阅读说明：本技术 一种基于酶法合成烟酰胺单核苷酸的方法 (Method for synthesizing nicotinamide mononucleotide based on enzyme method ) 是由 赵强 赵士敏 周晶辉 曾红宇 许岗 于 2021-01-28 设计创作，主要内容包括：本发明提供了一种基于酶法合成烟酰胺单核苷酸(NMN)的方法,其以D-核糖、烟酰胺、ATP为底物,在核糖激酶、磷酸核糖变位酶及烟酰胺核糖激酶的偶联催化作用下,一锅法合成得到烟酰胺单核苷酸。本发明制备NMN的方法开辟了一条酶法合成NMN的新途径。本发明中的三种酶可以循环利用、成本低、节能环保,适用于规模化工业生产。(The invention provides a method for synthesizing Nicotinamide Mononucleotide (NMN) based on an enzymatic method, which takes D-ribose, nicotinamide and ATP as substrates, and nicotinamide mononucleotide is synthesized by a one-pot method under the coupling catalysis action of ribokinase, phosphoribosyl mutase and nicotinamide ribokinase. The method for preparing NMN opens up a new way for synthesizing NMN by an enzyme method. The three enzymes in the invention can be recycled, have low cost, are energy-saving and environment-friendly, and are suitable for large-scale industrial production.)

1. A method for synthesizing nicotinamide mononucleotide based on an enzymatic method is characterized in that D-ribose, nicotinamide and ATP are used as substrates, and nicotinamide mononucleotide is synthesized by a one-pot method under the coupling catalysis action of ribokinase, phosphoribosyltransferase and nicotinamide ribokinase.

2. The method according to claim 1, wherein the concentration of D-ribose in the reaction system is 20 to 120mM, preferably 50 to 100mM, and more preferably 50 mM; nicotinamide concentration is 20-120mM, preferably 50-100mM, further preferably 50 mM; the ATP concentration is 20-150mM, preferably 60-120mM, and more preferably 60 mM.

3. The method according to claim 1, wherein the reaction temperature of the reaction system is 20 to 37 ℃, preferably 25 to 30 ℃, and more preferably 25 ℃; the pH of the reaction system is 4.5 to 7.0, preferably 5.0 to 6.0, further preferably 5.0; the reaction time is 3 to 8 hours, preferably 4 to 6 hours, and more preferably 4 hours.

4. The method according to claim 1 or 3, characterized in that the buffer of the reaction system is a phosphate buffer or a Tris-HCl buffer, preferably a phosphate buffer, at a concentration of 20-200mM, preferably 50-100mM, further preferably 50 mM.

5. The method according to claim 1, wherein Mg is added to the reaction system²⁺And Mn²⁺Ions, Mg in reaction²⁺A concentration of 10 to 50mM, preferably 20 to 40mM, further preferably 20 mM; mn in the reaction²⁺The concentration is 1 to 20mM, preferably 5 to 10mM, and more preferably 5 mM.

6. The method of claim 1, wherein the ribokinase is derived from at least one of Saccharomyces cerevisiae, Lactobacillus plantarum, and Escherichia coli, preferably Saccharomyces cerevisiae; the phosphoribosyl mutase is derived from at least one of Saccharomyces cerevisiae, Thermotoga maritima and Escherichia coli, preferably Thermotoga maritima; the nicotinamide ribokinase is derived from at least one of human and Saccharomyces cerevisiae, preferably human.

7. The enzyme according to claim 6, wherein the Saccharomyces cerevisiae-derived ribokinase has the amino acid sequence of SEQ ID No. 1; the amino acid sequence of phosphoribosyl mutase from Thermotoga maritima is SEQ ID NO. 2; the amino acid sequence of the human-derived nicotinamide ribokinase is SEQ ID NO. 3.

8. The method according to claim 6 or 7, wherein the expression hosts for ribokinase, phosphoribosyltransferase and nicotinamide ribokinase are microorganisms.

9. The method according to claim 8, wherein the microorganism comprises at least one of E.coli, Bacillus subtilis and Saccharomyces cerevisiae, preferably E.coli, further preferably E.coli BL21(DE 3); recombinant expression vectors for enzymes in microorganisms include: at least one of various plasmids, cosmids, phages and viral vectors, preferably the prokaryotic expression vector pET30a (+).

10. The method according to claim 6 or 7, wherein the ribokinase, phosphoribosyltransferase, and nicotinamide ribokinase comprise at least one of crude, purified, and immobilized enzymes, preferably immobilized enzymes.

Technical Field

The invention belongs to the technical field of biological pharmacy and biological catalysis, and relates to a novel method for synthesizing nicotinamide mononucleotide based on an enzymatic method.

Background

Nicotinamide mononucleotide (beta-Nicotinamide mononucleotide, referred to as beta-NMN or NMN for short) is a product of Nicotinamide phosphoribosyltransferase reaction, is a key precursor of NAD +, which becomes NAD + after adenylation by Nicotinamide nucleotide adenylyltransferase. Human extracellular NMN requires dephosphorylation to Nicotinamide Riboside (NR) for entry into the interior of hepatocytes, and then NR produces NMN under the action of nicotinamide riboside kinase 1. 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 researches show that the level of NAD + in vivo can be obviously improved by supplementing NMN, the composition has better treatment and repair effects on cardiovascular and cerebrovascular diseases, neurodegenerative diseases, aging degenerative diseases and the like, can also regulate endocrine, plays a role in protecting and repairing pancreatic islet functions, increases the secretion of insulin, and has a prevention effect on metabolic diseases such as diabetes, obesity and the like.

