Preparation method of nicotinamide adenine dinucleotide

Abstract

The invention relates to the technical field of biology, in particular to a preparation method of nicotinamide adenine dinucleotide. According to the invention, the nicotinamide adenine dinucleotide is synthesized under the action of nicotinamide mononucleotide adenosine transferase NMN-AT20 by taking ANK as a substrate. Cheap adenosine is used as a starting substrate, AMP and ADP are synthesized by ANK, and ATP is finally generated under the action of PPK, so that the cost is greatly reduced. Meanwhile, co-expression molecular construction is carried out on NMN-AT20 and adenosine kinase (ANK), so that the mutual influence of enzymes in a multi-enzyme system is reduced, the conversion rate is increased, the enzyme fermentation amount is reduced, the economical efficiency of the NAD production process is further enhanced, and the efficiency and sustainability of the whole process are improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing nicotinamide adenine dinucleotide. Background Art

[0002] Nicotinamide adenine dinucleotide (NAD + ) is a type of transferred electron ( )of , referred to as oxidized coenzyme I, participates in a variety of physiological activities such as cell metabolism, energy synthesis, and cell DNA repair. It is an essential coenzyme for cellular redox reactions in all life systems. Studies have found that NAD decreases with age and is considered to be one of the factors that lead to physical decline (aging phenomenon). Therefore, targeting NAD metabolism has become a potential treatment for improving aging-related diseases and extending human healthy lifespan. At the same time, NAD is also a popular "anti-aging" product in the health care product market.

[0003] With the development of life science and technology, the use of biological methods to synthesize NAD has become a research hotspot. Currently, NAD is produced mainly through two pathways: 1) The traditional method of direct yeast fermentation synthesis: fermentation of a culture medium configured with glucose, yeast extract, peptone, ammonium sulfate, etc. to obtain yeast, and then separation through processes such as cell wall breaking. 2) Bioenzyme catalysis method (based on the salvage pathway of mammals): using nicotinamide ribose (chemically synthesized) and ATP as substrates, NAD is synthesized under the action of nicotinamide ribokinase (NRK) and nicotinamide mononucleotide adenosine transferase (NMN-AT). Among them, the transferase NMN-AT is considered to be the rate-limiting enzyme in this pathway.

[0004] In the prior art, yeast fermentation mainly separates and obtains NAD through processes such as cell wall breaking, but the yield of NAD in the fermenter is low, and the crude NAD solution obtained by the fermentation method contains a large amount of impurities, with a purity of only 20-40%. Further separation and purification are required to obtain high-purity NAD products, which leads to a high market price of NAD. It is reported that Japan has improved the fermentation process for producing NAD, which has reduced the price. In addition, a Chinese patent also reported a method for preparing NAD fermentation liquid using golden apple snails and cassava as raw materials, and the accumulation of NAD can reach 4.5-6.5 g / L. But overall, the yeast fermentation method still needs further technical improvements and optimization to improve its competitiveness in industrial production.

[0005] The biocatalytic synthesis of NAD requires the direct use of ATP molecules to provide adenosine monophosphate. As a high-energy molecule, ATP is relatively expensive in the biosynthesis process. Especially in large-scale production, the continuous supply of ATP to meet the needs of enzyme-catalyzed reactions will increase the cost of the entire production process, which is challenging for enzyme-catalyzed reactions. In addition, the NMN-AT enzyme is also limited by the concentration of ATP, and there may be mutual interference between different enzymes, affecting enzyme activity and reducing the number of fermentation bacteria. Summary of the invention

[0006] In view of this, the present invention provides a method for preparing nicotinamide adenine dinucleotide. The present invention uses ANK as a substrate to synthesize nicotinamide adenine dinucleotide under the action of nicotinamide mononucleotide adenyltransferase NMN-AT20. Using cheap adenosine as a starting substrate, ANK is used to synthesize AMP and ADP, and then ATP is finally generated under the action of polyphosphatase PPK, which greatly reduces the cost. At the same time, NMN-AT20 and adenosine kinase (ANK) are co-expressed molecularly to reduce the mutual influence of enzymes between multi-enzyme systems, while improving the conversion rate and reducing the amount of enzyme fermentation, further enhancing the economy of the NAD production process, and improving the efficiency and sustainability of the overall process.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing nicotinamide adenine dinucleotide, comprising: using nicotinamide mononucleotide adenylyltransferase NMN-AT20 to catalyze nicotinamide mononucleotide and adenosine triphosphate to generate the nicotinamide adenine dinucleotide;

[0009] The nicotinamide mononucleotide adenylyltransferase NMN-AT20 has an amino acid sequence as shown in SEQ ID NO:5.

