WO2018023205A1 - Procédé de purification de mononucléotide nicotinamide - Google Patents

Procédé de purification de mononucléotide nicotinamide Download PDF

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Publication number
WO2018023205A1
WO2018023205A1 PCT/CN2016/092455 CN2016092455W WO2018023205A1 WO 2018023205 A1 WO2018023205 A1 WO 2018023205A1 CN 2016092455 W CN2016092455 W CN 2016092455W WO 2018023205 A1 WO2018023205 A1 WO 2018023205A1
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WO
WIPO (PCT)
Prior art keywords
nicotinamide mononucleotide
purifying
anion exchange
exchange resin
nmn
Prior art date
Application number
PCT/CN2016/092455
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English (en)
Chinese (zh)
Inventor
傅荣昭
张琦
张冬民
Original Assignee
邦泰生物工程(深圳)有限公司
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Application filed by 邦泰生物工程(深圳)有限公司 filed Critical 邦泰生物工程(深圳)有限公司
Priority to PCT/CN2016/092455 priority Critical patent/WO2018023205A1/fr
Priority to CN201680003982.7A priority patent/CN108026132B/zh
Publication of WO2018023205A1 publication Critical patent/WO2018023205A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/048—Pyridine radicals

Definitions

  • 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.
  • Nicotinamide mononucleotide 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).
  • NAD coenzyme I
  • the level of NMN in the body and the activity of nicotinamide nucleotide adenosyltransferase (NAMPT) directly affect the concentration of NAD.
  • NAMPT nicotinamide nucleotide adenosyltransferase
  • the same NMN is directly involved in adenosine transfer in vivo, which is an important synthetic substrate and function regulation in vivo. substance.
  • 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.
  • the preparation method of nicotinamide mononucleotide mainly includes the following three types: 1. yeast fermentation method; 2. chemical synthesis method; 3 , biocatalysis method.
  • 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.
  • 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).
  • PR PP 5'-phosphoribosyl-ruthenium-pyrophosphate
  • Preparation of NMN under the catalysis of Nampt Nicotinamide phosphoribosyltransferase
  • 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
  • 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. .
  • the present invention provides a method for purifying a nicotinamide mononucleotide, characterized in that
  • the method comprises the following steps:
  • A the biocatalytic preparation of the crude nicotinamide mononucleotide product on the anion exchange resin column, eluted with water, collecting the eluate;
  • step A the eluent of step A is subjected to nanofiltration concentration treatment, and the concentrated liquid is collected;
  • step B of the concentrated liquid chelating resin column, eluted with water, collecting the eluent;
  • step C the eluent of step C is concentrated and dried to obtain a purified nicotinamide single nucleotide product.
  • 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 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.
  • 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.
  • 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.
  • the anion exchange resin is a weakly basic anion exchange resin, including D201, D354 and A
  • the weakly basic anion exchange resin is an anion exchange resin containing a tertiary amine group, including
  • 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.
  • the nanofiltration membrane used in the nanofiltration concentration treatment in the method has a molecular weight cut off of 100-300.
  • 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.
  • 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.
  • 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%.
  • 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.
  • the method further comprises the step of regenerating the chelating resin, and the regenerant used is a 1.0 mol/L hydrochloric acid solution.
  • 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.
  • the purity of the purified NMN is as high as 97% or more.
  • 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.
  • 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.
  • 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. ;
  • 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;
  • 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);
  • 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;
  • 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;
  • 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;
  • step 11 Drying: The concentrated liquid of step 11 is lyophilized to obtain a purified NMN finished product.
  • Regeneration Sodium chloride regeneration liquid with a pH of 1.0 and 1.0 mol/L, 400-500 L, rotary regenerative valve
  • regeneration preparation of 1.0mol / L hydrochloric acid regeneration liquid 400-500L, rotary regeneration valve, snoring regeneration pump, with 20

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Saccharide Compounds (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention porte également sur un procédé de purification de mononucléotide de nicotinamide, concernant un procédé de purification d'un produit brut de NMN obtenu par un procédé de biocatalyse. Le but de la présente invention est de résoudre les problèmes dans les procédés existants de purification de NMN de faible rendement et de faible pureté du produit purifié. Le procédé comprend les étapes suivantes : A, placer un produit brut de mononucléotide de nicotinamide obtenu par un procédé de biocatalyse dans une colonne de résine échangeuse d'anions, éluer avec de l'eau, et recueillir un éluat; B, effectuer un traitement de concentration de nanofiltration sur l'éluat de l'étape A, et collecter un concentré; C, placer le concentré de l'étape B dans une colonne de résine de chélation, éluer avec de l'eau, et collecter un éluat; D, effectuer un traitement de concentration et de séchage sur l'éluat de l'étape C, et obtenir un produit de mononucléotide de nicotinamide purifié. Le présent procédé est universellement applicable pour la purification de produits bruts de NMN obtenus par des procédés de biocatalyse.
PCT/CN2016/092455 2016-07-30 2016-07-30 Procédé de purification de mononucléotide nicotinamide WO2018023205A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2016/092455 WO2018023205A1 (fr) 2016-07-30 2016-07-30 Procédé de purification de mononucléotide nicotinamide
CN201680003982.7A CN108026132B (zh) 2016-07-30 2016-07-30 一种烟酰胺单核苷酸的纯化方法

