理：首先以烟酰胺核糖进行微滤和纳滤,收集浓缩的粗品溶液。②生产工艺：粗品溶液在烟酰胺核糖激酶或含有烟酰胺核糖激酶的重组细胞的催化下反应生成β-烟酰胺单核苷酸。③纯化工艺：将生产的β-烟酰胺单核苷酸溶液pH值调至3-7,进样上反相高效液相色谱制备柱,进行梯度洗脱纯化,得到纯化的β-烟酰胺单核苷酸溶液。该基于酶法的高纯度NMN制备工艺,保证烟酰胺单核苷酸的生产率,提高了产品纯度,可以使烟酰胺单核苷酸在缓解和改善缺血性心脑组织损伤效果更具明显性,烟酰胺单核苷酸可以较好改善氧化相关的退行性疾病和身体机能障碍。(The invention relates to the technical field of NMN preparation, and discloses a high-purity NMN preparation process based on an enzyme method, which comprises the following steps of ① raw material pretreatment, namely microfiltration and nanofiltration are carried out on nicotinamide ribose, a concentrated crude product solution is collected, ② a production process is carried out, wherein the crude product solution reacts under the catalysis of nicotinamide ribokinase or recombinant cells containing nicotinamide ribokinase to generate β -nicotinamide mononucleotide, ③ a purification process is carried out, the pH value of the produced β -nicotinamide mononucleotide solution is adjusted to 3-7, a reversed-phase high-performance liquid chromatography preparation column is fed for sample injection, and gradient elution purification is carried out to obtain a purified β -nicotinamide mononucleotide solution.)

1. A preparation process of high-purity NMN based on an enzyme method is characterized by comprising the following steps:

① pretreatment of raw material, firstly, carrying out microfiltration and nanofiltration by using nicotinamide ribose, and collecting concentrated crude product solution.

② the production process comprises reacting the crude solution with nicotinamide ribokinase or recombinant cell containing nicotinamide ribokinase to generate β -nicotinamide mononucleotide.

③ and purifying by gradient elution to obtain β -nicotinamide mononucleotide solution, wherein the pH value of β -nicotinamide mononucleotide solution is adjusted to 3-7, a sample is fed into a reversed-phase high performance liquid chromatography column, the stationary phase is octadecylsilane chemically bonded silica, the mobile phase A is a solution with pH3-7 prepared from hydrochloric acid solution, and the mobile phase B is ethanol.

2. The enzymatic process of NMN according to claim 1, wherein the purified sample solution is nanofiltered by a membrane concentration device and then freeze-dried by a vacuum freeze-dryer to obtain purified β -nicotinamide mononucleotide in step ③.

3. The enzymatic based process of preparation of high purity NMN according to claim 1, characterized by: all the raw materials used in the reaction are added into a reaction kettle, mixed uniformly and placed at a set temperature for stirring reaction.

4. The enzymatic based process of preparation of high purity NMN according to claim 1, characterized by: the reaction is carried out under the catalysis of recombinant cells containing nicotinamide ribokinase, and the recombinant cells are microbial cells.

5. The enzymatic based process of preparation of high purity NMN according to claim 4, wherein: the microorganism is Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris.

6. The enzymatic based process of preparation of high purity NMN according to claim 1, characterized by: the reaction is carried out in an aqueous system at a temperature of 32-40 ℃ and a pH of 5.5-8.5.

7. The enzymatic based process of preparing NMN of high purity according to claim 1, wherein said step ③ is performed with a gradient elution time of 40-50 min.

Technical Field

The invention relates to the technical field of NMN preparation, in particular to a high-purity NMN preparation process based on an enzyme method.

Background

NMN (nicotinamide mononucleotide) is the product of the nicotinamide phosphoribosyltransferase reaction, one of the key precursors of NAD +. In mammals, NMN is produced by nicotinamide under catalysis of Nampt, followed by NMN producing NAD + under catalysis of nicotinamide mononucleotide adenyl transferase. Extracellular NMN requires dephosphorylation to nicotinamide riboside before it can enter the interior of hepatocytes, after entry into the cells, NR is phosphorylated by nicotinamide riboside kinase 1 to form NMN, which is then combined with ATP to form NAD +. NMN exerts its physiological functions in the human body by being converted to NAD +, such as activating NAD + substrate-dependent enzyme Sirt1 (histone deacetylase, also known as sirtuin), regulating cell survival and death, maintaining redox status, and the like. Recent research shows that the NMN in the organism is regulated to have better treatment and repair effects on cardiovascular and cerebrovascular diseases, neurodegenerative diseases, aging degenerative diseases and the like; in addition, NMN can also play a role in protecting and repairing pancreatic islet function, increasing insulin secretion, and preventing and treating metabolic diseases such as diabetes, obesity and the like by participating in and regulating endocrine of an organism.

Nicotinamide mononucleotide as intermediate in NAD + remedial pathway has the functions of resisting oxidation and reducing oxidative stress, and has excellent performance in treating some specific diseases, such as cerebral apoplexy, cardiac ischemia-reperfusion, Alzheimer disease, Parkinson disease, acute kidney injury, retinal degenerative disease, type 2 diabetes, etc. Particularly in the aspect of anti-aging, NMN can slow down physiological decline of organisms, enhance energy metabolism and prolong the life. Because NMN is a human endogenous substance, the safety is high, and the thermal stability is good, the NMN has a wide prospect in the field of functional food development as an active substance.

