Method for synthesizing beta-nicotinamide mononucleotide (NMN) through enzyme catalysis

Abstract

The invention belongs to the technical field of biological catalytic synthesis, and particularly relates to a method for synthesizing beta-nicotinamide mononucleotide (NMN) by enzyme catalysis, which comprises the following steps: by taking D-ribose as a starting substrate, and utilizing a three-step enzyme catalysis reaction system which is synergistically completed by an S-methyl-5-thioribose kinase mutant (SMTK), purine nucleotidase (PNP) and nicotinamide ribose kinase (NRK), synthesizing beta-nicotinamide mononucleotide (NMN) by enzyme catalysis. And efficient synthesis of the NMN is realized. In order to further optimize the cost, an ATP (adenosine triphosphate) regeneration system is innovatively introduced in the process, so that the consumption of ATP in the reaction process is effectively reduced. On the basis, by combining an enzyme directed evolution technology, an immobilized enzyme technology and a continuous reactor design, the catalytic reaction efficiency and the product purity level are remarkably improved. The whole process has the characteristics of greenness and environmental protection, the reaction conditions are mild and controllable, the product yield stably reaches more than or equal to 85%, and the requirements of industrial large-scale production are completely met.

Description

Technical Field

[0001] This invention relates to the field of biocatalytic synthesis technology, specifically to a method for the enzyme-catalyzed synthesis of β-nicotinamide mononucleotide (NMN), and particularly to the enzyme catalytic process route from D-ribose to NMN, optimization of key enzyme genes, and industrial application system. Background Technology

[0002] β-Nicotinamide mononucleotide (NMN) is an important bioactive molecule naturally found in living organisms. Catalyzed by nicotinamide adenosine transferase, NMN is converted into nicotinamide adenine dinucleotide (NAD+, also known as coenzyme I), a core substance for maintaining cellular life activities. This conversion plays a crucial role in cellular energy metabolism. Modern medical research has revealed that NMN possesses multiple physiological functions: significantly delaying the aging process, improving symptoms of neurodegenerative diseases, precisely regulating insulin secretion, modulating key gene expression, enhancing cell repair capabilities, and optimizing energy metabolism efficiency. These important findings have made NMN one of the most promising bioactive substances in current anti-aging research and health intervention fields, and its potential applications continue to expand. Currently, NMN synthesis methods mainly include chemical synthesis, microbial fermentation, and enzymatic catalysis.

[0003] Chemical synthesis was the primary method for early NMN production, with core patents focusing on optimizing reaction pathways and purification processes. This method uses nicotinamide, ribose, or nicotinamide nucleoside (NR) as raw materials, generating NMN through reactions such as phosphorylation and condensation. For example, patent US20150073043A1 synthesizes NMN via the phosphorylation of nicotinamide nucleoside and optimizes reaction conditions to improve yield; patent CN110256385A employs an enzyme-chemical coupling method, first synthesizing nicotinamide nucleoside chemically and then using enzyme-catalyzed phosphorylation to generate NMN. However, chemical synthesis has certain drawbacks. Its reaction conditions are relatively harsh, requiring high temperatures and an anhydrous environment, and the product contains mixed isomers, leading to high subsequent purification costs.

[0004] The patented technology of microbial fermentation involves modifying microorganisms (such as Escherichia coli and yeast) through genetic engineering to synthesize NMN. Patents primarily focus on strain construction and metabolic regulation. For example, patent US20200165554A1 modifies yeast strains to overexpress genes related to the NAD+ synthesis pathway (such as NAMPT and PNP), thereby increasing NMN production. Patent CN113249428A utilizes Bacillus subtilis fermentation, improving yield through optimized culture media and conditions. While microbial fermentation is relatively low-cost, it also presents several challenges: complex metabolic pathways, difficulty in product separation, low production concentrations, limited subsequent production capacity, and difficulties in purification.

[0005] Due to its green and efficient characteristics, enzymatic catalysis has become a research hotspot in recent years, with related patents mainly focusing on enzyme selection and immobilization technology. For example, patent WO2017197338A1 utilizes NAMPT enzyme to catalyze the direct synthesis of NMN from nicotinamide and phosphoribosyl pyrophosphate (PRPP); patent CN112280748A uses a multi-enzyme cascade reaction, with glucose as a substrate, to generate NMN through multiple conversion steps. Enzymatic synthesis has the advantages of mild reaction conditions and high selectivity, but traditional enzymatic methods also have shortcomings. When using NAMPT enzyme to catalyze the direct synthesis of NMN from nicotinamide and phosphoribosyl pyrophosphate, product inhibition occurs, resulting in low conversion rates and making industrial production difficult. When using NR kinase, enzyme instability is a problem, and the substrate NR is still synthesized chemically, resulting in complex components and uneconomical prices. Summary of the Invention

[0006] The purpose of this invention is to provide a method for the enzyme-catalyzed synthesis of β-nicotinamide mononucleotide (NMN) to solve the problems raised in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: Step 1: By constructing enzyme-engineered bacteria, glycerol kinase expression strains BL21(DE3)-TK-SMTK, BL21(DE3)-EcPNPm and BL21(DE3)-SC-NRK were obtained and stored in glycerol tubes; Step 2: The glycerol kinase expressing strains were cultured, expression was induced, and enzyme solutions were prepared to obtain TK-SMTK enzyme solution, EcPNPm enzyme solution and SC-NRK enzyme solution; Step 3: Using D-ribose as the starting substrate, an enzyme-catalyzed reaction was carried out using TK-SMTK enzyme solution, EcPNPm enzyme solution and SC-NRK enzyme solution, while ATP was added, to synthesize a solution containing β-nicotinamide mononucleotide.