Current methods for NMN synthesis include chemical and enzymatic methods. NMN is produced by chemical methods, which generally uses Nicotinamide Riboside (NR) as a raw material and is obtained by phosphorylation with phosphorus oxychloride and the like (chem. commun.1999,729-730, CN 107613990 a), but the obtained product has many impurities, low yield and high cost, and a large amount of toxic and harmful reagents are used, so that serious environmental pollution is caused, and the production application of the NMN in food grade is limited. The enzymatic production of NMN is now the mainstream mode of NMN production. For example, CN108026130A and CN110195089A disclose that nicotinamide mononucleotide is prepared by reacting nicotinamide, ATP and ribose as raw materials under the catalytic action of nicotinamide phosphoribosyltransferase, ribose-phosphate pyrophosphorykinase and ribose kinase; CN108949865A discloses that nicotinamide mononucleotide is synthesized by whole-cell catalysis of immobilized phosphoribosyl pyrophosphate synthetase and nicotinamide phosphoribosyl transferase by taking D-ribose-5-phosphate, ATP and nicotinamide as raw materials. CN106755209A discloses that nicotinamide riboside and ATP are used as substrates, and nicotinamide mononucleotide is generated under the catalysis of nicotinamide riboside kinase; CN108026535A discloses that nicotinamide mononucleotide is prepared by reacting nicotinamide, ATP and AMP as raw materials under the catalytic action of nicotinamide phosphoribosyltransferase, ribose phosphate pyrophosphorykinase and nucleosidase. PCT/CN2016/092457 discloses a method for preparing nicotinamide mononucleotide, which takes nicotinamide, ATP and xylose as raw materials, and takes reaction under the catalytic action of nicotinamide phosphoribosyl transferase, ribose phosphate pyrophosphorykinase, ribose-5-phosphate isomerase, ribulose-3-phosphate isomerase, xylulokinase and xylose isomerase to synthesize the nicotinamide mononucleotide.

Among the raw materials used in the above patent documents, the direct preparation method of nicotinamide riboside by the enzyme-less method requires chemical synthesis, which is expensive and not easy to purchase, and the D-ribose-5-phosphate is unstable in property and expensive; in the patent using cheap D-ribose as the raw material, NMN needs to be synthesized by multi-step reactions such as ribokinase, nicotinamide phosphoribosyltransferase and ribose phosphate pyrophosphorykinase, and the like, so that the conversion rate is low (about 50 percent), the purification is difficult and the cost is high; the invention discloses the double-layer function of phosphoribosyl kinase for the first time, namely, the phosphoribosyl kinase has phosphate-solubilizing effect on ribose-1-phosphate and phosphorylation effect on nicotinamide ribose, under the above effects of the phosphoribosyl kinase, ribose-1-phosphate, nicotinamide and ATP can be synthesized into NMN in one step, and further, the invention has a brand new NMN synthesis route, namely, nicotinamide mononucleotide is synthesized by a one-pot method under the coupling catalysis effect of ribokinase, phosphoribosyltransferase and nicotinamide ribokinase by taking D-ribose, nicotinamide and ATP as substrates, the method has high conversion rate (more than 90 percent), high yield (more than 80 percent) and low cost, and is suitable for large-scale industrial production.

Disclosure of Invention

In view of the reports related to the direct preparation of nicotinamide ribose by the temporary enzyme-free method, the invention aims to provide a novel method for synthesizing NMN by an enzyme method, which is based on cheap raw material D-ribose, firstly synthesizes nicotinamide ribose from D-ribose by the enzyme method, and then synthesizes nicotinamide mononucleotide, and particularly develops a novel method for synthesizing NMN by the enzyme method, wherein the novel method specifically utilizes the double-layer functions of nicotinamide ribose kinase for dephosphorylation of ribose-1-phosphate and phosphorylation of nicotinamide ribose to prepare NMN. The specific reaction process is as follows: synthesizing ribose-5-phosphate from the D-ribose under the action of ribose kinase; ribose-5-phosphate becomes ribose-1-phosphate under the action of phosphoribosyl mutase; the ribose-1-phosphate is synthesized into nicotinamide ribose under the phosphate-solubilizing action of nicotinamide ribose kinase; nicotinamide riboside is phosphorylated by nicotinamide ribokinase to synthesize nicotinamide mononucleotide. The present invention provides the following technical solutions.

A method for synthesizing nicotinamide mononucleotide based on an enzymatic method comprises the following steps:

taking D-ribose, nicotinamide and ATP as substrates, under the coupling catalysis of ribokinase, phosphoribosyl mutase and nicotinamide ribokinase, synthesizing nicotinamide mononucleotide by a one-pot method, wherein the process route is shown in figure 1.

In the above reaction system, the concentration of D-ribose is 20 to 120mM, preferably 50 to 100mM, and more preferably 50 mM; nicotinamide concentration is 20-120mM, preferably 50-100mM, further preferably 50 mM; the ATP concentration is 20-150mM, preferably 60-120mM, and more preferably 60 mM.

The reaction temperature of the above reaction system is 20 to 37 ℃, preferably 25 to 30 ℃, and more preferably 25 ℃; the pH of the reaction system is 4.5 to 7.0, preferably 5.0 to 6.0, further preferably 5.0; the reaction time is 3 to 8 hours, preferably 4 to 6 hours, and more preferably 4 hours.

