WO2021253476A1 - β-烟酰胺单核酸的合成方法及其中间体 - Google Patents
β-烟酰胺单核酸的合成方法及其中间体 Download PDFInfo
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- WO2021253476A1 WO2021253476A1 PCT/CN2020/098277 CN2020098277W WO2021253476A1 WO 2021253476 A1 WO2021253476 A1 WO 2021253476A1 CN 2020098277 W CN2020098277 W CN 2020098277W WO 2021253476 A1 WO2021253476 A1 WO 2021253476A1
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- phospholipase
- reaction
- nicotinamide
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- phospholipid
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- 230000002792 vascular Effects 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 108091022915 xylulokinase Proteins 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/04—Phosphoric diester hydrolases (3.1.4)
- C12Y301/04003—Phospholipase C (3.1.4.3)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/04—Phosphoric diester hydrolases (3.1.4)
- C12Y301/04011—Phosphoinositide phospholipase C (3.1.4.11)
Definitions
- the invention relates to a method for synthesizing ⁇ -nicotinamide mononucleic acid and its intermediates, in particular to a method for enzymatically catalyzed preparation of two products of ⁇ -nicotinamide mononucleic acid and phosphatidylnicotinamide ribose and the synthesis of ⁇ -nicotinamide mononucleic acid Intermediate phosphatidyl nicotinamide ribose.
- NMN ⁇ -Nicotinamide Mononucleotide
- NAD + nicotinamide adenine dinucleotide
- Coenzyme I nicotinamide adenine dinucleotide
- the vegetables edamame and broccoli that contain more NMN in the daily diet contain an average of less than 10mg of NMN per kilogram.
- the amount of NMN synthesized in the body of an adult is roughly the same every day A few hundred kilograms of fruits and vegetables! (Cell Metabolism, 2016, 24: 795-806)
- NMN health products are currently on the market. According to relevant statistics, the effects of individual cases reported by users are very diverse, including: energy improvement, physical strength improvement, fat reduction, muscle increase, exercise capacity enhancement, skin improvement, hair loss reduction, hair growth, and sleep Improvement, circadian clock regulation, immune regulation, allergy reduction, sexual function enhancement, appetite boost, visual fatigue reduction, vision improvement, mood enhancement, decrease in high blood sugar, decrease in hypertension and systolic blood pressure, normalization of hypotension, improvement in constipation, etc.
- NMN nicotinamide ribose
- NR nicotinamide ribose
- phosphorylated with phosphorus oxychloride Chem. Commun. 1999, 729-730, CN 107613990 A
- the obtained product has many impurities, difficult purification, low yield, high comprehensive production cost, and involves the use of a large number of toxic and harmful reagents, which causes heavy pollution and is not suitable for the production of food-grade NMN. Therefore, the production of NMN by an enzyme-catalyzed method has become the current mainstream method of NMN production.
- nicotinamide, ATP and ribose are used as raw materials, and NMN is produced through a multi-step catalytic reaction composed of nicotinamide phosphoribosyltransferase, ribose phosphopyrophosphate kinase and ribokinase.
- Patent CN 108026535 A uses nicotinamide, ATP and AMP as raw materials, and produces NMN through a multi-step catalytic reaction catalyzed by nicotinamide phosphoribosyltransferase, ribose phosphate pyrophosphate kinase, and AMP ribosidase.
- Patent PCT/CN2016/092457 uses nicotinamide, ATP and xylose as raw materials, through nicotinamide phosphoribosyltransferase, ribose phosphate pyrophosphate kinase, ribose-5-phosphate isomerase, ribulose-3-phosphate isomerase A multi-step reaction consisting of enzymes, xylulose kinase, and xylose isomerase is used to produce NMN.
- Patent PCT/CN2016/092459 uses nicotinamide, pyrophosphate and AMP as raw materials to produce NMN through the catalysis of nicotinamide phosphoribosyl transferase and adenine phosphoribosyl transferase.
- Patent PCT/CN016/092461 uses nicotinamide, pyrophosphate and inosinic acid as raw materials to produce NMN under the catalysis of nicotinamide phosphoribosyl transferase, hypoxanthine phosphoribosyl transferase and xanthine oxidase.
- Patent CN 108368493 A; CN 108949865 A uses D-5-phosphate ribose, ATP and nicotinamide as raw materials, and realizes the synthesis of NMN through phosphoribose pyrophosphate synthase and nicotinamide phosphoribosyl transferase.
- Patent CN 106755209 A uses NR and ATP as substrates to generate NMN under the catalysis of nicotinamide ribokinase.
- PRPP is not easily available, expensive, and not suitable for large-scale industrial production.
- the purpose of the present invention is to provide a new NMN enzymatic synthesis technology, which completely breaks away from the natural biocatalytic synthesis system of NMN, does not involve the use of the above-mentioned enzyme varieties, and does not use expensive substrates such as ATP or PRPP.
- the reaction steps Simple, low production cost, environmentally friendly and pollution-free, suitable for large-scale industrial production and other advantages.
- the technical solution of the present invention is like this.
- One of the objectives of the present invention is to provide a two-step enzyme-catalyzed reaction (ie, sequential reaction) to produce NMN:
- a1 Taking nicotinamide ribose and phospholipids as substrates, in the presence of calcium ions, catalyzed by phospholipase D to generate phosphatidyl nicotinamide ribose;
- a2 Using the phosphatidylnicotinamide ribose produced in step a1 as a substrate, in the presence of calcium ions, under the catalysis of phospholipase C, ⁇ -nicotinamide mononucleic acid is generated;
- NR nicotinamide ribose
- the transphosphatidylation function of phospholipase D can catalyze the reaction between phospholipids and NR to produce phosphatidyl nicotinamide ribose ( (Referred to as PNR)
- PNR is a new type of phospholipid molecule, which is easily separated from the water-soluble NR; subsequently, under the catalysis of phospholipase C, the PNR is hydrolyzed to generate diacylglycerol and ⁇ -nicotinamide mononucleic acid NMN.
- Fatty acylglycerol is a natural oil, insoluble in water, and easily separated from water-soluble NMN.
- the second objective of the present invention is to provide two ways to produce NMN through a one-pot method. Specifically:
- the first way is to use nicotinamide ribose (NR) and phospholipids as substrates, in the presence of calcium ions, first add phospholipase D to react, and after the reaction is complete, add phospholipase C to react to obtain NMN.
- NR nicotinamide ribose
- the second way is to use nicotinamide ribose (NR) and phospholipids as substrates, and in the presence of calcium ions, phospholipase D and phospholipase C are added together to react to obtain NMN.
- NR nicotinamide ribose
- phospholipase D and phospholipase C are added together to react to obtain NMN.