[0008] Furthermore, the specific steps for constructing the enzyme-engineered bacteria described in step 1 are as follows: EC-SMTK, EcPNPm, and SC-NRK were cloned into the Ndel and Baml restriction enzyme sites of plasmid pET28a(+), respectively. The plasmids were then transformed into BL21(DE3) competent cells using the calcium chloride method. The BL21(DE3) competent cells were spread onto plates containing kanamycin and cultured. Single colonies were picked and verified by enzyme digestion to obtain glycerol kinase expression strains BL21(DE3)-TK-SMTK, BL21(DE3)-EcPNPm, and BL21(DE3)-SC-NRK, respectively, which were then stored in glycerol tubes.

[0009] Furthermore, the EC-SMTK encoding gene is SEQ ID NO: 1, and the amino acid sequence is SEQ ID NO: 4; the EcPNPm encoding gene is SEQ ID NO: 2, and the amino acid sequence is SEQ ID NO: 5; and the SC-NRK encoding gene is SEQ ID NO: 3, and the amino acid sequence is SEQ ID NO: 6.

[0010] Furthermore, the steps for culturing the glycerol kinase-expressing strain, inducing expression, and preparing the enzyme solution in step 2 are as follows: Glycerol kinase expressing strains were taken from glycerol tubes and inoculated into LB medium containing 50 mg / L kanamycin at a 1% inoculation rate. The culture was incubated statically at 37°C for 12 hours to obtain activated bacterial solution. This activated bacterial solution was then transferred to LB medium containing 50 mg / L kanamycin at a 5% inoculation rate and incubated at 36–38°C with shaking at 200 rpm for 2–3 hours until OD (exponential growth) was achieved. 600nm When the concentration reaches 0.6-0.8, add 0.2 mM isopropyl-β-D-thiogalactoside to the culture medium and induce expression of the target protein at 29-31℃ and 200 rpm for 4-5 h. After induction, centrifuge the bacterial culture at 4℃ and 12000 rpm for 2 min, discard the supernatant, invert the centrifuge tube to drain the residual culture medium, resuspend the bacterial cells in a 1:4 volume ratio with pH 7.4 lysis buffer, and sonicate for 2 min to lyse the bacterial cells. After centrifugation, collect the supernatant to obtain TK-SMTK enzyme solution, EcPNPm enzyme solution and SC-NRK enzyme solution.

[0011] Furthermore, the specific steps for synthesizing β-nicotinamide mononucleotide using the enzyme solution of TK-SMTK, EcPNPm, and SC-NRK in step 3 are as follows: 1) Mix tris(hydroxymethyl)aminomethane hydrochloride buffer, D-ribose solution, magnesium chloride solution and sodium ATP solution at pH 7.2, preheat at 23~27℃ for 5~6 min, add TK-SMTK enzyme solution, and then incubate at 33~37℃ and 120 rpm in a water bath shaker for 1~1.5 h to obtain reaction solution 1; 2) Add nicotinamide solution and EcPNPm enzyme solution to reaction solution 1 and incubate at 33~37℃ for 85~95 min to obtain reaction solution 2; 3) Add SC-NRK enzyme solution to reaction solution 2, and add 2mM ATP sodium salt solution to maintain the concentration. React at 28~32℃ for 3~3.5h to obtain a solution containing β-nicotinamide mononucleotide.

[0012] Further: 1) The final concentrations of the tris(hydroxymethyl)aminomethane hydrochloride buffer and D-ribose solution at pH 7.2 are 50 mM, the final concentration of the magnesium chloride solution is 5 mM, the final concentration of the sodium ATP solution is 10 mM, and the final concentration of the TK-SMTK enzyme solution is 5 U / mL; the tris(hydroxymethyl)aminomethane hydrochloride buffer, D-ribose solution, magnesium chloride solution, sodium ATP solution, and TK-SMTK enzyme solution at pH 7.2 are mixed in a volume ratio of 1:1:1:1:1; 2) The final concentration of the nicotinamide solution is 20 mM, the final concentration of the EcPNPm enzyme solution is 2 U / mL, and reaction solution 1, nicotinamide solution, and EcPNPm enzyme solution are mixed in a volume ratio of 5:1:1; 3) The final concentration of the SC-NRK enzyme solution is 2 U / mL, and reaction solution 2 and SC-NRK enzyme solution are mixed in a volume ratio of 7:1.

[0013] Furthermore, during the water bath shaker culture described in 1), 100 μL of reaction solution 1 was taken every 10 min and 10 μL of 0.5 M ethylenediaminetetraacetic acid was added to terminate the reaction, and then the amount of ribose-1-phosphate generated was detected using a Dionex IonPac PA100 anion exchange column.

[0014] Furthermore, during the culture period described in 2), 50 μL of reaction solution 2 was added to 50 μL of methanol to precipitate the protein. After centrifugation, the supernatant was analyzed by HPLC to determine the amount of nicotinamide nucleoside produced. 18 The column and mobile phase consist of 10 mM potassium dihydrogen phosphate at pH 4 and methanol in a volume ratio of 95:5.