In the above method, the buffer solution of the reaction system is a phosphate buffer solution or a Tris-HCl buffer solution, preferably a phosphate buffer solution, and the concentration thereof is 20 to 200mM, preferably 50 to 100mM, and more preferably 50 mM.

The above method, adding Mg to the reaction system²⁺And Mn²⁺Ions, Mg in reaction²⁺A concentration of 10 to 50mM, preferably 20 to 40mM, further preferably 20 mM; mn in the reaction²⁺The concentration is 1 to 20mM, preferably 5 to 10mM, and more preferably 5 mM.

In the method, the ribokinase is derived from at least one of saccharomyces cerevisiae, lactobacillus plantarum and escherichia coli, preferably saccharomyces cerevisiae; the phosphoribosyl mutase is derived from at least one of Saccharomyces cerevisiae, Thermotoga maritima and Escherichia coli, preferably Thermotoga maritima; the nicotinamide ribokinase is derived from at least one of human and Saccharomyces cerevisiae, preferably human.

In the method, the amino acid sequence of the saccharomyces cerevisiae-derived ribokinase is SEQ ID NO. 1; the amino acid sequence of phosphoribosyl mutase from Thermotoga maritima is SEQ ID NO. 2; the amino acid sequence of the human-derived nicotinamide ribokinase is SEQ ID NO. 3.

In the above method, the expression hosts of ribokinase, phosphoribosyltransferase and nicotinamide ribokinase are microorganisms.

The above method, wherein the microorganism comprises at least one of Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae, preferably Escherichia coli, and further preferably Escherichia coli BL21(DE 3); recombinant expression vectors for enzymes in microorganisms are various vectors conventional in the art, including: at least one of various plasmids, cosmids, phages and viral vectors, preferably the prokaryotic expression vector pET30a (+).

In the above method, the ribokinase, phosphoribosyltransferase, and nicotinamide ribokinase include at least one of crude enzyme, purified enzyme, and immobilized enzyme, preferably immobilized enzyme.

The present invention adopts the above method, preferably clones the above gene encoding the enzyme into prokaryotic expression vector pET30a (+), constructs recombinant plasmid, introduces into Escherichia coli BL21(DE3) through electrotransformation, and induces expression according to conventional method.

In the invention, after the enzyme is preferably produced in Escherichia coli BL21(DE3) cells, crude enzyme is obtained by ultrasonic disruption, purified enzyme is obtained by further adopting immobilized metal chelating affinity chromatography (IMAC), and both crude enzyme and purified enzyme are prepared into protein powder by adopting vacuum freeze drying; further adopting an epoxy carrier ECEP for immobilization treatment to obtain immobilized enzyme.

When crude enzyme freeze-dried powder is selected in the reaction system, the dosage of the three enzymes is 50mM D-ribose, namely 500mg of crude enzyme freeze-dried powder of ribokinase, 200mg of crude enzyme freeze-dried powder of nicotinamide ribokinase, 500mg of crude enzyme freeze-dried powder of phosphoribosyl mutase, 400mg of crude enzyme freeze-dried powder of phosphoribosyl mutase, 800mg of nicotinamide ribokinase;

preferably: 50mM D-ribose, 300mg of ribokinase crude enzyme freeze-dried powder, 300mg of nicotinamide ribokinase crude enzyme freeze-dried powder and 600mg of phosphoribosyl mutase crude enzyme freeze-dried powder.

When purified enzyme freeze-dried powder is selected in the reaction system, the dosage of the three enzymes is 50mM D-ribose, 50-300mg of ribokinase purified enzyme freeze-dried powder, 50-300mg of nicotinamide ribokinase purified enzyme freeze-dried powder and 100-400mg of phosphoribosyl mutase purified enzyme freeze-dried powder;

preferably: 50mM D-ribose, 100mg of lyophilized powder of purified enzyme of ribokinase, 100mg of lyophilized powder of purified enzyme of nicotinamide ribokinase and 200mg of lyophilized powder of purified enzyme of phosphoribosyl mutase.

When the immobilized enzyme is selected in the reaction system, the dosage of the three enzymes is 50mM D-ribose, 8-32g of ribokinase immobilized enzyme, 8-32g of nicotinamide ribokinase immobilized enzyme and 12-48g of phosphoribosyl mutase immobilized enzyme;

preferably 50mM D-ribose, 16g of ribokinase immobilized enzyme, 16g of nicotinamide ribokinase immobilized enzyme and 24g of phosphoribosyl mutase immobilized enzyme.

The freeze-dried powder and the immobilized enzyme of the three enzymes are expressed in host cells, crude enzyme is obtained by ultrasonic crushing, purified enzyme is further obtained by immobilized metal chelating affinity chromatography (IMAC), and both crude enzyme and purified enzyme are prepared into protein powder by vacuum freeze drying; further adopting an epoxy carrier ECEP for immobilization treatment to obtain immobilized enzyme.

The dosages of the immobilized enzyme and the crude enzyme refer to the dosages of the purified enzyme.