- the phospholipid is a natural phospholipid or a synthetic phospholipid; further preferably, the main component of the phospholipid is phosphatidylcholine and/or phosphatidylethanolamine; further preferably, the phospholipid is lecithin.
- the phospholipids used are usually natural phospholipids, which can be plant sources, such as soybeans, sunflowers, rapeseeds, safflower seeds, and other oil plants; they can also be animal sources, such as cow’s milk, goat’s milk, deep-sea fish, etc. Deep-sea shrimp, deep-sea scallops, etc.; it can also be a source of microorganisms, etc.
- Phospholipids refer to a large class of compounds with similar phosphatidyl skeletons. Its structure is shown in Figure 1.
- the R1 and R2 parts of the phospholipid molecule are fatty acids. These fatty acids can be any fatty acids: short-chain, medium-chain, and long-chain. Chain fatty acid; can be saturated fatty acid, monounsaturated fatty acid or polyunsaturated fatty acid; R1 and R2 can be the same or different; other, not repeat them here.
- the X part of the phospholipid molecule is a polar head and is a molecule with a hydroxyl group. It also has diversity. The more common ones are choline, ethanolamine, inositol, glycerol, short-chain alcohols, and so on.
- the phospholipase C includes broad-spectrum phospholipase C and phosphatidylinositol-type phospholipase C; more preferably, phospholipase C is phosphatidylinositol-type phospholipase C.
- the phospholipase D is phospholipase D derived from animals, plants and microorganisms, and more preferably phospholipase D derived from microorganisms, such as phospholipase D derived from Streptomyces.
- the enzymes used in the above methods are: phospholipase D (Phospholipase D, abbreviated as PLD), which can catalyze the transphosphatidylation reaction, such as phosphatidylcholine and phosphatidylethanolamine Phospholipids and substances with hydroxyl groups produce corresponding phosphatidylation products.
- PLD phospholipase D
- the bond positions of the catalyzed phospholipids are shown in Figure 1.
- the enzyme can act on the PO bond or OX bond as shown in the figure, including but not limited to EC 3.1.4.4; Phospholipase C (Phospholipase C, abbreviated as PLC), there are many types of phospholipase C, including but not limited to EC 3.1.4.3, EC 3.1.4.10, EC 3.1.4.11, EC 4.6.1.13, etc., this enzyme It can hydrolyze the glycerophosphate bond of phospholipid compounds to generate diacylglycerol and corresponding phosphoric acid compounds.
- the bond position of its catalytic phospholipid is shown in Figure 1.
- the phospholipase D and phospholipase C used in the above methods can be derived from animals, plants or microorganisms, and usually microbial sources or microorganisms express enzymes are the best; the specific forms of phospholipase D and phospholipase C used include enzyme solution, enzyme freeze Dry powder, enzyme-containing cells and various immobilized enzymes and immobilized enzyme-containing cells can be in the form of unpurified crude enzymes, partially or completely purified forms, commercial enzymes, or Specially produced. There are many types of phospholipase C, among which broad-spectrum phospholipase C and phosphatidylinositol phospholipase C are preferred.
- the substrate nicotinamide ribose (NR) and phospholipid in the a1 coexist and can be mixed in any ratio, and the more preferred molar ratio is 1:10-10:1; more preferably, the molar amount of nicotinamide ribose and phospholipid The ratio is 1:5-5:1; more preferably, the molar ratio of nicotinamide ribose to phospholipid is 1:2-3:1.
- the a1 reaction needs to add calcium salt, usually a soluble calcium salt, such as calcium chloride, etc.
- the concentration of calcium ions of the a1 in water is 0.01-20 g/L; more preferably, the concentration of calcium ions in water 0.5-5g/L;
- the catalytic reaction temperature of phospholipase D is 20-70°C, and the reaction pH is 4.5-7.5; more preferably, the catalytic reaction temperature of phospholipase D is 40-60°C, and the reaction pH is 5.0-6.5 .
- auxiliary agents may be added, including but not limited to any one or a combination of two or more of n-hexane, n-heptane, and isopropanol.
- the content of the auxiliary agent is 0-50% when the auxiliary agent is n-hexane or n-heptane, and the content is 0-30% when the auxiliary agent is isopropanol.
- the usage amount of the reaction assistant n-hexane, n-heptane and/or isopropanol can be zero.
- the concentration of the a2 calcium ion in the water is 0.01-20 g/L; more preferably, the concentration of the calcium ion in the water is 0.1-2 g/L;
- the catalytic reaction temperature of phospholipase C is 20-70°C, and the reaction pH is 4.0-7.0; more preferably, the catalytic reaction temperature of phospholipase C is 40-55°C, and the reaction pH is 5.0-7.0 .
- alkanes and/or short-chain alcohols can also be added as auxiliary agents, wherein the alkanes can be selected from one or two of n-hexane and n-heptane, and the short-chain alcohols can be One or two selected from methanol, ethanol, propanol, isopropanol, butanol, and pentanol.
- the content of alkane can be 0-80%, and the addition amount of short-chain alcohol can be 0 -30%.
- the usage amount of the organic solvent-based reaction assistant may be zero.
- the materials and technological methods used in the preparation are basically the same as those of the two-step enzymatic hydrolysis method, which will not be redundant here.
- PNR phosphatidylnicotinamide ribose
- R 1 and R 2 are fatty acyl groups, and natural phospholipids R 1 and R 2 are mostly long-chain fatty acids, generally 14-24 carbon fatty acids, more preferably 16 and 18 carbon fatty acids, and further R 1 are common : -C 15 H 31 , -C 17 H 35 , -C 17 H 33 , R2 common are: -C 17 H 31 , -C 19 H 29 , -C 19 H 31 , -C 21 H 31 , -C 17 H 33 .
- the present invention uses a completely new enzyme-catalyzed reaction pathway to produce NMN, using nicotinamide ribose (NR), a conventional NMN precursor, as a raw material, and using phosphatidyl components in phospholipids as phosphatidyl donors.
- NR nicotinamide ribose
- NR nicotinamide ribose
- the present invention uses phospholipid metabolizing enzymes phospholipase D and phospholipase C, which are widely present in the biological world, as catalysts, and develops and utilizes their ability to catalyze unnatural substrates. , Production of NMN.
- the phosphate-based donor phospholipid used in the present invention is a very cheap and common industrial raw material, especially the most well-known as lecithin.
- the catalytic enzymes used are phospholipase D and phospholipase C, both of which are Industrialized enzyme preparations are mostly used in the refining of vegetable oils such as soybean oil. They are easy to express heterologously, have high enzyme activity and are cheap. It is easier for large-scale industrial production.
- Figure 1 The action sites of phospholipase D and phospholipase C of the present invention.