[0015] Furthermore, the specific steps for synthesizing β-nicotinamide mononucleotide using the enzyme solution of TK-SMTK, EcPNPm, and SC-NRK in step 3 are as follows: D-ribose, ATP, magnesium chloride, sodium hexametaphosphate, and nicotinamide were added to a three-necked round-bottom flask, dissolved in deionized water, and the pH was adjusted to 7.0–7.5 with 6 mol / L sodium hydroxide solution. The system was heated to 35–37 °C, and TK-SMTK, polyphosphokinase, EcPNPm, and SC-NRK were added sequentially to initiate the reaction. During the reaction, 6 mol / L hydrochloric acid was used to maintain the pH within the range of 7.0–7.5, and the temperature was maintained at 35–37 °C. After the pH of the reaction system stabilized, a sample was taken for HPLC analysis. The reaction was terminated after confirming that D-ribose was completely converted to cytidine 5'-phosphate with no residue, yielding a solution containing β-nicotinamide mononucleotide.

[0016] Furthermore, D-ribose, ATP, magnesium chloride, sodium hexametaphosphate, nicotinamide, and deionized water were mixed in a mass ratio of 4:0.4:1:8:3:200, and TK-SMTK, polyphosphokinase, EcPNPm, and SC-NRK were added in a mass ratio of 5:5:4:6.

[0017] Compared with the prior art, the beneficial effects of the present invention are: This invention uses D-ribose as a starting material and achieves efficient conversion through an optimized three-enzyme catalytic system, including an S-methyl-5-thioribokinase mutant, purine nucleotidase, and nicotinamide ribokinase, while integrating an ATP recycling mechanism to improve economic efficiency. This invention employs strategies such as protein engineering, carrier immobilization, and reactor optimization to give the entire production process the following outstanding characteristics: significantly improved catalytic efficiency, excellent product purity (≥85%), mild reaction conditions (room temperature and pressure), environmental friendliness, and suitability for large-scale application. This invention provides a competitive solution for the industrial production of NMN. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the cascade enzyme reaction for synthesizing NMN according to the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Unless otherwise stated, all materials and reagents used in the experiment can be obtained through commercial channels.

[0021] Reagents used in upstream genetic engineering: The genome extraction kit and plasmid extraction kit used in the embodiments of this invention were purchased from Nanjing Genscript Biotech Co., Ltd.; E. coli BL21(DE3), plasmid pET-28a(+), etc. were purchased from Qingke Biotechnology Co., Ltd.; primer synthesis and sequence sequencing were completed by Qingke Biotechnology Co., Ltd. The usage methods of the above reagents are as follows: refer to the product instructions.

[0022] LB culture medium: catalog number FT-LG0094, sourced from Shanghai Fantai Biotechnology Co., Ltd. Kanamycin: Product number SS10495, sourced from Shanghai Jingkang Biotechnology Co., Ltd.; Isopropyl-β-D-thiogalactoside: Product No. S11086, sourced from Shanghai Yuanye Biotechnology Co., Ltd.; Tris(hydroxymethyl)aminomethane hydrochloride buffer: Catalog number BH-S63674, sourced from Shanghai Bohu Biotechnology Co., Ltd.; D-ribose: Product number 965, sourced from Shijiazhuang Ruitian Biochemical Co., Ltd.; Magnesium chloride: Product number SS3708, sourced from Hubei Shishun Biotechnology Co., Ltd.; ATP (sodium salt): Product number XK6691, sourced from Hubei Xinkang Pharmaceutical Chemical Co., Ltd.; Ethylenediaminetetraacetic acid (EDTA): Product number S30020, sourced from Shanghai Yuanye Biotechnology Co., Ltd. Nicotinamide: Product number S13015, sourced from Shanghai Yuanye Biotechnology Co., Ltd.; Sodium hexametaphosphate: Product number xk526, sourced from Hubei Xinkang Pharmaceutical Chemical Co., Ltd.; Polyphosphokinase: derived from Xiamen Huijia Biotechnology Co., Ltd.

[0023] Unless otherwise stated, the experimental methods used in this embodiment are all conventional techniques in the art. For operations where specific experimental conditions are not explicitly stated, they are performed according to standard conditions, such as those described in Molecular Cloning: A Laboratory Manual (J. Sambrook, DW. Russell, translated by Huang Peitang, Wang Jiaxi, Zhu Houchu et al., 3rd edition, Beijing: Science Press, 2002).

