阅读说明：本技术 β-烟酰胺单核酸的合成方法及其中间体 (Method for synthesizing beta-nicotinamide mononucleotide and intermediate thereof ) 是由 袁永红 于 2020-06-19 设计创作，主要内容包括：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。(The invention relates to a method for preparing beta-nicotinamide mononucleotide and an intermediate thereof, which uses phospholipid metabolic enzymes phospholipase D and phospholipase C widely existing in biology as catalysts to prepare the beta-nicotinamide mononucleotide by a two-step enzymolysis method or a one-pot method; the intermediate, namely phosphatidyl nicotinamide ribose, is obtained in a two-step enzymatic hydrolysis method. Simple reaction steps, low production cost, environmental protection, no public nuisance, suitability for large-scale industrial production and the like.)

1. A method for preparing a β -nicotinamide mononucleotide, comprising: the method can be prepared by any one of (a) or (b):

(a) the method comprises the following steps Two-step enzymolysis method

a 1: taking nicotinamide ribose and phospholipid as substrates, and generating the phosphatidyl nicotinamide ribose under the catalysis of phospholipase D in the presence of calcium ions;

a 2: taking the phosphatidyl nicotinamide ribose generated in the step a1 as a substrate, and generating beta-nicotinamide mononucleotide under the catalysis of phospholipase C in the presence of calcium ions;

(b) the method comprises the following steps Preparing beta-nicotinamide mononucleotide by a one-pot method.

2. The method of claim 1, wherein the one-pot method for preparing the beta-nicotinamide mononucleotide can be any one of (1) or (2):

(1): taking nicotinamide ribose and phospholipid as substrates, adding phospholipase D for reaction in the presence of calcium ions, and adding phospholipase C for reaction after the reaction is finished to obtain beta-nicotinamide mononucleotide;

(2): taking nicotinamide ribose and phospholipid as substrates, and adding phospholipase D and phospholipase C together to react in the presence of calcium ions to obtain the beta-nicotinamide mononucleotide.

3. The method of claim 1, wherein said phosphatidylnicotinamide ribose is I:

wherein R is₁And R₂Is fatty acyl, natural phospholipid R₁And R₂Most are long chain fatty acids selected from fatty acids of 14 to 24 carbons, more preferably 16 and 18 carbons, and further more, R₁Is selected from-C₁₅H₃₁、－C₁₇H₃₅、－C₁₇H₃₃Any one of (1), R₂Is selected from-C₁₇H₃₁、－C₁₉H₂₉、－C₁₉H_31、－C₂₁H₃₁、－C₁₇H₃₃Any one of them.

4. The method according to claim 1 or 2, wherein the phospholipid is a natural phospholipid or an artificial phospholipid; more preferably, the main component of the phospholipid is phosphatidylcholine and/or phosphatidylethanolamine; more preferably, the phospholipid is lecithin.

5. The method according to any one of claims 1 and 2, wherein: the phospholipase C is ubiquitous phospholipase C in organisms; more preferably, the phospholipase C is a broad-spectrum phospholipase C; more preferably phosphatidylinositol-type phospholipase C.

6. The method according to any one of claims 1 and 2, wherein: the phospholipase D is a phospholipase D which is present in the organism, and is more preferably a phospholipase D derived from a microorganism.

7. The method according to claim 1 or 2, characterized in that:

the molar using amount ratio of the nicotinamide riboside to the phospholipid in the a1 is 1:10-10: 1; more preferably, the molar ratio of nicotinamide riboside to phospholipid in a1 is 1:5-5: 1; more preferably, the molar ratio of the nicotinamide riboside to the phospholipid in the a1 is 1:2-3: 1;

the concentration of the calcium ions in water is 0.01-20 g/L; more preferably, the concentration of calcium ions in water is 0.5-5 g/L;

the catalytic reaction temperature of the phospholipase D is 20-70 ℃, and the reaction pH is 4.5-7.5; more preferably, the reaction temperature is 40 to 60 ℃ and the reaction pH is 5.0 to 6.5.