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PCT/CN2016/092455 WO2018023205A1 (fr) 2016-07-30 2016-07-30 Procédé de purification de mononucléotide nicotinamide

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Cited By (9)

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Publication number Priority date Publication date Assignee Title
US10392416B2 (en) 2015-10-02 2019-08-27 Metro International Biotech, Llc Crystal forms of beta-nicotinamide mononucleotide
US10548913B2 (en) 2015-08-05 2020-02-04 Metro International Biotech, Llc Nicotinamide mononucleotide derivatives and their uses
US10618927B1 (en) 2019-03-22 2020-04-14 Metro International Biotech, Llc Compositions and methods for modulation of nicotinamide adenine dinucleotide
CN112979730A (zh) * 2021-02-23 2021-06-18 成都西域从容生物科技有限公司 一种nmn提取及纯化方法
CN113121629A (zh) * 2021-03-25 2021-07-16 沁浩膜技术(厦门)有限公司 一种从发酵液中提取烟酰胺单核苷酸的方法
US11180521B2 (en) 2018-01-30 2021-11-23 Metro International Biotech, Llc Nicotinamide riboside analogs, pharmaceutical compositions, and uses thereof
WO2022114105A1 (fr) * 2020-11-27 2022-06-02 ミライラボバイオサイエンス株式会社 MONONUCLÉOTIDE β-NICOTINAMIDE DE GRANDE PURETÉ (NMN) ET SON PROCÉDÉ DE PRODUCTION
US11787830B2 (en) 2021-05-27 2023-10-17 Metro International Biotech, Llc Crystalline solids of nicotinic acid mononucleotide and esters thereof and methods of making and use
US11939348B2 (en) 2019-03-22 2024-03-26 Metro International Biotech, Llc Compositions comprising a phosphorus derivative of nicotinamide riboside and methods for modulation of nicotinamide adenine dinucleotide

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CN112870979B (zh) * 2020-12-31 2022-12-23 内蒙古金达威药业有限公司 一种β-烟酰胺单核苷酸的分离纯化方法
CN112772814A (zh) * 2021-01-05 2021-05-11 金华贝塔生物科技有限公司 一种含nmn的香菇提取物的制备方法
CN114262355A (zh) * 2021-12-27 2022-04-01 江苏诚信药业有限公司 一种烟酰胺腺嘌呤二核苷酸的纯化方法
CN114835768A (zh) * 2022-05-19 2022-08-02 浙江省农业科学院 从果蔬中提取β-烟酰胺单核苷酸的方法及其纯化方法

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Cited By (13)

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Publication number Priority date Publication date Assignee Title
US11878027B2 (en) 2015-08-05 2024-01-23 Metro International Biotech, Llc Nicotinamide mononucleotide derivatives and their uses
US10548913B2 (en) 2015-08-05 2020-02-04 Metro International Biotech, Llc Nicotinamide mononucleotide derivatives and their uses
US11464796B2 (en) 2015-08-05 2022-10-11 Metro International Biotech, Llc Nicotinamide mononucleotide derivatives and their uses
US10392416B2 (en) 2015-10-02 2019-08-27 Metro International Biotech, Llc Crystal forms of beta-nicotinamide mononucleotide
US11059847B2 (en) 2015-10-02 2021-07-13 Metro International Biotech, Llc Crystal forms of β-nicotinamide mononucleotide
US11180521B2 (en) 2018-01-30 2021-11-23 Metro International Biotech, Llc Nicotinamide riboside analogs, pharmaceutical compositions, and uses thereof
US10618927B1 (en) 2019-03-22 2020-04-14 Metro International Biotech, Llc Compositions and methods for modulation of nicotinamide adenine dinucleotide
US11939348B2 (en) 2019-03-22 2024-03-26 Metro International Biotech, Llc Compositions comprising a phosphorus derivative of nicotinamide riboside and methods for modulation of nicotinamide adenine dinucleotide
WO2022114105A1 (fr) * 2020-11-27 2022-06-02 ミライラボバイオサイエンス株式会社 MONONUCLÉOTIDE β-NICOTINAMIDE DE GRANDE PURETÉ (NMN) ET SON PROCÉDÉ DE PRODUCTION
CN112979730A (zh) * 2021-02-23 2021-06-18 成都西域从容生物科技有限公司 一种nmn提取及纯化方法
CN113121629A (zh) * 2021-03-25 2021-07-16 沁浩膜技术(厦门)有限公司 一种从发酵液中提取烟酰胺单核苷酸的方法
US11787830B2 (en) 2021-05-27 2023-10-17 Metro International Biotech, Llc Crystalline solids of nicotinic acid mononucleotide and esters thereof and methods of making and use
US11952396B1 (en) 2021-05-27 2024-04-09 Metro International Biotech, Llc Crystalline solids of nicotinic acid mononucleotide and esters thereof and methods of making and use

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CN108026132B (zh) 2021-08-13

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