The construction of the engineered strain for gene expression of the present invention further preferably comprises the following steps:

the ribokinase, phosphoribosyl mutase and nicotinamide ribokinase amino acid sequence (SEQ ID NO.1-3) related by the invention is sent to a gene synthesis company for carrying out codon optimization of escherichia coli and artificially synthesizing a coding gene, the coding gene is respectively cloned between NdeI and XhoI enzyme cutting sites of a prokaryotic expression vector pET30a (+), and 6 histidine coding sequences are added at the C end of the gene, so that the subsequent protein purification is facilitated. The recombinant expression vector containing the 3 enzyme genes is introduced into escherichia coli BL21(DE3) through electrotransformation to obtain a corresponding enzyme gene expression engineering strain. The engineering strain is fermented by adopting a conventional fermentation culture medium TB, and OD is cultured under the conditions of 37 ℃ and 200rpm₆₀₀Reaching 0.6-0.8, inducing expression with final concentration of 0.5mM IPTG or 0.1g/L lactose at 25 deg.C for 10 hr, collecting thallus, breaking cell, and detecting the expression of target protein.

The preparation of the immobilized enzyme of the invention further preferably comprises the steps of:

collecting ribokinase, phosphoribosyl mutase and nicotinamide ribokinase fermentation thalli after induction for 10h by using a low-temperature high-speed centrifuge (10000rpm, 4 ℃ and 10min), repeatedly washing the thalli twice by using a phosphate buffer solution with the pH value of 8.0 and the concentration of 0.1mol/L, re-suspending the thalli with the concentration of 20 times in 50ml of the phosphate buffer solution, and ultrasonically crushing in an ice bath (the ultrasonic condition is that the ultrasonic operation is carried out for 2s, and the interval is 5s) until the thalli is clarified. Centrifuging the above crushed solution in a low-temperature high-speed centrifuge (10000rpm, 4 deg.C, 20min), collecting supernatant to obtain crude enzyme solution of ribokinase, phosphoribosyl mutase and nicotinamide ribokinase, and vacuum freeze drying to obtain crude enzyme protein powder. Further injecting the crude enzyme protein solution onto the activated IDA resin combined with Ni +, sequentially carrying out gradient elution and impurity removal by using 10 and 30mM low-concentration imidazole solutions with the volume of 1-2 times of the column volume, eluting the target protein by using 300mM high-concentration imidazole with the volume of 1 time of the column volume, removing imidazole from the protein eluent by adopting a conventional dialysis or ultrafiltration method, thus obtaining the purified enzyme of the enzyme, and further preparing the purified enzyme protein powder by adopting vacuum freeze drying. Taking a proper amount of the purified enzyme protein powder, adding the purified enzyme protein powder into a phosphate buffer solution with the pH value of 8.0 and the mol/L of 0.1, then adding 50g of the activated epoxy-based carrier ECEP, stirring and immobilizing for 48 hours at a low speed under the conditions of 30 ℃ and 120rpm, finally washing the obtained immobilized enzyme with deionized water for 2-3 times, and carrying out vacuum filtration to obtain the immobilized enzyme of the enzyme.

The invention utilizes the reaction conditions to carry out catalytic reaction, and has the following gain effects: the invention utilizes the double-layer function of nicotinamide ribokinase to catalyze and synthesize NMN, thus indirectly solving the problem of enzymatic preparation of nicotinamide riboside; compared with other conventional NMN synthesis methods, the immobilized multi-enzyme system one-pot catalytic reaction has the advantages of mild reaction conditions, simplicity in operation, high conversion rate and the like, the reaction conversion rate is greater than 90%, the total yield is greater than 80%, the purity of NMN crystal powder is greater than 99.5%, the product purity is high, and the quality is reliable.

The method creatively utilizes the double-layer function of the nicotinamide ribokinase, takes D-ribose as a raw material, synthesizes nicotinamide ribose firstly, then synthesizes NMN, does not need to directly use expensive nicotinamide ribose as the raw material, and creates a new NMN enzyme method synthesis way. The three enzymes in the invention can be recycled, and the invention has low cost, energy saving and environmental protection. The method for preparing the NMN adopts a one-pot reaction, so the method has the advantages of simple operation, high reaction yield, less impurities and obvious cost reduction, and is suitable for large-scale industrial production.

Drawings

FIG. 1 is a process route diagram of nicotinamide mononucleotide NMN synthesized by the one-pot method of the invention;

FIG. 2 is an electrophoretogram of the protein expressed by ribokinase BL21(DE3) of the present invention;

FIG. 3 is an electrophoretogram of phosphoribosyltransferase BL21(DE3) expression protein of the present invention;

FIG. 4 is an electrophoretogram of the expression protein of nicotinamide ribokinase BL21(DE3) of the invention;

FIG. 5 is a diagram showing the analysis of the result of NMN production by immobilized enzyme in one pot at a substrate concentration of 25 mM;

FIG. 6 is a diagram showing the analysis of the result of NMN production by immobilized enzyme in one pot at a substrate concentration of 50mM according to the present invention;

FIG. 7 is a graph showing the analysis of the crystal powder results of NMN production by immobilized enzyme in one pot at a substrate concentration of 50 mM;

FIG. 8 is a graph showing the analysis of the results of NMN production by the immobilized enzyme one-pot method at a substrate concentration of 100 mM.

Detailed Description

In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below with reference to embodiments, and the described embodiments are only some embodiments, not all embodiments, of the present invention. Therefore, all other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention and without any inventive work are within the scope of the present invention.

The ribokinase, phosphoribosyltransferase and nicotinamide ribokinase related to the following embodiments of the invention are constructed and expressed by a prokaryotic expression vector pET30a (+) and produced in Escherichia coli BL21(DE3) cells.