[0024] Example 1: As Figure 1 As shown, the present invention provides a specific implementation step of a method for enzyme-catalyzed synthesis of β-nicotinamide mononucleotide (NMN): Step 1: Construction of enzyme-engineered bacteria Based on the amino acid sequences of EC-SMTK (L123P mutant), EcPNPm (C20A), and SC-NRK, Nanjing Genscript Biotech Co., Ltd. was commissioned to optimize their nucleotide sequences to improve their expression efficiency in *E. coli*. The encoding gene (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 4) for EC-SMTK (L123P mutant), the encoding gene (SEQ ID NO: 2) and amino acid sequence (SEQ ID NO: 5) for EcPNPm (C20A), and the encoding gene (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 4) for SC-NRK were analyzed. NO: 6) was cloned into the Ndel and Baml restriction enzyme sites of the pET28a(+) plasmid, respectively; the recombinant plasmid was introduced into BL21(DE3) competent cells by calcium chloride transformation, and screened on plates containing 50 mg / L kanamycin. After single colony picking and enzyme digestion verification, three glycerol kinase expression strains were successfully constructed: BL21(DE3)-TK-SMTK (L123P), BL21(DE3)-EcPNPm (C20A) and BL21(DE3)-SC-NRK. Each strain was preserved in glycerol tubes. Step 2: Culture of enzyme-engineered strains, induction of expression, and preparation of enzyme solution. Glycerol kinase expressing strains were taken from glycerol tubes and inoculated into LB medium containing 50 mg / L kanamycin at a 1% inoculation rate. The culture was incubated statically at 37°C for 12 h to obtain activated bacterial solution. This activated bacterial solution was then transferred to LB medium containing 50 mg / L kanamycin at a 5% inoculation rate and cultured at 37°C with shaking at 200 rpm for 2.5 h until OD (exponential growth) was achieved. 600nm When the concentration reaches 0.7, add 0.2 mM isopropyl-β-D-thiogalactoside to the culture medium and induce expression of the target protein at 30℃ and 200 rpm for 4.5 h. After induction, centrifuge the bacterial culture at 4℃ and 12000 rpm for 2 min, discard the supernatant, invert the centrifuge tube to drain the residual culture medium, resuspend the bacterial cells in a 1:4 volume ratio with pH 7.4 lysis buffer, and sonicate for 2 min to lyse the bacterial cells. After centrifugation, collect the supernatant to obtain TK-SMTK enzyme solution, EcPNPm enzyme solution and SC-NRK enzyme solution. Step 3: Stepwise synthesis of β-nicotinamide mononucleotide Mix 50 mL of 50 mM tris(hydroxymethyl)aminomethane hydrochloride buffer (pH 7.2), 50 mL of 50 mM D-ribose solution, 50 mL of 5 mM magnesium chloride solution, and 50 mL of 10 mM ATP (sodium salt) solution. Preheat the mixture to 25 °C for 5 min. Add 50 mL of 5 U / mL TK-SMTK enzyme solution and immediately take a sample as T0 (for HPLC baseline control). Then, incubate the mixture in a water bath at 35 °C and 120 rpm for 1 h to obtain reaction solution 1. During this period, take 100 μL of reaction solution 1 every 10 min and add 10 μL of 0.5 M ethylenediaminetetraacetic acid to terminate the reaction. Finally, use a Dionex IonPac PA100 anion exchange column to detect the amount of ribose-1-phosphate generated. Add 50 mL of nicotinamide solution (final concentration 20 mM) and 50 mL of EcPNPm enzyme solution (final concentration 2 U / mL) directly to reaction solution 1 and incubate at 35 °C for 90 min to obtain reaction solution 2. During this period, take 50 μL of reaction solution 2 and add 50 μL of methanol to precipitate the protein. After centrifugation, the supernatant is analyzed by HPLC (C10-C20). 18 Column, mobile phase: composed of 10mM potassium dihydrogen phosphate at pH 4 and methanol in a volume ratio of 95:5) Nicotinamide nucleoside production: Add 50mL of SC-NRK enzyme solution with a final concentration of 2U / mL to reaction solution 2, and add 2mM ATP (sodium salt) solution to maintain the concentration. React at 30℃ for 3h to obtain a solution containing β-nicotinamide mononucleotide.

[0025] Example 2: Synthesis of β-nicotinamide mononucleotide via cascade reaction 20g D-ribose, 2g ATP, 5g magnesium chloride, 40g sodium hexametaphosphate, and 15g nicotinamide were added to a three-necked round-bottom flask and dissolved in 1L deionized water. The pH was adjusted to 7.3 with 6mol / L sodium hydroxide solution to ensure complete dissolution. The system was then heated to 36℃, and 5mL TK-SMTK, 5mL polyphosphokinase, 4mL EcPNPm, and 6mL SC-NRK were added sequentially to initiate the reaction. During the reaction, 6mol / L hydrochloric acid was used to maintain the pH at 7.3 and the temperature at 36℃. After the pH of the reaction system stabilized, a sample was taken for HPLC analysis. The reaction was terminated after confirming that D-ribose was completely converted to cytidine 5'-phosphate with no residue, yielding a solution containing β-nicotinamide mononucleotide.

[0026] Experiment: The reaction process monitoring and product analysis of this invention were performed using high-performance liquid chromatography (HPLC). Specific analytical conditions were as follows: AQ-C was used. 18 The chromatographic column was used for separation at a column temperature of 30℃ and a flow rate of 1 mL / min; the detector wavelength was set to 254 nm, and the mobile phase was a 20 mM sodium dihydrogen phosphate buffer solution with a pH of 6.

[0027] Take 200 μL of the β-nicotinamide mononucleotide solution from the example, boil it in a water bath for 5 min, then cool it rapidly in an ice bath, filter it through a 0.22 μm filter membrane, and analyze the final product by HPLC.

[0028] The concentration of β-nicotinamide mononucleotide in the product of Example 2 was determined to be 31.4 g / L by HPLC.