[0010] In some specific embodiments of the present invention, the preparation method comprises: taking nicotinamide mononucleotide, adenosine triphosphate, and magnesium chloride solution, and then adding the remaining adenosine triphosphate and magnesium chloride solution at a uniform rate within 2 hours by a flow addition method, so that the molar ratio of nicotinamide mononucleotide, adenosine triphosphate, and magnesium chloride is 1:1.2:1.2, adjusting the pH of the reaction system to 7, and mixing with 692.3 U / mL of nicotinamide mononucleotide adenylyltransferase NMN-AT20, stirring the reaction at a temperature of 37° C. and a pH of 7.0 for 3 h to obtain the nicotinamide adenine dinucleotide.

[0011] In some specific embodiments of the present invention, the preparation method further comprises the step of synthesizing adenosine triphosphate using adenosine.

[0012] In some specific embodiments of the present invention, the step of synthesizing adenosine triphosphate using adenosine specifically comprises:

[0013] Step 1, adenosine kinase catalyzes adenosine and adenosine triphosphate to generate adenosine monophosphate and adenosine diphosphate;

[0014] Step 2: polyphosphatase catalyzes the adenosine monophosphate, the adenosine diphosphate, and sodium hexametaphosphate to generate the adenosine triphosphate.

[0015] In some specific embodiments of the present invention, the preparation method comprises: taking nicotinamide mononucleotide, adenosine, adenosine triphosphate, magnesium chloride, and sodium hexametaphosphate, and then adding the remaining magnesium chloride, adenosine, and sodium hexametaphosphate solution at a uniform rate within 2 hours by a feeding method, so that nicotinamide mononucleotide, adenosine, adenosine triphosphate, magnesium chloride, and sodium hexametaphosphate are mixed in a molar ratio of 60:32:1:72:36, adjusting the pH of the reaction system to 7, and mixing with nicotinamide mononucleotide adenylyltransferase NMN-AT20, adenosine kinase ANK, and polyphosphatase PPK, stirring and reacting at a temperature of 37° C. and a pH of 7.0 for 3 h to obtain the nicotinamide adenine dinucleotide;

[0016] The enzyme activity ratios of nicotinamide mononucleotide adenylyltransferase NMN-AT20, adenosine kinase ANK, and polyphosphatase PPK include (736.7-712.3): (628.3-712.3): 525.9.

[0017] In some specific embodiments of the present invention, the preparation method further comprises a purification step of obtaining the nicotinamide adenine dinucleotide by removing enzymes in the reaction system, removing impurities with anion exchange resin, removing salt with a reverse osmosis membrane, concentrating, and crystallizing.

[0018] In some specific embodiments of the present invention, the purification step specifically includes: adjusting the pH in the reaction system to 2.0 to denature the enzyme and precipitate it, and then removing it by centrifugation; then adjusting the pH of the reaction solution to 7.0 and directly loading it with D201 anion exchange resin to remove the remaining nicotinamide mononucleotide, adenosine triphosphate, adenosine diphosphate and free phosphate impurities; removing salt by reverse osmosis membrane; and finally concentrating and crystallizing the product to obtain the nicotinamide adenine dinucleotide.

[0019] The present invention also provides the use of nicotinamide mononucleotide adenine transferase NMN-AT20 in the preparation of nicotinamide adenine dinucleotide; the nicotinamide mononucleotide adenine transferase NMN-AT20 has an amino acid sequence as shown in SEQ ID NO:5.

[0020] The present invention also provides a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO:8.

[0021] The present invention also provides a co-expression element, comprising the nucleotide sequences shown in SEQ ID NO:7 and SEQ ID NO:8.

[0022] The present invention also provides an expression vector comprising the co-expression element.

[0023] In some specific embodiments of the present invention, the expression vector includes pRSFDuet-1.

[0024] The present invention also provides a host, comprising the expression vector.

[0025] The present invention also provides a kit, comprising nicotinamide mononucleotide adenylyltransferase, nicotinamide mononucleotide, magnesium chloride and adenosine triphosphate.

[0026] Preferably, the kit further comprises adenosine kinase, polyphosphatase and sodium hexametaphosphate.

[0027] The present invention provides the following beneficial effects:

[0028] The present invention provides an economical and efficient NAD synthesis method, discovers and verifies a highly efficient new enzyme NMN-AT20, and at the same time, the present invention uses cheap adenosine as a starting substrate, uses ANK to synthesize AMP and ADP, and then finally generates ATP under the action of polyphosphatase PPK. Compared with the direct addition of ATP, costs are saved. By co-expressing NMN-AT20 and ANK in one strain, the optimization of the multi-enzyme catalytic system is achieved. The co-expression design reduces the potential interference between different enzyme components, improves the conversion efficiency of the enzyme catalytic process, and reduces the number of enzyme strains required for fermentation. Through this strategy, not only the economy of the NAD production process is enhanced, but also the efficiency and sustainability of the overall process are improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0030] Figure 1 Show technical route 1;