- FIG. 2 Schematic diagram of the phospholipase D and phospholipase C catalyzed reactions of the present invention in sequence.
- Fig. 3 The liquid phase analysis diagram of the phospholipase D of the present invention catalyzing the reaction of nicotinamide ribose and phospholipids to produce phosphatidyl nicotinamide ribose (before reaction and after reaction).
- Figure 4 The liquid phase analysis pattern of NMN produced by the reaction product of phospholipase C catalyzed by phospholipase C to hydrolyze phospholipase D (phosphatidyl nicotinamide ribose) (NR, NMN standard product spectrum, NMN reaction solution plus standard product).
- phospholipase C catalyzed by phospholipase C to hydrolyze phospholipase D (phosphatidyl nicotinamide ribose)
- NR NMN standard product spectrum
- NMN reaction solution plus standard product phosphatidyl nicotinamide ribose
- the substrate NR and the product NMN are water-soluble compounds with strong polarity and need to use reversed-phase column analysis.
- the chromatographic column is Agilent SB-C18 (5 ⁇ m, 4.6 ⁇ 250mm), and the detection wavelength is 254nm.
- Mobile phase Combine mobile phase A and mobile phase B according to the process shown in Table 1, and perform gradient elution. The initial flow rate is 0.8 mL/min, and the column temperature is 30°C.
- Figure 4 shows the analysis of substrate phospholipids and PNR by sampling during the reaction.
- the substrate phospholipid and the product PNR are water-insoluble compounds, which are weak in polarity and require normal phase column analysis.
- the high performance liquid phase analysis method of the two is as follows: the chromatographic column is a silica gel column Si60 (5 ⁇ m, 4.5 ⁇ 250mm), the detection wavelength is 205nm, the analysis is at room temperature, and the mobile phase is acetonitrile-methanol-85% phosphoric acid aqueous solution (100:10:1.8 , V/V/V), isocratic constant rate analysis, flow rate 1.0mL/min.
- Example 7 Synthesis method of ⁇ -nicotinamide mononucleic acid
- Example 8 Synthesis method of ⁇ -nicotinamide mononucleic acid
- Example 11 Synthesis method of ⁇ -nicotinamide mononucleic acid
- Example 12 Synthesis method of ⁇ -nicotinamide mononucleic acid
- Example 13 Preparation of ⁇ -nicotinamide mononucleic acid by adding enzymes successively in one-pot method
- Example 14 Preparation of ⁇ -nicotinamide mononucleic acid by one-pot method followed by enzymatic reaction
- Example 15 Preparation of ⁇ -Nicotinamide Mononucleic Acid by Enzyme Reaction in One-Pot Method
- phospholipase C was added for the reaction, the temperature was adjusted to 43-48°C, the pH was adjusted to 6.0-6.5, and the reaction was carried out for about 6 hours. After the reaction, 83kg NMN was obtained.
- Example 16 Preparation of ⁇ -nicotinamide mononucleic acid by one-pot method followed by enzymatic reaction
- Example 17 Preparation of ⁇ -Nicotinamide Mononucleic Acid by Enzyme Reaction in One-Pot Method
- phospholipase C was added for the reaction, the temperature was adjusted to 43-48°C, the pH was adjusted to 6.0-6.5, and the reaction was carried out for about 1 hour. After the reaction, 86kg NMN was obtained.
- Example 18 Preparation of ⁇ -Nicotinamide Mononucleic Acid by Enzyme Reaction in One-Pot Method
- phospholipase C was added for the reaction, the temperature was adjusted to 43-48°C, the pH was adjusted to 6.0-6.5, and the reaction was carried out for about 6 hours. After the reaction, 157kg NMN was obtained.
- Example 19 Preparation of ⁇ -nicotinamide mononucleic acid by adding enzyme reaction simultaneously in one-pot method
- Example 20 Preparation of ⁇ -Nicotinamide Mononucleic Acid by One-Pot Method Simultaneously Adding Enzyme Reaction
- Example 21 Preparation of ⁇ -nicotinamide mononucleic acid by simultaneous addition of enzymes in one-pot method
- Example 22 Preparation of ⁇ -nicotinamide mononucleic acid by adding enzyme reaction simultaneously in one-pot method
- Example 23 Preparation of ⁇ -nicotinamide mononucleic acid by simultaneous addition of enzymes in one-pot method
- Example 24 Preparation of ⁇ -nicotinamide mononucleic acid by simultaneous addition of enzymes in one pot
- Example 25 Preparation of ⁇ -nicotinamide mononucleic acid by one-pot method followed by enzymatic reaction
- Example 26 Preparation of ⁇ -Nicotinamide Mononucleic Acid by Enzyme Reaction in One-Pot Method
- Example 27 Preparation of ⁇ -nicotinamide mononucleic acid by adding enzyme reaction simultaneously in one-pot method
- Example 28 Preparation of ⁇ -nicotinamide mononucleic acid by simultaneous addition of enzymes in one pot
Abstract
本发明涉及β-烟酰胺单核酸的方法及其中间体,本发明使用了生物界广泛存在的磷脂代谢酶磷脂酶D和磷脂酶C作为催化剂,两步酶解法或一锅法制备β-烟酰胺单核酸;在两步酶解法中获得中间体,即磷脂酰烟酰胺核糖。反应步骤简单、生产成本低、绿色环保无公害、适宜大规模工业化生产等优势。
Description
本发明涉及β-烟酰胺单核酸的合成方法及其中间体,特别涉及β-烟酰胺单核酸和磷脂酰烟酰胺核糖两种产品的酶法催化制备方法和合成β-烟酰胺单核酸所得的中间体磷脂酰烟酰胺核糖。
β-烟酰胺单核酸(Nicotinamide Mononucleotide,NMN)是生命体代谢的关键辅因子烟酰胺腺嘌呤二核苷酸(NAD
+,辅酶I)合成的直接前体,存在于几乎所有的生命体中。主导人体内数百项生命活动,是细胞能量代谢不可或缺的关键性因子,催化产生95%以上生命活动所需的能量。为了维持生命体的正常功能,人体每天需要大量合成NMN,再转化为对于辅酶I。食物中的NMN极少,远远不能满足人体需求,比如日常饮食中含NMN较多的蔬菜毛豆和西兰花每公斤含有的NMN平均不到10mg,一个成年人身体中每天合成的NMN数量大致相当于几百公斤的水果蔬菜!(Cell Metabolism,2016,24:795-806)
许多研究表明NAD
+的水平常常伴随机体衰老而降低,特别是中年后,动物体内的辅酶I(NAD
+)数量急剧减少,由此会导致人体出现各种衰老症状,例如记忆力衰退、心血管功能弱化、免疫力低、抵抗力差、睡眠质量下降、精力衰退、便秘、脱发和食欲不振等等。特别是近几年来,国际权威的学术杂志持续不断发表人体和动物研究,反复证明补充NMN可有效地增加和恢复体内辅酶I水平,大幅延缓衰老和防止老年痴呆症等多种神经元退化疾病,并由此从根本上调理和改善衰老的各种症状。其它研究还涉及癌症、不孕不育、肥 胖、脑出血、心脏衰竭、心脏损伤、血管老化、急性肾衰竭、糖尿病等,表明补充NMN具有多方面的医疗和保健潜力(Cell Metabolism,2011,14:528-536;2016,24:795-806)。而外源补充NAD
+的前体NMN可显著提升细菌、哺乳动物体内NAD
+水平,延缓衰老的进程、提高生命代谢的活力(Developmental Cell,2020,53:240-252)。
目前已有数种NMN保健品上市,根据有关统计,使用者反馈的个案效果非常多样化,包括:精力改善、体力改善、脂肪减少肌肉增加、运动能力增强、皮肤改善、脱发减少、头发增生、睡眠改善、生物钟调节、免疫调节、过敏减轻、性功能增强、食欲提振、视觉疲劳减轻、视力改善、情绪提升、高血糖下降、高血压收缩压下降、低血压恢复正常、便秘改善等等。
最早人们试图通过化学法来生产NMN,该方法能常是以烟酰胺核糖(NR)为原料,用三氯氧磷等进行磷酸化得到(Chem.Commun.1999,729-730,CN 107613990 A),但所获产品杂质多、纯化困难、得率低,综合生产成本高,且涉及大量有毒有害试剂的使用,污染重,不宜用于食品级NMN的生产。因而以酶催化的方法来生产NMN就成为当前NMN生产的主流方式。酶法催化合成NMN的方法有多种,但这些方法大多与生物体内NMN代谢合成有关,需要多种酶参与、且需要以ATP等价格昂贵的生物分子作为NMN的磷酸基供体,或者需要使用价格极其昂贵的底物。
例如:专利CN 108026130 A和CN 110195089 A中以烟酰胺、ATP和核糖为原料,通过烟酰胺磷酸核糖转移酶、核糖磷酸焦磷酸激酶和核糖激酶组成多步催化反应,来生产NMN。
专利CN 108026535 A中以烟酰胺、ATP和AMP为原料,通过烟酰胺磷酸核糖转移酶、核糖磷酸焦磷酸激酶以及AMP核苷酶催化组成的多步催化反应,来生产NMN。
专利PCT/CN2016/092457中以烟酰胺、ATP和木糖为原料,通过烟酰胺磷酸核糖转移酶、核糖磷酸焦磷酸激酶、核糖-5-磷酸异构酶、核酮糖-3-磷酸异构酶、木酮糖激酶、以及 木糖异构酶组成的多步反应,来生产NMN。
专利PCT/CN2016/092459中以烟酰胺、焦磷酸和AMP为原料,通过烟酰胺磷酸核糖转移酶和腺嘌呤磷酸核糖转移酶催化,生产NMN。
专利PCT/CN016/092461中以烟酰胺、焦磷酸和肌苷酸为原料,在烟酰胺磷酸核糖转移酶、次黄嘌呤磷酸核糖转移酶和黄嘌呤氧化酶的催化下,生产NMN。
专利CN 108368493 A;CN 108949865 A以D-5-磷酸核糖、ATP和烟酰胺为原料,通过磷酸核糖焦磷酸合成酶和烟酰胺磷酸核糖转移酶,实现NMN的合成。
专利CN 106755209 A中以NR和ATP为底物,在烟酰胺核糖激酶催化下生成NMN。
上述这些NMN的酶法合成方法,除了需要ATP等多种价格昂贵的生物活性分子做底物之外,还需要多种非常规酶,这些酶不易生产,价格昂贵,至今尚未有可商品化的酶制剂出现,需要NMN生产企业专门制备,这使得NMN酶法生产过程十分复杂、成本高昂。此外,也有以单酶催化生产NMN的方法,例如以烟酰胺和5’-磷酸核糖基-1’-焦磷酸(PRPP)为底物,在烟酰胺磷酸核糖转移酶的催化下,生产NMN,但PRPP不易得,价格昂贵,也不适于大规模工业化生产。
发明内容
针对上述背景技术中提到的现有NMN生产技术所存在的诸多问题。本发明的目的在于提供一种全新的NMN酶法合成技术,该方法完全脱离了NMN的天然生物催化合成体系,完全不涉及使用上述酶品种,也不使用ATP或PRPP等昂贵底物,反应步骤简单、生产成本低、绿色环保无公害、适宜大规模工业化生产等优势。
具体而言,本发明的技术方案是这样的。
为了实现上述目的,发明人经过多年的工作积累和大量的实验探索,最终实现了以烟酰胺核糖(NR)为底物,创造性地发明了以廉价的磷脂为磷基供体,以磷脂酶D和磷脂酶 C为催化剂的NMN酶法合成路线(参见图1和图2),这是一条合成NMN的非天然酶法催化合成路线,磷脂酶D和磷脂酶C廉价易得且有商品化酶制剂,整个反应过程简单快速、无污染、产品收率高、生产成本低,适宜规模化工业化生产。
本发明的目的之一在于提供了一种两步酶催化反应(即按次序反应)来生产NMN:
(a):两步酶解法
a1:以烟酰胺核糖和磷脂为底物,在钙离子存在下,经磷脂酶D催化,生成磷脂酰烟酰胺核糖;
a2:以步骤a1生成的磷脂酰烟酰胺核糖为底物,在钙离子存在下,在磷脂酶C的催化下,生成β-烟酰胺单核酸;
更进一步地,以烟酰胺核糖(NR)为底物,以磷脂类物质为磷脂酰基供体,通过磷脂酶D的转磷脂酰化功能,催化磷脂与NR发生反应,生成磷脂酰烟酰胺核糖(简称PNR),PNR是一种新型磷脂分子,易与水溶性的NR分离;随后,在磷脂酶C的催化下,使PNR发生水解,生成二脂酰甘油和β-烟酰胺单核酸NMN,二脂酰甘油是一种天然油脂,不溶于水,易与水溶性NMN分离。
本发明的目的之二在于提供了一种通过一锅法来生产NMN的两种途径。具体为:
第一种途径是:以烟酰胺核糖(NR)和磷脂为底物,在钙离子存在下,先加入磷脂酶D进行反应,反应结束后,再加入磷脂酶C进行反应,获得NMN。
第二种途径是:以烟酰胺核糖(NR)和磷脂为底物,在钙离子存在下,一并加入磷脂酶D和磷脂酶C进行反应,获得NMN。
所述的磷脂为天然磷脂或人工合成磷脂;进一步优选为,所述的磷脂的主要成分为磷脂酰胆碱和/或磷脂酰乙醇胺;进一步优选为,所述的磷脂为卵磷脂。
所用磷脂通常以天然磷脂为佳,可为植物来源,例如,来源于大豆、葵花、油菜籽、红花籽等油料植物;也可以为动物来源,例如,来源于牛奶、羊奶、深海鱼、深海虾、深 海扇贝等;也可为微生物来源等。
磷脂指的是一大类具有相似磷脂酰骨架的化合物,其结构与图1所示,磷脂分子中的R1和R2部分为脂肪酸,这些脂肪酸可为任何脂肪酸:可以为短链、中链、长链脂肪酸;可为饱和脂肪酸、单不饱和脂肪酸或多不饱和脂肪酸;R1和R2可以相同,也可以不同;其它,这里不再赘述。磷脂分子中的X部分为极性头,为带羟基的分子,也具有多样性,较为常见的有胆碱、乙醇胺、肌醇、甘油、短链醇等等。
所述的磷脂酶C包括广谱型磷脂酶C和磷脂酰肌醇型磷脂酶C;进一步优选为,磷脂酶C为磷脂酰肌醇型磷脂酶C。
所述的磷脂酶D为动植物和微生物来源的磷脂酶D,进一步优选为微生物来源的磷脂酶D,例如链霉菌来源的磷脂酶D。
根据酶的国际系统命名法,上述方法所使用的酶分别为:磷脂酶D(Phospholipase D,简写为PLD),该酶能够催化转磷脂酰化反应,例如能够催化磷脂酰胆碱、磷脂酰乙醇胺等磷脂与带有羟基的物质,生成对应的磷脂酰化产物,其催化磷脂的键位如图1所示,该酶可以作用于如图所示的P-O键或者O-X键,包括但并不限于EC 3.1.4.4;磷脂酶C(Phospholipase C,简写为PLC),磷脂酶C种类很多,包括但不限于EC 3.1.4.3,EC 3.1.4.10,EC 3.1.4.11,EC 4.6.1.13等,该酶能够水解磷脂类化合物的甘油磷酸酯键,生成二脂酰甘油和对应磷酸化合物,其催化磷脂的键位如图1所示。
上述方法中所使用的磷脂酶D和磷脂酶C可以来源于动植物或微生物,通常以微生物来源或微生物表达酶为最佳;所用磷脂酶D和磷脂酶C的具体形式包括酶液、酶冻干粉、含酶细胞以及各种固定化酶和固定化含酶细胞,可以是未经纯化的粗酶形式,也可以是经部分纯化或完全纯化的形式,可以是商品化的酶,也可以专门生产。磷脂酶C种类多,其中以广谱型磷脂酶C和磷脂酰肌醇型磷脂酶C为佳。
所述a1中的底物烟酰胺核糖(NR)和磷脂同时存在,且可以任意比例混合,较为优 选的摩尔用量比为1∶10-10∶1;进一步优选为,烟酰胺核糖和磷脂摩尔用量比为1∶5-5∶1;更进一步优选为,烟酰胺核糖和磷脂摩尔用量比为1∶2-3∶1。
所述a1反应中需要加入钙盐,通常采用可溶性钙盐,例如为氯化钙等,所述a1的钙离子在水中的浓度为0.01-20g/L;进一步优选为,钙离子在水中的浓度为0.5-5g/L;
所述a1反应过程中的磷脂酶D的催化反应温度为20-70℃,反应pH为4.5-7.5;进一步优选为,磷脂酶D的催化反应温度为40-60℃,反应pH为5.0-6.5。
所述a1反应过程中的磷脂酶D催化反应中还可以加入助剂,包括但不限于正己烷、正庚烷、异丙醇任意一种或二种以上的组合。
进一步优选的,其中,以重量用量比计算,当助剂为正己烷或正庚烷时的含量可为0-50%,当助剂为异丙醇时的含量可为0-30%。
所述磷脂酶D催化反应,为了更适于高品质食品生产的需求,反应助剂正己烷、正庚烷和/或异丙醇的使用量可为0。
所述a2钙离子在水中的浓度为0.01-20g/L;进一步优选为,钙离子在水中的浓度为0.1-2g/L;
所述a2反应过程中的磷脂酶C的催化反应温度为20-70℃,反应pH为4.0-7.0;进一步优选为,磷脂酶C的催化反应温度为40-55℃,反应pH为5.0-7.0。
所述a2反应过程中的磷脂酶C催化反应中还可以加入烷烃和/或短链醇做助剂,其中烷烃可选自正己烷、正庚烷中的一种或二种,短链醇可选自甲醇、乙醇、丙醇异丙醇、丁醇、戊醇中的一种或二种,以重量用量比计算,烷烃的含量可为0-80%,短链醇的加量可为0-30%。
所述磷脂酶C催化反应,为了更适于高品质食品生产的需求,有机溶剂类反应助剂使用量可为0。
对于一锅法制备NMN的两种途径,其制备中所使用的物料和工艺手段与两步酶解法 基本相同,此处不再累赘。
本发明所述的磷脂酰烟酰胺核糖(简称PNR),结构式如下所示:
其中,R
1和R
2为脂肪酰基,天然磷脂R
1和R
2多为长链脂肪酸,一般是14-24个碳的脂肪酸,进一步优选为16和18碳脂肪酸,更进一步R
1常见的有:-C
15H
31、-C
17H
35、-C
17H
33,R2常见的有:-C
17H
31、-C
19H
29、-C
19H
31、-C
21H
31、-C
17H
33。
与现有技术相比,本发明的优点在于:
(1)本发明通过一种全新的酶催化反应途径,来生产NMN,以烟酰胺核糖(NR)这种常规的NMN前体为原料,以磷脂中的磷脂酰组分为磷脂酰基供体,通过两步酶催化,对NR进行磷酸基化,来生产NMN。
(2)本发明使用了生物界广泛存在的磷脂代谢酶磷脂酶D和磷脂酶C作为催化剂,开发利用了它们催化非天然底物的能力,从而经磷脂酰烟酰胺核糖PNR这种新中间产物,生产NMN。
(3)本发明所采用的磷酸基供体磷脂则是一种十分廉价常见的工业原料,尤其是最为人们所熟知为卵磷脂,所使用的催化酶为磷脂酶D和磷脂酶C,均为工业化酶制剂,多用于大豆油等植物油的精炼,易于异源表达、酶活高,价格便宜。更易于大规模工业化生产。
图1本发明磷脂酶D和磷脂酶C的作用部位。
图2本发明磷脂酶D和磷脂酶C次序催化反应的示意图。
图3本发明磷脂酶D催化烟酰胺核糖与磷脂反应生成磷脂酰烟酰胺核糖的液相分析图谱(反应前、反应后)。
图4本发明磷脂酶C催化水解磷脂酶D的反应产物磷脂酰烟酰胺核糖生成NMN的液相分析图谱(NR、NMN的标准品图谱、NMN反应液加点标品)。
对于上述涉及的物质进行检测。
1、在反应过程中对NR和NMN的取样分析。
在上述反应过程中,底物NR和产物NMN为水易溶性化合物,极性强,需要使用反相柱分析。
NR和产物NMN的高效液相分析方法如下:
色谱柱为Agilent SB-C18(5μm,4.6×250mm),检测波长254nm。
流动相:由流动相A和流动相B,按表1所示过程组合,进行梯度洗脱,初始流速0.8mL/min,柱温30℃。流动相A:水(1.36g磷酸二氢钾溶于1L水中,加磷酸调节pH=2.5);流动相B:甲醇。
时间/min | A% | B% |
0.01 | 95 | 5 |
5 | 95 | 5 |
11 | 5 | 95 |
15 | 5 | 95 |
16 | 95 | 5 |
25 | 95 | 5 |
2、在反应过程中对磷脂和PNR的取样分析。
图4是在反应过程中取样对底物磷脂和PNR的分析。在上述反应过程中,底物磷脂和产物PNR为水不溶性化合物,极性弱,需要使用正相柱分析。这两者的高效液相分析方法 如下:色谱柱为硅胶柱Si60(5μm,4.5×250mm),检测波长205nm,室温下分析,流动相为乙腈-甲醇-85%磷酸水溶液(100∶10∶1.8,V/V/V),等度恒速分析,流速为1.0mL/min。
实施例1:磷脂酰烟酰胺核糖PNR的制备
向反应釜中加入水、正庚烷和异丙醇的混合物共1000L,比例分别为5∶4∶1,投150g磷脂(以含50%磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入50kg烟酰胺核糖(NR),随后加入0.8kg氯化钙。搅拌升温至均匀乳化状态,升温50-55℃。用盐酸或氢氧化钠溶液,调pH为5.0-6.0。加入适量磷脂酶D开始反应,反应约2个小时。反应结束后,静置,分出水不溶相,获得83kg PNR。
实施例2:磷脂酰烟酰胺核糖PNR的制备
向反应釜中加入水和异丙醇的混合物共2000L,比例分别为8.5∶1.5,投500g磷脂(以含50%的磷脂酰胆碱的卵磷脂为例),乳化溶解,随后加入120kg烟酰胺核糖(NR),随后加入10kg氯化钙。搅拌升温至均匀乳化状态,升温45-50℃。用盐酸或氢氧化钠溶液,调pH为5.6-6.4。加入适量磷脂酶D开始反应,反应约1个小时。反应结束后,静置,分出水不溶相,获得264kg PNR.
实施例3:磷脂酰烟酰胺核糖PNR的制备
向反应釜中加入2000L饮用水,投加400kg磷脂(以含50%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入140kg烟酰胺核糖(NR),随后加入3kg氯化钙。搅拌升温至均匀乳化状态,升温至45-50℃。用盐酸或氢氧化钠溶液,调pH为5.5-6.0。加入适量磷脂酶D开始反应,反应约以4个小时。反应结束后,静置,分出水不溶相或水不溶物,获得218kg PNR。
实施例4:磷脂酰烟酰胺核糖PNR的制备
向反应釜中加入水、正己烷和异丙醇的混合物共2000L,比例分别为6∶3∶1,投240g磷脂(以含50%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入20kg烟酰胺核糖(NR),随后加入4kg氯化钙。搅拌升温至均匀乳化状态,升温55-60℃。用盐酸或氢氧化钠溶液,调pH为5.0-6.0。加入适量磷脂酶D开始反应,反应约1个小时。反应结束后,静置,分出水不溶相,获得63kg PNR.
实施例5:磷脂酰烟酰胺核糖PNR的制备
向反应釜中加入1000L饮用水,投加200kg磷脂(以含60%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入150kg烟酰胺核糖(NR),随后加入0.5kg氯化钙。搅拌升温至均匀乳化状态,升温至45-50℃。用盐酸或氢氧化钠溶液,调pH为5.5-6.0。加入适量磷脂酶D开始反应,反应约以4个小时。反应结束后,静置,分出水不溶相或水不溶物,获得115kg PNR。
实施例6:磷脂酰烟酰胺核糖PNR的制备
向反应釜中加入水、正庚烷和异丙醇的混合物共3000L,比例分别为7.5∶2∶0.5,投500kg磷脂(以含30%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入100kg烟酰胺核糖(NR),随后加入20kg氯化钙。搅拌升温至均匀乳化状态,升温47-52℃。调pH为5.5-6.0。加入适量磷脂酶D开始反应,反应约12个小时。反应结束后,静置,分出水不溶相,获得163kg PNR.
实施例7:β-烟酰胺单核酸的合成方法
把来自实施例1和6的150kg的PNR,加入100kg的水,加入10kg的正庚烷和5kg的异丙醇。200g氯化钙。搅拌均匀,升温至43-48℃。调pH为5.5-6.0。加入适量磷脂酶C开始反应,反应约2个小时。反应结束后,得52kg的NMN。
实施例8:β-烟酰胺单核酸的合成方法
把来自实施例3和5的100kg PNR,加入200kg的饮用水,加入0.5kg的氯化钙,搅 拌均匀,升温至45-50℃。调pH为6.0至6.5,加入适量磷脂酶C开始反应,反应约4个小时。反应结束后,得到34kg的NMN。
实施例9:β-烟酰胺单核酸的合成方法
把来自实施例2的200kg PNR,加入880kg的饮用水和120kg的异丙醇,加入2kg的氯化钙,搅拌均匀,升温至43-48℃。调pH为6.0至6.5,加入适量磷脂酶C开始反应,反应约8个小时。反应结束后,得到70kg的NMN。
实施例10:β-烟酰胺单核酸的合成方法
把来自实施例4的50kg的PNR,加入100kg的水,加入7kg的正己烷和3kg的异丙醇。80g氯化钙。搅拌均匀,升温至45-50℃。调pH为6.0-6.5。加入适量磷脂酶C开始反应,反应约1个小时。反应结束后,得16kg的NMN。
实施例11:β-烟酰胺单核酸的合成方法
把来自实施例3和5的100kg PNR,加入300kg的饮用水和30kg的丁醇,加入2kg的氯化钙,搅拌均匀,升温至45-50℃。调pH为6.5至7.0,加入适量磷脂酶C开始反应,反应约2个小时。反应结束后,得到34kg的NMN。
实施例12:β-烟酰胺单核酸的合成方法
把来自实施例3和5的100kg PNR,加入300kg的饮用水和30kg的乙醇,加入2kg的氯化钙,搅拌均匀,升温至45-50℃。调pH为6.5至7.0,加入适量磷脂酶C开始反应,反应约2个小时。反应结束后,得到33kg的NMN。
实施例13:一锅法先后加酶制备β-烟酰胺单核酸
向反应釜中加入水、正庚烷和异丙醇的混合物共2000L,比例分别为6∶4∶1,投360g磷脂(以含50%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入120kg烟酰胺核糖(NR),随后加入2kg氯化钙。搅拌升温至均匀状态,升温47-52℃。调pH为5.5-6.0。加入适量磷脂酶D开始反应,反应约2个小时。随后,再加入磷脂酶C进行反应,调温度 为43-48℃,反应约4个小时。反应结束后,得71kg NMN。
实施例14:一锅法先后加酶反应制备β-烟酰胺单核酸
向反应釜中加入水和异丙醇的混合物共3000L,比例分别为8∶2,投450g磷脂(以含50%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入90kg烟酰胺核糖(NR),随后加入6kg氯化钙。搅拌升温至均匀乳化状态,升温48-53℃。调pH为5.5-6.0。加入适量磷脂酶D开始反应,反应约2个小时。随后,再加入磷脂酶C进行反应,调pH为6.0-6.5,反应约4个小时。反应结束后,得92kg NMN。
实施例15:一锅法先后加酶反应制备β-烟酰胺单核酸
向反应釜中加入2000L饮用水,投加400kg磷脂(以含50%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入90kg烟酰胺核糖(NR),随后加入1.5kg氯化钙。搅拌升温至均匀乳化状态,升温至45-50℃。用盐酸或氢氧化钠溶液,调pH为5.5-6.0。加入适量磷脂酶D开始反应,反应约以3个小时。随后,再加入磷脂酶C进行反应,调温度为43-48℃,调pH为6.0-6.5,反应约6个小时。反应结束后,得83kg NMN。
实施例16:一锅法先后加酶反应制备β-烟酰胺单核酸
向反应釜中加入水、正己烷和异丙醇的混合物共3000L,比例分别为6∶3∶1,投500kg磷脂(以含50%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入120kg烟酰胺核糖(NR),随后加入5kg氯化钙。搅拌升温至均匀乳化状态,升温55-60℃。用盐酸或氢氧化钠溶液,调pH为5.5-6.0。加入适量磷脂酶D开始反应,反应约2个小时。随后,再加入磷脂酶C进行反应,调温度为43-48℃,调pH为6.0-6.5,反应约3个小时。反应结束后,得99kg NMN。
实施例17:一锅法先后加酶反应制备β-烟酰胺单核酸
向反应釜中加入2000L饮用水,投加350kg磷脂(以含60%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入120kg烟酰胺核糖(NR),随后加入1kg氯化钙。搅拌 升温至均匀乳化状态,升温至48-53℃。用盐酸或氢氧化钠溶液,调pH为5.5-6.0。加入适量磷脂酶D开始反应,反应约以4个小时。随后,再加入磷脂酶C进行反应,调温度为43-48℃,调pH为6.0-6.5,反应约1个小时。反应结束后,得86kg NMN。
实施例18:一锅法先后加酶反应制备β-烟酰胺单核酸
向反应釜中加入水、正庚烷和异丙醇的混合物共5000L,比例分别为7.5∶2∶0.5,投1000kg磷脂(以含40%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入150kg烟酰胺核糖(NR),随后加入20kg氯化钙。搅拌升温至均匀乳化状态,升温47-52℃。调pH为5.5-6.0。加入适量磷脂酶D开始反应,反应约6个小时。随后,再加入磷脂酶C进行反应,调温度为43-48℃,调pH为6.0-6.5,反应约6个小时。反应结束后,得157kg NMN。
实施例19:一锅法同时加酶反应制备β-烟酰胺单核酸
向反应釜中加入水和异丙醇的混合物共3000L,比例分别为9∶1,投500g磷脂(以含50%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入100kg烟酰胺核糖(NR),随后加入3kg氯化钙。搅拌升温至均匀乳化状态,升温45-50℃。调pH为5.8-6.3。同时加入磷脂酶D和磷脂酶C开始反应,反应约4个小时。反应结束后,得100kg NMN。
实施例20:一锅法同时加酶反应制备β-烟酰胺单核酸
向反应釜中加入2000L饮用水,投加400kg磷脂(以含50%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入80kg烟酰胺核糖(NR),随后加入2kg氯化钙。搅拌升温至均匀乳化状态,升温至45-50℃。用盐酸或氢氧化钠溶液,调pH为5.8-6.3。同时加入磷脂酶D和磷脂酶C开始反应,反应约5个小时。反应结束后,得82kg NMN。
实施例21:一锅法同时加酶反应制备β-烟酰胺单核酸
向反应釜中加入2000L饮用水,投加300kg磷脂(以含60%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入72kg烟酰胺核糖(NR),随后加入1kg氯化钙。搅拌升温至均匀乳化状态,升温至45-50℃。用盐酸或氢氧化钠溶液,调pH为5.8-6.3。同时加入 磷脂酶D和磷脂酶C开始反应,反应约3个小时。反应结束后,得74kg NMN。
实施例22:一锅法同时加酶反应制备β-烟酰胺单核酸
向反应釜中加入5000L饮用水,投加1000kg磷脂(以含40%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入175kg烟酰胺核糖(NR),随后加入10kg氯化钙。搅拌升温至均匀乳化状态,升温至45-50℃。用盐酸或氢氧化钠溶液,调pH为5.8-6.3。同时加入磷脂酶D和磷脂酶C开始反应,反应约8个小时。反应结束后,得167kg NMN。