[0029] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0030] sequence list <120> Shanghai Riguan Biotechnology Co., Ltd. <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1260 <212> DNA <213> Artificial Sequence <400> 1 atgacggatt caattccatc aggttacaag ccgctgacat gtgacacgct gccgggttat 60 ctctcgtcca gactgacccc ttcatgcgaa ccgggagggt tacctgaaga gtggaaagtt 120 tcagaagtgg gggacggaaa cctgaacatg gtgtttatcg ttgaggggac acataaaacc 180 atcattgtaa aacaagctct gccctggctt cgtgccgggg gcgaaggatg gcctttatct 240 ctgagccgtg cgggctttga gtacaacgtc ttatgtcagg aagccaagta cgcgggtcac 300 acactgattc cgcaggtcta tttttacgac ccggaaatgg cgctgtttgc catggagtat 360 ctgactcctc acgtgattct gcgtaaggaa ctgattaacg gtaaaaaatt ccctaaacta 420 gctgaagata tcggcagatt tttagcacag actctcttca atacgtctga cattggcatg 480 tcagcagaac agaaaaaagc gcttactgcc gagttcgcgt tgaatcatga gctgtgcaaa 540 attacggaag atctgatctt cacagagccc tattacaacg ctgaacggaa taactggact 600 tctcctgagc tggacgatgc cgtccataag gcctgggctg atgtagagat gatccaggtt 660 gccatgcgtt ataagtacaa atttatgaca gaagcgcagg cattgttaca tggcgacctt 720 cattcaggct caatcatggt gaccgacacg gataccaaag tgattgatcc ggagttcggt 780 ttcatggggc caatggcgtt tgatatcggc aactatattg gcaacctcct gctggcgtac 840 ttctcacgcc ctgggtggga tgcgaatgag caacgtcgcg ctgactatca ggaatggctg 900 cttcagcaga ttgtccaaac ctggtccgtt ttcacccggg agttccgcca gctctgggac 960 aacaaaacac agggcgacgc gtggtcgaca gaaatgtatc aacagaacag ggccgctctt1020 gaggacgcac aggatcagtt ttttgccacg ctgcttgaag attccctggt gaatgccggc1080 atagaaatga atcgccggat cattggtttt gctggcgttg ccgagctgaa acagattgaa1140 aatacagagc ttcgcgcagg atgtgaacga cgtgcattga ccatggcgcg cgatcttatc1200 gtcaatgccc gccagtttaa aaatatggat tccgtcatcc agtctgcgaa ggttaagtaa1260 <210> 2 <211> 720 <212> DNA <213> Artificial Sequence <400> 2 atggctaccc cacacattaa tgcagaaatg ggcgatttcg ctgacgtagt tttgatgcca 60 ggcgacccgc tgcgtgcgaa gtatattgct gaaactttcc ttgaagatgc ccgtgaagtg 120 aacaacgttc gcggtatgct gggcttcacc ggtacttaca aaggccgcaa aatttccgta 180 atgggtcacg gtatgggtat cccgtcctgc tccatctaca ccaaagaact gatcaccgat 240 ttcggcgtga agaaaattat ccgcgtaggc tcctgtggcg ctgttctgcc gcacgtaaaa 3 hundred ctgcgcgacg tcgttatcgg tatgggtgcc tgcaccgatt ccaaagttaa ccgtatccgt 360 tttaaagacc atgactttgc cgctatcgct gactttgaca tggtgcgtaa cgcggtagac 420 gcggctaaag cactgggcat tgatgctcgc gttggtaacc tgttctccgc tgacctgttc 480 tactctccgg acggcgaaat gttcgacgtg atggaaaaat acggcatcct cggcgtggaa 540 atggaagcgg ctggtatcta cggcgtcgct gcagaatttg gcgcgaaagc cctgaccatc 600 tgtaccgtgt ctgaccacat ccgcactcac gagcagacca ctgccgctga gcgtcagacc 660 accttcaacg acatgatcaa agtcgcactg gaatccgttc tgctgggcga taaagagtaa 720 <210> 3 <211> 723 <212> DNA <213> Artificial Sequence <400> 3 atgacttcga aaaaagtgat attagttgca ttgagtggat gctcctccag tggtaagacg 60 acaattgcga aacttacagc aagtttattc acgaaggcta cattaattca tgaagatgac 120 ttttacaaac atgataatga agtgccagta gatgctaaat ataacattca aaattgggat 180 tcgccagaag ctcttgattt taaacttttc ggtaaagaat tagatgtgat caaacaaact 240 ggtaaaatag ccaccaaact tatacacaat aacaacgtag atgatccctt tacaaagttc 300 cacattgata gacaagtttg ggacgagtta aaggctaagt atgactctat taatgacgac 360 aaatatgaag ttgtaattgt agatgggttt atgattttca ataatactgg aatatcaaaa 420 aaatttgatt tgaagatatt agtgcgtgct ccctatgaag tactaaaaaa aaggagggct 480 tccagaaaag gataccagac tttggattct ttctgggtgg atccgccgta ttatttcgac 540 gaatttgtgt atgaatctta tcgtgcaaat catgcgcagt tatttgttaa tggagacgta 600 gaaggtttac tagacccaag gaagtcaaag aatataaaag agttcataaa tgatgatgac 660 actccaattg cgaaaccttt aagctgggtg tgccaagaga ttctaaagct ttgtaaggat 720 tag 723 <210> 4 ​​​​​​​​​​​​Leu Pro Gly Tyr Leu Ser Ser Arg Leu Thr Pro Ser Cys Glu Pro Gly 20 25 30 Gly Leu Pro Glu Glu Trp Lys Val Ser Glu Val Gly Asp Gly Asn Leu 35 40 45 Asn Met Val Phe Ile Val Glu Gly Thr His Lys Thr Ile Ile Val Lys 50 55 60 Gln Ala Leu Pro Trp Leu Arg Ala Gly Gly Glu Gly Trp Pro Leu Ser 65 70 75 80 Leu Ser Arg Ala Gly Phe Glu Tyr Asn Val Leu Cys Gln Glu Ala Lys 85 90 95 Tyr Ala