[0031] Figure 2 Show technical route 2;

[0032] Figure 3 The liquid phase results of the reaction in Example 1 are shown;

[0033] Figure 4 The liquid phase results of the reaction in Example 2 are shown;

[0034] Figure 5 The liquid phase results of the reaction of Example 3 are shown;

[0035] Figure 6 Shows the liquid phase results of NAD purification samples;

[0036] Figure 7 shows the mass spectrum of NAD purified sample;

[0037] Figure 8 The reaction liquid phase results of Comparative Example 1 are shown;

[0038] Figure 9 The reaction liquid phase results of Comparative Example 2 are shown. DETAILED DESCRIPTION

[0039] The present invention discloses a method for preparing nicotinamide adenine dinucleotide. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0040] This patent adopts a bio-enzyme catalysis method, using nicotinamide mononucleotide (NMN) and adenosine triphosphate (ATP) as substrates to generate NAD. Among them, a new high-efficiency transferase NMN-AT20 was discovered and verified, which can improve the conversion rate while tolerating high substrate concentrations. It is worth noting that the present invention uses adenosine as a substrate to synthesize ATP, which greatly reduces the cost. At the same time, NMN-AT20 and adenosine kinase (ANK) were co-expressed molecularly to reduce the mutual influence of enzymes between multi-enzyme systems, while improving the conversion rate and reducing the amount of enzyme fermentation, further reducing production costs.

[0041] The technical route adopted by the present invention is:

[0042] Route 1:

[0043] Nicotinamide adenine dinucleotide (NAD) is generated by nicotinamide mononucleotide (NMN) and adenosine triphosphate (ATP) as substrates under the action of nicotinamide mononucleotide adenylyltransferase (NMNAT). + ) and pyrophosphate ( Figure 1 ).

[0044] Route 2:

[0045] Due to the high cost of ATP, a two-step enzyme-catalyzed reaction of synthesizing ATP from adenosine is used instead of directly inputting ATP.

[0046] Step 1: If Figure 2As shown, adenosine and ATP generate adenosine monophosphate (AMP) and adenosine diphosphate (ADP) under the action of adenosine kinase.

[0047] Step 2: Add sodium hexametaphosphate to AMP and ADP, and finally generate ATP under the action of polyphosphatase PPK.

[0048] Step 3: Using nicotinamide mononucleotide (NMN) and adenosine triphosphate (ATP) as substrates, nicotinamide adenine dinucleotide (NAD + ) and pyrophosphate.

[0049] The present invention utilizes novel nicotinamide mononucleotide adenylyltransferases (NMN-AT04, 15, 20). NMN-AT04 is derived from Pseudomonas aeruginosa (Uniprot#: Q9HX21), which is a known enzyme in the literature. NMN-AT15 is derived from Azomonas agilis (Uniprot#: A0A562I1N9), which has not been reported in the literature for the preparation of NAD; NMN-AT20 is derived from Halopseudomonas salegens (Uniprot#: A0A1H2GGL8), which has not been reported in the literature. Polyphosphate kinase (PPK), first expressed for enzyme catalysis: polyphosphate kinase derived from a salmon fish bacterium: Renibacterium salmoninarum (Uniprot ID: A9WPD3). Adenosine Kinase (ANK): derived from Zygosacchatomyces rouxii (Candidamogii), Uniprot#: A0A1Q2ZYY0.

[0050] The amino acid sequence of the enzyme NMN-AT20 with the best reaction effect is shown in Table 1, and the DNA sequence is shown in Table 2.