After the enzyme is produced in Escherichia coli BL21(DE3) cells, crude enzyme is obtained by ultrasonic disruption, purified enzyme is obtained by further adopting immobilized metal chelate affinity chromatography (IMAC), and both crude enzyme and purified enzyme are prepared into protein powder by adopting vacuum freeze drying; further adopting an epoxy carrier ECEP for immobilization treatment to obtain immobilized enzyme.

EXAMPLE 1 construction of engineered Strain for Gene expression

The amino acid sequences of the ribokinase, the phosphoribosyl mutase and the nicotinamide ribokinase related to the invention are sent to a gene synthesis company for carrying out codon optimization of escherichia coli and artificially synthesizing coding genes, and the coding genes are respectively cloned between NdeI and XhoI enzyme cutting sites of a prokaryotic expression vector pET30a (+), and 6 histidine coding sequences are added at the C end of the genes, so that the subsequent protein purification is convenient. The recombinant expression vector containing the 3 enzyme genes is introduced into escherichia coli BL21(DE3) through electrotransformation to obtain a corresponding enzyme gene expression engineering strain. The engineering strain is fermented by adopting a conventional fermentation culture medium TB, and OD is cultured under the conditions of 37 ℃ and 200rpm₆₀₀Reaching 0.6-0.8, inducing expression with final concentration of 0.5mM IPTG or 0.1g/L lactose at 25 deg.C for 10 hr, collecting thallus, breaking cell, and detecting the expression of target protein (see FIG. 2, FIG. 3, and FIG. 4).

Example 2 preparation of immobilized enzyme

Collecting ribokinase, phosphoribosyl mutase and nicotinamide ribokinase fermentation thalli after induction for 10h by using a low-temperature high-speed centrifuge (10000rpm, 4 ℃ and 10min), repeatedly washing the thalli twice by using a phosphate buffer solution with the pH value of 8.0 and the concentration of 0.1mol/L, re-suspending the thalli with the concentration of 20 times in 50ml of the phosphate buffer solution, and ultrasonically crushing in an ice bath (the ultrasonic condition is that the ultrasonic operation is carried out for 2s, and the interval is 5s) until the thalli is clarified. Centrifuging the above crushed solution in a low-temperature high-speed centrifuge (10000rpm, 4 deg.C, 20min), collecting supernatant to obtain crude enzyme solution of ribokinase, phosphoribosyl mutase and nicotinamide ribokinase, and vacuum freeze drying to obtain crude enzyme protein powder. Further injecting the crude enzyme protein solution onto the activated IDA resin combined with Ni +, sequentially carrying out gradient elution and impurity removal by using 10 and 30mM low-concentration imidazole solutions with the volume of 1-2 times of the column volume, eluting the target protein by using 300mM high-concentration imidazole with the volume of 1 time of the column volume, removing imidazole from the protein eluent by adopting a conventional dialysis or ultrafiltration method, thus obtaining the purified enzyme of the enzyme, and further preparing the purified enzyme protein powder by adopting vacuum freeze drying. Taking a proper amount of the purified enzyme protein powder, adding the purified enzyme protein powder into a phosphate buffer solution with the pH value of 8.0 and the mol/L of 0.1, then adding 50g of the activated epoxy-based carrier ECEP, stirring and immobilizing for 48 hours at a low speed under the conditions of 30 ℃ and 120rpm, finally washing the obtained immobilized enzyme with deionized water for 2-3 times, and carrying out vacuum filtration to obtain the immobilized enzyme of the enzyme.

Example 3 production of NMN Using D-ribose as substrate and crude enzyme one-pot method

D-ribose at a final concentration of 50mM, nicotinamide at a final concentration of 50mM, 60mM ATP, 5mM manganese chloride, and 20mM magnesium chloride were added to the reaction system in this order, and dissolved in 1L of a phosphate buffer solution at pH 6.0 and 50mM with sufficient shaking, followed by adjustment of pH 5.00. Adding 300mg of crude ribokinase enzyme freeze-dried powder, 300mg of crude ribokinase enzyme freeze-dried powder and 600mg of crude ribomutase phosphate freeze-dried powder prepared in the embodiment 2, uniformly mixing, controlling the reaction temperature to be 25 ℃, stirring at 300rpm for reaction, adopting an automatic titrator, controlling the pH to be 5.0 by using 1mol/l of sodium hydroxide in the whole process, detecting the generation concentration of NMN by using liquid chromatography HPLC in the reaction process, finishing the reaction within 4 hours to obtain 14.8g of NMN and 88.62% of reaction yield, carrying out microfiltration, high performance liquid chromatography separation and purification, nanofiltration and concentration, vacuum freeze drying and other post-treatment on the reaction liquid to obtain 12.87g of crystal powder, wherein the total yield is 77.06%, and the purity of the crystal powder is more than 99.5%.

Example 4 production of NMN Using D-ribose as substrate and purified enzyme one-pot method

D-ribose at a final concentration of 50mM, nicotinamide at a final concentration of 50mM, 60mM ATP, 5mM manganese chloride, and 20mM magnesium chloride were added to the reaction system in this order, and dissolved in 1L of a phosphate buffer solution at pH 6.0 and 50mM with sufficient shaking, followed by adjustment of pH 5.00. Adding 100mg of ribokinase purified enzyme freeze-dried powder, 100mg of nicotinamide ribokinase purified enzyme freeze-dried powder and 200mg of phosphoribosyl mutase purified enzyme freeze-dried powder prepared in the embodiment 2, uniformly mixing, controlling the reaction temperature to be 25 ℃, stirring at 300rpm for reaction, adopting an automatic titrator, controlling the pH to be 5.0 by using 1mol/l of sodium hydroxide in the whole process, detecting the generation concentration of NMN by using liquid chromatography HPLC (high performance liquid chromatography) in the reaction process, finishing the reaction within 4 hours to obtain 15.1g of NMN, wherein the reaction yield is 90.42%, carrying out post-treatment on reaction liquid by membrane equipment microfiltration, high performance liquid chromatography separation and purification, nanofiltration concentration, vacuum freeze drying and the like to obtain 13.07g of crystal powder, wherein the total yield is 78.26%, and the purity of the crystal powder is more than 99.

Example 5 production of NMN Using D-ribose as a substrate and immobilized enzyme in one pot

D-ribose at a final concentration of 25mM, nicotinamide at a final concentration of 25mM, ATP at a final concentration of 30mM, manganese chloride at a final concentration of 2.5mM, and magnesium chloride at a final concentration of 10mM were added to the reaction system, and the reaction system was dissolved in 1L of phosphate buffer solution at pH 6.0 and 50mM with sufficient shaking, and then adjusted to pH 5.00. Adding 8g of the ribokinase immobilized enzyme prepared in the example 2, 8g of the nicotinamide ribokinase immobilized enzyme and 12g of the phosphoribosyl mutase immobilized enzyme, uniformly mixing, controlling the reaction temperature to be 25 ℃, stirring at 300rpm for reaction, adopting an automatic titrator, controlling the pH to be 5.0 by using 1mol/l of sodium hydroxide in the whole process, detecting the generation concentration of NMN by using liquid chromatography in the reaction process, finishing the reaction within 4 hours to obtain 7.7g of NMN with the reaction yield of 92.21 percent (shown in figure 5), carrying out post-treatment on the reaction liquid by screen filtration, high performance liquid chromatography separation and purification, nanofiltration concentration, vacuum freeze drying and the like to obtain 6.7g of crystal powder, wherein the total yield is 80.23 percent, and the purity of the crystal powder is more than 99.5 percent.

Example 6 production of NMN Using D-ribose as a substrate and immobilized enzyme in one pot

D-ribose at a final concentration of 50mM, nicotinamide at a final concentration of 50mM, 60mM ATP, 5mM manganese chloride and 20mM magnesium chloride were added to the reaction system in this order, and dissolved in 1L of a phosphate buffer solution at pH 6.0 and 50mM with sufficient shaking, and then the pH was adjusted to 5.00 with 1mol/L sodium hydroxide. Adding 16g of the ribokinase immobilized enzyme, 16g of the nicotinamide ribokinase immobilized enzyme and 24g of the phosphoribosyl mutase immobilized enzyme prepared in the example 2, uniformly mixing, controlling the reaction temperature to be 25 ℃, stirring at 300rpm for reaction, adopting an automatic titrator, controlling the pH to be 5.0 by using 1mol/l of sodium hydroxide in the whole process, detecting the generation concentration of NMN by using liquid chromatography HPLC (high performance liquid chromatography) in the reaction process, finishing the reaction within 4 hours to obtain 15.9g of NMN with the reaction yield of 95.20 percent (shown in figure 6), carrying out post-treatment on the reaction liquid by screen filtration, high performance liquid chromatography separation and purification, nanofiltration concentration, vacuum freeze drying and the like to obtain 14.07g of crystal powder, wherein the total yield is 84.25 percent, and the purity of the crystal powder is more than 99.5.

Example 7 production of NMN Using D-ribose as a substrate and immobilized enzyme in one pot

To the reaction system were added D-ribose at a final concentration of 100mM, nicotinamide at a final concentration of 100mM, 120mM ATP, 10mM manganese chloride and 40mM magnesium chloride in this order, and the mixture was dissolved in 1L of a phosphate buffer solution of pH 6.0 and 50mM with sufficient shaking, and then adjusted to pH 5.00 with 1mol/L sodium hydroxide. Adding 32g of the ribokinase immobilized enzyme, 32g of the nicotinamide ribokinase immobilized enzyme and 48g of the phosphoribosyl mutase immobilized enzyme prepared in the example 2, uniformly mixing, controlling the reaction temperature to be 25 ℃, stirring at 300rpm, reacting, adopting an automatic titrator, controlling the pH to be 5.0 by using 1mol/l of sodium hydroxide in the whole process, detecting the generation concentration of NMN by using liquid chromatography HPLC (high performance liquid chromatography) in the reaction process, finishing the reaction within 4 hours to obtain 30.9g of NMN with the reaction yield of 92.51 percent (shown in figure 8), carrying out post-treatment on the reaction liquid by screen filtration, high performance liquid chromatography separation and purification, nanofiltration concentration, vacuum freeze drying and the like to obtain 28.06g of crystal powder, wherein the total yield is 84.01 percent, and the purity of the crystal powder is more than. Saccharomyces cerevisiae ribokinase amino acid sequence

SEQ ID NO.1

MGITVIGSLNYDLDTFTDRLPNAGETFRANHFETHAGGKGLNQAAAIGKLKNPSSRYSVRMIGNVGNDTFGKQLKDTLSDCGVDITHVGTYEGINTGTATILIEEKAGGQNRILIVEGANSKTIYDPKQLCEIFPEGKEEEEYVVFQHEIPDPLSIIKWIHANRPNFQIVYNPSPFKAMPKKDWELVDLLVVNEIEGLQIVESVFDNELVEEIREKIKDDFLGEYRKICELLYEKLMNRKKRGIVVMTLGSRGVLFCSHESPEVQFLPAIQNVSVVDTTGAGDTFLGGLVTQLYQGETLSTAIKFSTLASSLTIQRKGAAESMPLYKDVQKDA

Phosphoribosyl mutase amino acid sequence from Thermotoga maritima

SEQ ID NO.2

MRVVLIVLDSVGIGEMPDAHLYGDEGSNTIVNTAKAVSGLHLPNMAKLGLGNLDDIPGVEPVKPAEGIYGKMMEKSPGKDTTTGHWEIAGVILKKPFDLFPEGFPKELIEEFERRTGRKVIGNKPASGTEIIKELGPIHEKTGALIVYTSADSVFQIAAKKEIVPLEELYRYCEIARELLNEMGYKVARVIARPFTGEWPNYVRTPERKDFSLEPEGKTLLDVLTENGIPVYGVGKIADIFAGRGVTENYKTKDNNDGIDKTISLMKEKNHDCLIFTNLVDFDTKYGHRNDPVSYAKALEEFDARLPEIMHNLNEDDVLFITADHGCDPTTPSTDHSREMVPLLGYGGRLKKDVYVGIRETFADLGQTIADIFGVPPLENGTSFKNLIWE

Amino acid sequence of nicotinamide ribokinase of human origin

SEQ ID NO.3

MKLIVGIGGMTNGGKTTLTNSLLRALPNCCVIHQDDFFKPQDQIAVGEDGFKQWDVLESLDMEAMLDTVQAWLSSPQKFARAHGVSVQPEASDTHILLLEGFLLYSYKPLVDLYSRRYFLTVPYEECKWRRSTRNYTVPDPPGLFDGHVWPMYQKYRQEMEANGVEVVYLDGMKSREELFREVLEDIQNSLLNRSQESAPSPARPARTQGPGRGCGHRTARPAASQQDSM

Sequence listing

<110> Hunan Fulaige Biotechnology Ltd

<120> method for synthesizing nicotinamide mononucleotide based on enzyme method

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 333

<212> PRT

<213> Saccharomyces cerevisiae

<400> 1

Met Gly Ile Thr Val Ile Gly Ser Leu Asn Tyr Asp Leu Asp Thr Phe

1 5 10 15

Thr Asp Arg Leu Pro Asn Ala Gly Glu Thr Phe Arg Ala Asn His Phe

20 25 30

Glu Thr His Ala Gly Gly Lys Gly Leu Asn Gln Ala Ala Ala Ile Gly

35 40 45

Lys Leu Lys Asn Pro Ser Ser Arg Tyr Ser Val Arg Met Ile Gly Asn

50 55 60

Val Gly Asn Asp Thr Phe Gly Lys Gln Leu Lys Asp Thr Leu Ser Asp

65 70 75 80

Cys Gly Val Asp Ile Thr His Val Gly Thr Tyr Glu Gly Ile Asn Thr

85 90 95

Gly Thr Ala Thr Ile Leu Ile Glu Glu Lys Ala Gly Gly Gln Asn Arg

100 105 110

Ile Leu Ile Val Glu Gly Ala Asn Ser Lys Thr Ile Tyr Asp Pro Lys

115 120 125

Gln Leu Cys Glu Ile Phe Pro Glu Gly Lys Glu Glu Glu Glu Tyr Val

130 135 140

Val Phe Gln His Glu Ile Pro Asp Pro Leu Ser Ile Ile Lys Trp Ile

145 150 155 160

His Ala Asn Arg Pro Asn Phe Gln Ile Val Tyr Asn Pro Ser Pro Phe

165 170 175

Lys Ala Met Pro Lys Lys Asp Trp Glu Leu Val Asp Leu Leu Val Val

180 185 190

Asn Glu Ile Glu Gly Leu Gln Ile Val Glu Ser Val Phe Asp Asn Glu

195 200 205

Leu Val Glu Glu Ile Arg Glu Lys Ile Lys Asp Asp Phe Leu Gly Glu

210 215 220

Tyr Arg Lys Ile Cys Glu Leu Leu Tyr Glu Lys Leu Met Asn Arg Lys

225 230 235 240

Lys Arg Gly Ile Val Val Met Thr Leu Gly Ser Arg Gly Val Leu Phe

245 250 255

Cys Ser His Glu Ser Pro Glu Val Gln Phe Leu Pro Ala Ile Gln Asn

260 265 270

Val Ser Val Val Asp Thr Thr Gly Ala Gly Asp Thr Phe Leu Gly Gly

275 280 285

Leu Val Thr Gln Leu Tyr Gln Gly Glu Thr Leu Ser Thr Ala Ile Lys

290 295 300

Phe Ser Thr Leu Ala Ser Ser Leu Thr Ile Gln Arg Lys Gly Ala Ala

305 310 315 320

Glu Ser Met Pro Leu Tyr Lys Asp Val Gln Lys Asp Ala

325 330

<210> 2

<211> 390

<212> PRT

<213> Thermotoga maritima (Thermus aquaticus)

<400> 2

Met Arg Val Val Leu Ile Val Leu Asp Ser Val Gly Ile Gly Glu Met

1 5 10 15

Pro Asp Ala His Leu Tyr Gly Asp Glu Gly Ser Asn Thr Ile Val Asn

20 25 30

Thr Ala Lys Ala Val Ser Gly Leu His Leu Pro Asn Met Ala Lys Leu

35 40 45

Gly Leu Gly Asn Leu Asp Asp Ile Pro Gly Val Glu Pro Val Lys Pro

50 55 60

Ala Glu Gly Ile Tyr Gly Lys Met Met Glu Lys Ser Pro Gly Lys Asp

65 70 75 80

Thr Thr Thr Gly His Trp Glu Ile Ala Gly Val Ile Leu Lys Lys Pro

85 90 95

Phe Asp Leu Phe Pro Glu Gly Phe Pro Lys Glu Leu Ile Glu Glu Phe

100 105 110

Glu Arg Arg Thr Gly Arg Lys Val Ile Gly Asn Lys Pro Ala Ser Gly

115 120 125

Thr Glu Ile Ile Lys Glu Leu Gly Pro Ile His Glu Lys Thr Gly Ala

130 135 140

Leu Ile Val Tyr Thr Ser Ala Asp Ser Val Phe Gln Ile Ala Ala Lys

145 150 155 160

Lys Glu Ile Val Pro Leu Glu Glu Leu Tyr Arg Tyr Cys Glu Ile Ala

165 170 175

Arg Glu Leu Leu Asn Glu Met Gly Tyr Lys Val Ala Arg Val Ile Ala

180 185 190

Arg Pro Phe Thr Gly Glu Trp Pro Asn Tyr Val Arg Thr Pro Glu Arg

195 200 205

Lys Asp Phe Ser Leu Glu Pro Glu Gly Lys Thr Leu Leu Asp Val Leu

210 215 220

Thr Glu Asn Gly Ile Pro Val Tyr Gly Val Gly Lys Ile Ala Asp Ile

225 230 235 240

Phe Ala Gly Arg Gly Val Thr Glu Asn Tyr Lys Thr Lys Asp Asn Asn

245 250 255

Asp Gly Ile Asp Lys Thr Ile Ser Leu Met Lys Glu Lys Asn His Asp

260 265 270

Cys Leu Ile Phe Thr Asn Leu Val Asp Phe Asp Thr Lys Tyr Gly His

275 280 285

Arg Asn Asp Pro Val Ser Tyr Ala Lys Ala Leu Glu Glu Phe Asp Ala

290 295 300

Arg Leu Pro Glu Ile Met His Asn Leu Asn Glu Asp Asp Val Leu Phe

305 310 315 320

Ile Thr Ala Asp His Gly Cys Asp Pro Thr Thr Pro Ser Thr Asp His

325 330 335

Ser Arg Glu Met Val Pro Leu Leu Gly Tyr Gly Gly Arg Leu Lys Lys

340 345 350

Asp Val Tyr Val Gly Ile Arg Glu Thr Phe Ala Asp Leu Gly Gln Thr

355 360 365

Ile Ala Asp Ile Phe Gly Val Pro Pro Leu Glu Asn Gly Thr Ser Phe

370 375 380

Lys Asn Leu Ile Trp Glu

385 390

<210> 3

<211> 230

<212> PRT

<213> Homo sapiens

<400> 3

Met Lys Leu Ile Val Gly Ile Gly Gly Met Thr Asn Gly Gly Lys Thr

1 5 10 15

Thr Leu Thr Asn Ser Leu Leu Arg Ala Leu Pro Asn Cys Cys Val Ile

20 25 30

His Gln Asp Asp Phe Phe Lys Pro Gln Asp Gln Ile Ala Val Gly Glu

35 40 45

Asp Gly Phe Lys Gln Trp Asp Val Leu Glu Ser Leu Asp Met Glu Ala

50 55 60

Met Leu Asp Thr Val Gln Ala Trp Leu Ser Ser Pro Gln Lys Phe Ala

65 70 75 80

Arg Ala His Gly Val Ser Val Gln Pro Glu Ala Ser Asp Thr His Ile

85 90 95

Leu Leu Leu Glu Gly Phe Leu Leu Tyr Ser Tyr Lys Pro Leu Val Asp

100 105 110

Leu Tyr Ser Arg Arg Tyr Phe Leu Thr Val Pro Tyr Glu Glu Cys Lys

115 120 125

Trp Arg Arg Ser Thr Arg Asn Tyr Thr Val Pro Asp Pro Pro Gly Leu

130 135 140

Phe Asp Gly His Val Trp Pro Met Tyr Gln Lys Tyr Arg Gln Glu Met

145 150 155 160

Glu Ala Asn Gly Val Glu Val Val Tyr Leu Asp Gly Met Lys Ser Arg

165 170 175

Glu Glu Leu Phe Arg Glu Val Leu Glu Asp Ile Gln Asn Ser Leu Leu

180 185 190

Asn Arg Ser Gln Glu Ser Ala Pro Ser Pro Ala Arg Pro Ala Arg Thr

195 200 205

Gln Gly Pro Gly Arg Gly Cys Gly His Arg Thr Ala Arg Pro Ala Ala

210 215 220

Ser Gln Gln Asp Ser Met

225 230