Gly His Thr Leu Ile Pro Gln Val Tyr Phe Tyr Asp Pro Glu 100 105 110 Met Ala Leu Phe Ala Met Glu Tyr Leu Thr Pro His Val Ile Leu Arg 115 120 125 Lys Glu Leu Ile Asn Gly Lys Lys Phe Pro Lys Leu Ala Glu Asp Ile 130 135 140 Gly Arg Phe Leu Ala Gln Thr Leu Phe Asn Thr Ser Asp Ile Gly Met 145 150 155 160 Ser Ala Glu Gln Lys Lys Ala Leu Thr Ala Glu Phe Ala Leu Asn His 165 170 175 Glu Leu Cys Lys Ile Thr Glu Asp Leu Ile Phe Thr Glu Pro Tyr Tyr 180 185 190 Asn Ala Glu Arg Asn Asn Trp Thr Ser Pro Glu Leu Asp Asp Ala Val 195 200 205 His Lys Ala Trp Ala Asp Val Glu Met Ile Gln Val Ala Met Arg Tyr 210 215 220 Lys Tyr Lys Phe Met Thr Glu Ala Gln Ala Leu Leu His Gly Asp Leu 225 230 235 240 His Ser Gly Ser Ile Met Val Thr Asp Thr Asp Thr Lys Val Ile Asp 245 250 255 Pro Glu Phe Gly Phe Met Gly Pro Met Ala Phe Asp Ile Gly Asn Tyr 260 265 270 Ile Gly Asn Leu Leu Leu Ala Tyr Phe Ser Arg Pro Gly Trp Asp Ala 275 280 285 Asn Glu Gln Arg Arg Ala Asp Tyr Gln Glu Trp Leu Leu Gln Gln Ile 290 295 300 Val Gln Thr Trp Ser Val Phe Thr Arg Glu Phe Arg Gln Leu Trp Asp 305 310 315 320 Asn Lys Thr Gln Gly Asp Ala Trp Ser Thr Glu Met Tyr Gln Gln Asn 325 330 335 Arg Ala Ala Leu Glu Asp Ala Gln Asp Gln Phe Phe Ala Thr Leu Leu 340 345 350 Glu Asp Ser Leu Val Asn Ala Gly Ile Glu Met Asn Arg Arg Ile Ile 355 360 365 Gly Phe Ala Gly Val Ala Glu Leu Lys Gln Ile Glu Asn Thr Glu Leu 370 375 380 Arg Ala Gly Cys Glu Arg Arg Ala Leu Thr Met Ala Arg Asp Leu Ile 385 390 395 400 Val Asn Ala Arg Gln Phe Lys Asn Met Asp Ser Val Ile Gln Ser Ala 405 410 415 Lys Val Lys <210> 5 <211> 239 <212> PRT <213> Artificial Sequence <400> 5 Met Ala Thr Pro His Ile Asn Ala Glu Met Gly Asp Phe Ala Asp Val 1 5 10 15 Val Leu Met Pro Gly Asp Pro Leu Arg Ala Lys Tyr Ile Ala Glu Thr 20 25 30 Phe Leu Glu Asp Ala Arg Glu Val Asn Asn Val Arg Gly Met Leu Gly 35 40 45 Phe Thr Gly Thr Tyr Lys Gly Arg Lys Ile Ser Val Met Gly His Gly 50 55 60 Met Gly Ile Pro Ser Cys Ser Ile Tyr Thr Lys Glu Leu Ile Thr Asp 65 70 75 80 Phe Gly Val Lys Lys Ile Ile Arg Val Gly Ser Cys Gly Ala Val Leu 85 90 95 Pro His Val Lys Leu Arg Asp Val Val Ile Gly Met Gly Ala Cys Thr 100 105 110 Asp Ser Lys Val Asn Arg Ile Arg Phe Lys Asp His Asp Phe Ala Ala 115 120 125 Ile Ala Asp Phe Asp Met Val Arg Asn Ala Val Asp Ala Ala Lys Ala 130 135 140 Leu Gly Ile Asp Ala Arg Val Gly Asn Leu Phe Ser Ala Asp Leu Phe 145 150 155 160 Tyr Ser Pro Asp Gly Glu Met Phe Asp Val Met Glu Lys Tyr Gly Ile 165 170 175 Leu Gly Val Glu Met Glu Ala Ala Gly Ile Tyr Gly Val Ala Ala Glu 180 185 190 Phe Gly Ala Lys Ala Leu Thr Ile Cys Thr Val Ser Asp His Ile Arg 195 200 205 Thr His Glu Gln Thr Thr Ala Ala Glu Arg Gln Thr Thr Phe Asn Asp 210 215 220 Met Ile Lys Val Ala Leu Glu Ser Val Leu Leu Gly Asp Lys Glu 225 230 235 <210> 6 <211> 240 <212> PRT <213> Artificial Sequence <400> 6 Met Thr Ser Lys Lys Val Ile Leu Val Ala Leu Ser Gly Cys Ser Ser 1 5 10 15 Ser Gly Lys Thr Thr Ile Ala Lys Leu Thr Ala Ser Leu Phe Thr Lys 20 25 30 Ala Thr Leu Ile His Glu Asp Asp Phe Tyr Lys His Asp Asn Glu Val 35 40 45 Pro Val Asp Ala Lys Tyr Asn Ile Gln Asn Trp Asp Ser Pro Glu Ala 50 55 60 Leu Asp Phe Lys Leu Phe Gly Lys Glu Leu Asp Val Ile Lys Gln Thr 65 70 75 80 Gly Lys Ile Ala Thr Lys Leu Ile His Asn Asn Asn Val Asp Asp Pro 85 90 95 Phe Thr Lys Phe His Ile Asp Arg Gln Val Trp Asp Glu Leu Lys Ala 100 105 110 Lys Tyr Asp Ser Ile Asn Asp Asp Lys Tyr Glu Val Val Ile Val Asp 115 120 125 Gly Phe Met Ile Phe Asn Asn Thr Gly Ser Lys Phe Asp Leu 130 135 140 Lys Ile Leu Val Arg Ala Pro Tyr Glu Val Leu Lys Lys Arg Ala 145 150 155 160 Ser Arg Lys Gly Tyr Gln Thr Leu Asp Ser Phe Trp Val Asp Pro Pro 165 170 175 Tyr Tyr Phe Asp Glu Phe Val Tyr Glu Ser Tyr Arg Ala Asn His Ala 180 185 190 Gln Leu Phe Val Asn Gly Asp Val Glu Gly Leu Leu Asp Pro Arg Lys 195 200 205 Serving Lys Asn Ile Lys Glu Phe Ile Asn Asp Asp Asp Thr Pro Ile Ala 210 215 220 Lys Pro Leu Ser Trp Val Cys Gln Glu Ile Lys Leu Cys Lys Asp 225 230 235 240