[0051] Table 1 Amino acid sequences of enzymes

[0052] Abbreviations and English Sequence number Amino acid sequence of the enzyme PPK SEQ ID NO:1 MSLGRPAMASKHGFKKSPAELLVVGPEFNLDQVDTSGAPGFSGKKRDAEQVLAEGERKLSVLQEQLFAQSKFGGKHSVLLVLQAMDTAGKGGIVSHVVGSVDPQGMQLHAFKAPTDEEKQHDFLWRIEKGVPKAGMLGVFDRSHYEDVLIHRVHGWADETELERRYQAINDFEEQLTSNGTKIVKVMLDISPETQKERLTARLAEPTKHWKYSPTDVTEREFWPQYMEAYQLAFEKTSTKIAPWHVVPADKKWYARLAVQELLLDAFESLDLSWPEGDYDLALERKRVAAS ANK SEQ ID NO:2 MILSSIKSGIKLKRSSITTVQKSIRRSITANMSHSPQVICLGNPLLDIQADVDAAYLEKYALKANDAILVDANSGDKRMEIYEEVIKKPNVHFVAGGAAQNTARGAAYVLGPQKVGYFGSVGQDTYADKLLAENETAGVASFYQVQKSVGTGKCAALITGHNRSLVTDLGAANHFTPDHLDAHWDKVEAAKLFYIGGFHLTVSPDAICKLGKHAQESGKPFILNLSAPFIPQFFKSALDQVLPYTTYVIANESEAASYAESYGLTCSKDDLEAIAKHIVGDSTQRTVIFTHGLEPTVVVSNQGTKSVPVKPIAGEKIVDTNGAGDAFAGGFLAGLAQGFDLLKSIDLGQWLAALSLQEIGPSFPKTKVQYNN NMN-AT04 SEQ ID NO:3 MGKRIGLFGGTFDPVHIGHMRSAVEMAEQFALDELRLLPNARPPHRETPQVSAAQRLAMVERAVAGVERLTVDPRELQRDKPSYTIDTLESVRAELAADDQLFMLIGWDAFCGLPTWHRWEALLDHCHIVVLQRPDADSEPPESLRDLLAARSVADPQALKGPGGQITFVWQTPLAVSATQIRALLGAGRSVRFLVPDAVLNYIEAHHLYRAPH NMN-AT15 SEQ ID NO:4 MSRPLRIGILGGTFDPVHIGHLRSALEVAELLELDEMRLVPCARPPHREAPNRSADHRLAMVRCAVGGQLPLRVDDRELRREKPSYTLDTLESLRAELGPEPKLYLLLGWDAFCGLPSWHRWLELLDHCHILVLQRPDADTELPEALRNLLAARSVADPRALSGSAGQIAFVWQTPLAVSATQIRQLLAAGRSVRFLVPDSVLAYINTHGLYRAPH NMN-AT20 SEQ ID NO:5 MSRRIGLLGGTFNPVHFGHLRSAIEVRERLALDELRLVPSARPPHREAPGVSAEQRLAMVHLALGNTAELQVDDRELQRDKPSWTVDTLTSLRDELGVEAPLFFILGWDAFCGLPGWQRWQELLELAHLVILQRPDFDQEAPEALKDLLAARTIASLDALQGPAGQIITLEQTPLAISATQIRHLIGQGQSARFLLPDPVLDYIHTHGLYRPDRQD