8. The method according to claim 1 or 2, characterized in that: the phospholipase D catalytic reaction can be further added with an auxiliary agent, including but not limited to any one or a combination of more than two of n-hexane, n-heptane and isopropanol, wherein the auxiliary agent is n-hexane or n-heptane with the content of 0-50% and the auxiliary agent is isopropanol with the content of 0-30% in terms of weight and dosage ratio.

9. The method according to claim 1 or 2, characterized in that: the concentration of the a2 calcium ions in water is 0.01-20 g/L; more preferably, the concentration of calcium ions in water is 0.1-2 g/L; the catalytic reaction temperature of the phospholipase C is 20-70 ℃, and the reaction pH of the phospholipase C is 4.0-7.0; more preferably, the reaction temperature is 40 to 55 ℃ and the reaction pH of phospholipase C is 5.0 to 7.0.

10. The method of claim 9, wherein: alkane and/or short-chain alcohol can be added into the catalytic reaction of the phospholipase C as an auxiliary agent, wherein the alkane is selected from one or two of n-hexane and n-heptane, the short-chain alcohol is selected from one or two of methanol, ethanol, propanol, isopropanol, butanol and pentanol, and the auxiliary agent is alkane with the content of 0-80 percent and the auxiliary agent is short-chain alcohol with the content of 0-30 percent in terms of weight and dosage ratio.

Technical Field

The invention relates to a synthesis method of beta-nicotinamide mononucleotide and an intermediate thereof, in particular to an enzymatic catalysis preparation method of beta-nicotinamide mononucleotide and phosphatidyl nicotinamide ribose and an intermediate phosphatidyl nicotinamide ribose obtained by synthesizing the beta-nicotinamide mononucleotide.

Background

β Nicotinamide Mononucleotide (NMN) is key cofactor for metabolism of living body⁺Coenzyme I), the direct precursor of synthesis, is present in almost all living bodies. The biological energy-saving agent is mainly used for hundreds of life activities in human bodies, is an indispensable key factor for cell energy metabolism, and catalyzes and generates more than 95 percent of energy required by the life activities. In order to maintain the normal functions of the living body, the human body needs to synthesize NMN in large quantities every day and then convert it into coenzyme I. The NMN in food is very little and far from meeting the human needs, for example, the vegetable green beans and broccoli containing much NMN in daily diet contain less than 10mg of NMN per kilogram, and the amount of NMN synthesized in an adult per day is approximately equivalent to several hundred kilograms of fruit and vegetables! (Cell Metabolism,2016,24:795-806)

A number of studies have shown NAD⁺Often accompanied byCoenzyme I (NAD) in animals decreases with aging, particularly after middle age⁺) The number is drastically reduced, thereby causing various aging symptoms such as memory deterioration, cardiovascular function weakening, low immunity, poor resistance, sleep quality deterioration, energy deterioration, constipation, hair loss, and poor appetite, etc. in the human body. Especially, in recent years, the international authoritative journal of academic continually releases human and animal research, and repeatedly proves that the supplement of NMN can effectively increase and recover the level of coenzyme I in vivo, greatly delay senility, prevent various neuronal degeneration diseases such as senile dementia and the like, and fundamentally condition and improve various symptoms of senility. Other studies have also implicated cancer, infertility, obesity, cerebral hemorrhage, heart failure, heart injury, vascular aging, acute renal failure, diabetes, etc., suggesting that NMN supplementation has a wide range of medical and health potential (Cell Metabolism,2011, 14: 528-. While exogenous supplementation NAD⁺The precursor NMN can obviously promote NAD in bacteria and mammals⁺Level, delaying the aging process, and improving the vitality of the metabolism of life (2020, 53: 240-.