The traditional nicotinamide mononucleotide production method is characterized in that nicotinamide ribose is used as a raw material, phosphorus oxychloride is used for phosphorylation to obtain the nicotinamide mononucleotide, the production rate is low, the product purity is low, a large amount of organic solvent is used, and the environmental damage is serious, so that the nicotinamide mononucleotide has an unobvious effect of relieving and improving ischemic heart and brain tissue damage, and the nicotinamide mononucleotide cannot improve oxidative related degenerative diseases and physical dysfunction to a large extent, and therefore, the high-purity NMN preparation process based on the enzyme method is provided.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a high-purity NMN preparation process based on an enzyme method, which has the advantages of ensuring the productivity of nicotinamide mononucleotide, improving the product purity, avoiding serious damage to the environment due to a large amount of organic solvent, can lead the effect of the nicotinamide mononucleotide on relieving and improving ischemic heart and cerebral tissue injury to be more explicit, the nicotinamide mononucleotide can better improve oxidative related degenerative diseases and physical dysfunction and the like, solves the problems that the traditional nicotinamide mononucleotide production method takes nicotinamide ribose as a raw material, uses phosphorus oxychloride for phosphorylation, has lower production rate and low product purity, uses a large amount of organic solvent, the damage to the environment is serious, so that the effect of the nicotinamide mononucleotide on relieving and improving ischemic heart and cerebral tissue injury is not obvious, and the nicotinamide mononucleotide cannot improve oxidative related degenerative diseases and physical dysfunction to a greater extent.

The invention provides a preparation process of high-purity NMN based on an enzyme method, which comprises the following steps of ① raw material pretreatment, namely microfiltration and nanofiltration are carried out on nicotinamide ribose, concentrated crude product solution is collected, ② production process, wherein the crude product solution reacts under the catalysis of nicotinamide ribokinase or recombinant cells containing nicotinamide ribokinase to generate β -nicotinamide mononucleotide, ③ purification process comprises the steps of adjusting the pH value of the produced β -nicotinamide mononucleotide solution to 3-7, feeding a sample into a reverse phase high performance liquid chromatography preparation column, using octadecylsilane bonded silica gel as a stationary phase, using a mobile phase A as a solution prepared from hydrochloric acid solution to obtain a solution with pH of 3-7, using ethanol as a mobile phase B for purification, and carrying out elution gradient β - β purification to obtain a nicotinamide mononucleotide solution.

Preferably, in the step ③, the purified sample solution is subjected to nanofiltration by a membrane concentration device and then lyophilized by a vacuum freeze dryer to obtain purified β -nicotinamide mononucleotide.

Preferably, all the raw materials used for the reaction are added into a reaction kettle, mixed uniformly, placed at a set temperature and stirred for reaction.

Preferably, the reaction is carried out under catalysis of recombinant cells containing nicotinamide ribokinase, and the recombinant cells are microbial cells.

Preferably, the microorganism is Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris.

Preferably, the reaction is carried out in an aqueous system at a temperature of 32 to 40 ℃ and a pH of 5.5 to 8.5.

Preferably, the elution time in the step ③ is 40-50 min.

Compared with the prior art, the invention provides an enzymatic method-based high-purity NMN preparation process, which has the following beneficial effects:

the preparation process of high-purity NMN based on the enzyme method comprises the steps of adjusting the pH value of a produced β -nicotinamide mononucleotide solution to 3-7, feeding a sample into a reverse phase high performance liquid chromatography preparation column, wherein the stationary phase is octadecylsilane chemically bonded silica, the mobile phase A is a solution with the pH value of 3-7 prepared from a hydrochloric acid solution, and the mobile phase B is ethanol, and performing gradient elution purification to obtain a purified β -nicotinamide mononucleotide solution.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

A preparation process of high-purity NMN based on an enzyme method comprises the following steps:

① pre-treating raw material, firstly carrying out microfiltration and nanofiltration on nicotinamide riboside, collecting concentrated crude product solution, ② production process, namely, carrying out reaction on the crude product solution under catalysis of nicotinamide ribokinase or recombinant cells containing nicotinamide ribokinase to generate β -nicotinamide mononucleotide, ③ purification process, namely, adjusting the pH value of the produced β -nicotinamide mononucleotide solution to 3-7, feeding the solution into a reverse phase high performance liquid chromatography preparation column, wherein a stationary phase is octadecylsilane chemically bonded silica, a mobile phase A is a solution with the pH value of 3-7 prepared by hydrochloric acid solution, a mobile phase B is ethanol, carrying out gradient elution purification to obtain purified β -nicotinamide mononucleotide solution, ③, carrying out nanofiltration on the purified sample solution by using membrane concentration equipment, freeze-drying by using a vacuum freeze-drying machine to obtain purified β -nicotinamide mononucleotide, adding all raw materials used for the reaction into a reaction kettle, uniformly mixing, carrying out stirring reaction at a set temperature, carrying out stirring reaction under catalysis of recombinant cells containing nicotinamide ribokinase, wherein the recombinant cells are microbial yeast cells of the yeast, the yeast is a yeast micro-yeast cell, and the yeast is a yeast aqueous phase elution temperature of 40-368, and the temperature of the pichia temperature is 40.5-368.

The preparation process of high-purity NMN based on the enzyme method comprises the following steps of firstly carrying out microfiltration and nanofiltration on nicotinamide ribose, collecting a concentrated crude product solution, then carrying out a reaction on the crude product solution under the catalysis of nicotinamide ribokinase or recombinant cells containing nicotinamide ribokinase to generate β -nicotinamide mononucleotide, adjusting the pH value of the produced β -nicotinamide mononucleotide solution to 3-7, feeding the sample into a reverse phase high performance liquid chromatography preparation column, carrying out nanofiltration on the purified sample solution by using membrane concentration equipment, carrying out air-cooling and freeze-drying by using a vacuum freeze-drying machine to obtain purified β -nicotinamide mononucleotide, wherein the reaction is carried out in an aqueous phase system with the temperature of 32-40 ℃ and the pH of 5.5-8.5, and the elution time in the step ③ is 40-50 min.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.