Claims

1. A method for enzyme-catalyzed synthesis of β-nicotinamide mononucleotide (NMN), characterized in that: Includes the following steps: Step 1: By constructing enzyme-engineered bacteria, glycerol kinase expression strains BL21(DE3)-TK-SMTK, BL21(DE3)-EcPNPm and BL21(DE3)-SC-NRK were obtained and stored in glycerol tubes; Step 2: The glycerol kinase expressing strains were cultured, expression was induced, and enzyme solutions were prepared to obtain TK-SMTK enzyme solution, EcPNPm enzyme solution and SC-NRK enzyme solution; Step 3: Using D-ribose as the starting substrate, an enzyme-catalyzed reaction was carried out using TK-SMTK enzyme solution, EcPNPm enzyme solution and SC-NRK enzyme solution, while ATP was added, to synthesize a solution containing β-nicotinamide mononucleotide.

2. The method for enzyme-catalyzed synthesis of β-nicotinamide mononucleotide (NMN) according to claim 1, characterized in that: The specific steps for constructing the enzyme-engineered bacteria described in step 1 are as follows: EC-SMTK, EcPNPm, and SC-NRK were cloned into the Ndel and Baml restriction enzyme sites of plasmid pET28a(+), respectively. The plasmids were then transformed into BL21(DE3) competent cells using the calcium chloride method. The BL21(DE3) competent cells were spread onto plates containing kanamycin and cultured. Single colonies were picked and verified by enzyme digestion to obtain glycerol kinase expression strains BL21(DE3)-TK-SMTK, BL21(DE3)-EcPNPm, and BL21(DE3)-SC-NRK, respectively, which were then stored in glycerol tubes.

3. The method for enzyme-catalyzed synthesis of β-nicotinamide mononucleotide (NMN) according to claim 2, characterized in that: The EC-SMTK encoding gene is SEQ ID NO: 1, and the amino acid sequence is SEQ ID NO: 4; the EcPNPm encoding gene is SEQ ID NO: 2, and the amino acid sequence is SEQ ID NO: 5; the SC-NRK encoding gene is SEQ ID NO: 3, and the amino acid sequence is SEQ ID NO:

6.

4. The method for enzyme-catalyzed synthesis of β-nicotinamide mononucleotide (NMN) according to claim 1, characterized in that: The steps for culturing the glycerol kinase expression strain, inducing expression, and preparing the enzyme solution in step 2 are as follows: Glycerol kinase expressing strains were taken from glycerol tubes and inoculated into LB medium containing 50 mg / L kanamycin at a 1% inoculation rate. The culture was incubated statically at 37°C for 12 hours to obtain activated bacterial solution. This activated bacterial solution was then transferred to LB medium containing 50 mg / L kanamycin at a 5% inoculation rate and incubated at 36–38°C with shaking at 200 rpm for 2–3 hours until OD (exponential growth) was achieved. 600nm When the concentration reaches 0.6-0.8, add 0.2 mM isopropyl-β-D-thiogalactoside to the culture medium and induce expression of the target protein at 29-31℃ and 200 rpm for 4-5 h. After induction, centrifuge the bacterial culture at 4℃ and 12000 rpm for 2 min, discard the supernatant, invert the centrifuge tube to drain the residual culture medium, resuspend the bacterial cells in a 1:4 volume ratio with pH 7.4 lysis buffer, and sonicate for 2 min to lyse the bacterial cells. After centrifugation, collect the supernatant to obtain TK-SMTK enzyme solution, EcPNPm enzyme solution and SC-NRK enzyme solution.