Several NMN health products are on the market at present, and according to relevant statistics, individual case effects fed back by users are very diverse, including: improved energy, improved physical strength, reduced fat, increased muscle mass, enhanced exercise capacity, improved skin, reduced hair loss, hair growth, improved sleep, improved biological clock, immune regulation, reduced allergies, enhanced sexual function, increased appetite, reduced visual fatigue, improved vision, improved mood, reduced hyperglycemia, reduced systolic blood pressure, reduced hypotension return to normal, improved constipation, and the like.

The first attempts to produce NMN by chemical methods, which usually uses Nicotinamide Riboside (NR) as raw material and is obtained by phosphorylation with phosphorus oxychloride (chem. commun.1999,729-730, CN 107613990 a), but the obtained product has many impurities, is difficult to purify, has low yield, high comprehensive production cost, involves the use of a large amount of toxic and harmful reagents, has heavy pollution, and is not suitable for the production of food grade NMN. Therefore, the enzyme-catalyzed production of NMN is the mainstream method for NMN production. There are many enzymatic methods for synthesizing NMN, but these methods are often involved in the metabolic synthesis of NMN in vivo, and require various enzymes, require expensive biomolecules such as ATP as a phosphate group donor of NMN, and require the use of an extremely expensive substrate.

For example: in patents CN 108026130 a and CN 110195089 a, nicotinamide, ATP and ribose are used as raw materials, and NMN is produced by multi-step catalytic reaction of nicotinamide phosphoribosyltransferase, ribose phosphate pyrophosphorykinase and ribose kinase.

In patent CN 108026535 a, nicotinamide, ATP and AMP are used as raw materials, and NMN is produced by multi-step catalytic reaction catalyzed by nicotinamide phosphoribosyltransferase, ribophosphoribosyl pyrophosphate kinase and AMP nucleosidase.

In PCT/CN2016/092457, NMN is produced from nicotinamide, ATP and xylose as raw materials by a multi-step reaction involving nicotinamide phosphoribosyl transferase, ribose phosphate pyrophosphokinase, ribose-5-phosphate isomerase, ribulose-3-phosphate isomerase, xylulokinase and xylose isomerase.

In PCT/CN2016/092459, NMN is produced from nicotinamide, pyrophosphoric acid and AMP as raw materials by the catalysis of nicotinamide phosphoribosyltransferase and adenine phosphoribosyltransferase.

In PCT/CN016/092461, NMN is produced by using nicotinamide, pyrophosphoric acid and inosinic acid as raw materials under the catalysis of nicotinamide phosphoribosyl transferase, hypoxanthine phosphoribosyl transferase and xanthine oxidase.

Patent CN 108368493 a; CN 108949865A takes D-5-phosphoribose, ATP and nicotinamide as raw materials, and realizes the synthesis of NMN through phosphoribosyl pyrophosphate synthetase and nicotinamide phosphoribosyl transferase.

In patent CN 106755209 a, NR and ATP are used as substrates, and NMN is generated under the catalysis of nicotinamide ribokinase.

The enzymatic synthesis method of the NMN needs various expensive bioactive molecules such as ATP and the like as substrates, also needs various unconventional enzymes, is difficult to produce and expensive, has no commercialized enzyme preparation so far, needs special preparation of NMN production enterprises, and leads the enzymatic production process of the NMN to be very complex and expensive. In addition, there is also a method for producing NMN by a single enzyme catalysis, for example, NMN is produced by nicotinamide and 5 '-phosphoribosyl-1' -pyrophosphate (PRPP) as substrates and under the catalysis of nicotinamide phosphoribosyltransferase, but PRPP is not readily available, expensive, and not suitable for large-scale industrial production.

Disclosure of Invention

The method aims at solving the problems of the prior NMN production technology mentioned in the background technology. The invention aims to provide a brand-new NMN enzymatic synthesis technology, which completely breaks away from a natural biological catalytic synthesis system of NMN, does not relate to the use of the enzyme varieties at all, does not use expensive substrates such as ATP or PRPP, and has the advantages of simple reaction steps, low production cost, environmental protection, no pollution, suitability for large-scale industrial production and the like.