[0053] Table 2 DNA sequences of enzymes

[0054] Abbreviations and English Sequence number DNA sequence of the enzyme PPK SEQIDNO:6 ATGAGCCTGGGCCGTCCGGCAATGGCCAGTAAACATGGCTTTAAAAAAAGTCCGGCAGAACTGCTGGTGGTTGGCCCGGAATTTAATCTGGATCAGGTTGATACCAGTGGTGCACCTGGTTTTAGCGGCAAAAAACGTGATGCAGAACAGGTTCTGGCCGAAGGCGAACGTAAACTGAGCGTTCTGCAGGAACAGCTGTTTGCCCAGAGTAAATTTGGTGGCAAACATAGTGTGCTGCTGGTGCTGCAGGCAATGGATACCGCCGGCAAAGGCGGTATTGTGAGCCATGTGGTGGGCAGTGTTGATCCGCAGGGTATGCAGCTGCATGCATTTAAAGCACCTACCGATGAAGAAAAACAGCATGATTTTCTGTGGCGCATTGAAAAAGGCGTTCCGAAAGCAGGTATGCTGGGCGTTTTTGATCGTAGCCATTATGAAGATGTGCTGATTCATCGTGTTCATGGTTGGGCAGATGAAACCGAACTGGAACGTCGTTATCAGGCCATTAATGATTTTGAAGAACAGCTGACCAGCAATGGTACCAAAATTGTGAAAGTGATGCTGGATATTAGCCCGGAAACCCAGAAAGAACGTCTGACCGCCCGCCTGGCAGAACCGACCAAACATTGGAAATATAGTCCGACCGATGTGACCGAACGCGAATTTTGGCCGCAGTATATGGAAGCCTATCAGCTGGCCTTTGAAAAAACCAGCACCAAAATTGCACCGTGGCATGTTGTTCCGGCCGATAAAAAATGGTATGCCCGCCTGGCGGTTCAGGAACTGCTGCTGGATGCCTTTGAAAGCCTGGATCTGAGTTGGCCGGAAGGTGATTATGATCTGGCACTGGAACGTAAACGCGTTGCAGCAAGTTAA ANK SEQIDNO:7 ATGTTCAAGAACCGTAAAAACATCCTGGTTCTGGCAGATCTGGGCGGTTGCCCGCCGCATTATTTTTATGAAAATATTGCCCAGAACTACAACATTGTTAGTTATATTCCGCGCCCGTTTGCAATTACCAAAGATCATTTTGATCTGATTAACAAGTACAGTATCGCCGTTATTCAGGAAGAAAGCTATTTTGAAAACATCAGTAGCTTTACCAAGACCAATGATGTTTATTGGGCCATGGATGAACATAATTTTAGCCTGGAAGAAGTTGTGAATAAGCTGATTGAACTGAGCAAAATGTTTAATATCCAGGCCATTACCACCAATAATGAACTGTTTGTGGAACCGGTTGCCGTGGCAAGTAAAAAACTGGATCTGAAAGGTGCAGGCAGTAAAGGTGCCAAAAATGCACGTGATAAATTTCTGATGCGTCAGTGTTTTAATAACAGCAAAGCACGCACCGTGAATTGTAAAAAAGTTGATGATCTGAACGACCTGATTATTGCAAGTAAAGAACTGGGCTTTCCGTTTATTCTGAAACCGACCTATCTGGCCAGCAGCCTGGGCGTGACCCTGTTTAAAGATAGTAGCGAACTGGAACAGAAATTCAATGAACTGAAAAAATTCGTGGACACCCTGACCGTTCCGAAAAGCGTGAAACCGAGCTGTATTTTTATTGCCGAAGAATATCTGGTGGGTAATAGTAGCGAATGGTATGGCGTTGAAGGCTATGGCGATTATGTTAGTATTGAAGGTATTATGAAGGACGGCCAGTATTATCCGATTGCAATTACCGATAAAAGTCCGCAGATTGGCTTTACCGAAACCAGCCATATTAGCACCAGTGTTCTGAGCGCCGATAAACAGAAACTGATTATTGAAGCCGCCAAATATGCAAATGAAAGCCTGGATCTGGAAAATTGTGCAACCCATACCGAAATGAAACTGCTGCCGAATCTGGAAGTTGGCCTGATTGAAACCGCAGCACGTTATGGTGGCTGGAATATTATTCCGAATACCAGCAAAGTTCTGGATATTAATTACCCGAAACTGCTGATTGATGTTCTGACCAGCAATGTTAGCGATCAGTATCTGGAAAATATTATTGCCAAAAAGCCGAAAAAGTTCATTAGCGATTTTCATATCTATCTGAAGGATGGTGAAAATGTGAATACCGATATTATTTTCGAGGGCCTGGAAGATACCAGTCAGTATATTAATCAGGATGTGAAAGTGAATAAGTACGATGAAATTCCGAAAAAGACTATTCTGAAATACGAAGATTTCACCGCATTCAATAGTGTGGCATTTTATGAACTGGAAAGCGCAAATAGTAATAGTCTGGTTCAGACCATTAATAATATCCGCAAAAATATCAACTTCAAGTAA NMN-AT20 SEQIDNO:8 ATGAGCCGGCGGATTGGTTTACTGGGGGGGACGTTTAATCCGGTGCACTTTGGCCACCTGCGCAGCGCGATTGAAGTTCGCGAACGGCTGGCGCTGGACGAGCTGAGACTGGTTCCGAGCGCAAGACCTCCGCATCGCGAAGCGCCGGGCGTTAGCGCAGAACAACGTTTAGCAATGGTTCACCTGGCGCTGGGGAACACAGCAGAACTGCAAGTTGATGATAGAGAACTGCAGCGGGATAAACCGTCATGGACAGTGGATACCCTGACCAGCTTACGGGACGAACTGGGAGTTGAGGCACCGCTGTTTTTTATTCTGGGTTGGGATGCATTTTGTGGTCTGCCGGGTTGGCAGCGTTGGCAGGAACTGCTGGAACTGGCCCATTTAGTTATTTTACAGAGACCTGATTTCGATCAAGAGGCCCCGGAGGCGTTAAAGGATTTATTAGCGGCGCGTACCATTGCGAGCTTAGATGCGTTACAGGGTCCTGCAGGTCAGATTATTACCCTGGAGCAGACACCACTGGCCATTAGCGCAACCCAAATTCGTCATCTGATTGGCCAGGGACAGAGCGCACGCTTTCTGCTGCCGGATCCGGTTTTAGATTATATTCACACCCACGGCCTTTATCGTCCGGACAGACAAGATTAA

[0055] Unless otherwise specified, the raw materials and reagents used in the method for preparing nicotinamide adenine dinucleotide provided by the present invention can be purchased from the market.

[0056] The present invention will be further described below in conjunction with embodiments:

[0057] Example 1 Preparation of NAD

[0058] 1. The enzymes required by the present invention are all produced by E. coli fermentation; it specifically includes the following steps: transferring the plasmid containing the target gene into E. coli (BL21) competent cells for plate culture and single clone screening, and small-scale liquid culture of single clones, and finally amplifying the liquid culture of bacteria with correct protein expression step by step. It specifically includes transferring a single colony into 5mL LB culture solution containing 50 μM kanamycin (37 o C) culture, when the cells grow to the logarithmic phase, inoculate them into 250 mL LB culture medium containing the same antibiotics, and when they grow to the logarithmic phase, transfer them to a 5 L culture fermenter for culture and final protein expression. In the 5 L fermenter culture, when the cell OD is ~20, add 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 25 o C was used to induce protein expression for 6 hours, and finally the cells were collected by high-speed centrifugation (4000 rpm, 20 min) to obtain 25-50 g of enzyme-overexpressing wet cells. A small amount of cells was first mixed evenly with Tris.HCl buffer (50mM, pH 8.0) on an ice basin, and then the cells were broken by freeze-thaw method, and the supernatant was run on SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to determine protein expression. The LB medium consists of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate, and 5% glycerol.