5. The method for enzyme-catalyzed synthesis of β-nicotinamide mononucleotide (NMN) according to claim 1, characterized in that: The specific steps for synthesizing β-nicotinamide mononucleotide using the enzyme solution of Tk-SMTK, EcPNPm, and SC-NRK in step 3 are as follows: 1) Mix tris(hydroxymethyl)aminomethane hydrochloride buffer, D-ribose solution, magnesium chloride solution and sodium ATP solution at pH 7.2, preheat at 23~27℃ for 5~6 min, add TK-SMTK enzyme solution, and then incubate at 33~37℃ and 120 rpm in a water bath shaker for 1~1.5 h to obtain reaction solution 1; 2) Add nicotinamide solution and EcPNPm enzyme solution to reaction solution 1 and incubate at 33~37℃ for 85~95 min to obtain reaction solution 2; 3) Add SC-NRK enzyme solution to reaction solution 2, and add 2mM ATP sodium salt solution to maintain the concentration. React at 28~32℃ for 3~3.5h to obtain a solution containing β-nicotinamide mononucleotide.

6. The method for enzyme-catalyzed synthesis of β-nicotinamide mononucleotide (NMN) according to claim 5, characterized in that: 1) The final concentrations of the tris(hydroxymethyl)aminomethane hydrochloride buffer and D-ribose solution at pH 7.2 are 50 mM, the final concentration of the magnesium chloride solution is 5 mM, the final concentration of the sodium ATP solution is 10 mM, and the final concentration of the TK-SMTK enzyme solution is 5 U / mL; the tris(hydroxymethyl)aminomethane hydrochloride buffer, D-ribose solution, magnesium chloride solution, sodium ATP solution, and TK-SMTK enzyme solution at pH 7.2 are mixed in a volume ratio of 1:1:1:1:1; 2) The final concentration of the nicotinamide solution is 20 mM, the final concentration of the EcPNPm enzyme solution is 2 U / mL, and reaction solution 1, nicotinamide solution, and EcPNPm enzyme solution are mixed in a volume ratio of 5:1:1; 3) The final concentration of the SC-NRK enzyme solution is 2 U / mL, and reaction solution 2 and SC-NRK enzyme solution are mixed in a volume ratio of 7:

1.

7. The method for enzyme-catalyzed synthesis of β-nicotinamide mononucleotide (NMN) according to claim 5, characterized in that: During the water bath shaker culture described in section 1), 100 μL of reaction solution 1 was taken every 10 min and 10 μL of 0.5 M ethylenediaminetetraacetic acid was added to terminate the reaction. The amount of ribose-1-phosphate generated was then detected using a Dionex IonPac PA100 anion exchange column.

8. The method for enzyme-catalyzed synthesis of β-nicotinamide mononucleotide (NMN) according to claim 5, characterized in that: 2) During the culture period described, 50 μL of reaction solution 2 was added to 50 μL of methanol to precipitate the protein. After centrifugation, the supernatant was analyzed by HPLC to determine the amount of nicotinamide nucleoside produced. 18 The column and mobile phase consisted of 10 mM potassium dihydrogen phosphate at pH 4 and methanol in a volume ratio of 95:

5.

9. The method for enzyme-catalyzed synthesis of β-nicotinamide mononucleotide (NMN) according to claim 1, characterized in that: The specific steps for synthesizing β-nicotinamide mononucleotide using the enzyme solution of TK-SMTK, EcPNPm, and SC-NRK in step 3 are as follows: D-ribose, ATP, magnesium chloride, sodium hexametaphosphate, and nicotinamide were added to a three-necked round-bottom flask, dissolved in deionized water, and the pH was adjusted to 7.0–7.5 with 6 mol / L sodium hydroxide solution. The system was heated to 35–37 °C, and TK-SMTK, polyphosphokinase, EcPNPm, and SC-NRK were added sequentially to initiate the reaction. During the reaction, 6 mol / L hydrochloric acid was used to maintain the pH within the range of 7.0–7.5, and the temperature was maintained at 35–37 °C. After the pH of the reaction system stabilized, a sample was taken for HPLC analysis. The reaction was terminated after confirming that D-ribose was completely converted to cytidine 5'-phosphate with no residue, yielding a solution containing β-nicotinamide mononucleotide.

10. The method for enzyme-catalyzed synthesis of β-nicotinamide mononucleotide (NMN) according to claim 9, characterized in that: D-ribose, ATP, magnesium chloride, sodium hexametaphosphate, nicotinamide, and deionized water are mixed in a mass ratio of 4:0.4:1:8:3:

200. TK-SMTK, polyphosphokinase, EcPNPm, and SC-NRK are added in a mass ratio of 5:5:4:6.

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