Specifically, the technical solution of the present invention is as follows.

In order to achieve the purpose, through working accumulation for many years and a large number of experimental researches, the inventor finally realizes an NMN enzyme method synthesis route (see a figure 1 and a figure 2) which takes cheap phospholipid as a phosphorus-based donor and takes phospholipase D and phospholipase C as catalysts, and the NMN enzyme method synthesis route is a non-natural enzyme method catalytic synthesis route for synthesizing NMN.

It is an object of the present invention to provide a two-step enzyme-catalyzed reaction (i.e., an in-order reaction) to produce NMN:

(a) the method comprises the following steps Two-step enzymolysis method

a 1: taking nicotinamide ribose and phospholipid as substrates, and generating the phosphatidyl nicotinamide ribose under the catalysis of phospholipase D in the presence of calcium ions;

a 2: taking the phosphatidyl nicotinamide ribose generated in the step a1 as a substrate, and generating beta-nicotinamide mononucleotide under the catalysis of phospholipase C in the presence of calcium ions;

furthermore, taking Nicotinamide Riboside (NR) as a substrate, taking phospholipids as a phosphatidyl donor, catalyzing phospholipid to react with NR through the transphosphatidylation function of phospholipase D to generate Phosphatidyl Nicotinamide Riboside (PNR), wherein the PNR is a novel phospholipid molecule and is easy to separate from water-soluble NR; then, under the catalysis of phospholipase C, PNR is hydrolyzed to generate diacylglycerol and beta-nicotinamide mononucleotide NMN, wherein the diacylglycerol is natural oil, is insoluble in water and is easy to separate from the water-soluble NMN.

The invention also aims to provide two ways for producing NMN by a one-pot method. The method specifically comprises the following steps:

the first approach is: taking Nicotinamide Ribose (NR) and phospholipid as substrates, firstly adding phospholipase D for reaction in the presence of calcium ions, and then adding phospholipase C for reaction after the reaction is finished to obtain NMN.

The second approach is: taking Nicotinamide Ribose (NR) and phospholipid as substrates, and adding phospholipase D and phospholipase C together to react in the presence of calcium ions to obtain the NMN.

The phospholipid is natural phospholipid or artificially synthesized phospholipid; more preferably, the main component of the phospholipid is phosphatidylcholine and/or phosphatidylethanolamine; more preferably, the phospholipid is lecithin.

The phospholipids used are preferably natural phospholipids and can be of vegetable origin, for example from oil plants such as soybean, sunflower, rapeseed, safflower seeds; or of animal origin, e.g., from cow's milk, goat's milk, deep sea fish, deep sea shrimp, deep sea scallop, etc.; and may be of microbial origin.

Phospholipids refer to a large group of compounds with similar phosphatidyl skeletons, whose structures are shown in fig. 1, and the R1 and R2 moieties in the phospholipid molecule are fatty acids, which can be any fatty acid: can be short chain, medium chain, long chain fatty acids; can be saturated fatty acid, monounsaturated fatty acid or polyunsaturated fatty acid; r1 and R2 may be the same or different; otherwise, it will not be described herein. The X part in phospholipid molecule is polar head, is molecule with hydroxyl, and has diversity, and common choline, ethanolamine, inositol, glycerol, short chain alcohol, etc.

The phospholipase C comprises broad-spectrum phospholipase C and phosphatidylinositol phospholipase C; more preferably, the phospholipase C is a phosphatidylinositol-type phospholipase C.

The phospholipase D is derived from animals, plants and microorganisms, and more preferably from microorganisms, such as Streptomyces.