[0059] 2. Preparation of Nicotinamide Mononucleotide Adenylyltransferase Supernatant:

[0060] Prepare 100 mM Tris (hydroxymethylaminomethane) and adjust the pH to 8 with hydrochloric acid (HCl) to obtain 100 mM Tris-HCl buffer (pH=8).

[0061] First, weigh an appropriate amount of nicotinamide mononucleotide adenylyltransferase bacteria at a mass ratio of 1:9 and resuspend them in 100 mM Tris-HCl. Use a cell disruptor at 35% power for 15 minutes to dissolve the intracellular proteins in the buffer, then centrifuge and take the supernatant for later use. Dilute the supernatant 200 times when measuring activity.

[0062] Nicotinamide mononucleotide adenylyltransferase activity assay method:

[0063] Add 150 μL 50 mM ATP, 150 μL 50 mM glucose 6-phosphate, and 150 μL 100 mM MgCl2 to a disposable cuvette. Then add 0.5 Units of glucose 6-phosphate dehydrogenase and 100 μL of nicotinamide mononucleotide adenylyltransferase supernatant diluted 200 times, and add the prepared Tris-HCl buffer to the volume to 1350 μL. Finally, add 150 μL 50 mM NMN at room temperature and the reaction starts immediately. Use a UV spectrophotometer to detect at 340 nm and read the change in absorbance within a certain period of time. Finally, the enzyme activity of glucose dehydrogenase is calculated according to the Lambert-Beer law A=ε·b·c. The unit of enzyme activity is 1 min to convert 1 μ The required amount of enzyme for the substrate, i.e. 1IU = 1 μmol / min.

[0064] Prepare 100 mM Tris (Tris), and adjust the pH to 8 with hydrochloric acid (HCl). First, weigh an appropriate amount of nicotinamide mononucleotide adenylyltransferase (NMN-AT20) according to the enzyme-to-buffer mass ratio of 1:9 and resuspend it in 100 mM Tris-HCl. Dissolve the intracellular proteins in the buffer by cell disruption in a cell disruptor (Lichen LC-JY92) at 40% power for 15 minutes, and then centrifuge to obtain the supernatant for use.

[0065] 3. Preparation of NAD:

[0066] In a 60 mM Tris-HCl pH=8 buffer system, the concentration of the reaction starting substrate NMN is 300 mM, and the molar ratio of the final added concentration of ATP to the starting substrate is 1.2:1. First, 80 mM ATP and 100 mM magnesium chloride solution are added, and then the remaining 280 mM ATP and 260 mM magnesium chloride solution are added at a uniform rate within 2 hours as the reaction proceeds. After the reaction, the final concentrations of ATP and magnesium chloride are both 360 mM. The use of a flow addition method is beneficial to reduce the inhibitory effect of high concentrations of ATP and magnesium chloride, while increasing the reaction rate and yield of NAD. After adjusting the pH of the reaction system to about 7 with ammonia water, 200 μL / mL of NMN-AT20 with an enzyme activity of 692.3 U / mL was added. The reaction was stirred for 3 hours at a temperature of 37°C and a pH of 7.0. After the reaction, liquid phase detection ( Figure 3 ), the yield of NAD is about 88.3%.

[0067] The liquid phase detection method is shown in Table 3:

[0068] Table 3 Liquid phase detection method

[0069]

[0070] Example 2 Preparation of NAD

[0071] Nicotinamide mononucleotide adenyltransferase NMN-AT20, ANK and PPK were prepared according to the method described in Example 1. Tris (Tris) was prepared at a concentration of 100 mM, and the pH value was adjusted to 8 with hydrochloric acid (HCl). First, appropriate amounts of nicotinamide mononucleotide adenyltransferase NMN-AT20, ANK and PPK were weighed according to the mass ratio of enzyme to buffer of 1:9, and resuspended in 100 mM Tris-HCl, respectively, and the cell wall was broken in a cell disruptor (Lichen LC-JY92) at 40% power for 15 minutes to dissolve the intracellular protein in the buffer, and then the supernatant was centrifuged for use.