实施例23:一锅法同时加酶反应制备β-烟酰胺单核酸
向反应釜中加入水、正庚烷和异丙醇的混合物共3000L,比例分别为6∶3∶1,投750kg磷脂(以含50%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入200kg烟酰胺核糖(NR),随后加入7kg氯化钙。升温至45-50℃。用盐酸或氢氧化钠溶液,调pH为5.8-6.3。同时加入磷脂酶D和磷脂酶C开始反应,反应约12个小时。反应结束后,得128kg NMN。
实施例24:一锅法同时加酶反应制备β-烟酰胺单核酸
向反应釜中加入3000L饮用水,投加550kg磷脂(以含55%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入300kg烟酰胺核糖(NR),随后加入2kg氯化钙。搅拌升温至均匀乳化状态,升温至45-50℃。用盐酸或氢氧化钠溶液,调pH为5.8-6.3。同时加入磷脂酶D和磷脂酶C开始反应,反应约4个小时。反应结束后,得121kg NMN。
实施例25:一锅法先后加酶反应制备β-烟酰胺单核酸
向反应釜中加入1000L饮用水,投加20kg磷脂(以含60%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入36kg烟酰胺核糖(NR),随后加入0.1kg氯化钙。搅拌升温至均匀乳化状态,升温至45-50℃。用盐酸或氢氧化钠溶液,调pH为5.8-6.3。同时加入磷脂酶D和磷脂酶C开始反应,反应约2个小时。反应结束后,得4.1kg NMN。
实施例26:一锅法先后加酶反应制备β-烟酰胺单核酸
向反应釜中加入水、正庚烷和异丙醇的混合物共5000L,比例分别为7.5∶2∶0.5,投800 kg磷脂(以含70%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入250kg烟酰胺核糖(NR),随后加入100kg氯化钙。搅拌升温至均匀乳化状态,升温至45-50℃。用盐酸或氢氧化钠溶液,调pH为5.8-6.3。同时加入磷脂酶D和磷脂酶C开始反应,反应约6个小时。反应结束后,得222kg NMN。
实施例27:一锅法同时加酶反应制备β-烟酰胺单核酸
向反应釜中加入水和异丙醇的混合物共3000L,比例分别为7∶3,投600g磷脂(以含50%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入100kg烟酰胺核糖(NR),随后加入2kg氯化钙。搅拌升温至均匀乳化状态,升温至45-50℃。用盐酸或氢氧化钠溶液,调pH为5.8-6.3。同时加入磷脂酶D和磷脂酶C开始反应,反应约5个小时。反应结束后,得105kg NMN。
实施例28:一锅法同时加酶反应制备β-烟酰胺单核酸
向反应釜中加入水的混合物共3000L,投600g磷脂(以含50%的磷脂酰胆碱规格的卵磷脂为例),乳化溶解,随后加入45kg烟酰胺核糖(NR),随后加入1kg氯化钙。用盐酸或氢氧化钠溶液,调pH为5.8-6.3。同时加入磷脂酶D和磷脂酶C开始反应,反应约2个小时。反应结束后,得26kg NMN。
Claims (10)
- 一种制备β-烟酰胺单核酸的方法,其特征在于:所述的方法可以由(a)或(b)的任意一种制得:(a):两步酶解法a1:以烟酰胺核糖和磷脂为底物,在钙离子存在下,经磷脂酶D催化,生成磷脂酰烟酰胺核糖;a2:以步骤a1生成的磷脂酰烟酰胺核糖为底物,在钙离子存在下,在磷脂酶C的催化下,生成β-烟酰胺单核酸;(b):一锅法制备β-烟酰胺单核酸。
- 根据权利要求1所述的方法,其特征在于:所述的一锅法制备β-烟酰胺单核酸可以是(1)或(2)的任意一种:(1):以烟酰胺核糖和磷脂为底物,在钙离子存在下,先加入磷脂酶D进行反应,反应结束后,再加入磷脂酶C进行反应,获得β-烟酰胺单核酸;(2):以烟酰胺核糖和磷脂为底物,在钙离子存在下,一并加入磷脂酶D和磷脂酶C进行反应,获得β-烟酰胺单核酸。
- 根据权利要求1或2任一项所述的方法,其特征在于:所述的磷脂为天然磷脂或人工合成磷脂;进一步优选为,所述的磷脂的主要成分为磷脂酰胆碱和/或磷脂酰乙醇胺;进一步优选为,所述的磷脂为卵磷脂。
- 根据权利要求1和2任一项所述的方法,其特征在于:所述的磷脂酶C为生物体内普遍存在的磷脂酶C;进一步优选为,磷脂酶C为广谱型磷脂酶C;更优选为磷脂酰肌醇型磷脂酶C。
- 根据权利要求1和2任一项所述的方法,其特征在于:所述的磷脂酶D为生物体内普遍存在的磷脂酶D,进一步优选为微生物来源的磷脂酶D。
- 根据权利要求1或2所述的方法,其特征在于:所述a1中的烟酰胺核糖和磷脂摩尔用量比为1∶10-10∶1;进一步优选为,所述a1中的烟酰胺核糖和磷脂摩尔用量比为1∶5-5∶1;更进一步优选为,所述a1中的烟酰胺核糖和磷脂摩尔用量比为1∶2-3∶1;所述钙离子在水中的浓度为0.01-20g/L;进一步优选为,钙离子在水中的浓度为0.5-5g/L;所述磷脂酶D的催化反应温度为20-70℃,反应pH为4.5-7.5;进一步优选为,反应温度为40-60℃,反应pH为5.0-6.5。
- 根据权利要求1或2所述的方法,其特征在于:所述磷脂酶D催化反应中还可以加入助剂,包括但不限于正己烷、正庚烷、异丙醇任意一种或二种以上的组合,其中,以重量用量比计算,助剂为正己烷或正庚烷,含量为0-50%,助剂为异丙醇,含量为0-30%。
- 根据权利要求1或2所述的方法,其特征在于:所述a2钙离子在水中的浓度为 0.01-20g/L;进一步优选为,钙离子在水中的浓度为0.1-2g/L;所述磷脂酶C的催化反应温度为20-70℃,磷脂酶C的反应pH为4.0-7.0;进一步优选为,反应温度为40-55℃,磷脂酶C的反应pH为5.0-7.0。
- 根据权利要求9所述的方法,其特征在于:所述磷脂酶C催化反应中还可以加入烷烃和/或短链醇做助剂,其中烷烃选自正己烷、正庚烷中的一种或二种,短链醇选自甲醇、乙醇、丙醇、异丙醇、丁醇、戊醇中的一种或二种,以重量用量比计算,助剂为烷烃,含量可为0-80%,助剂为短链醇,含量可为0-30%。
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CN114672530B (zh) * | 2022-04-22 | 2024-01-23 | 江苏信佑生物有限公司 | 一种制备β-烟酰胺单核苷酸的方法 |
CN115227705A (zh) * | 2022-08-01 | 2022-10-25 | 成都川宇健维生物科技有限公司 | β-烟酰胺单核苷酸在制备预防和/或治疗便秘的药物中的应用 |
CN116875578A (zh) * | 2023-08-25 | 2023-10-13 | 康盈红莓(中山)生物科技有限公司 | 一种三联体融合酶及其制备方法和以之制备烟酰胺单核苷酸的方法 |
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US20230357811A1 (en) | 2023-11-09 |
CN111647635A (zh) | 2020-09-11 |
CN111647635B (zh) | 2021-09-21 |
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