According to the international system nomenclature of enzymes, the enzymes used in the above method are respectively: phospholipase D (PLD for short) which catalyzes a transphosphatidylation reaction, for example, a reaction of a phospholipid such as phosphatidylcholine or phosphatidylethanolamine with a substance having a hydroxyl group to produce a corresponding phospholipid acylation product, and the bonding position of the phospholipid is shown in FIG. 1, and the enzyme may act on the P-O bond or O-X bond as shown in the figure, including but not limited to EC 3.1.4.4; phospholipase C (abbreviated as PLC), which is capable of hydrolyzing the glycerophosphate bond of phospholipid compounds to generate diacylglycerol and corresponding phosphate compounds, and catalyzes the bond of phospholipid as shown in FIG. 1, is widely used, and includes but is not limited to EC 3.1.4.3, EC3.1.4.10, EC 3.1.4.11, EC 4.6.1.13, etc.

The phospholipase D and phospholipase C used in the above method can be derived from animals, plants or microorganisms, and it is usually preferable that the enzymes are derived from microorganisms or are expressed by microorganisms; the specific forms of the phospholipase D and the phospholipase C comprise enzyme liquid, enzyme freeze-dried powder, enzyme-containing cells and various immobilized enzymes and immobilized enzyme-containing cells, and the enzyme can be in an unpurified crude enzyme form, can also be in a partially purified or completely purified form, can be a commercial enzyme, and can also be specially produced. The phospholipase C enzymes are of various types, and among them, broad-spectrum phospholipase C enzymes and phosphatidylinositol phospholipase C enzymes are preferred.

The Nicotinamide Riboside (NR) as the substrate in the a1 and the phospholipid exist at the same time and can be mixed in any proportion, and the more preferable molar ratio is 1:10-10: 1; more preferably, the molar ratio of the nicotinamide riboside to the phospholipid is 1:5-5: 1; more preferably, the molar ratio of nicotinamide riboside to phospholipid is 1:2-3: 1.

Calcium salt is required to be added in the reaction of the a1, soluble calcium salt is generally adopted, such as calcium chloride and the like, and the concentration of the calcium ions of the a1 in water is 0.01-20 g/L; more preferably, the concentration of calcium ions in water is 0.5-5 g/L;

the catalytic reaction temperature of the phospholipase D in the reaction process of the a1 is 20-70 ℃, and the reaction pH is 4.5-7.5; more preferably, the temperature of the catalytic reaction of the phospholipase D is 40-60 ℃ and the reaction pH is 5.0-6.5.

In the phospholipase D catalytic reaction in the reaction process of the a1, auxiliaries can be added, including but not limited to any one or a combination of more than two of n-hexane, n-heptane and isopropanol.

Further preferred, wherein the content of the assistant is 0 to 50% when n-hexane or n-heptane and 0 to 30% when isopropanol is used as the assistant, calculated by weight ratio.

The phospholipase D catalyzes the reaction, and in order to be more suitable for the requirement of high-quality food production, the using amount of reaction auxiliary agents of n-hexane, n-heptane and/or isopropanol can be 0.

The concentration of the a2 calcium ions in water is 0.01-20 g/L; more preferably, the concentration of calcium ions in water is 0.1-2 g/L;

the catalytic reaction temperature of the phospholipase C in the reaction process of the a2 is 20-70 ℃, and the reaction pH is 4.0-7.0; more preferably, the temperature of the catalytic reaction of the phospholipase C enzyme is 40-55 ℃ and the reaction pH is 5.0-7.0.

Alkane and/or short chain alcohol can be added as auxiliary agent in the phospholipase C catalytic reaction in the a2 reaction process, wherein the alkane can be selected from one or two of n-hexane and n-heptane, the short chain alcohol can be selected from one or two of methanol, ethanol, propanol isopropanol, butanol and pentanol, the alkane content can be 0-80% and the short chain alcohol content can be 0-30% by weight ratio.

The phospholipase C catalyzes the reaction, and in order to meet the requirement of high-quality food production, the usage amount of the organic solvent reaction auxiliary agent can be 0.