[0072] In a 60 mM Tris-HCl pH=8 buffer system, 300 mM NMN, 30 mM adenosine, and 5 mM ATP were added at the beginning of the reaction. Then 100 mM magnesium chloride and 60 mM sodium hexametaphosphate were added, and the remaining magnesium chloride, adenosine, and sodium hexametaphosphate solutions were added at a uniform rate within 2 hours by a flow addition method, and the final concentration of magnesium chloride was 360 mM, the final concentration of adenosine was 160 mM, and the final concentration of sodium hexametaphosphate was 180 mM. The flow addition method is conducive to reducing the inhibitory effect of high concentrations of magnesium chloride and sodium hexametaphosphate, and at the same time, it can overcome the limitation of the low solubility of adenosine to increase the reaction rate and yield of NAD. After adjusting the pH of the reaction system to about 7 with ammonia water, 200 μL / mL of NMN-AT20 with an enzyme activity of 692.3 U / mL, 150 μL / mL of ANK with an enzyme activity of 628.3U / mL, and 100 μL / mL of PPK with an enzyme activity of 525.9 U / mL were added. The reaction was stirred at 37°C and pH 7.0 for 3 h. After the reaction, liquid phase detection ( Figure 4 ), the yield of NAD is about 96.6%.

[0073] Example 3 Preparation of NAD

[0074] To further optimize the process, reduce the number of fermentation bacteria, shorten the process route, and reduce the fermentation cost, the present invention uses a vector to co-express two key enzymes: nicotinamide mononucleotide adenylyltransferase (NMN-AT20) and adenosine kinase (ANK). The dual-promoter expression vector pRSFDuet-1 (Miaoling Biological P0051) was used to obtain the pRSFDuet-T7-[NMN-AT20]-T7-[ANK]-1 expression plasmid (T7 promoter is carried on the plasmid, and the sequence is reused). According to the enzyme preparation method described in Example 1, the pRSFDuet-T7-[NMN-AT20]-T7-[ANK]-1 expression plasmid was transfected to obtain a strain, and two enzymes were produced by fermentation.

[0075] Prepare 100 mM Tris (Tris), adjust the pH to 8 with hydrochloric acid (HCl) to obtain 100 mM Tris-HCl buffer with pH = 8. First, weigh appropriate amounts of co-expressed enzymes NMN-AT20-ANK and PPK according to the enzyme to buffer mass ratio of 1:9, and resuspend them in 100 mM Tris-HCl, respectively, and break the cell wall in a cell disruptor (Lichen LC-JY92) at 40% power for 15 minutes to dissolve the intracellular proteins in the buffer, and then centrifuge to obtain the supernatant for use.

[0076] In a 60 mM Tris-HCl pH=8 buffer system, 300 mM NMN, 30 mM adenosine, and 5 mM ATP were added at the beginning of the reaction. Then 100 mM magnesium chloride and 60 mM sodium hexametaphosphate were added, and the remaining magnesium chloride solution, adenosine solution, and sodium hexametaphosphate solution were added at a uniform rate within 2 hours by a flow addition method, and the total concentration of magnesium chloride added was 360 mM, the total concentration of adenosine was 160 mM, and the total concentration of sodium hexametaphosphate was 180 mM. The flow addition method is conducive to reducing the inhibitory effect of high concentrations of magnesium chloride and sodium hexametaphosphate, and at the same time, it can overcome the limitation of the low solubility of adenosine to increase the reaction rate and yield of NAD. After adjusting the pH of the reaction system to about 7 with ammonia water, NMN-AT20-ANK200 μL / mL with an enzyme activity of 712.3 U / mL and PPK 100 μL / mL with an enzyme activity of 525.9 U / mL were added. The reaction was stirred at 37°C and pH 7.0 for 3 h. After the reaction, liquid phase detection ( Figure 5 ), the yield of NAD is about 99.6%.

[0077] Take 1L of reaction solution, adjust the solution pH to 2.0 to denature the enzyme in the reaction system and remove the precipitate by centrifugation, then adjust the pH of the reaction solution to 7.0 and directly load D201 anion exchange resin to remove the remaining NMN, ATP, adenosine diphosphate and free phosphate impurities, then use reverse osmosis membrane to desalinate the crude product, and finally concentrate and crystallize the product (crystallize in a mixed solvent of ethanol and water) to obtain 141 grams of white solid (yield 80%). The obtained pure product was verified by HPLC to have a purity of 99.8% for NAD, and confirmed by mass spectrometry. Figure 6 , Figure 7 .

[0078] Mass spectrometry method:

[0079] Table 4 Mass spectrometry methods

[0080]

[0081] Comparative Example 1

[0082] Prepare 100 mM Tris (Tris), and adjust the pH to 8 with hydrochloric acid (HCl). First, weigh an appropriate amount of nicotinamide mononucleotide adenylyltransferase (NMN-AT04) according to the enzyme to buffer mass ratio of 1:9 and resuspend it in 100 mM Tris-HCl. Disrupt the cell wall in a cell disruptor (Lichen LC-JY92) at 40% power for 15 minutes to dissolve the intracellular proteins in the buffer, and then centrifuge to obtain the supernatant for use.

[0083] In a 60 mM Tris-HCl pH=8 buffer system, the concentration of the reaction starting substrate NMN is 300 mM, and the molar ratio of the final added concentration of ATP to the starting substrate is 1.2:1. First, add 80 mM ATP and 100 mM magnesium chloride solution, and then add the remaining 280 mM ATP and 260 mM magnesium chloride solution at a uniform rate within 2 hours as the reaction proceeds. After the reaction, the final concentrations of ATP and magnesium chloride are both 360 mM. The use of a flow addition method is beneficial to reduce the inhibitory effects of high concentrations of ATP and magnesium chloride, while increasing the reaction rate and yield of NAD. The pH of the reaction system was adjusted to about 7 with ammonia water, and 200 μL / mL of an enzyme solution with the same protein content as NMN-AT20 in Example 1 (NMN-AT04 with an enzyme activity of 412.6 U / mL) was added. The reaction was stirred for 3 hours at a temperature of 37°C and a pH of 7.0. After the reaction, liquid phase detection ( Figure 8 ), the yield of NAD is about 52.9%.

[0084] Comparative Example 2

[0085] Prepare 100 mM Tris (Tris), and adjust the pH to 8 with hydrochloric acid (HCl). First, weigh an appropriate amount of nicotinamide mononucleotide adenylyltransferase (NMN-AT15) according to the enzyme to buffer mass ratio of 1:9 and resuspend it in 100 mM Tris-HCl. Disrupt the cell wall in a cell disruptor (Lichen LC-JY92) at 40% power for 15 minutes to dissolve the intracellular proteins in the buffer, and then centrifuge to obtain the supernatant for use.

[0086] In a 60 mM Tris-HCl pH=8 buffer system, the concentration of the reaction starting substrate NMN is 300 mM, and the molar ratio of the final added concentration of ATP to the starting substrate is 1.2:1. First, 80 mM ATP and 100 mM magnesium chloride solution are added, and then the remaining 280 mM ATP and 260 mM magnesium chloride solution are added at a uniform rate within 2 hours as the reaction proceeds. After the reaction, the final concentrations of ATP and magnesium chloride are both 360 mM. The use of a flow addition method is beneficial to reduce the inhibitory effects of high concentrations of ATP and magnesium chloride, while increasing the reaction rate and yield of NAD. After adjusting the pH of the reaction system to about 7 with ammonia water, 200 μL / mL of enzyme solution with the same protein content as NMN-AT20 in Example 1 (NMN-AT15 with an enzyme activity of 648.5 U / mL) was added. The reaction was stirred for 3 hours at a temperature of 37°C and a pH of 7.0. After the reaction, liquid phase detection ( Figure 9 ), the yield of NAD is about 84.6%.

[0087] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing nicotinamide adenine dinucleotide, characterized in that: include: Using nicotinamide mononucleotide adenylyltransferase NMN-AT20 to catalyze nicotinamide mononucleotide and adenosine triphosphate to generate the nicotinamide adenine dinucleotide; The nicotinamide mononucleotide adenylyltransferase NMN-AT20 has an amino acid sequence as shown in SEQ ID NO:

5.

2. The preparation method according to claim 1, characterized in that Also included is the step of synthesizing adenosine triphosphate using adenosine.

3. The preparation method according to claim 2, characterized in that: The step of synthesizing adenosine triphosphate using adenosine specifically comprises: Step 1, adenosine kinase catalyzes adenosine and adenosine triphosphate to generate adenosine monophosphate and adenosine diphosphate; Step 2: polyphosphatase catalyzes the adenosine monophosphate, the adenosine diphosphate, and sodium hexametaphosphate to generate the adenosine triphosphate.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The preparation method also includes a purification step of obtaining the nicotinamide adenine dinucleotide by removing enzymes in the reaction system, removing impurities with anion exchange resin, removing salt with a reverse osmosis membrane, concentrating, and crystallizing.

5. Use of nicotinamide mononucleotide adenylyltransferase NMN-AT20 in the preparation of nicotinamide adenine dinucleotide; the nicotinamide mononucleotide adenylyltransferase NMN-AT20 has an amino acid sequence as shown in SEQ ID NO:

5.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:

8.

7. A co-expression element, characterized in that Including having the nucleotide sequences shown as SEQ ID NO:7 and SEQ ID NO:

8.

8. An expression vector, characterized in that Comprising the co-expression element as described in claim 7.

9. A host, characterized in that Comprising the expression vector as claimed in claim 8.

10. A kit, characterized in that Includes nicotinamide mononucleotide adenylyltransferase, nicotinamide mononucleotide, magnesium chloride and adenosine triphosphate.

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