For the two routes of NMN preparation by the one-pot method, the materials and process used in the preparation are basically the same as those of the two-step enzymolysis method, and the two routes are not redundant.

The phosphatidyl nicotinamide ribose (PNR for short) has the following structural formula:

wherein R is₁And R₂Is fatty acyl, natural phospholipid R₁And R₂Most of them are long-chain fatty acids, generally fatty acids having 14 to 24 carbon atoms, more preferably fatty acids having 16 and 18 carbon atoms, and further R₁The following are common: -C₁₅H₃₁、－C₁₇H₃₅、－C₁₇H₃₃Common for R2 are: -C₁₇H₃₁、－C₁₉H₂₉、－C₁₉H_31、－C₂₁H₃₁、－C₁₇H₃₃。

Compared with the prior art, the invention has the advantages that:

(1) the invention produces NMN by a brand-new enzyme catalytic reaction way, takes Nicotinamide Ribose (NR) which is a conventional NMN precursor as a raw material, takes phosphatidyl components in phospholipid as phosphatidyl donors, and performs phosphate group conversion on NR by two-step enzyme catalysis to produce NMN.

(2) The invention uses the phospholipid metabolic enzymes phospholipase D and phospholipase C which are widely existed in the biological world as catalysts, develops and utilizes the capability of catalyzing unnatural substrates, and produces NMN through a new intermediate product, namely phosphatidyl nicotinamide ribose PNR.

(3) The adopted phosphate group donor phospholipid is a cheap common industrial raw material, particularly is most known as lecithin, and the used catalytic enzymes are phospholipase D and phospholipase C which are industrial enzyme preparations, are mostly used for refining vegetable oil such as soybean oil, and are easy for heterologous expression, high in enzyme activity and low in price. Is easy for large-scale industrial production.

Drawings

FIG. 1 shows the action sites of phospholipase D and phospholipase C of the present invention.

FIG. 2 is a schematic diagram showing sequential catalytic reactions of phospholipase D and phospholipase C of the present invention.

FIG. 3 is a liquid phase analysis chart of the phospholipase D of the present invention catalyzing the reaction of nicotinamide riboside with phospholipid to form phosphatidylnicotinamide riboside (before and after the reaction).

FIG. 4 is a liquid phase analysis spectrum of NMN generated by hydrolyzing phospholipase D with phospholipase C (NR, NMN standard spectra, NMN reaction solution and point standard).

The substance mentioned above is detected.

1. Samples of NR and NMN were analyzed during the reaction.

In the reaction process, the substrate NR and the product NMN are water-soluble compounds, have strong polarity, and need to be analyzed by using a reverse phase column.

The high performance liquid phase analysis method of NR and product NMN is as follows:

the column was Agilent SB-C18(5 μm, 4.6X 250mm) with a detection wavelength of 254 nm.

Mobile phase: gradient elution was performed from mobile phase A and mobile phase B according to the procedure shown in Table 1, with an initial flow rate of 0.8mL/min and a column temperature of 30 ℃. Mobile phase A: water (1.36g potassium dihydrogen phosphate in 1L water, pH 2.5 adjusted with phosphoric acid); mobile phase B: methanol.

2. Samples of phospholipids and PNR were analyzed during the reaction.

FIG. 4 is an analysis of the substrates phospholipid and PNR during the reaction sampling. In the above reaction process, the substrate phospholipid and the product PNR are water-insoluble compounds, and have weak polarity, and need to be analyzed by using a normal phase column. The high performance liquid analysis methods of the two are as follows: the chromatographic column is silica gel column Si60(5 μm, 4.5X 250mm), the detection wavelength is 205nm, the analysis is carried out at room temperature, the mobile phase is acetonitrile-methanol-85% phosphoric acid water solution (100:10:1.8, V/V/V), the isocratic constant-speed analysis is carried out, and the flow rate is 1.0 mL/min.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION